中性粒细胞网的研究进展

中性粒细胞通过吞噬、脱颗粒和形成中性粒细胞网(NET)等方式抵抗微生物感染。自NET被发现后，研究者们即从一个全新的视角来诠释固有免疫控制和杀伤微生物的机制。本文就NET的结构、形成和在疾病中的作用以及微生物逃逸NET的作用作一综述。     

中性粒细胞在牙周组织炎症中的调节作用

中性粒细胞是人体重要的免疫细胞之一，其在微生物杀伤、炎症和免疫调节中的作用一直广受关注。作为第一个对牙周组织感染部位作出反应的先天免疫细胞，中性粒细胞进入牙周袋成为宿主反应的一部分，以对抗微生物的挑战并维持口腔内的稳态。文章对中性粒细胞在牙周组织炎症中的调节作用做一综述。     

中性粒细胞在牙周炎发生发展中的作用的研究进展

龈沟内的中性粒细胞是抵御牙周致病菌入侵的第一道防线。炎症感染时,中性粒细胞最早被募集到炎症部位,通过吞噬、脱颗粒、呼吸爆发等方式杀灭细菌。本文拟就中性粒细胞在牙周炎发生发展中的作用进行综述。     

肿瘤相关中性粒细胞诱导人粘液表皮样癌细胞上皮-间质转变和侵袭转移的功能及机制研究

目的:研究肿瘤相关中性粒细胞对人粘液表皮样癌MC3细胞上皮-间质转变(EMT)和侵袭转移过程调控的功能及机制。方法:分离培养C57小鼠骨髓中性粒细胞,Transwell检测肿瘤相关中性粒细胞对MC3侵袭和迁移的影响;Western印迹和Real-time PCR检测Vimentin等mRNA和蛋白表达;免疫荧光等检测Vimentin表达。结果:肿瘤相关中性粒细胞中TGF-β表达增加;MC3细胞与中性粒细胞共培养后Vimentin和Snail表达增加;裸鼠体内实验表明中性粒细胞浸润程度与Vimentin表达相关。结论:肿瘤相关中性粒细胞通过TGF-β诱导MC3细胞中Snail表达,并促进肿瘤侵袭和转移。     

Chemerin对口腔鳞癌中性粒细胞浸润的影响及机制探讨

目的：探讨Chemerin对口腔鳞癌（OSCC）组织中性粒细胞浸润的影响及其可能的分子机制。方法：应用免疫组织化学双染色方法，探讨中性粒细胞浸润与Chemerin表达之间的关系。利用Transwell实验、实时定量PCR(qRTPCR)、蛋白质印迹法（Western blot）、酶联免疫吸附法（ELISA）和流式细胞术检测Chemerin对中性粒细胞的趋化作用。采用SPSS 23.0软件包对数据进行统计学分析。Chemerin表达和中性粒细胞密度之间的关系采用Spearman等级相关分析法，ChemR23的敲除效率、趋化指数的计算采用方差分析，Chemerin表达和中性粒细胞密度与临床病理因素的关系采用Mann-Whitney检验，生存分析采用Kaplan-Meier法和Log rank检验，OSCC生存的危险因素采用Cox回归模型。结果：免疫组织化学双染色结果显示，Chemerin高表达与口腔鳞癌组织内中性粒细胞浸润密度呈正相关（P=0.023）,Chemerin高表达且中性粒细胞高密度与临床分期（P<0.001）、颈淋巴结转移（P<0.001）和肿瘤复发相关（P=0.0...     

Rac1在小鼠中性粒细胞抗白假丝酵母菌感染中的作用

目的评价Rac1基因在小鼠抗白假丝酵母菌感染中的作用。方法 (1)β-actin作为内参,Western blot半定量法检测两种小鼠中性粒细胞Rac1蛋白表达量;(2)趋化实验用Zigmond法经甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)诱导中性粒细胞移动,延时显微镜观察中性粒细胞移动并拍照,细胞追踪软件分析小鼠中性粒细胞趋化功能;(3)fMLP诱导小鼠中性粒细胞过氧化物的产生应用异氨基苯二酰肼化学发光分析法通过微孔板发光检测仪检测过氧化物酶含量,采用细胞色素c还原法分析豆蔻酸-佛波醇-乙酸酯(PMA)引发中性粒细胞产生的过氧化物。结果假丝酵母菌耐受BALB/c小鼠中性粒细胞Rac1蛋白表达量高于假丝酵母菌易感CBA/CaH小鼠;BALB/c小鼠中性粒细胞趋化运动功能较CBA/CaH小鼠强(P<0.05);经fMLP和PMA诱导后,BALB/c小鼠中性粒细胞过氧化物的产生量高于CBA/CaH小鼠(P<0.05)。结论在小鼠中性粒细胞抗白假丝酵母菌感染过程中,Rac1能增强小鼠中性粒细胞的杀伤能力。     

