Invasion of vascular cells in vitro by Porphyromonas endodontalis

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.     

Bacterial virulence in the etiology of periodontal diseases

Strong relationships have been very often described between various form of periodontal disease (PD) and certain bacterial species, so that nowadays periodontal disease is recognized as an infectious disease. Destruction of periodontal supporting tissues happens as a response to very intricate host-parasite interactions. When the clinician will be able to fully understand and identify such phenomena it would be possible to succeed in a properly diagnosis and control of the active phase of periodontal disease. The first step in such a direction would be to analyze the common characteristic of some bacterial species, the so called suspected periodontopathogens. Such species namely Gram-negative, associated with the outbreak of periodontal disease have in common the capacity to disrupt the integrity of the host defences by means of the so called virulence factors. These factors may enhance the bacterial colonization or may interfere with the host response that ultimately results in periodontal support breakdown. The present review focuses on the virulence factors of the main suspected periodontopathogens evaluating the effects on the host immune response and directly on the periodontal tissues.     

慢性牙周炎致病菌与冠状动脉粥样硬化相关性研究

目的 探讨慢性牙周炎与冠状动脉粥样硬化性心脏病(简称冠心病)发病的相关性机制。方法 收集36例行冠状动脉搭桥手术并伴有慢性牙周炎患者的冠状动脉粥样硬化斑块和龈下菌斑,50例慢性牙周炎患者的龈下菌斑,采用Chelex-100法提取细菌DNA,以聚合酶链反应(PCR)法检测冠状动脉粥样硬化斑块和龈下菌斑中牙龈卟啉单胞菌(Porphyromonasgingivalis,Pg)、伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)、具核梭杆菌(Fusobacterium nucleatum,Fn)、中间普氏菌(Preuotella intermedia,Pi)、福赛斯坦纳菌(Tannerella forsythensis,Tf)5种慢性牙周炎相关致病菌。结果 36例患者冠状动脉粥样硬化斑块中,各种牙周炎相关致病菌的检出率分别为:Pg 36%,Aa 3%,Pi 14%,Fn 25%,Tf 47%;同一患者冠状动脉粥样硬化斑块及龈下菌斑中同时检出5种牙周炎相关致病菌的一致的例数:Tf 15例,Pg 13例,Fn 5例,Pi 3例,Aa 0例;冠心病组的Pi检出率高于对照组,二者差异具有统计学意义(P<0.05)。结论 在冠状动脉粥样硬化斑块中慢性牙周炎相关致病菌DNA的检出,提示慢性牙周炎相关致病菌在冠心病的发生、发展中可能起着重要的作用;中间普氏菌可能是冠心病的危险因素。     

牙周致病菌在冠心病患者龈下菌斑中的分布

目的:分析牙周致病菌在冠心病患者龈下菌斑中的分布情况,及冠心病患者龈下菌斑中牙周致病菌的分布与慢性牙周炎(chronic periodontitis,CP)病变程度之间的关系。方法:收集44例患冠心病并伴有CP患者的龈下菌斑,采用Chelex-100法提取细菌DNA,以聚合酶链反应(polymerase chain reaction,PCR)法检测龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、伴放线菌嗜血菌(Haemophilus actinomycetemcomitans,Ha)、具核梭杆菌(Fusobacterium nucleatum,Fn)、中间普氏菌(Prevotella intermedia,Pi)、福赛斯坦纳菌(Tannerella forsythensis,Tf)五种牙周炎相关致病菌。结果:44例冠心病患者的龈下菌斑中各牙周致病菌的检出率分别为:Pg 19(43.18%)、Ha 9(20.45%)、Pi 27(61.36%)、Fn 38(86.36%)、Tf 41(93.18%);轻度CP患者1例,Fn 1例(100%);中度CP患9例,其中Pg6(66.67%)、Ha 2(22.22%)、Pi 7(77.78%)、Fn 9(100%)、Tf 8(88.89%);重度CP患者34例,其中Pg13(38.24%)、Ha 7(20.59%)、Pi 20(58.82%)、Fn 28(82.35%)、Tf 33(97%)。结论:Tf、Fn可能在冠心病的发生发展中起着重要作用,冠心病患者中CP的发病和进展可能有其特定的细菌学病因。     

Bacterial invasion and persistence: critical events in the pathogenesis of periodontitis?

