钩端螺旋体内毒素的实验研究 Ⅴ 钩端螺旋体脂多糖增强机体特异性抗感染能力的作用

波摩那群波摩那型56608株钩端螺旋体脂多糖(L-LPS)免疫的豚鼠能抵抗同株钩端螺旋体的攻击而免于发病和死亡,旦其心血和肾脏培养阳性率也明显下降。L-LPS免疫动物后可产生以IgM为主的群特异性抗体。该抗体当有补体存在时,能在体外裂解钩端旋螺体。此外,L-LPS尚能特异性地抑制豚鼠腹腔巨噬细胞的粘附作用。实验结果提示L-LPS为一种保护性抗原,可特异性地增强机体抗感染的能力。     

多抗甲素治疗重症肝炎疗效及甲襞微循环观察

免疫制剂多抗甲素是具有高度免疫活性的多糖类药物,它能改善和增强机体的免疫功能,它具有高度的抗应激能力,具有解毒作用,具有改善微循环,改善血液流态的作用,它治疗病毒性肝炎有一定效果。本组选     

糖皮质激素受体对大鼠中性粒细胞粘附的影响

本文通过Percoll梯度离心法分离获得大鼠中性粒细胞(PMNS)后,采用PMNS与玻璃珠粘附的模型,通过给予Dex及糖皮质激素受体(GR)阻断剂Mifepristone(RU_(38486))研究大鼠PMNS粘附过程中GR的作用。结果显示,Dex可以明显抑制PMNS的粘附,其作用随着Dex浓度的增大而增强;单纯给予不同浓度的RU_(38486)未发现明显的PMNS粘附增强,说明RU_(38486)本身对离体的PMNS粘附没有明显的作用;若同时给予Dex和RU_(38486),则Dex抑制PMNS粘附的作用逐渐减小,直至完全逆转。该结果强烈提示:糖皮质激素(GC)具有抑制PMNS粘附的作用,其作用是通过GR介导的,当GR被阻断时,这一抑制作用减弱,甚至消失。     

肺炎链球菌粘附肺Ⅱ型上皮细胞触发宿主细胞酪氨酸蛋白磷酸化

目的 探讨细胞内信号传导与肺炎球菌侵袭、致病的关系,在体外研究Ⅱ型肺炎链球菌粘附肺Ⅱ型上皮细胞（A549）是否能触发细胞内酪氨酸蛋白激酶（TPK）信号传导途径 ,以及触发该信号传导可能的细菌亚组分。方法 用FTTC荧光标记肺炎链球菌,在体外观察肺炎链球菌粘附肺Ⅱ型上皮细胞的粘附动力学特征;用免疫组织化学和ELISA方法观察完整细菌触发的细胞内酪氨酸蛋白磷酸化,用各种因素预处理肺炎链球菌后,观察触发细胞内酪氨酸蛋白磷酸化可能的细菌亚组分。结果 证实了上述粘附过程存在剂量依赖和时间的依赖关系,而且是特异的过程;细菌粘附使细胞内酪氨酸磷酸化由细菌表面蛋白质介导。结论 肺炎链球菌粘附肺Ⅱ型上皮细胞能触发细胞内信号传导,且细菌表面蛋白质在触发胞内酪氨酸蛋白磷酸化传导中起着重要作用。     

S-O_2-1菌苗对小鼠淋巴细胞的作用

动物实验和初步临床应用表明有抑制肿瘤生长作用的S-O_2-1菌苗,在体外能直接刺激小鼠脾细胞淋巴细胞的增殖,并协同增强脾细胞的Con A增殖反应、S-O_2-1菌苗诱导和增强小鼠腹腔粘附细胞的抑制活性,后者表现在直接抑制脾细胞和肠系膜淋巴结细胞对Con A和S-O_2-1菌苗的增殖反应。腹腔转移菌苗活化的腹腔粘附细胞能抑制受体小鼠对SRBC的PFC应答。提示:S-O_2-1菌苗诱导增强巨噬细胞的抑制活性,以致间接抑制淋巴细胞的免疫应答。     

