成人牙周健康状况与fimA基因型牙龈卟啉单胞菌的相关性

目的分析不同fimA基因型牙龈卟啉单胞菌（P.gingivalis）在牙周健康人群和慢性牙周炎人群中的分布,探讨不同fimA基因型P.gingivalis与成人牙周状况的相关关系。方法收集牙周健康组（136例）和慢性牙周炎组（115例）的龈下菌斑样本,采用16S rRNA PCR法检测P.gingivalis,并根据各fimA基因型（Ⅰ～Ⅴ和Ⅰb）的特异性引物检测不同fimA基因型P.gingivalis菌株的分布,计算OR值和95%可信区间。结果牙周健康组和慢性牙周炎组龈下菌斑样本中P.gingivalis阳性率分别为22.1%和81.7%,多数样本中只检测到1种fimA基因型。牙周健康组中ⅠfimA型的检出率最高（占66.7%）;慢性牙周炎组中则为ⅡfimA基因型（占43.6%）,其次为Ⅳ和Ⅰb fimA基因型。慢性牙周炎的发生与P.gingivalis的关系密切（OR=16.36）,Ⅰ、Ⅰb、Ⅱ、Ⅲ、Ⅳ、ⅤfimA基因型P.gingivalis与慢性牙周炎相关性的OR值分别为0.97、13.26、36.62、4.57、22.86、1.19;ⅡfimA基因型P.gingivalis与慢性牙周炎的相关性最强,其次为Ⅳ和Ⅰb型。结论P.gingivalis菌株的fimA基因型存在差异,特异性fimA基因型P.gingivalis可能与成人慢性牙周炎的发生关系密切。     

Periodontal Treatment Decreases Levels of Antibodies to Porphyromonas gingivalis and Citrulline in Patients With Rheumatoid Arthritis and Periodontitis

Background: Porphyromonas gingivalis has been implicated as an etiologic agent of rheumatoid arthritis (RA) because of the expression of peptidylarginine deiminase. The present study evaluates whether periodontal treatment may affect serum antibodies to P. gingivalis and citrulline levels in relation to disease activity of RA. Methods: Fifty‐five patients with RA were randomly assigned to receive oral hygiene instruction and supragingival scaling (treatment group, n = 26) or no periodontal treatment (control group, n = 29). Periodontal and rheumatologic parameters and serum levels of cytokine and inflammatory markers citrulline and immunoglobulin (Ig)G to P. gingivalis were examined at baseline and 8 weeks later. Results: Both groups did not differ statistically in any parameters except percentage of sites with probing depth and clinical attachment level ≥4 mm at baseline. The treatment group exhibited a significantly greater decrease in disease activity score including 28 joints using C‐reactive protein (DAS28‐CRP) (P = 0.02), serum levels of IgG to P. gingivalis hemin binding protein (HBP)35 (P = 0.04), and citrulline (P = 0.02) than the control group. Serum levels of IgG to P. gingivalis HBP35 were significantly correlated positively with those of anti‐cyclic citrullinated peptide antibodies (P = 0.0002). The same correlation was obtained between serum levels of IgG to P. gingivalis–sonicated extracts and those of rheumatoid factor (P = 0.02). Conclusions: These results suggest that supragingival scaling decreases DAS28‐CRP and serum levels of IgG to P. gingivalis HBP35 and citrulline in patients with RA. These observations may reflect a role of P. gingivalis in the protein citrullination, which is related to the pathogenesis of RA.     