Periodontitis is chronic inflammation of the periodontium caused by the host's inflammatory response to plaque biofilm, which destroys tooth‐supporting soft and hard tissues. Periodontitis is a complex disease that involves interactions among three main features – microbial challenge, the host immune response, and environmental and genetic risk factors – in its pathogenesis. Although periodontitis has been regarded as the result of hyperimmune or hyperinflammatory responses to plaque bacteria, recent studies indicate that periodontal pathogens are rather poor activators and/or suppressors of the host immune response. This raises the question of how periodontal pathogens cause inflammation. To resolve this issue, in the present review we propose that bacterial invasion into gingival tissue is a key event in the initiation of periodontitis and that the persistence of these bacteria within host tissue results in chronic inflammation. In support of this hypothesis, we present the ways in which microbial, environmental and genetic risk factors contribute to bacterial invasion. It is hoped that the current model will instigate active discussion and new research to complete the puzzle of this complex disease process.     

Evidence for local production of antibodies to auto and non-self antigens in periodontal disease

Autoimmune mechanisms may contribute to periodontal disease (PD) pathogenesis; autoantibody to collagen type 1 is produced at the periodontal site and local levels are found to be higher than in serum. OBJECTIVES: To find any evidence of autoimmune destruction in diseased periodontal tissues in patients with periodontitis. The study examines the relationship of antibodies to a self antigen collagen Type 1 and antigens from two periodontal pathogens namely Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa) and a non-oral bacterium Bacteroides fragilis (Bf) in disease sites and in serum. MATERIALS AND METHODS: Granulomatous tissues from periodontally diseased sites and serum samples were obtained from 13 patients (15 sites) undergoing surgical therapy. Tissues were homogenized at 4 degrees C on Tris saline buffer [1 g (5 ml)-1], homogenate was centrifuged and the resultant supernatants were used in assays. Antibody to collagen and Aa, Pg and Bf in tissue eluates and serum were determined by competitive enzyme linked immunosorbant assay (ELISA) and conventional ELISA respectively using an alkaline phosphatase/p-nitrophenyl phosphate enzyme-substrate system. Sera from age and sex matched healthy subjects and pooled human serum were used as controls. Antibody (Ab) levels in tissues and serum were standardized by concomitant albumin assay. RESULTS: Level of antibodies to collagen type 1 in tissue was significantly higher than in serum (P = 0.0001). Antibody levels in tissue to Pg were significantly higher than in serum (P = 0.0271). Ab levels to both Aa and Bf in tissues and serum were not significantly different from each other. CONCLUSIONS: These findings confirm the process of the local production of antibodies to autoantigen namely collagen type-1 and to bacterial antigens in the granulomatous tissues housed within the periodontal lesions in patients with periodontitis.     

Subgingival colonization by Porphyromonas gingivalis

Porphyromonas gingivalis, a gram-negative anaerobe, is a major causative agent in the initiation and progression of severe forms of periodontal disease. In order to cause periodontal disease, P. gingivalis must colonize the subgingival region, a process that involves several distinct steps and multiple gene products. The organism must first navigate within the oral fluids in order to reach the hard or soft tissues of the mouth. Retention and growth of bacteria on these surfaces is facilitated by a repertoire of adhesins including fimbriae, hemagglutinins and proteinases. Once established subgingivally, P. gingivalis cells participate in intercellular communication networks with other oral prokaryotic cells and with eukaryotic cells. The establishment of these multiple interactive interfaces can lead to biofilm formation, invasion of root dentin and internalization within gingival epithelial cells. The resulting bacterial and host cellular locations, products and fate contribute to the success of P. gingivalis in colonizing the periodontal region.     

