伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的体外研究

目的研究伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的作用。方法选取10名全身及牙周组织健康受试者,分离外周血淋巴细胞,在有／无伴放线放线杆菌情况下培养0—96h,用荧光探针（AnnexinV—FITC、PI、CD69-TC7）进行标记,并进行流式细胞仪检测。结果全淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为13．42±2．88、22．74±2．18、46．92±4．28,全淋巴细胞组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为8．46±2．53、6．36±2．36、9．36±2．67,2组间存在明显差异（P〈0．01）。CD69加淋巴细胞加伴放线放线杆菌组和CD69＋淋巴细胞组AnnexinV＋／PI-细胞百分数除48h外的4个时间点上都无明显差异（P〉0．05）。CD69＋淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在各个时间点上都明显高于全淋巴细胞加伴放线放线杆菌组（P〈0．01）。结论伴放线放线杆菌能够诱导人外周血淋巴细胞活化,并且能够通过活化促进其凋亡。     

伴放线放线杆菌产生的细胞致死膨胀毒素及其与牙周病的关系

伴放线放线杆菌能产生一种热不稳定蛋白——细胞致死膨胀毒素。该毒素主要由三个相邻的基因编码，分别为cdtA，cdtB，cdtC，对应的蛋白产物分别是CDTA，CDTB，CDTC，它们构成三聚体的全毒素。它能引起细胞体积膨胀，使细胞停滞在G2／M周期而不能进入分裂期，通过线粒体途径导致淋巴细胞凋亡，诱导细胞因子的合成和分泌。由于该毒素具有多种细胞毒性，在牙周炎的发生发展中可能具有重要的致病作用。     

伴放线放线杆菌产生的细胞致死膨胀毒素及其与牙周病的关系

伴放线放线杆菌能产生一种热不稳定蛋白——细胞致死膨胀毒素。该毒素主要由三个相邻的基因编码,分别为cdtA,cdtB,cdtC,对应的蛋白产物分别是CDTA,CDTB,CDTC,它们构成三聚体的全毒素。它能引起细胞体积膨胀,使细胞停滞在G2/M周期而不能进入分裂期,通过线粒体途径导致淋巴细胞凋亡,诱导细胞因子的合成和分泌。由于该毒素具有多种细胞毒性,在牙周炎的发生发展中可能具有重要的致病作用。     

伴放线放线杆菌细胞致死性膨胀毒素研究进展

伴放线放线杆菌产生一种毒性蛋白成分——细胞致死性膨胀毒素（cytolethal distending toxin,CDT）。该毒素由3个相邻基因cdtA（669 bp）、cdtB（852 bp）、cdtC（561 bp）编码的蛋白亚基CdtA（28 000 Da）、CdtB（32 000 Da）、CdtC（20 000 Da）组成异源三聚体全毒素。CDT引起细胞膨胀,导致细胞周期阻滞,抑制细胞增殖,还能诱导淋巴细胞凋亡,影响宿主免疫功能,诱导细胞因子分泌等,在牙周病的发生发展过程中发挥至关重要的作用。本文就伴放线放线杆菌CDT各亚基及其致病机制作一综述。     

伴放线放线杆菌与致龋菌生长关系的体外研究

目的:观察伴放线放线杆菌(A.actinomycetemcomitans)与4种致龋菌,即变异链球菌(S.mutans)、嗜酸乳杆菌(L.acidophilus)、内氏放线菌(A.naeslundii)、粘性放线菌(A.viscosus)在体外共培养时相互间的生长抑制作用.方法:改良琼脂扩散法和双菌种液体培养观察A.actinomycetemcomitans与S.mutans、Lacidophilus、A.,naeslundii、A.viscosus生长的相互关系;扫描电镜及菌落计数观察双菌种生物膜形态和细菌比例的差异.实验数据用SPSS10.0软件包进行两样本均数的t检验.结果:琼脂扩散法抑菌实验表明,A.actinomycetemcomitans对S.mutans、L.acidophilus、A.naeslundii、A.viscosus的生长无抑制作用,S.mutans、L.acidophilus、A.naeslundii、A.viscosus可抑制,A.actinomycetemcomitans的生长.在双菌种液体培养中,A.actinomycetemcomitans所占比例随时间逐渐下降,差异具有显著性(P＜0.05):A.actinomycetemcomitans培养12h后,所占比例呈下降趋势,24h在S.mutans和L.acidophilus组中的比例下降为0.生物膜的观察表明,单菌种A.actinomycetemcomitans成块状散在黏附,无完整生物膜形成,4种致龋菌在48h均形成结构完整的生物膜,具有三维立体结构.与单菌种相比,加入A.actinomycetemcomitans后,致龋菌的生物膜形态未发生明显改变.更换培养液后,A.actinomycetemcomitans在混合菌种生物膜中的比例仍随时间延长下降,72h降低到最低点,各时间点的差异具有显著性(P＜0.05).结论:体外培养4种致龋菌可抑制A.actirtomycetemcomitans的生长.     