牙龈卟啉单胞菌FimA蛋白和白细胞介素-15共表达质粒免疫小鼠的实验研究

目的观察重组质粒pIRES- fimA:IL15经滴鼻免疫BABL/c小鼠后诱导产生的血清和唾液抗体反应及白细胞介素- 15（IL- 15）对sIgA反应的调节作用。方法重组质粒pIRES- fimA:IL15与pIRES- fimA经滴鼻免疫和肌肉注射免疫BABL/c小鼠,以间接ELISA方法检测血清中IgG和唾液中sIgA抗体水平。结果重组质粒pIRES- fimA:IL15与pIRES- fimA经滴鼻免疫可诱导产生唾液sIgA反应,且该反应明显高于肌肉注射组,血清IgG水平与肌肉注射组无统计学差异。pIRES- fimA:IL15经滴鼻免疫产生的唾液sIgA滴度显著高于pIRES- fimA组,且有统计学差异（P<0.05）。结论滴鼻免疫可以作为抗牙龈卟啉单胞菌DNA疫苗有效的黏膜免疫途径,诱导循环和口腔局部抗体反应;IL- 15可以作为细胞因子佐剂通过滴鼻途径增强该疫苗诱导的sIgA反应强度。     

牙龈卟啉单胞菌与Toll样受体的研究进展

牙龈卟啉单胞菌是公认的牙周致病菌，能分泌大量的毒力因子。Toll样受体是一种识别病原相关分子模式的跨膜蛋白，在机体的先天性免疫中具有重要的作用。本文就近年来牙龈卟啉单胞菌与Toll样受体的研究进展作一综述。     

慢性牙周炎患者龈下菌斑中不同基因型牙龈卟啉单胞菌的检测

摘要 目的:建立临床标本中牙龈卟啉单胞菌(P.g)的PCR检测方法,探讨慢性牙周炎患者不同牙位的龈下菌斑中P.g基因型的差异。方法:采用培养法分离鉴定慢性牙周炎患者不同牙位龈下菌斑中P.g,同时采用PCR检测 P.g16SrDNA、prtC和fimA基因。部分扩增产物测定了核苷酸序列。结果:在66例患者的127个龈下菌斑标本中, P.g16SrDNA、prtC和fimA多重引物扩增的阳性率为9814%;PCR阳性率显著高于培养法P.g的检出率(P< 0101)。3010%的患者(18/60)同时感染了不同基因型的P.g菌株。P.g16SrDNA、prtC和fimA扩增片段的核苷酸序列同源性在98162%~100%之间。结论:本文所建立的P.g的PCR检测方法具有较高的敏感性和特异性,适用于P.g的快速临床诊断。同一患者可被不同感染来源的多株P.g同时感染。     

Porphyromonas gingivalis HmuY‐Induced Production of Interleukin‐6 and IL‐6 Polymorphism in Chronic Periodontitis

Background: In chronic periodontitis (CP), the gene polymorphism of interleukin‐6 (IL‐6) to 174C/G has been associated with the altered production of this cytokine. The aim of this pilot study is to compare the allelic and genotypic frequencies in patients with CP with control individuals without periodontitis (NP) and to measure the production of IL‐6 by whole blood cells stimulated with Porphyromonas gingivalis HmuY protein. Methods: DNA was isolated from peripheral blood cells of 49 patients with CP and 60 control individuals classified as NP, and genotyping was performed by polymerase chain reaction using sequence‐specific primers. Whole blood cells from 29 patients with CP and 30 control individuals were stimulated for 48 hours with HmuY, and IL‐6 levels were measured using enzyme‐linked immunosorbent assay. Results: The proportion of individuals carrying the G allele at position –174 of the IL‐6 gene was higher in the group with CP (85.7%) than in the normal control group (73.3%; P <0.03). P. gingivalis HmuY‐induced production of IL‐6 was higher in the group with CP (P <0.05). Conclusions: Our findings suggest that P. gingivalis HmuY may be associated with increased IL‐6 production during CP. Furthermore, patients with periodontitis and individuals with higher HmuY‐induced production of IL‐6 show a high frequency of the G allele at position –174.     