兔体内细菌对人工心脏瓣膜的粘附及清除研究

目的探讨细菌在兔体内对人工心脏瓣膜材料的粘附情况及机体对细菌的清除能力。方法将H-胸腺嘧啶3脱氧核苷H-TDR标记的表皮葡萄球菌菌液等量分别与人工心脏瓣膜材料涤纶、热解碳、聚四氟乙烯植入日本长耳白3兔双侧腹膜后间隙,观察术后1、3、5、7天细菌在体内对不同材料的粘附。将球菌在体外粘附于不同人工心脏瓣膜材料,植入腹膜后间隙,观察和比较不同时间兔对已粘附在不同材料上球菌的清除能力。将人工心脏瓣膜材料植入腹膜腔,同时经静脉注入表皮葡萄球菌菌液造成菌血症或败血症,比较细菌对腹膜腔内不同材料的粘附情况。结果当将细菌与材料一起放入兔体内,细菌对材料粘附在第3天、第5天最多。对涤纶的粘附较强P<0.01。热解碳在第5天、第7天的细菌粘附明显增强,与聚四氟乙烯相比差异有显著性P<0.05。兔对材料上粘附细菌的清除能力以3天内最快,7天取出材料作细菌培养,仍有细菌生长。静脉注入表皮葡萄球菌产生菌血症或败血症,腹膜腔内生物材料的细菌培养阳性率以第5天、第7天最多,涤纶细菌培养阳性率最高。结论在机体内,同一种细菌对不同的人工心脏瓣膜材料有不同粘附能力,机体对不同材料上粘附细菌的清除能力不同,不易完全清除材料上粘附的细菌。     

肝再生磷酸酶2缺失促进巨噬细胞的杀菌功能

肝再生磷酸酶2(protein phosphatase of regenerating liver 2,PRL-2)是肝再生磷酸酶家族成员,其在免疫系统中的作用未有研究。文章对免疫器官组织中PRL-2的表达及其在巨噬细胞中的作用进行了初探。研究发现,PRL-2在免疫组织与器官中均高表达,PRL-2~(-/-)小鼠的脾脏与胸腺中免疫细胞的类群及比例与WT小鼠相比无显著差异。体外研究显示,PRL-2的缺失不影响小鼠原代巨噬细胞细胞因子的产生及细菌吞噬能力,但PRL-2缺失后巨噬细胞杀伤细菌的能力显著增强。综上,PRL-2可能通过调控固有免疫细胞的杀菌能力在抗感染免疫中发挥作用。     

尼古丁对中性粒细胞活化及细胞间粘附分子基因表达的影响

目的:观察尼古丁对中性粒细胞(PMNs)的活化,PMNs与内皮细胞的粘附及内皮细胞表达ICAM-1mRNA,有助于阐明尼古丁在慢性阻塞性肺疾患(COPD)炎症发病中的作用。方法:测定β-葡萄糖醛酸苷酶及溶菌酶活性,以反映PMNs的活化;培养人脐静脉内皮细胞,观察PMNs与内皮细胞的粘附;制备探针,提取总RNA,Northern杂交测细胞间粘附分子-1(ICAM-1)mRNA。结果:尼古丁可活化PMNs,增加PMNs－内皮细胞粘附;增强ICAM-1mRNA表达,764-3可明显抑制尼古丁的上述作用。结论:尼古丁通过活化PMNs,促进PMNs－内皮细胞粘附,在COPD慢性炎症发病中起重要作用。而这种粘附作用的增加与粘附分子表达增强有关;抑制尼古丁的上述作用可能是764-3抗炎作用的部分机理。     

促炎刺激物对淋巴细胞粘附和穿透内皮细胞的影响

本文观察了促炎刺激物（LPS、类脂A、TNFα、IL-1β、IL-6）对淋巴细胞粘附于内皮细胞及对穿透内皮细胞能力的影响,结果显示:LPS、类脂A、TNFα、IL-1β均能明显增加淋巴细胞与内皮细胞之间的粘附并能增强淋巴细胞穿透内皮细胞的能力（P均<0.05）,其中以IL-1β的作用最为明显,而IL-6对淋巴细胞粘附于内皮细胞及淋巴细胞穿透内皮细胞的能力无明显影响（P>0.20）。     

凝血酶、花生四烯酸及阿司匹林对中性粒细胞与血小板之间粘附的影响

目的: 探讨不同浓度的凝血酶、花生四烯酸(AA)和阿司匹林对大鼠血小板与中性粒细胞之间粘附作用的影响。方法: 应用改良的Hamburger等和沈志强等方法观察大鼠血小板与中性粒细胞之间的粘附作用。结果: 凝血酶在50U/L就能促进血小板中性粒细胞之间的粘附,且随浓度增加而增大,在300U/L时达最大作用,再随着浓度的增加,粘附率反而下降。25μmol/L的AA即表现为促进细胞间粘附作用,并随浓度的增加而增强,于100μmol/L时达最大作用,若再增加浓度,则粘附率明显下降;阿司匹林对上述两种血小板激活剂引起的血小板-中性粒细胞间的粘附具有明显抑制作用,且呈浓度相关性,其IC₅₀分别为62.9和51.4μmol/L。结论: 凝血酶和AA在一定范围内呈浓度依赖性促进血小板-中性粒细胞的粘附作用,但浓度再增加,则粘附率反而下降;阿司匹林可明显抑制凝血酶和AA引起的血小板-中性粒细胞粘附作用。     

Effect of carbohydrates on adherence of Escherichica coli to human urinary tract epithelial cells.