牙龈卟啉单胞菌红细胞凝集素A黏附和入侵人牙龈上皮细胞的研究

目的:检测牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)膜表面红细胞凝集素A(hemagglutinin A,HagA)黏附和入侵人牙龈上皮细胞的功能。方法:构建P.g381hagA基因的变异菌株,连接hagA基因到pYA292质粒上,并克隆到无毒性的沙门杆菌x4072菌中,通过比较变异菌株、野生菌株和表达hagA基因的沙门杆菌对人牙龈上皮细胞的黏附和入侵功能,检测HagA在此过程中的作用。结果:P.g381hagA基因变异菌株和野生菌株在对细胞的黏附和入侵过程中没有显著差别,但沙门杆菌x4072HagA表达菌株对细胞的黏附性和入侵性比对照组分别提高了3倍和4倍。结论:HagA参与了P.g381黏附和入侵牙龈上皮细胞的过程。     

Porphyromonas gingivalis-epithelial cell interactions in periodontitis

Emerging data on the consequences of the interactions between invasive oral bacteria and host cells have provided new insights into the pathogenesis of periodontal disease. Indeed, modulation of the mucosal epithelial barrier by pathogenic bacteria appears to be a critical step in the initiation and progression of periodontal disease. Periodontopathogens such as Porphyromonas gingivalis have developed different strategies to perturb the structural and functional integrity of the gingival epithelium. P. gingivalis adheres to, invades, and replicates within human epithelial cells. Adhesion of P. gingivalis to host cells is multimodal and involves the interaction of bacterial cell-surface adhesins with receptors expressed on the surfaces of epithelial cells. Internalization of P. gingivalis within host cells is rapid and requires both bacterial contact-dependent components and host-induced signaling pathways. P. gingivalis also subverts host responses to bacterial challenges by inactivating immune cells and molecules and by activating host processes leading to tissue destruction. The adaptive ability of these pathogens that allows them to survive within host cells and degrade periodontal tissue constituents may contribute to the initiation and progression of periodontitis. In this paper, we review current knowledge on the molecular cross-talk between P. gingivalis and gingival epithelial cells in the development of periodontitis.     

Invasion of Porphyromonas gingivalis into human gingival fibroblasts in vitro

Porphyromonas gingivalis, one of the important periodontal pathogens, exhibits many virulence properties. Among these, the adhesion to and invasion into host tissues are crucial for the initiation and progression of periodontal diseases. While evidence indicating the ability of this organism to adhere to and invade into epithelial cells as well as endothelial cells has accumulated, that involving the gingival fibroblasts is very limited. Therefore, this study aimed to determine the ability of P. gingivalis to invade primary cultures of human gingival fibroblasts using the antibiotic protection assay. In addition, interactions between P. gingivalis and the gingival fibroblasts were investigated using electron microscopy. The results demonstrated that P. gingivalis 381 could invade human gingival fibroblasts with an invasion efficiency of 0.17%. Using the scanning electron microscopic study, numerous filopodia were seen on the surfaces of gingival fibroblasts after P. gingivalis adhesion. The transmission electron microscopy revealed the presence of an intracellular bacterium. After 90 min incubation, the bacterium was found in the cytoplasm of the gingival fibroblasts, without membrane surrounding. Some fibroblasts contained a number of vacuoles and dilated rough endoplasmic reticulum even when bacteria were not found intracellularly. Thus, the invasion of this organism into the gingival fibroblasts may play a direct role in the destruction of the periodontal tissues and may also relate to the difficulties of eradicating the bacteria from periodontitis lesions.     

冠状动脉粥样硬化斑块中牙周炎致病菌的检测

目的探讨牙周炎与冠状动脉粥样硬化性心脏病（以下简称冠心病）发病的相关机制。方法收集31例行冠状动脉搭桥手术并伴有牙周炎的患者冠状动脉粥样硬化斑块和龈下菌斑，采用Chelex-100法提取细菌DNA，以聚合酶链反应（PCR）法检测冠状动脉粥样硬化斑块和龈下菌斑中牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）、伴放线放线杆菌（Actinobacillus actinomycetemcomitans，Aa）、具核梭杆菌（Fusobacterium nucleatum，Fn）、中间普氏菌（Prevotella intermedia，Pi）、福赛拟杆菌（Bacteroides forsythus，Bf）5种牙周炎相关致病菌。结果在31例患者冠状动脉粥样硬化斑块中，各种牙周炎相关致病菌的检出率分别为：Pg 38．7％、Aa 0％、Pi 12．9％、Fn 22．6％、Bf 38．7％；在本组同一患者冠状动脉粥样硬化斑块及龈下菌斑中同时检出Pg 5例、Aa 0例、Pi 2例、Fn 4例、Bf 8例，一致率分别为Pg 16．1％、Aa 0％、Pi 6．5％、Fn 12．9％、Bf 25．8％。结论在冠状动脉粥样硬化斑块中牙周炎相关致病菌DNA的检出，提示牙周炎相关致病菌在冠心病的发生、发展中可能起着重要的作用，为冠心病危险因素的研究和冠心病防治策略的制定提供了参考依据。     