Periodontal status and serum antibody titers for Porphyromonas gingivalis fimbriae in a rural population in Japan

BACKGROUND, AIMS: The present study was undertaken to assess the periodontal status of a rural Japanese population and to study the correlation between the periodontal status and the serum antibody titers for Porphyromonas gingivalis (Pg) fimbriae. METHOD: A total of 236 individuals were examined for their periodontal conditions by the use of the community periodontal index for treatment needs (CPITN), and serum antibody titers for Pg fimbriae in their peripheral blood samples were evaluated using the enzyme-linked immunosorbent assay. RESULTS: There was a substantially larger proportion of edentulous subjects in the age group older than 60 years. The remaining teeth were 24.1, 23.2, 11.1 and 10.1 per person in the 40-49, 50-59, 60-69 and > or = 70 age groups, respectively. The % of sextants with a CPITN code of missing sextant (MS) increased towards elderly and reached >60% in the age group of > or = 70 years, as the % of the CPITN 2, 1 or 0 sextant decreased. The % of CPITN 4 and 3 sextants did not differ between different age groups and were about 6-8% and 15-20%, respectively. The % of CPITN 1 or 0 sextants was higher in female subjects than in male subjects in the 60-69 and > or = 70 age groups, while the % of CPITN 4 or 3 sextants was higher in male subjects than in female subjects in all age groups. There was no significant difference between various age groups in the mean serum antibody titers for Pg fimbriae. The mean anti-Pg fimbriae antibody titers was significantly higher for the subjects with a maximum CPITN code 4 (max.-CPITN 4 subject) than for the subjects with lower maximum CPITN codes. The antibody titers varied extensively among the max.-CPITN 4 or 3 subjects, but not among the max.-CPITN 2/1/0 or MS subjects. CONCLUSIONS: The present study demonstrated that tooth loss is a remarkable event in elderly subjects and that oral prophylaxis and mechanical debridement should be mandatory in the population examined. It was also demonstrated that the serum antibody titers against Pg fimbriae could be useful for screening individuals with moderate to severe periodontitis.     

牙龈卟啉单胞菌在牙周病防治中的应用

牙周病是口腔两大类主要疾病之一,具有较高的发病率,因此,探索预防牙周病的有效途径十分重要。本文介绍了国外学者以牙龈卟啉单胞菌不同形式的抗原进行免疫学防治牙周炎的试验,其中包括对牙龈卟啉单胞菌菌毛、血凝素、牙龈素和外膜蛋白的研究。     

牙龈卟啉单胞菌在龈下菌斑和颊黏膜中的检测

目的　检测牙周健康者及牙周炎患者在颊黏膜和龈下菌斑中牙龈卟啉单胞菌的阳性率,探讨其与牙周炎发生和发展的关系。方法　选取40例牙周健康者和39例慢性牙周炎患者,分别收集颊黏膜和龈下菌斑样本,提取细菌DNA,设计细菌通用引物和牙龈卟啉单胞菌的特异引物用于PCR扩增,检测牙龈卟啉单胞菌的阳性率。结果　牙周健康组菌斑样本和颊黏膜样本牙龈卟啉单胞菌的阳性率分别为37·5%和32·5%,而牙周炎组菌斑样本和颊黏膜样本牙龈卟啉单胞菌的阳性率分别为69·23%和46·15%。牙周炎组菌斑的牙龈卟啉单胞菌阳性率高于牙周健康组,颊黏膜的牙龈卟啉单胞菌阳性率在组间无统计学差异;牙周炎组菌斑牙龈卟啉单胞菌阳性率高于颊黏膜, 牙周健康组两部位阳性率无统计学差异。结论　牙龈卟啉单胞菌除在菌斑中有高检出率外,在颊黏膜中也有较高的检出率,提示颊黏膜也是牙周细菌在口腔定植的重要部位,牙龈卟啉单胞菌也可在健康人群中检出,提示其有可能是口腔内固有菌群之一。     

免疫低下豚鼠伴放线放线杆菌和牙龈卟啉单胞菌感染特征

目的　研究免疫低下对青少年牙周炎(JP)特异性病原菌感染的影响。方法　二次60Co全身照射制备免疫低下豚鼠及正常豚鼠各36只,随机分组,下前牙龈沟接种伴放线放线杆菌(Aa)和/或牙龈卟啉单胞菌(Pg),于2、3、 6周分批处死,进行组织病理学及破骨细胞计数对比研究。结果　免疫低下Aa、Pg、Aa+Pg组2、3周时牙周破坏明显,重于免疫正常组,破骨细胞计数高于免疫正常组,有统计学差异(P<0·05)。结论　免疫低下加重Aa、Pg对牙周组织破坏,机体免疫状态是JP发生发展的重要环节。     

