膀胱上皮细胞清除胞内大肠杆菌

目的:了解大肠杆菌与膀胱上皮细胞的相互关系,观察大肠杆菌在人膀胱上皮细胞株T24中的动态变化。方法:采用大肠杆菌标准株K12和大肠杆菌临床分离株299侵袭T24细胞,并用庆大霉素杀死细胞外的细菌,分别于细菌侵袭细胞后的4、24及48 h用TritonX-100裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌数。结果:两株大肠杆菌在T24细胞内都不能生长,随着时间的推移,细胞内大肠杆菌活菌数量呈不断减少趋势。24 h细胞内活菌数下降为4 h活菌数的15%。48h细胞内活菌数进一步减少。结论:膀胱上皮细胞清除进入细胞内的大肠杆菌,这可能是泌尿道天然免疫防御的一个重要防御机     

肺炎克雷伯杆菌在T24细胞内存活的动态观察

目的：了解肺炎克雷伯杆菌与膀胱上皮细胞的相互关系，观察肺炎克雷伯杆菌在人膀胱上皮细胞抹T24中生存的动态变化。方法：采用肺炎克雷伯杆菌临床分离抹03138侵袭T24细胞，并用庆大霉素杀死细胞外的细菌，分别于细菌进入细胞后的4、24、48及72h裂解细胞，释放出细胞内的活细菌，用平板菌落计数法计数胞内活菌数。结果：T24细胞内的肺炎克雷伯杆菌03138抹在实验48h内有一定生长，试验72h细胞内活菌数量明显减少。加入细胞因子（TNF-αd和INF-γ）可以促进上皮细胞清除胞内细菌。结论：膀胱上皮细胞清除进入细胞内的肺炎克雷伯杆菌，可能是泌尿道天然免疫的一种防御机制，而细胞因子可以调控上皮细胞的抗菌作用。     

TNF-α增强膀胱上皮细胞清除细胞内肺炎克雷伯杆菌

研究TNF-α对膀胱上皮细胞内肺炎克雷伯杆菌生存的影响。方法：以肺炎克雷伯杆菌侵入体外培养的人膀胱上皮细胞（T24细胞）为模型,观察在TNF-α等细胞因子处理条件下,不同时间点细胞内细菌数量变化。结果：单独使用TNF-α使T24细胞内的肺炎克雷伯杆菌K5株细菌数量明显减少,联合使用TNF-α与IFN-γ使胞内活菌数量更显著的减少。而IL-1β对细胞内活菌数量无明显影响。过氧化氢酶可以有效抑制TNF-α与IFN-γ刺激的＂1＂24细胞抗菌作用,而一氧化氮合酶抑制剂L-NAME无抑制作用。结论：TNF-α能够增强膀胱上皮细胞对抗细胞内肺炎克雷伯杆菌,抗菌机制与细胞产生活性氧（ROS）有关。     

腺病毒介导突变Rip2基因对膀胱上皮细胞清除克雷伯杆菌的影响

目的探讨受体相互作用蛋白2（Rip2）在克雷伯杆菌所致的尿路感染中的作用。方法使用含有突变Rip2基因（mRip2）、绿色荧光蛋白（GFP）基因的复制缺陷腺病毒（Ad-mRip2-GFP）感染人膀胱上皮癌细胞T24细胞，表达GFP为转染成功标志，观察转染率，同时感染含有GFP基因腺病毒做对照，利用克雷伯杆菌临床分离株Kp5与细胞37℃孵育4、24、48小时，用平板培养菌落计数活菌数。结臬刺激后，在感染Ad-mRip2-GFP的T24细胞清除胞内Kp5能力下降，与Ad-GFP组比较P〈0．01。结论Rip2可以阻断膀胱上皮细胞的清除克雷伯杆菌作用，提示Rip2在膀胱上皮细胞的天然免疫中可能起重要作用。     

人气管上皮细胞对绿脓杆菌抗菌作用观察

目的：探讨呼吸道上皮细胞与呼吸道致病菌之间的相互关系,观察呼吸道上皮细胞对绿脓杆菌的抗菌作用。方法：（1）贴壁生长的人呼吸道上皮细胞株HBE-16与绿脓杆菌标准株ATCC27853共孵育,庆大霉素杀死胞外菌,动态观察上皮细胞内活细菌数;（2）HBE-16细胞与绿脓杆菌共同悬浮于细胞培养基,在不同孵育时间点用平板菌落计数法计数活菌数。结果：绿脓杆菌不能在HBE-16细胞内生长,并被细胞逐渐清楚。悬浮状态下的HBE-16细胞对绿脓杆菌有一定杀菌作用。结论：呼吸道上皮细胞对胞内外绿脓杆菌的抗菌作用,可能是呼吸道抵抗细菌感染的一种天然免疫防御机制。     