伴放线放线杆菌诱导T淋巴细胞通过Fas-FasL途径的细胞凋亡的研究

目的 :检测与Aa诱导T淋巴细胞凋亡有关的Fas(CD95 )介导的细胞凋亡途径。方法 :选取 10名全身及牙周组织健康受试者 ,分离外周血单核细胞 (PBMC) ,Aa体外刺激PBMC ,用特殊的单克隆抗体 (Fas、FasL)标记 ,并进行流式细胞仪检测。结果 :Fas和FasL的表达量明显上调。实验组Fas:2 4h - 2 7.2 2 %± 4 .10 ,96h - 6 8.2 6 %± 3.14 ;FasL :2 4h 6 .18%± 1.37,96h - 2 2 .6 4 %± 2 .82。用抗Fas单克隆抗体阻滞Fas-FasL相互作用导致明显的T细胞凋亡减少 ,百分比为 2 2 .72 %± 3.5 4 ,未加抗体的为 5 1.4 7%± 3.75。 (P <0 .0 0 0 5 ,但残余的细胞凋亡活动比阴性对照仍高。结论 :Aa诱导T淋巴细胞主要通过Fas -FasL途径凋亡 ,在细胞凋亡的分子机制上得到了数据证明 ,且有时间依赖性     

白介素10对伴放线放线杆菌内毒素诱导兔肺巨噬细胞凋亡的影响

目的：探讨白介素10（IL-10）对伴放线放线杆菌内毒素（Aa—LPS）体外诱导兔肺巨噬细胞凋亡作用的影响。方法：经兔气管肺泡灌洗获得肺泡巨噬细胞,随机分为空白对照组、Aa—LPS组、Aa—LPS＋IL-10组。按实验分组加入Aa—LPS（1汕g／mL）、IL-lo（o．1p．g／mL）,24h后裂解细胞,荧光定量PCR法检测促凋亡基因bax、p53和caspase-3的表达。结果：Aa—LPS组bax、caspase-3的表达较空白对照组明显升高（P〈0．05）,p53的表达与空白对照组比较差异无统计学意义（P〉0．05）。Aa—LPS＋IL-10组bax、p53、caspase．3的表达较Aa—LPS组降低（P〈0．05）。结论：Aa—LPS体外对肺巨噬细胞有促凋亡作用,IL-10可抑制Aa—LPS的促凋亡作用,其机制可能与细胞凋亡的线粒体途径有关。     

伴放线放线杆菌细胞致死性扩张毒素的细胞毒性研究

目的:体外诱导表达伴放线放线杆菌(Aqqregatibacter actinomycetemcomitans,Aa)细胞致死性扩张毒素(cytolethal distending toxin,CDT),观察其对中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO cell)和人宫颈癌上皮细胞(HeLa cell)的毒性作用。方法:诱导重组蛋白Aa CdtA、CdtB、CdtC的体外表达,采用镍亲和层析柱法纯化目的蛋白,体外重构Aa CDT全毒素。通过体外酶活性实验检测重组蛋白CdtB的生物学活性,并通过细胞克隆形成实验(Colony-forming experiment)及3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide,MTT]比色法进一步观察CDT对CHO细胞和HeLa细胞的毒性作用。结果:由重组蛋白Aa CdtA、CdtB、CdtC体外重构获得的Aa CDT全毒素不仅抑制CHO细胞增殖,导致CHO细胞克隆形成单位(Colony-Forming Unit,CFU)数目减少甚至消失。也可抑制He-La细胞增殖并引起包括细胞体积增大及细胞核增大增多的细胞形态学改变,并且此增殖抑制作用呈浓度依赖性。结论:体外成功构建了具有生物学活性的Aa CDT全毒素,且Aa CDT毒性发挥需要三个亚基CdtA、CdtB、CdtC同时存在并构成三聚体。     