牙龈卟啉单胞菌脂蛋白基因在慢性牙周炎及牙周健康者中的检测

目的 应用基因芯片技术检测3种脂蛋白基因PG0717、PG0183、PG2135在慢性牙周炎患者和牙周健康者牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)中的分布,探讨这些基因与牙周临床指数之间的关系,为研究脂蛋白在Pg致病过程中的作用提供依据.方法 选取41例慢性牙周炎患者(牙周炎组)及76例牙周健康者(健康对照组),记录探诊深度、附着丧失、探诊出血及牙齿松动度,取龈下菌斑进行细菌分离培养,以临床采集的样本提取的DNA为探针,以抑制消减杂交技术获得的PgW83特异基因片段PG0717、PG0183、PG2135为目标序列,采用Cy5荧光标记目标序列.应用基因芯片技术检测PG0717、PG0183、PG2135基因在牙周炎组病变部位、非病变部位和健康对照组Pg中的分布.结果在牙周炎组病变部位PG0717、PG0183、PG2135基因的检出率分别为90%(18/20)、70%(14/20)、70%(14/20),非病变部位的检出率分别为60%(12/20)、45%(9/20)、40%(8/20),而在健康对照组PG0717、PG0183、PG2135的检出率分别为55%(11/20)、25%(5/20)、30%(6/20).3种脂蛋白基因在牙周炎组病变部位和健康对照组中的检出率差异均有统计学意义(P＜0.05),并且与牙周探诊深度、临床附着丧失、探诊出血及牙齿松动度相关.结论 带有PG0717、PG0183、PG2135基因的Pg菌株致病力强.     

牙龈卟啉单胞菌PG1055基因在临床菌株中的表达

目的应用基因芯片技术检测PG1055基因在不同人群的牙龈卟啉单胞菌(P.gingivalis)中分布,探讨这些基因与牙周临床指数之间的关系。方法取龈下菌斑进行细菌分离培养,以临床采集样本提取的DNA为探针,以抑制消减杂交技术获得P.gingivalisW83的特异基因片段PG1055为目标序列,采用Cy5荧光标记目标序列。应用基因芯片技术检测PG1055基因在牙周病患者及健康人群的牙龈卟啉单胞菌中的分布。结果PG1055基因在牙周病患者及健康人群中的检出率有统计学差异,并且与牙周临床指数相关。结论PG1055基因与P.gingivalis的致病性有关。     

大鼠实验性牙周炎中牙龈卟啉单胞菌rgpB和kgp变化的研究

目的:通过建立大鼠实验性牙周炎模型,检测不同时段大鼠上颌第一磨牙龈下人工定植的牙龈卟啉单胞菌、牙龈素rgpB和kgp相对含量的改变,动态观察牙周炎发展不同阶段牙龈卟啉单胞菌致病基因的变化。方法:选择6周的成年雄性大鼠13只,应用钢丝结扎法和细菌种植法建立大鼠实验性牙周炎模型。分别在4周和8周取上颌第一磨牙龈下菌斑,提取细菌DNA,应用PCR方法进行牙龈卟啉单胞菌、牙龈素基因rgpB和kgp特异引物扩增,应用SPSS13.0统计软件,分析大鼠牙周炎不同时段rgpB和kgp的相对变化。结果:大鼠实验性牙周炎模型,龈下菌斑中牙龈卟啉单胞菌的相对含量实验组明显高于对照组,8周组高于4周组;实验组牙龈卟啉单胞菌牙龈素rgpB和kgp明显高于对照组,4周组和8周组rgpB和kgp相对含量无显著性差异。结论:rgpB基因和kgp基因与牙周炎的致病性有关而与牙周炎的严重程度可能无直接相关。     

Proteases of Porphyromonas gingivalis: what don't they do?