呼吸道上皮细胞与致病菌相互作用--上皮细胞内吞细菌实验研究

目的：呼吸道上皮细胞在防御这类机会致病菌感染时发挥了重要的作用。本研究旨在探讨上皮细胞能否清除胞内的绿脓杆菌，胞内模式识别受体Nods蛋白家族是否参与胞内杀菌，防御素是否能通过促经克雷伯杆菌粘附到上皮细胞而被上皮细胞内在化而清除。方法：首先用绿脓杆菌活菌刺激支气管原代上皮细胞及A549细胞，活菌细胞共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续孵育4小时，24小时后，用TritonX-100溶解细胞，采用平板菌落计数法计数胞内活菌数。绿脓杆菌与细胞按不同比例共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续培养24小时后收集细胞培养上清，酶联免疫吸附法（ELISA）检测IL-8的表达量。为进一步研究Nods蛋白是否在胞内杀菌及活菌在胞内引起IL-8分泌中发挥作用，我们用超声处理的绿脓杆菌菌体成分刺激使细胞膜通透性增加的温和去污剂digitonin处理和未使用digitonin处理A549细胞，ELISA检测细胞IL-8表达水平。胞内模式识别受体Nod1，Nod2可以识别菌体成分，用RT-PCR检测肺上皮细胞Nodl，Nod2的表达。其次，     

HNP对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响

目的：探讨呼吸道抗感染防御机制，观察人中性粒细胞α-防御素（HNP）对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响。方法：从人中性粒细胞分离纯化HNP。A549细胞与HNP（20μg·ml^-1）及两株肺炎克雷伯杆菌临床分离株共同孵育4小时，用平板培养茵落计数法测定与细胞粘附的活茵数。结果：在HNP存在的情况下，Kp03.33株细菌对肺上皮细胞的粘附提高了12倍（P〈0.01），Kp03116株细菌对上皮细胞的粘附提高了5倍（p〈0.01）结论：HNP显著增强肺炎克雷伯杆菌粘附于呼吸道上皮细胞，可能有利于呼吸道清除细菌。     

铜绿假单胞菌活菌对呼吸道上皮细胞表达IL-8的影响

目的:探讨铜绿假单胞菌活菌与人呼吸道上皮细胞的相互关系,细菌对呼吸道上皮炎症反应的影响。方法:采用PAO1及ATCC 27853两株铜绿假单胞菌,在体外与培养的呼吸道上皮细胞株A549及无血清培养的人支气管上皮原代细胞相互作用,收集细胞培养上清,ELISA检测上清IL-8浓度。结果:两株绿脓杆菌均能诱导呼吸道上皮细胞IL-8分泌增加,在细菌刺激下,A549细胞IL-8分泌比对照高出5倍(P<0.05),原代上皮细胞IL-8分泌比对照高出8倍(P<0.05)。结论:铜绿假单胞菌呼吸道感染的过程中,细菌与上皮细胞的直接作用可能是呼吸道炎症反应的重要原因。铜绿假单胞菌刺激上皮细胞炎症的分子机制和信号传导值得进一步探讨。     

志贺菌福氏5a M90T热休克蛋白70(DnaK)多克隆抗体的制备和鉴定

目的制备小鼠抗志贺菌福氏5a M90T热休克蛋白70(Dna K)的多克隆抗体,并鉴定其特异性和效价。方法 PCR扩增M90T中的基因dna K插入到原核表达载体p ET-24a中,获得p ET24a-dna K重组质粒,然后转化到大肠杆菌BL21(DE3)中表达,通过亲和柱纯化融合蛋白Dna K-His。将纯化的融合蛋白作为抗原免疫BALB/c小鼠,采集抗血清。用免疫印迹法、ELISA和免疫荧光技术鉴定抗血清的特异性和效价。结果重组质粒p ET24a-dna K在大肠杆菌BL21(DE3)中可以高效表达。用纯化的融合蛋白Dna K-His免疫小鼠制备得到的多克隆抗体能特异性低检测志贺菌福氏5a M90T Dna K蛋白,能有效地用于免疫印迹法、ELISA和免疫荧光等试验。结论成功制备了抗志贺菌福氏5a M90T Dna K蛋白的小鼠多克隆抗体。     

肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL-8的研究

目的 :探讨肺炎克雷伯杆菌 (Klebsiellapneumoniae ,Kp)分泌因子及活菌诱导肺上皮细胞株表达和分泌IL 8的状况。方法 :用临床分离株Kp0 3 1 1 6、Kp0 3 1 83的细菌培养上清或活菌刺激肺上皮细胞株A5 49和SPC A 1 ,酶联免疫吸附实验 (ELISA)检测细胞IL 8表达量。结果 :①两株Kp培养上清分别刺激A5 49或SPC A 1后 ,IL 8表达量均有显著增高 (P <0 .0 1 ) ,且随上清刺激浓度增加而上升。②两株Kp及DH5 (活菌分别与SPC A 1共孵育 2h ,用庆大霉素杀死胞外菌 ,继续孵育 2 4h后两株Kp诱导细胞IL 8表达量均显著高于DH5α(P <0 .0 1 ) ,且随细菌数 /细胞数比例增大而上升。结论 :Kp分泌因子及活菌都能够诱导肺上皮细胞IL 8表达 ,而活菌上调IL 8的效应较培育上清分泌因子更显著 ,提示肺上皮细胞在Kp感染刺激的肺部炎症反应中起重要作用。     

In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.     