伴放线放线杆菌血清型分析

目的 PCR法检测伴放线放线杆菌（Actinobacillus actinomy＇etemcomitans，Aa）临床分离菌株血清型，分析其与flp-1基因型的关系。方法用血清型特异性引物，通过普通PCR和多重PCR的方法对60株Aa临床分离菌株的血清型进行鉴定，并分析其与flp-1基因型的关系。结果 60株Aa临床分离菌株中血清型c型63，33％，e型23．33％，b型6.67％，a，f型各占3．33％，未检测到d型菌株；在24名被检测者中，15名检测到c型An菌株，3名检测到b型菌株，各有2名分别检测到a、e、f型菌株。fip-1基因型Ⅰ型菌株的血清型均为a型，40株Ⅱ型菌株中38株为c型，Ⅳ型菌株均为b型，11株Ⅴ型菌株中9株为e型，Ⅵ型菌株均为e型。结论 Aa血清型分布以c型为主，fip-1基因型与菌株血清型存在一定对应关系。     

伴放线共生放线杆菌表面相关物质的骨吸收活性研究

目的：通过对伴放线共生放线杆菌（Aa)表面相关物质（SAM）的提取，纯化，研究其潜在的骨吸收活性，以揭示SAM在牙周病变过程中的作用。方法：Aa国际标准菌株培养，SAM提取，过Sepharyl-S200层析柱和DEAE－Sephacel层析柱，所有的管用酚硫法测其碳水化合物含量，纯化后鉴定其骨吸收活性.结果：冻干的Aa(1.0g)产生0．1375g粗提物，通过Sepharyl-S200时出现单峰，通过DEAE－Sephacel出现两个峰，骨吸收实验证明SAM有骨吸收活性。结论：Aa的SAM在骨吸收方面具有极大潜能，提示SAM在牙周病骨损害上起到重要作用。     

Periodontitis in the primary dentition associated with Actinobacillus actinomycetemcomitans infection and leukocyte dysfunction

2 siblings, aged 5 years (girl) and 7 years (boy), with periodontitis in the primary dentition were studied. Actinobacillus actinomycetemcomitans was isolated from the gingival pockets of both children. The titers of IgG antibodies against A. actinomycetemcomitans were elevated, and the polymorphonuclear leukocytes had a decreased capacity to ingest IgG-coated latex particles. Other functions of the polymorphonuclear leukocytes were normal. After antibiotic therapy and for the 7-year-old boy, debridement of the gingival pockets A. actinomycetemcomitans was no longer detectable in the gingival pockets, and the phagocytic capacity of the polymorphonuclear leukocytes was normalized. After 2 1/2 years, A. actinomycetemcomitans was found again in the gingival pockets of the boy, and once more his polymorphonuclear leukocytes had a decreased capacity to ingest IgG-coated latex particles. The antibiotic therapy was repeated and 6 months later, the number of A. actinomycetemcomitans was very low and the phagocytic capacity of the polymorphonuclear leukocytes was back to normal. This case report suggests that A. actinomycetemcomitans may be involved in the etiology of periodontitis in the primary dentition, possibly by triggering a phagocytic dysfunction of polymorphonuclear leukocytes.     

伴放线放线杆菌外膜蛋白的研究进展

伴放线放线杆菌与牙周炎,特别是与局限性侵袭性牙周炎有着密切的关系.伴放线放线杆菌外膜蛋白作为其重要毒力因子,在牙周病的发病中起着重要的作用.本文就近年来有关伴放线放线杆菌外膜蛋白的结构特征、外膜蛋白的表现型、外膜蛋白与血清型、外膜蛋白的致病性等研究进展作一综述.     

