质粒蛋白pORF5在沙眼衣原体感染细胞中的定位及免疫原性研究(英文)

目的分析沙眼衣原体隐蔽性质粒编码的质粒蛋白pORF5在感染细胞中的定位并初步探讨免疫原性特征。方法 PCR扩增pORF5质粒蛋白编码基因,构建原核表达重组体pGEX-6p/pORF5,重组体转化大肠杆菌XL1-blue中诱导表达融合蛋白;融合蛋白经Glutathione Sepharose亲和层析纯化后免疫小鼠,制备单克隆抗体和多克隆抗体,间接免疫荧光技术及免疫印迹鉴定抗体的特异性,并分析pORF5质粒蛋白在感染细胞中的分布特征。采用ELISA方法分析pORF5质粒蛋白的免疫原性。结果 pORF5原核表达重组体成功构建,融合蛋白在大肠杆菌中可溶性表达;pORF5主要分布于宿主细胞胞浆,但也少量分布在EB、RB上;pORF5能与衣原体患者血清及鼠免疫血清发生强烈地免疫反应。结论 pORF5为衣原体分泌蛋白,并具有很强的免疫原性。     

Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (Medac) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci

A commercial test (rELISA) based on a recombinant chlamydial lipopolysaccharide (LPS) antigen has been evaluated for the diagnosis of acute infections caused by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci. This test and a microimmunofluorescence test (MIF) were compared in 160 patients with community-acquired pneumonia. Seventeen of nineteen cases with significant titre changes detected by rELISA were confirmed by MIF. The two remaining cases not confirmed by MIF were considered false-positive reactions. One case positive by MIF only was judged not to be a true-positive reaction. All three cases occurred in patients with Mycoplasma pneumoniae infection and may be the result of a mitogenic effect. High antibody titres have been used to indicate acute C. pneumoniae infection. We found high MIF or rELISA titres to be equally common in patients and controls; no association between the two tests was detected. An unexpected cross-reactivity between the rELISA antigen and parvovirus was observed, which might have diagnostic implications. Both MIF and rELISA detected acute C. pneumoniae and C. psittaci infection, and there was good agreement between the tests. Single serum diagnosis was generally not feasible with either MIF or rELISA.     

Frequent contamination of Chlamydia trachomatis and Chlamydia pneumoniae strains with mycoplasma. Biological relevance and selective eradication of mycoplasma from chlamydial cultures with mupirocin

Several strains of Chlamydia trachomatis (CT) and C. pneumoniae (CP) from different sources were screened for mycoplasma contamination using a sensitive nested 16S rDNA polymerase chain reaction-specific for a broad range of mycoplasma species. Five of nine CT and 5/16 CP isolates were contaminated by mycoplasma. Mycoplasma fermentans, M. hyorhinis and M. hominis were found as contaminating agents. To our knowledge no data are available on whether coinfection of chlamydia with mycoplasma alters the biological behavior of chlamydia. Analysis of the biological effect of mycoplasma on chlamydial infection showed a profound mycoplasma-induced reduction of chlamydial growth. Mycoplasma were efficiently eliminated from chlamydial cultures in HEp-2 cells by treatment with mupirocin without affecting chlamydial replication or host cell growth. Two chlamydial strains, C. trachomatis serovar K and one clinical isolate of C. pneumoniae were purged by this method. Received: 17 March 2000     

Localization of Chlamydia trachomatis hypothetical protein CT311 in host cell cytoplasm

The chlamydia-specific hypothetical protein CT311 was detected both inside and outside of the chlamydial inclusions in Chlamydia trachomatis-infected cells. The extra-inclusion CT311 molecules were distributed in the host cell cytoplasm with a pattern similar to that of CPAF, a known Chlamydia-secreted protease. The detection of CT311 was specific since the anti-CT311 antibody labeling was only removed by absorption with CT311 but not CPAF fusion proteins. In addition, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Although both CT311 and CPAF proteins were first detected 12 h after infection, localization of CT311 into host cell cytosol was delayed until 24 h while CPAF secretion into host cell cytosol was already obvious by 18 h after infection. The host cell cytosolic localization of CT311 was further confirmed in human primary cells. CT311 was predicted to contain an N-terminal secretion signal sequence and the CT311 signal sequence directed secretion of PhoA into bacterial periplasmic region in a heterologous assay system, suggesting that a sec-dependent pathway may play a role in the secretion of CT311 into host cell cytosol. This hypothesis is further supported by the observation that secretion of CT311 in Chlamydia-infected cells was blocked by a C16 compound known to inhibit signal peptidase I. These findings have provided important molecular information for further understanding the C. trachomatis pathogenic mechanisms.     

Chlamydia trachomatis induces anti-inflammatory effect in human macrophages by attenuation of immune mediators in Jurkat T-cells

The chronic course of Chlamydia trachomatis infection is subtle with no obvious unusual inflammatory change. The reason for this is not clear. The data reported here explain how macrophage usual inflammatory response switches to anti-inflammatory response during C. trachomatis infection of mixed culture of macrophages and Jurkat T-cells. We assessed the establishment of productive infection in individual or mixed cell culture models, determined the status of C. trachomatis in the cells by monitoring HSP-60:MOMP or the proportions of the estimated IFUs that shed HSP-60 or MOMP. Also, the specific time-course expression of IL-12, IL-10 and IFN-γ or IL-12R, IL-10R, and IFN-γ-R during infection of cell models was assessed. Finally, the early events in cytokine elaboration in circumstances of varying intracellular Ca²⁺ levels were determined. There was evidence of productive infection in all individual and mixed cell culture models. The shedding of HSP-60 was highest in THP-1/Jurkat mixed cell culture model. The proportions of IFU that shed HSP-60 was heightened in infected THP-1/Jurkat mixed culture model, while the proportion of IFU that shed MOMP was higher in infected macrophage/Jurkat mixed culture and infected macrophages only. There was profound early elaboration of IL-10, varying significantly from IL-12 and IFN-γ in all infected individual or mixed cell culture models except in the case of Jurkat; where all cytokine elaboration was downregulated. The receptor to IL-10 was upregulated in infected macrophage/Jurkat cells and THP-1/Jurkat cells compared with other models in which IL-12 and IFN-γ receptors were more expressed. There was no observed significant change in cytokine in any model following the impairment of intracellular Ca²⁺ except in the case of macrophage/Jurkat cell model in which IL-12 and IL-10 were upregulated in 1 h or 3 h, respectively. The implication of these findings is that C. trachomatis mediates a switch from inflammatory to anti-inflammatory function in macrophages due to downregulation of the regulatory cytokine, IFN-γ in Jurkat cells, culminating in C. trachomatis chronic course.     