Inhibition of Porphyromonas gingivalis hemagglutinating activity by IgY against a truncated HagA

Porphyromonas gingivalis has been implicated as an important pathogen in the development of periodontitis. Hemagglutinins have been identified as important adhesion molecules, allowing Porphyromonas gingivalis to adhere to gingival tissue cells, and to attach and lyse erythrocytes in order to uptake Fe ions as essential nutrition. One hemagglutinin, hemagglutinin A (HagA), has been molecularly cloned via functional screening for hemagglutinating activity. We previously cloned the gene encoding the 200-kDa cell-surface antigenic protein that was reacted by sera from periodontitis patients and was identified as a truncated protein of HagA by nucleotide sequence analysis. We further subcloned the gene encoding an 122-kDa protein (122k-HagA) which is a fusion protein composed of an 80-kDa truncated HagA containing the functional motif PVQNLT and a 42-kDa maltose binding protein. Passive immunization against infectious pathogens by specific antibodies produced from hen egg yolk antibody (IgY) has been extensively developed. In the present study, to develop passive immunotherapy against periodontal disease, we purified the recombinant 122k-HagA and used this to immunize hens and produce IgY. The purified IgY reacted with the recombinant 122k-HagA and the synthetic peptide containing PVQNLT, and inhibited hemagglutinating activity of Porphyromonas gingivalis. Thus, the novel IgY may be useful in the development of a passive immunization against periodontal diseases caused by P. gingivalis infection.     

Periodontal disease: modulation of the inflammatory cascade by dietary n‐3 polyunsaturated fatty acids

Periodontal disease, including gingivitis and periodontitis, is caused by the interaction between pathogenic bacteria and the host immune system. The ensuing oxidative stress and inflammatory cascade result in the destruction of gingival tissue, alveolar bone and periodontal ligament. This article reviews the underlying mechanisms and host–bacteria interactions responsible for periodontal disease and evidence that nutritional supplementation with fish oil may provide a protective effect. Historical investigations of diet and disease have highlighted an inverse relationship between ingestion of fish oil, which is high in n‐3 polyunsaturated fatty acids, and the incidence of typical inflammatory diseases such as arthritis and coronary heart disease. Ingestion of n‐3 polyunsaturated fatty acids, such as docosahexaenoic acid and eicosapentaenoic acid, results in their incorporation into membrane phospholipids, which can alter eicosanoid production after stimulation during the immune response. These eicosanoids promote a reduction in chronic inflammation, which has led to the proposal that fish oil is a possible modulator of inflammation and may reduce the severity of periodontal diseases. Tentative animal and human studies have provided an indication of this effect. Further human investigation is needed to establish the protective effects of fish oil in relation to periodontal disease.     

Cellular and soluble mediator components of the local immune response to dental plaque

The host immune response to dental plaque products leads to altered levels of activity by several types of cells involved in host defence. Cells of the periodontium which contribute to the supporting tissues of the teeth may be directly damaged by cellular attack, or their activity may be modulated by a range of soluble mediators released by host immune cells. These changes contribute to the modification of the tooth-supporting tissue which is seen in chronic inflammatory periodontal disease.     