Relationship of Actinobacillus actinomycetemcomitans serotypes to periodontal condition: prevalence and proportions in subgingival plaque

No study available has utilized the new classification scheme (the consensus report of the American Academy of Periodontology 1999) to determine the prevalence of Actinobacillus actinomycetemcomitans in different periodontal conditions. The purpose of this study was to investigate prevalence and proportions of A. actinomycetemcomitans serotypes in subgingival plaque samples from a young Taiwanese population with aggressive periodontitis, chronic periodontitis and no periodontal disease. A total of 221 subgingival plaque samples from 171 diseased subjects (70 had aggressive periodontitis, and 101 had chronic periodontitis) (mean age 25.0 +/- 8.2 yr) and 50 periodontally healthy subjects (mean age 18.4 +/- 9.5 yr) were screened for A. actinomycetemcomitans. Serotypes of A. actinomycetemcomitans were determined by an indirect immunofluorescence assay using serotype-specific polyclonal antisera to A. actinomycetemcomitans strains ATCC 29523 (serotype a), ATCC 43728 (serotype b) and ATCC 33384 (serotype c). Prevalence (% of positive samples) of A. actinomycetemcomitans was 84.3% in aggressive periodontitis, 60.4% in chronic periodontitis, and 64.0% in periodontally healthy subjects. Proportions of A. actinomycetemcomitans (mean percentage per total bacteria) in periodontally healthy subjects were significantly lower than in aggressive periodontitis subjects. The proportion of serotype b in subjects with aggressive periodontitis and subjects with chronic periodontitis were significantly greater than that in periodontally healthy subjects. The proportion of serotype c in periodontally healthy subjects was much higher than that in chronic periodontitis subjects. The results of this study suggest that prevalence and proportions of A. actinomycetemcomitans are significantly greater in patients with aggressive periodontitis than in those with chronic periodontitis. Serotype b is the predominant serotype of A. actinomycetemcomitans in patients with diseased periodontal conditions. Serotype c is a more common serotype detected in periodontally healthy subjects.     

Prevalence of Actinobacillus actinomycetemcomitans in an ethnic adult Chinese population

AIM: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population. METHOD: Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon. RESULTS: A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A. actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652-like promoter. CONCLUSIONS: The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis.     

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.     

Immunodominant antigens of Actinobacillus actinomycetemcomitans serotypes a and c in high-responder patients

This study was undertaken to examine the characteristics of the immunodominant antigens of Actinobacillus actinomycetemcomitans serotypes a and c. The top responders for A. actinomycetemcomitans serotypes a and c were selected (19 for serotype a and 21 for serotype c) from 150 clinically characterized patients. Competition assays revealed that 9 of 19 of these patients were reacting specifically to serotype a and 12 of 21 for serotype c. Limiting dilution analysis on Western blots revealed that most antigen bands apparent at low dilution disappeared as the patient's serum was diluted. The antigen band(s) remaining at the endpoint or the dilution corresponding to the antibody titer were defined as immunodominant. For serotype a there were several different immunodominant antigens but none was present in more than half of the subjects. For serotype c the immunodominant antigens included a number of discrete bands and a diffuse smeared polysaccharide band. Only 2 of these antigens were present in the majority of the high-responders: 92% had the smeared antigen and 67% had a 15 kDa antigen. The 15 kDa band was a protein common to all A. actinomycetemcomitans serotypes. The smeared antigen was unaffected by protease K treatment and gave a reaction of identity with the serotype c specific rabbit antiserum. This rabbit antiserum is specific for a mannan carbohydrate and does not react with LPS (23). Therefore, the smeared immunodominant antigen appears to be a polysaccharide containing mannan.     

Quantitative aspects of the subgingival distribution of Actinobacillus actinomycetemcomitans in a patient with localized juvenile periodontitis

Abstract The subgingival microflora in a patient with localized juvenile periodontitis was studied. Of the 97 sites investigated, 28 (29%) showed attachment loss. A correlation was found between the number of Actinobacillus actinomycetemcomitans cells and the clinical attachment level and probing pocket depth. Of the 97 test sites, 70 (73%) were positive for A. actinomycetemcomitans. Of the total number of A. actinomycetemcomitans cells isolated from this patient, more than 99% were found at sites with attachment loss, <1 % being present at sites without attachment loss. The mean percentage of A. actinomycetemcomitans was 21.2% at sites with attachment loss and 0.45% at sites without attachment loss. The distribution of Porphyromonas gingilis showed a symmetrical pattern, being present at the 1st molar and 2nd premolar sites in all quadrants and at the lower incisor sites. This species was absent at multiple sites showing overt attachment loss.     

