Tissue localization of Actinobacillus actinomycetemcomitans in human periodontitis. II. Correlation between immunofluorescence and culture techniques

Recent immunohistological studies have suggested that Actinobacillus actinomycetemcomitans is present in the gingival tissues in juvenile periodontitis lesions. The present study examined tissue bound A. actinomycetemcomitans by bacterial culture and immunohistological demonstration of antigen in tissue. A total of 14 periodontitis lesions were examined. Eleven biopsies were obtained from gingiva adjacent to A. actinomycetemcomitans infected pockets, while the remaining three control biopsies were obtained from gingiva adjacent to pockets where subgingival A. actinomycetemcomitans infection could not be detected. Each biopsy was hemisected, one half was used for immunofluorescence microscopic examination while the other half was processed for culture of A. actinomycetemcomitans. The latter section was surface-disinfected, repeatedly washed and then minced to release bacteria from within the tissues. Aliquots from the serial washings and the minced tissue suspension were cultured on medium selective for A. actinomycetemcomitans. Surface disinfection and serial washings gradually decreased cultivable A. actinomycetemcomitans in the washings aliquots. Following tissue disruption, an increase in colony-forming units of A. actinomycetemcomitans was seen from eight of the 11 test biopsies. This bacterium could not be detected in washings or minced tissue suspensions from the control biopsies obtained from lesions in which subgingival A. actinomycetemcomitans was previously not detected. A positive correlation was seen between the presence of A. actinomycetemcomitans antigens in the gingival biopsies and; (1) A. actinomycetemcomitans colony-forming units released from the minced tissues (r = 0.90, p = 0.000), as well as; (2) the colony-forming units from the periodontal pocket (r = 0.62, P = 0.017).(ABSTRACT TRUNCATED AT 250 WORDS)     

Safety and immunogenicity of whole-virus,alum-adjuvanted whole-virus,virosomal, and whole-virus intradermal influenza A/H9N2 vaccine formulations

Avian influenza H9N2 viruses are considered as a pandemic threat. We assessed the safety and immunogenicity of fourteen H9N2 vaccine formulations. A randomized, phase I trial was done in 353 adults, aged 18–82 years. Subjects received two doses of A/Hong Kong/1073/99 (H9N2) whole-virus, alum-adjuvanted whole-virus, virosomal, or intradermal whole-virus vaccine at four doses (1.7, 5, 15 or 45 μg haemagglutinin). Sera were obtained before and three weeks after each vaccination (days 0, 21, and 42) for haemagglutination–inhibition (HAI) and neutralization assays. All formulations were well tolerated. Pre-vaccination sera from subjects aged below or above 40 years had baseline antibody to H9N2 in 1% and 16% of samples. Compared to intramuscular whole-virus vaccine, alum-adjuvanted vaccine was more immunogenic, intradermal vaccine was comparable, and virosomal vaccine less immunogenic. Among subjects under 40 years, two doses (45, 15, and 5 μg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 41%, and 39% respectively, and neutralization seroconversions in 83%, 82%, and 78% of recipients. Among subjects over 40 years, one dose (45, 15, and 5 μg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 25% and 0% respectively, and neutralization seroconversions in 88%, 63% and 63% of recipients. Among immunologically naive subjects under 40 years, two doses of vaccine are required and alum-adjuvanted vaccines were most immunogenic. Among immunologically primed subjects over 40 years, one dose of whole-virus or alum-adjuvanted vaccine induced immune responses; the second dose provided less additional benefit. However, no vaccine formulation satisfied all European regulatory criteria for pandemic vaccines.     

Periodontal bone level and gingival proteinase activity in gnotobiotic rats immunized with Bacteroides gingivalis