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.     

Attachment of Escherichia coli to urinary sediment epithelial cells from urinary tract infection-prone and healthy children.

Escherichia coli isolated from patients with recurrent urinary tract infections were tested for ability to attach to human urinary sediment epithelial cells in vitro. Higher mean capacity to bind bacteria was found for epithelial cells of the patient from whom the E. coli strain had been isolated than for epithelial cells from subjects without a history of urinary tract infection. The two populations were age matched. No relation was found between the age of the cell donor (0 to 15 years) and the capacity for E. coli attachement.     

Enzyme-linked immunosorbent assay for adherence of bacteria to animal cells.

Epithelial cells scraped from human oral mucosa and from pig intestines were immobilized onto the flat bottom surfaces of microtiter plates to study the adherence of various bacterial species to host cells. Bacterial adherence was quantitated either by an enzyme-linked immunosorbent assay technique with specific antibacterial serum as the first antibody followed by peroxidase-conjugated second antibody or by using biotinylated bacteria and avidin-peroxidase as the detecting agent. Unlabeled Escherichia coli and purified E. coli 987P fimbriae inhibited the adherence of biotinylated E. coli to immobilized enterocytes. The adherence of a mannose-sensitive strain of E. coli to immobilized oral epithelial cells was inhibited by mannose derivatives. The adherence of fimbriated E. coli 987P to immobilized enterocytes was approximately four times higher than the adherence of a nonfimbriated variant of the same strain. The adherence of Streptococcus pyogenes to oral cells was detected in the range of 10 to 150 bacteria per cell and was inhibited by lipoteichoic acid and albumin. The data suggest that the putative receptors which bind bacteria on the immobilized cells retain a functional form similar to that of native cells in suspension. The proposed adherence assay is easy to perform, allows the detection of specific adherence of test bacteria, and provides objective quantitation of adherence with a sensitivity of 10 bacteria per cell. Most importantly, the assay allows the testing of many variables in the same day.     

Comparison of Escherichia coli fimbrial antigen F7 with type 1 fimbriae.

Two Escherichia coli O6:K2:H1 strains, C1212 and C1214, isolated from urinary tract infections, were compared for their capacity to adhere to various cells. After growth on solid medium, only C1212 bacteria agglutinate human erythrocytes and attach to urinary epithelial cells. Both of these reactions are mannose resistant. In contrast, C1214 bacteria cause a mannose-sensitive agglutination of guinea pig erythrocytes, show a mannose-sensitive attachment to buccal epithelial cells, and attach to urinary mucus. Immunoelectron microscopy revealed that C1214 bacteria possess type 1 fimbriae (mannose sensitive), which are not present in C1212 bacteria when this strain is grown on solid medium. The fimbriae of C1212 (mannose resistant) were also demonstrated by immunoelectron microscopy. We call these fimbriae demonstrated in C1212 the E. coli F7 antigen. Urinary mucus, and probably mucous material elsewhere, may function as a trap for Enterobacteriaceae with type 1 fimbriae by the specific adherence of such bacteria. We consider this a nonimmune resistance mechanism against disease caused by Enterobacteriaceae.     

Invasion processes of pathogenic Escherichia coli

Pathogenic Escherichia coli causes extraintestinal infections such as urinary tract infection and meningitis, which are prevalent and associated with considerable morbidity. Previous investigations have identified common strategies evolved by pathogenic E. coli to exploit host cell function and cause extraintestinal infections, which include the invasion into non-phagocytic eukaryotic cells such as epithelial and endothelial cells and associated host cell actin cytoskeletal rearrangements. However, the mechanisms involved in pathogenic E. coli invasion of eukaryotic cells are shown to differ depending upon types of host tissues and microbial determinants. In this mini-review, invasion processes of pathogenic E. coli are discussed using E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) as a paradigm. E. coli K1 is the most common Gram-negative organism causing neonatal meningitis, and E. coli invasion of HBMEC is shown to be a prerequisite for E. coli traversal of the blood-brain barrier in vivo. Previous studies have demonstrated that E. coli translocation of the blood-brain barrier is the result of specific E. coli host interactions including specific signal transduction pathways and modulation of endocytic pathways. Recent studies using functional genomics have identified additional microbial determinants contributing to E. coli K1 invasion of HBMEC. Complete understanding of microbial-host interactions that are involved in E. coli K1 invasion of HBMEC should help in the development of new strategies to prevent E. coli meningitis.     

Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells.

We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells. Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively). Prematurity and serious illness did not alter adherence in newborns. The increased susceptibility of premature newborns to E. coli K1 cannot be explained by enhanced epithelial cell adherence.