衣原体支原体弓形虫和巨细胞病毒感染与自然流产的关系

目的研究衣原体（CT）、支原体（UU）、弓形虫（TOX）、巨细胞病毒（HCMV）对流产的影响。方法应用聚合酶链反应（PCR）技术分别对164例自然流产患者（流产组）和100例人工流产和引产孕妇（对照组）的宫颈分泌物进行CT、UU、TOX、HCMV检测。结果流产组CT、UU、TOX、HCMV的检出阳性率分别为41.46%、34.15%、18.29%、30.39%,与对照组比较,差异有显著意义（P（0.01,P（0.05）。流产次数随UU阳性出率的增高而增多。结论CT、UU、TOX、HCMV是引起自然流产的重要感染源,其中CT、UU是主要病原体。     

E-选择素、CD14及VCAM1水平与生殖道沙眼衣原体感染的相关性分析

目的 探讨E-选择素、CD14及血管细胞粘附分子(vascular cell adhesion molecule 1,VCAM1)水平与生殖道沙眼衣原体感染的相关性.方法 选取86例生殖道沙眼衣原体感染患者为观察组;根据生殖道沙眼衣原体感染患者是否伴有炎症,分为感染伴炎症组和感染无炎症组,各43例;同时,选取36例健康体检者为对照组;采用双抗体夹心酶联免疫吸附法(ELISA)测定所有研究对象的血清E-选择素、CD14及VCAM1水平,并作对比分析;观察组患者采取炎克宁冲剂结合阿奇霉素治疗,对比治疗前后的血清E-选择素、CD14及VCAM1水平,并与对照组作对比分析.结果 观察组的血清E-选择素、CD14及VCAM1水平均显著高于对照组;差异具有统计学意义(P＜0.05);感染伴炎症组与感染无炎症组的血清E-选择素、CD14及VCAM1水平的差异具有统计学意义(P＜0.05);观察组患者采取炎克宁冲剂结合阿奇霉素治疗后,临床总有效率为94.19％,生殖道沙眼衣原体转阴率为87.21％;治疗后的血清E-选择素、CD14及VCAM1水平显著低于治疗前水平,差异具有统计学意义(P＜0.05);与对照组对比,差异无统计学意义(P＞0.05).结论 生殖道沙眼衣原体感染的病情进展与E-选择素、CD14及VCAM1水平密切相关,通过监测E-选择素、CD14及VCAM1水平,可为生殖道沙眼衣原体感染的治疗、评估疗效及预后而提供依据.     

Does infection with Chlamydia pneumoniae and/or Helicobacter pylori increase the expression of endothelial cell adhesion molecules in humans?

Objective   To investigate if Chlamydia pneumoniae and/or Helicobacter pylori seropositivity is associated with elevated levels of soluble endothelial cell adhesion molecules (sCAMs) as markers of atherosclerotic activity.
Methods   Immunoglobulin A (IgA) and IgG antibodies to the two bacteria, soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin were measured in coronary heart disease (CHD) patients ( n  = 193) and age- and sex-matched controls ( n  = 193). Two different serological methods were used for the detection of Chlamydia antibodies: Labsystems microimmunofluorescence to detect species-specific C. pneumoniae antibodies and Medac's recombinant enzyme-linked immunosorbent assay to detect genus-specific lipopolysaccharide antibodies.
Results   The concentrations of sICAM-1 and E-selectin were higher in CHD patients with positive vs. negative Chlamydia lipopolysaccharide IgA ( P  = 0.044 for both). H. pylori antibodies alone did not predict raised levels of sCAMs, but in CHD patients sICAM-1 was increased with IgA seropositivity to both bacteria compared to double seronegativity ( P  = 0.034). Concentrations of sVCAM-1 were elevated in CHD patients with double IgA seropositivity compared to those with Chlamydia lipopolysaccharide IgA seropositivity alone ( P  = 0.018).
Conclusion   Our results may indicate that C. pneumoniae contributes to increased inflammation in CHD, and that this contribution is even more pronounced when present in combination with H. pylori IgA antibodies.     

间接免疫荧光法检测肺炎衣原体抗体的临床意义

本文采用间接免疫荧光试验 ,对 132例 (呼吸系统感染性疾病、冠心病和心肌梗塞 )患者和 40名正常人的血清进行了Cpn特异性IgG、IgA和IgM抗体的检测 ,以研究Cpn感染的状况和探讨检测Cpn抗体的临床意义。结果 :观察组CpnIgG抗体阳性率虽高于对照组 ,但无显著性差异 (P >0 0 5 ) ;观察组GMT明显高于对照组 (P <0 0 5或P <0 0 1) ;呼吸系统感染性疾病的CpnIgA抗体阳性率显著高于对照组 (P <0 0 5 )。因此 ,测定Cpn抗体滴度有助于Cpn感染与疾病关系的研究。