Bacteroides gingivalis is associated with various forms of periodontal disease. To assess the role of the immune response in modulating B. gingivalis-associated periodontal disease, the effect of immunization of B. gingivalis-induced periodontal bone loss was evaluated in gnotobiotic rats. Male Sprague-Dawley rats immunized with various doses of whole cells or sham-immunized with incomplete Freund's adjuvant were monoinfected with B. gingivalis in carboxymethylcellulose by gavage. Two additional groups served as either sham-immunized or untreated germ-free controls. Forty-two days after infection, all rats were killed, periodontal bone level was assessed morphometrically and radiographically, and gingival proteinase (mammalian collagenase and acid cathepsin) activity was assessed biochemically. B. gingivalis was present in oral samples from all monoinfected rats, and no contaminating bacteria were detected in any oral or fecal sample. Animals immunized with B. gingivalis cells had elevated serum and saliva antibodies to whole cells and partially purified fimbriae from B. gingivalis. Infected sham-immunized rats had significantly more periodontal bone loss than noninfected controls, whereas the periodontal bone level in infected rats immunized with 10(10) B. gingivalis cells was similar to that of the noninfected controls. The activities of gingival collagenase and cathepsin B and L were high in sham-immunized infected rats and low in all other animal groups. In conclusion, it is possible to reduce B. gingivalis-induced periodontal tissue loss in gnotobiotic rats by immunization.     

抗角蛋白抗体、抗核周因子和抗环瓜氨酸肽抗体联合检测幼年类风湿关节炎：早期诊断及治疗评估意义

目的:评价抗角蛋白抗体、抗核周因子和抗环瓜氨酸肽抗体联合检测在幼年类风湿关节炎诊断及病情评估中的意义。方法:①观察对象及分组:选择2003-01/2005-12首都医科大学附属北京儿童医院风湿免疫病房住院治疗的76例幼年类风湿关节炎患儿及54例非幼年类风湿关节炎患儿,正常对照30例(家属均知情同意)。②检测上述人员血清抗角蛋白抗体、抗核周因子抗体和抗环瓜氨酸肽抗体水平;观察两组患儿出现临床症状、体征例数及实验室检测数据。③对幼年类风湿关节炎诊断的敏感性、特异性,阳性似然比、阴性似然比进行评价,并对幼年类风湿关节炎患儿中3种抗体联合检测阳性组阴性组的临床症状、体征及实验室检查方面的指标进行比较,资料作统计学分析。结果:两组患儿130例,正常儿童30例,全部进入结果分析。①两组患儿临床症状、体征例数及实验室检测值差异没有显著性意义。②抗角蛋白抗体、抗核周因子抗体和抗环瓜氨酸肽抗体联合检测对幼年类风湿关节炎组早期诊断缺乏有效性。③抗角蛋白抗体( )/抗核周因子抗体( )/抗环瓜氨酸肽抗体( )病例与抗角蛋白抗体(-)/抗核周因子抗体(-)/抗环瓜氨酸肽抗体(-)病例相比,关节强直病例明显增多,差异有显著性(较正χ2=3.902,P=0.048),抗链球菌溶血素“O”和C-反应蛋白均显著增高,差异有显著性(χ2=2.616,3.557,P=0.025,0.001)。结论:抗角蛋白抗体、抗核周因子抗体、抗环瓜氨酸肽抗体联合检测对幼年类风湿关节炎缺乏早期诊断意义及特异性,联合检测对判断疾病的活动性、病理损害程度和预后有临床意义。     

丹参酮ⅡA对神经祖细胞系C17．2的保护作用

目的:了解丹参酮ⅡA对神经祖细胞系C17.2的保护作用,探讨其可能的作用机制。方法:本实验于2005年起在广州血液中心器官移植配型中心实验室进行。C17.2祖细胞系由澳大利亚新南威尔士大学解剖教研室David Walsh博士惠赠。将C17.2细胞以1×109L-1的密度接种,用含10%胎牛血清IMDM,37℃、体积分数为0.05CO2、饱和湿度的CO2培养箱培养,接近融合的C17.2细胞用含0.1mmol/LEDTA的胰酶室温消化,按1∶3的比例传代。C17.2细胞以5×107L-1的密度接种于96孔板或25cm2的培养瓶中,用含10%胎牛血清IMDM培养过夜后,加入含4g/L AAPH(水溶性偶氮引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐)无血清的IMDM培养基培养建立神经细胞凋亡模型。C17.2细胞以5×103/孔的密度接种于96孔板中,用含10%胎牛血清IMDM培养过夜后,加入含4g/LAAPH无血清的IMDM培养基培养。对照组不加入丹参酮ⅡA,实验组分别加入0.02,0.05,0.1,0.2mg/L丹参酮ⅡA培养8h,噻唑蓝法检测细胞活性:细胞活性的相对值=(实验组吸光度值/对照组吸光度值)×100%,流式细胞仪检测细胞凋亡。结果:①AAPH处理8h后,C17.2细胞被过氧化损害,大多数细胞失去正常的形态,细胞呈圆形,脱落。加入丹参酮ⅡA后,细胞形态基本保持正常,少数细胞呈圆形。②C17.2细胞在IMDM的培养液中,细胞数量是含4g/L AAPH无血清的IMDM培养基条件下的2.5～3倍。浓度为0.02,0.05,0.1mg/L的丹参酮ⅡA对C17.2细胞有保护作用,质量浓度大于0.2mg/L丹参酮ⅡA对C17.2细胞保护作用降低。③AAPH作用前大部分C17.2细胞的线粒体完整,有少量的早期凋亡细胞和凋亡细胞,AAPH作用后凋亡细胞总数、凋亡细胞明显增加。丹参酮ⅡA处理组可以明显减少早期凋亡细胞。结论:在体外丹参酮ⅡA对神经细胞具有抗凋亡的作用,可以保护神经细胞。     