Bacterial adhesins--their role in tubule invasion and endodontic disease

Bacterial invasion of dentinal tubules is critical to the progression of dental caries and the development of pulp and periapical disease, and may also influence the progression of periodontal disease. However, little is known about the host or bacterial mechanisms involved in tubule invasion. Recent work has demonstrated that bacterial interactions with dentine, and salivary and tissue molecules influence invasion. Salivary molecules such as mucin and immunoglobulin G (IgG) co-aggregate with bacterial cells, which inhibits dentine invasion, while deposition of dentinal tubule fluid molecules e.g. albumin, IgG, or fibrinogen within dentinal tubules also inhibits invasion. Dentine invasion by streptococci has been shown to be associated with a bacterial growth response and adhesion to unmineralized collagen, which are mediated by streptococcal cell-surface antigen I/II polypeptides. These adhesins possess diverse binding properties including binding to salivary glycoprotein, other bacteria, and to collagen. Additionally, some antigen I/II polypeptides facilitate species-specific co-invasion between streptococci and obligate anaerobes that lack the ability to invade by themselves. An understanding of the mechanisms involved in bacterial invasion of dentine should allow development of new control strategies.     

Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175

Introduction: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. Methods: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). Results: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. Discussion: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.     

The role of salivary cytokines in the etiology and progression of periodontal disease

Periodontal disease is a common oral disease in all segments of the population, regardless of their systemic health status. The disease is generalized or localized and the severity of lesions can vary at different sites. It is possible to recognize a number of different types of periodontal disease on the basis of clinical and pathological criteria, which include the extent of periodontal destruction, the age of onset and severity of disease, the distribution of lesions, the microflora involved, and the variation in host responses. The ultimate outcome of periodontal disease in adults, whether treated or untreated, depends on patient susceptibility. While periodontopathic bacteria are recognized as the etiologic agents in periodontal disease, their interaction with and modulation of the host immune response is of fundamental importance. An interplay of protection and pathology active in periodontal disease is that of T-lymphocyte cells and the subsequent production of cytokines within the periodontal tissues.     

The microbiology of periodontal disease

Periodontal disease result from mixed bacterial infections, in which both host resistance barriers and bacterial interactions are important. Approximately ten bacterial species are strongly implicated with various forms of periodontal disease, although species that cannot yet be cultivated are likely also to be relevant. New technologies have shown that pathogenic bacterial species are present in defined complexes within subgingival plaque, thus identifying specific targets for therapeutic intervention. In light of increasing antibiotic resistance amongst oral bacteria, new strategies for control of periodontal bacterial complexes must be developed that inhibit the bacterial factors necessary for colonization and destruction of host tissues.     

EMMPRIN与牙周炎的相关性研究

目的：研究EMMPRIN在牙周组织中的表达情况,分析其可能发挥的作用。方法：免疫组化、RT-PCR和western blot检测牙龈组织中EMMPRIN mRNA和蛋白表达情况。结果：临床健康和炎性牙龈组织中检测到EMMPRIN蛋白的表达,在炎性牙龈组织中的蛋白表达水平显著高于正常牙龈（P〈0.05）。Pg LPS处理后的牙龈上皮细胞（HGEC）中EMMPRIN和MMPs mRNA的表达存在个体差异：来源于临床健康个体、牙龈炎和轻度牙周炎患者的HGEC在作用前后EMMPRIN和MMPs mRNA的表达无显著差异,而来源于重度牙周炎患者的HGEC在Pg LPS刺激后mRNA的水平显著上调（P〈0.05）。结论：EMMPRIN可能参与牙周组织正常的生理改建;在炎症状态下EMMPRIN可能作为MMPs的诱导因子参与了对牙周组织的破坏,且与牙周炎的严重程度存在一定相关性。     

Unveiling the role of stress in periodontal etiopathogenesis: an evidence‐based review

Periodontal disease is a multifactorial disease affecting the supporting tissues of the teeth, resulting in progressive attachment loss and bone loss. Periodontal disease is influenced by various systemic, environmental, and psychologic factors that have the potential to alter periodontal tissues and host immune response, resulting in more severe periodontal destruction. Several studies have documented the relationship between psychosocial stress and chronic forms of periodontal disease. Stress impairs periodontal health through changes in behavior and complex interactions among the nervous, endocrine, and immune systems. The influence of stress on periodontal disease is affected by the emotional coping skills of the individual. Psychologic stress and depression may also influence the outcome of periodontal therapy. Stressful life events and coping skills are factors to consider in the risk of periodontal disease destruction and the potential for successful periodontal therapy. Therefore psychosocial stress is considered as an important risk factor for periodontal disease, and emotional stress in periodontitis patients should be diagnosed and treatment should be administered for the proper management and successful outcome of periodontal disease.