Racl在小鼠中性粒细胞抗白假丝酵母菌感染中的作用

目的 评价Racl基因在小鼠抗白假丝酵母菌感染中的作用.方法 (1)β-actin作为内参,Western blot半定量法检测两种小鼠中性粒细胞Rael蛋白表达量;(2)趋化实验用Zigmond法经甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)诱导中性粒细胞移动,延时显微镜观察中性粒细胞移动并拍照,细胞追踪软件分析小鼠中性粒细胞趋化功能;(3)fMLP诱导小鼠中性粒细胞过氧化物的产生应用异氨基苯二酰肼化学发光分析法通过微孔板发光检测仪检测过氧化物酶含量,采用细胞色素c还原法分析豆蔻酸-佛波醇-乙酸酯(PMA)引发中性粒细胞产生的过氧化物.结果 假丝酵母菌耐受BALB/c小鼠中性粒细胞Racl蛋白表达量高于假丝酵母菌易感CBA/CaH小鼠:BALB/c小鼠中性粒细胞趋化运动功能较CBA/CaH小鼠强(P<0.05):经fMLP和PMA诱导后,BALB/c小鼠中性粒细胞过氧化物的产生量高于CBA/CaH小鼠(P<0.05).结论 在小鼠中性粒细胞抗白假丝酵母菌感染过程中,Racl能增强小鼠中性粒细胞的杀伤能力.     

中性粒细胞胞外诱捕网与牙周炎高凝状态的相关性研究

目的 探究中性粒细胞胞外诱捕网（neutrophil extracellular traps, NETs）对重度牙周炎患者凝血指标的影响。方法 纳入20例重度牙周炎患者作为试验组,20例健康人作为对照组,记录牙周指标、凝血指标。ELISA测定外周血中NETs标志物cf-DNA和MPO-DNA的含量,免疫荧光显微镜观察不同来源中性粒细胞受到刺激后产生NETs的能力。分析NETs标志物与凝血指标之间的相关性。结果 重度牙周炎组的牙周指标、NETs标志物、中性粒细胞的活性、凝血指标均显著高于对照组（P＜0.05）。结论 NETs在重度牙周炎患者高凝状态的形成中发挥重要作用。     

周期性中性粒细胞减少症一例

周期性中性粒细胞减少症临床表现为周期性中性粒细胞减少伴反复感染,典型临床表现包括发热、口腔溃疡、淋巴结肿大、严重感染等,发作周期约为21 d。口腔黏膜反复发作的口腔溃疡及牙龈坏死是周期性中性粒细胞减少症常见临床表现。本文报道1例周期性中性粒细胞减少症病例。     

内毒素耐受对共培养的中性粒细胞和单核细胞分泌TNF-α和IL-8的影响

目的 观察脂多糖（lipopolysaccharides,LPS）诱导的耐受对共培养的中性粒细胞和人单核细胞株THP-1细胞表达抗炎因子肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）和趋化因子白介素-8（Inter leukin-8, IL-8)水平的影响。 方法 采用1 μg/mL牙龈卟啉单胞菌（Porphyromonas gingivalis, P. gingivalis）LPS 或1 μg /mL大肠杆菌（Escherichia coli,E.coli）LPS分别刺激共培养的中性粒细胞和THP-1细胞24 h,洗脱后,再次刺激24 h,诱导细胞产生耐受。采用ELISA技术检测细胞条件培养液中 TNF-α 和 IL-8 表达水平的变化。 结果 P.gingivalis LPS或E.coli LPS单次刺激后,共培养的中性粒细胞和THP-1细胞分泌TNF-α和IL-8的水平均较刺激前明显增高（P＜0.05）;P.gingivalis LPS和E.coli LPS重复刺激后,共培养的中性粒细胞和THP-1细胞分泌的TNF-α水平较单次刺激后明显降低（P＜0.05）,但IL-8水平较单次刺激后明显增高（P＜0.05）。 结论 共培养的中性粒细胞和单核细胞产生的内毒素耐受可能抑制TNF-α表达,促进IL-8表达,进而可能影响牙周组织的炎症和免疫反应。     

Extracellular deoxyribonuclease production by periodontal bacteria

Palmer LJ, Chapple ILC, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodont Res 2012; 47: 439–445. © 2011 John Wiley & Sons A/S Background and Objective: Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host’s immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. Material and Methods: To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA‐supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Results: Twenty‐seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. Conclusion: The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.     

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net‐like fibers comprising DNA and antimicrobial components such as histones, LL‐37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram‐negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA‐ and nucD‐encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg²⁺ and Ca²⁺ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.     