The gram-negative anaerobic bacterium Porphyromonas gingivalis has been strongly associated with the causation of human periodontal diseases. One distinguishing property of these organisms that has been implicated in periodontal destruction is the expression of potent protease activity. Recent biochemical and genetic approaches have clearly demonstrated that at least five distinct proteases are elaborated by these organisms. The utilization of monospecific mutants defective in individual proteases has demonstrated that protease activity is important in virulence but also has suggested the complexity of the functions of the enzymes in the physiology of these microorganisms. This review summarizes current progress in assessing the role of these enzymes in periodontal inflammation and discusses some unresolved issues relevant to the significance of P. gingivalis proteases in virulence.     

Altered Antigenic Profiling and Infectivity of Porphyromonas gingivalis in Smokers and Non‐Smokers With Periodontitis

Background: Cigarette smokers are more susceptible to periodontal diseases and are more likely to be infected with Porphyromonas gingivalis than non‐smokers. Furthermore, smoking is known to alter the expression of P. gingivalis surface components and compromise immunoglobulin (Ig)G generation. The aim of this study is to evaluate whether the overall IgG response to P. gingivalis is suppressed in smokers in vivo and whether previously established in vitro tobacco‐induced phenotypic P. gingivalis changes would be reflected in vivo. Methods: The authors examined the humoral response to several P. gingivalis strains as well as specific tobacco‐regulated outer membrane proteins (FimA and RagB) by enzyme‐linked immunosorbent assay in biochemically validated (salivary cotinine) smokers and non‐smokers with chronic periodontitis (CP: n = 13) or aggressive periodontitis (AgP: n = 20). The local and systemic presence of P. gingivalis DNA was also monitored by polymerase chain reaction. Results: Smoking was associated with decreased total IgG responses against clinical (10512, 5607, and 10208C; all P <0.05) but not laboratory (ATCC 33277, W83) P. gingivalis strains. Smoking did not influence IgG produced against specific cell‐surface proteins, although a non‐significant pattern toward increased total FimA‐specific IgG in patients with CP, but not AgP, was observed. Seropositive smokers were more likely to be infected orally and systemically with P. gingivalis (P <0.001), as determined by 16S RNA analysis. Conclusion: Smoking alters the humoral response against P. gingivalis and may increase P. gingivalis infectivity, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from those of non‐smokers with the same disease classification.     

牙龈卟啉菌、中间普氏菌的分离、培养和鉴定

目的：采用厌氧培养和鉴定技术，分离牙周炎患者龈下菌斑中的牙龈卟啉菌和中间普氏菌。方法：采集牙周炎患者的龈下菌斑，厌氧培养，分离产黑色素菌。经长波长紫外灯观察，各种生化分析和间接免疫荧光染色，分离和鉴定牙龈卟啉菌，中间普氏菌。结果：在受检的33例牙周炎患者中，24例患者检出了牙龈卟啉菌，总检出率为72．77％，共分离了79株。18例患者检出了中间普氏菌，总检出率为54．54％。共分离了32株。结论：厌氧培养，生化反应鉴定技术的发展，使牙龈卟啉菌和中间普氏菌的分离与培养准确可靠，为今后在分子水平上了解各菌株的致病机理打下了基础。     