Actinobacillus actinomycetemcomitans in a rural adult population in southern Thailand

The prevalence of Actinobacillus actinomycetemcomitans isolates was examined in a rural population of southern Thailand. Sixty individuals aged 30-39 and 50-59 years were randomly selected from a group of 363 persons, living in four villages, who had been clinically examined previously. A subgingival plaque sample was taken with a curette from the mesial aspect of the two upper and lower first molars. Each sample was dispersed in 3.3 ml of VMGA III transport medium and spread onto Trypticase Soy Broth with Bacitracin and Vancomycin (TSBV)-agar plates on the same day. After incubation in 10% CO2 for 5 days the plates were examined for typical A. actinomycetemcomitans colonies which were tested for catalase activity. Each strain was further tested for biochemical characteristics, serotyped against serotype-specific antisera a-e and ribotyped after DNA digestion using the restriction endonucleases HindIII and EcoRI. For 53 (88%) of the 60 individuals, A. actinomycetemcomitans was present in at least one subgingival sample, which is considerably higher than the prevalence in Western European adults. In 11 individuals, two or three different strains were found. Serotypes a and c were the most prevalent, and serotype b was found only once among 46 tested isolates. Eleven ribotypes were found among the 46 strains. While the same ribotype could be found among individuals of the same village, no ribotype of A. actinomycetemcomitans was unique for individuals of any one village. The study demonstrated a high prevalence of A. actinomycetemcomitans among adults of the rural population of southern Thailand and indicates that this species is present as part of the resident oral flora in this population.     

Antigenic cross-reactivity and sequence homology between Actinobacillus actinomycetemcomitans GroEL protein and human fibronectin

The immunologic cross-reactivity between human fibronectin and Actinobacillus actinomycetemcomitans GroEL was examined. Analyses by SDS-PAGE/Western immunoblotting and ELISA showed that a polyclonal antibody directed against the purified GroEL protein of A. actinomycetemcomitans, but not against the Escherichia coli GroEL, cross-reacts with human fibronectin. No antigenic cross-reactivity was observed between anti-A. actinomycetemcomitans GroEL antibody and type IV collagen, another important constituent of the basement membrane. A comparative analysis of the amino acid sequences of A. actinomycetemcomitans GroEL and human fibronectin revealed eight instances of four-amino acid sequence homology between the two proteins. Six of these tetrapeptide sequences were also shared with E. coli GroEL, suggesting that the remaining two tetrapeptides, GQLI (Glycine-Glutamine-Leucine-Isoleucine) and TGLE (Threonine-Glycine-Leucine-Glutamic acid), may be associated with the epitope that the anti-A. actinomycetemcomitans GroEL antibody specifically recognizes. Reactivity between TGLE, but not GQLI, with anti-A. actinomycetemcomitans GroEL antibody was confirmed by a biospecific interaction analysis using a biosensor technology. Although additional investigations are required, the observed phenomenon may lead to an autoimmune response and thus contribute to tissue destruction during periodontitis.     

伴放线放线杆菌在龈下菌斑和颊黏膜分布研究

目的:比较伴放线放线杆菌(actinobac illus actinomycetem com itans,A.a)在不同类型牙周炎患者龈下菌斑和颊黏膜中的分布。方法:通过聚合酶链反应(polym erase chain reaction,PCR)对侵袭性牙周炎患者(AgP)、慢性牙周炎患者(CP)、牙周健康者口腔龈下菌斑和颊黏膜中的A.a进行检测,分析该菌分别在两部位的相对含量。结果:AgP组菌斑和颊黏膜样本中A.a阳性检出率均为41.7%,分别高于CP组(菌斑16.7%、颊黏膜10.0%)和牙周健康组(菌斑和颊黏膜均为0%)。AgP组A.a在菌斑和颊黏膜的相对含量分别为38.5%和22.2%,高于CP组(菌斑19%、颊黏膜12.75%)。结论:A.a不仅存在于龈下菌斑中,也能够粘附于颊黏膜;A.a是AgP的主要优势菌也参与了CP的菌群组成。