The etiological consideration of oxidized low-density lipoprotein in periodontitis

BackgroundEpidemiologically, correlations between periodontal disease activity and CVD/serum lipid-related condition have been reported. Known mediators of these links include triglycerides, oxidized LDL (oxLDL) and inflammatory cytokines such as TNF-α supplied by adipocytes as well as oxidative degeneration products of these lipids. In this review, we focused on oxidized LDL and considered the relationship between periodontal disease and systemic conditions.HighlightThe degree of oxidation in the periodontal pocket can be evaluated by analyzing the Gingival Cervicular Fluid (GCF), which can be easily collected with paperpoint. The oxLDL/LDL ratio in GCF has been shown to be 17 times as high as that in blood, and IL-8 and IL-1β were also abundantly found in GCF. Periodontal treatment significantly lowers oxLDL levels in not only GCF but also plasma. In addition, there has been growing body of evidence that periodontal infections by periodontopathic bacteria affect arteriosclerosis. On the other hands, neutrophil extracellular traps (NETs), a form of innate immune responses, reportedly play a role as a defense mechanism in the periodontal pockets. However, the regulatory mechanism of NETs in periodontal pocket is still unknown. Recently, NETs induced by oxidized cholesterol have been reported to be involved in inflammatory damage to vascular endothelial cells.ConclusionFurther understanding of the newly discovered roles of oxLDL in the defense and destruction of periodontal tissues are anticipated.     

Neutrophil Extracellular Traps Exert Potential Cytotoxic and Proinflammatory Effects in the Dental Pulp

IntroductionNeutrophil extracellular traps (NETs) are an important innate immune mechanism aimed at limiting the dissemination of bacteria within tissues and localizing antibacterial killing mechanisms. There is significant interest in the role of NETs in a range of infectious and inflammatory diseases; however, their role in diseased pulp has yet to be explored. Our aim was to determine their relevance to infected pulp and how their components affect human dental pulp cell (HDPC) responses.MethodsDiseased pulp tissue was stained for the presence of extracellular DNA and elastase to detect the presence of NETs. Bacteria known to infect pulp were also assayed to determine their ability to stimulate NETs. Coculture studies and NET component challenge were used to determine the effect of extracellular NET release on HDPC viability and inflammatory response. NET-stimulated HDPC secretomes were assessed for their chemotactic activity for lymphocytes and macrophages.ResultsData indicate that NETs are present in infected pulp tissue and whole NETs, and their histone components, particularly H2A, decreased HDPC viability and stimulated chemokine release, resulting in an attraction of lymphocyte populations.ConclusionsNETs are likely important in pulpal pathogenesis with injurious and chronic inflammatory effects on HDPCs, which may contribute to disease progression. Macrophages are chemoattracted to NET-induced apoptotic HDPCs, facilitating cellular debris removal. NETs and histones may provide novel prognostic markers and/or therapeutic targets for pulpal diseases.     

Nitric oxide synthesis and severity of human periodontal disease

The expression of the inducible nitric oxide synthase enzyme (iNOS) is a response to an inflammatory stimulus and produces a large amount of nitric oxide (NO), which may act as a cytotoxic molecule against the invading microorganism and may be related to both harmful and beneficial effects to tissues. OBJECTIVE AND MATERIAL AND METHODS: In order to further characterize the presence of NO in human periodontal disease, we undertook a quantitative study of iNOS positive cells in samples of clinically healthy gingival tissues, plaque-induced gingivitis and localized chronic periodontitis using immunohistochemistry. RESULTS: A significant increase in the number of iNOS+ cells mm-2 was found in the samples of the gingivitis and periodontitis compared with those of the control. In all groups most of the polymorphonuclear cells showed intense immunoreactivity for iNOS independent of the disease stage, and the percentage of iNOS+ polymorphonuclear cells increased significantly in periodontal disease when compared with the control. CONCLUSION: Our results indicate that iNOS increases in the presence of periodontal disease. In addition, our findings suggest that polymorphonuclear cells present an additional activation pathway in periodontal disease, expressing significant iNOS and probably representing an important source of NO in human periodontal disease that has not been previously reported.     

Neutrophil elastase in crevicular fluid: comparison of a middle-aged general population with healthy and periodontitis groups

Abstract Neutrophil elastase (NE) was measured in crevicular fluid (GCF) collected from 3 subject groups. GCF was harvested at a single visit of subjects with periodontal health (n=21) and with periodontitis (n=28). Samples were obtained from 132 middle-aged, middle-class health conscious patients of a health maintenance organization (HMO) at baseline and 1 year later. GCF NE was higher in periodontitis than in health. Mean GCF NE of HMO subjects was much closer to health than to periodontitis. Few members of the HMO population had enzyme levels typical of periodontitis. Subjects and sites of the HMO population were segregated into 3 categories based on enzyme levels of the healthy and periodontitis subjects. Most HMO subjects and sites were in the activity category corresponding to healthy subjects. Only a small portion were in the activity category common in periodontitis. Enzyme levels in the highest activity category at both samplings were infrequent. High enzyme levels in the HMO population were not associated with attachment loss. Thus, assay of GCF NB provided little evidence of disease in a middle-aged, middle-class health conscious general population. This finding confirms an analysis of epidemiological surveys which concluded that a population such as studied here would not benefit from periodontal diagnostic testing.     