ME-PCR for the identification of mutated K-ras in serum and bile of pancreatic cancer patients: an unsatisfactory technique for clinical applications

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.     

Antigastric autoantibodies inHelicobacter pylori infection: role in gastric mucosal inflammation

The aim of the study was to ascertain whether there is an association between the presence of serum parietal cell autoantibodies (PCA) and: (1) Helicobacter pylori infection; (2) the presence and degree of gastritis and intestinal metaplasia; and (3) the H. pylori infecting strain. Gastric mucosal biopsies were obtained from 49 consecutive patients in order to assess and grade gastritis, make a histological diagnosis, and culture and genotype H. pylori. H. pylori infection was present in 26 patients (group 1), had been present in 17 patients (group 2), and the remaining 6 (group 3) had never had the infection. The infecting strain was cagA positive in 21 of 26 group 1 patients. Positive PCA results were found in 84%, 76%, and 14% of patients in groups 1, 2, and 3, respectively. PCA results were correlated with anti-H. pylori antibody titers (P<0.05). In group 2 patients, PCA were associated with the degree of antral gastritis (Fisher's exact test P<0.05). cagA status was not associated with the presence of PCA (chi2=0.68, NS). The frequency of positive findings for PCA in group 2 was higher in patients with (90%) than in those without (50%) intestinal metaplasia. In conclusion: (1) H. pylori infection is associated with the production of PCA, which, after eradication of the infection, persist and might contribute to the persistent antral chronic gastritis and intestinal metaplasia; (2) the gastric lesions associated with infections sustained by the more-virulent H. pylori strains do not appear to be due to the induction of antigastric autoantibodies.     

The role of white cells in the transmission of Yersinia enterocolitica in blood components

The mechanism for the transmission of Yersinia enterocolitica in blood components has been studied experimentally. One hypothesis is that, during a Yersinia infection in the blood donor, bacteria are phagocytosed by white cells (WBCs), but are not killed. After collection of blood from such a donor and component production, the bacteria are present in WBCs for some time, during which the unit appears sterile. Later, when the WBCs disintegrate, the bacteria are released and multiply in the unit. Aliquots of whole blood and buffy coat were inoculated with 100 colony-forming units (CFU) per mL of a Y. enterocolitica strain of type O:3 and left at room temperature for 5 hours. Some aliquots were then WBC-reduced by filtration, while others retained their WBC contents. All aliquots were kept at 4 degrees C for 6 weeks. Meat extract broth culture medium was used as a control. Growth in the range of 2000 CFU per mL was obtained in the broth control by 24 hours, whereas the whole blood and buffy coat units appeared sterile for the first days of storage. After 1 week, a trace of bacteria and, after 4 weeks, massive growth were found in the WBC-containing units but not in the WBC-reduced units. The likely explanation is that the bacteria had been phagocytosed by the WBCs and were thereby hidden and not available for bacterial culture during the first phase of storage. When the WBCs spontaneously disintegrated, bacteria were released and multiplied in the blood units.(ABSTRACT TRUNCATED AT 250 WORDS)