Prevalence of Porphyromonas gingivalis fimA genotypes in Caucasians

The aim of the present study was to determine the prevalence of Porphyromonas gingivalis fimA genotypes in Caucasian patients with periodontitis. A total of 102 patients harboring P. gingivalis subgingivally were enrolled into the study. Pooled subgingival plaque samples of the six most severely affected sites were taken and analysed by fimA-specific polymerase chain reaction (PCR) and restriction analysis. Moreover, 26 P. gingivalis isolates were analysed by sequence analysis of the fimA gene. Sequence analysis revealed five major fimA genotypes (fimA types I-V) and allowed further subtyping of fimA genotypes II and IV into two subgroups each. The overall prevalences of fimA genotypes as assessed by PCR and restriction analysis among the P. gingivalis-positive patients with periodontitis were: type I, 25.5%; type II, 38.2%; type III, 4.9%; type IV, 18.6%; type V, 3.9%; and non-typable, 6.9%. Two patients were colonized by both type II and type IV, or type III and type IV fimA genotypes, respectively. Patients harboring different fimA genotypes showed no significant difference in severity of periodontal disease, as assessed by pocket probing depth and bleeding on probing following adjustment for smoking habit and age. The results indicate that predominant fimA genotypes in Caucasian periodontitis patients are types I, II, and IV. However, there was no difference in the association of the various fimA genotypes with disease severity.     

Distribution of fimA genotypes of Porphyromonas gingivalis in subjects with various periodontal conditions

Fimbria encoded by the gene fimA is considered one of the main factors in the colonization of the oral cavity by Porphyromonas gingivalis. Allelic variation in fimA led to the classification of strains of P. gingivalis into six genotypes. The occurrence of P. gingivalis was determined by polymerase chain reaction using 16S rRNA primers in 302 subgingival samples obtained from 102 Brazilian subjects exhibiting different periodontal conditions. Distribution of fimA genotypes was assessed in 146 P. gingivalis positive samples by polymerase chain reaction using primers pairs homologous to the different fimA genes. P. gingivalis was detected in 51 of 57 (89.4%) patients with periodontal attachment loss, in six of 20 gingivitis patients (30.0%) and in two of 25 (8.0%) subjects with a healthy periodontium. Variant type II was the only type detected in 53 sites (39.3%), distributed among 19 periodontitis patients (37.3%) and in one patient with no periodontal destruction. Type Ib was the second most prevalent genotype in periodontitis patients (19.6%). Genotype V was not detected in the studied population. Type IV was the most commonly type found among gingivitis patients, either alone or in combination with other genotypes. Multiple genotypes were detected in nine sites (6.1%). A fimA genotype was not identified in 26 sites (17.8%) of 146 sites positive for P. gingivalis, suggesting that other alleles of fimA not yet sequenced may be prevalent in this population. These data demonstrated that P. gingivalis type II strains followed by type Ib are more prevalent in periodontitis patients from a multiracial population in Brazil, suggesting an increased pathogenic potential of these types.     

Immunodominant antigens of Porphyromonas gingivalis in patients with rapidly progressive periodontitis

We studied 4 isolates of Porphorymonas gingivalis , ATCC 33277, 381, A7A1-28, and W50, to identify major cell surface antigens and select the best strain from which to obtain antigen for a test vaccine. Immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay using whole-cell sonicates as antigen were significantly elevated for the sera of 64 rapidly progressive periodontitis patients relative to sera of 30 normal control subjects for each of the 4 strains studied. Western blots were prepared for all 4 strains and developed using sera from 22 patients and 20 control subjects to identify and determine the frequency of antibody-binding components. The intensity of binding by patient sera was greatest for the 75-kDa and 55-kDa components. The 43-kDa component was also widely recognized. Strains ATCC 33277 and 381 appeared to be antigenically similar. Because of the higher serum antibody titers, the larger proportion of seropositive patients and higher frequency of binding to specific protein components in Western blots, our efforts were focused on strain ATCC 33277. Whole-cell sonicates, proteinase K-digested sonicate, lipopolysaccharide, capsular polysaccharide, and whole-cell protein fractions were prepared and evaluated for anti-genie activity. By dot immunoblot, most of the antibody binding activity was found in the whole-cell protein fraction, with much lesser amounts in lipopolysaccharide and none in capsular polysaccharide. The antibody-binding activity was accessible on the cell surface, since 98.9% of P. gingivalis -specific antibody, including antibody binding to the 43-kDa, 55-kDa and 75-kDa components on Western blot, was removed by whole-cell adsorption. Furthermore, the 43-kDa and 55-kDa but not the 75-kDa component on intact cells were accessible for labeling with 125_I, confirming their cell surface location and accessibility.