Oral periopathogens and systemic effects

Management of oral biofilms allows dentists to help control the pathogens responsible for periodontal disease and decay. Increasing evidence indicates that the oral system is a portal for pathogenic microorganisms. This is a cumulative situation with systemic effects that can overcome an individual's resistance threshold, culminating in systemic sequela. New evidence indicates that controlling these oral pathogens has systemic benefits, as oral pathology is related to cardiovascular and respiratory disease, diabetes, and systemic inflammatory responses, as well as low birth weight and pre-term deliveries. Some insurance companies now cover periodontal scaling for gingivitis and periodontal disease for pregnant women and patients at risk for pregnancy.     

The periodontal abscess: a review

BACKGROUND/AIMS: The periodontal abscess is a frequent periodontal condition in which periodontal tissues may be rapidly destroyed. Its importance is based on the possible need of urgent care, the affectation of tooth prognosis, and the possibility of infection spreading. There is scant information in the scientific literature regarding this condition and most of it has been published as case reports and text books, where conclusions are not evidence-based, but rather empirical observations made by recognised clinicians. The aim of this review was to critically analyse all available information on this subject in the dental and medical literature, including information on its prevalence, proposed etiologies and pathogenesis, diagnosis, microbiology and treatment alternatives. SUMMARY: The periodontal abscess is the 3rd most frequent dental emergency, and it is specially prevalent among untreated periodontal patients and periodontal patients during maintenance. Different etiologies have been proposed, and 2 main groups can be distinguished, depending on its relation with periodontal pockets. In the case of a periodontitis-related abscess, the condition may appear as an exacerbation of a non-treated periodontitis or during the course of periodontal therapy. In non-periodontitis related abscesses, impaction of foreign objects, and radicular abnormalities are the 2 main causes. The abscess microflora seems to be similar to that of adult periodontitis, and it is dominated by gram-negative anaerobic rods, including well-known periodontal pathogens. Complications and consequences include tooth loss and the spread of the infection to other body sites. Diagnosis and treatment is mainly based on empiricism, since evidence-based data are not available. The role of systemic antibiotics, in the treatment of periodontal abscesses, is especially controversial.     

Delayed neutrophil apoptosis in chronic periodontitis patients

BACKGROUND, AIMS: Neutrophil cells constitute the first defense barrier against the oral bacterial challenge in the periodontium. Reduction of neutrophils could impair this response against periopathogenic bacteria such as Porphyromonas gingivalis. Our previous work implicates the apoptosis of neutrophils in the pathogenesis of periodontitis. We now demonstrate that granulocyte monocyte-colony stimulating factor (GM-CSF) present in the gingival crevicular fluid (GCF) and secreted during the immune response reduces the apoptosis of neutrophils. METHOD: In this study, the presence of GM-CSF and tumor necrosis factor-alpha (TNF-alpha) in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva. GCF was collected for 30 s using Periopaper(R) strips, and cytokines were quantified by ELISA. We used ex vivo culture of gingival tissue biopsies for 2 and 4 days in the presence of GM-CSF. Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique, and expression of Bax by immunohistochemistry. RESULTS: The presence of GM-CSF and TNF-alpha was detected in the majority of sites from periodontal patients (83.3% and 63.3%, respectively), presenting a total amount of 27.65 and 42.38 pg, respectively. GM-CSF reduces the neutrophil apoptosis determined by double staining with TUNEL and myeloperoxidase and by a reduction of Bax expression. CONCLUSIONS: These findings suggest a novel mechanism by which neutrophils specifically accumulate in adult patients with periodontitis.     

Links between periodontal disease and general health. 1. Pneumonia and cardiovascular disease

The possible link between oral and general health is based on an old concept. This paper summarizes current ideas on the role of translocation of oral pathogens to other parts of the body in the development of systemic disease. It appears that colonisation of the oral cavity by respiratory pathogens is a risk factor in the development of pneumonia in institutionalised elderly and intensive care patients. Using a chloorhexidine oral rinse may reduce the risk of pneumonia. Furthermore, periodontal disease is associated with an increased risk of cardiovascular disease. Translocation of oral microorganisms and an increase in serum concentrations of inflammatory mediators are considered to play a role in the development of cardiovascular disease. Potentially, preventive oral health care and periodontal intervention could play a part in preventing cardiovascular disease.