Adherence of Escherichia coli cells to voided uroepithelial cells from healthy women was measured by use of [3H]uridine-labeled bacteria filtered through a polycarbonate membrane filter (5-micrometer pore size). At a concentration of 2.5% (wt/vol), D-mannose, D-mannitol, alpha-methyl-D-mannoside, and yeast mannan completely inhibited adherence of the bacteria to the epithelial cells. At this same concentration, D-fructose, D-lyxose, D-arabinose, and D-glyceraldehyde partially inhibited adherence. Reducing the concentration of D-mannose, or its derivatives, to between 1.0 and 0.1% resulted in partial inhibition in the adherence of the bacteria; a further reduction in the concentration to between 0.01 and 0.001% caused an enhancement of adherence up to 160% of the control level. Bacterial preincubation in 2.5% D-mannose for 1 min before epithelial cells were added completely inhibited adherence; similar treatment of the epithelial cells had no significant effect on subsequent adherence of the bacteria. Bacteria that were preincubated for 1 h with D-mannose at concentrations between 0.1 and 0.75% showed enhanced adherence. The inhibitory effect of D-mannose was decreased if bacterial adhesive ability, or cell receptivity, increased. A variety of other carbohydrates tested had no effect on the adherence of E. coli to the uroepithelial cells. These results suggest that adherence can be altered by interaction(s) between specific carbohydrate molecules and receptors on the bacterial surface.     

Correlation between adherence to HeLa cells and serogroups, serotypes, and bioserotypes of Escherichia coli.

Four hundred fifty Escherichia coli strains of 45 O serogroups and subgroups and 112 serotypes were studied to determine their patterns of adherence to HeLa cells. Adherence was exhibited by strains of 17 O serogroups and subgroups, but within these groups more than one adherence pattern was frequently observed. However, within each serotype, the adherence pattern was highly consistent. Localized adherence (LA) was observed much more frequently in serotypes that we considered to be enteropathogenic E. coli serotypes (93%) than in other serotypes (14%), whereas diffuse adherence (DA) occurred predominantly among nonenteropathogenic E. coli strains. Determination of biochemical characteristics showed that within O serogroups, nonmotile strains tended to have the same behavior as motile strains with the LA adherence pattern, suggesting that they were derived from these motile strains. LA and non-LA strains of the same serotype differed biochemically. LA appears to be a property of most E. coli commonly considered to be enteropathogenic and should assist attempts to determine which E. coli are enteropathogenic and to elucidate their pathogenic mechanisms.     

Direct effect of nitroxoline in the inhibition of bacterial adherence to urinary catheters

Nitroxolin or 5-nitro-8-hydroxyquinoline, used in the treatment of acute or recurrent uncomplicated urinary tract infection, has been investigated to demonstrate direct inhibitory effect on Escherichia coli adherence to solid surfaces. First of all, influence of growth medium on bacterial adherence was studied. No relation occurs between growth media enhancing production of adhesins and the ability to adhere to solid surfaces. While bacteria are grown on minimal medium, nitroxolin (MIC/16 to MIC/4) can significantly reduce bacterial adherence to urinary catheter of uropathogenic strains of Escherichia coli AL52 and 382. Increasing the concentration of nitroxolin does not proportionally modify this decrease. When growth is realised on LB broth or agar, nitroxolin does not affect bacterial adherence of strain AL52 and higher doses (8 to 32 mg.l-1) are necessary to obtain the same inhibition of adherence of strain 382. Nitroxolin, in certain conditions, can, directly and rapidly, reduce bacterial adherence to solid surfaces.     

Inhibition of bacterial adherence by nitroxoline on cellular adhesion and on urinary catheter surfaces

Nitroxolin or 5-nitro-8-hydroxyquinoline, used in the treatment of acute or recurrent uncomplicated urinary tract infection (UTI), has been investigated to demonstrate inhibitory effect on bacterial adherence to epithelial cells or solid surfaces. Nitroxolin in vitro and in urine inhibits bacterial adherence of E. coli 38 (MS/MS) on HeLa cells and epithelial cells from human bladder mucosa. In the same conditions, norfloxacin has no effect. Nitroxolin (MIC/8) decreases with a statistically significant difference (p less than 0.001) the bacterial attachment to a urinary catheter surface made in siliconated latex. These results justify the performance of a clinical trial in the prophylaxis of recurrent UTI and the outcome of a bacteriuria associated with indwelling or intermittent bladder catheter.     

Pathoadaptive mutation that mediates adherence of shiga toxin-producing Escherichia coli O111

The adherence of pathogenic Escherichia coli strains to intestinal epithelium is essential for initiation of infection. The cad operon encodes the lysine decarboxylase (LDC) system responsible for metabolizing lysine, and this operon has been proposed as an antivirulence mechanism in enteroinvasive E. coli and Shigella flexneri and as a factor mediating E. coli O157:H7 adherence. We sought to determine whether the LDC activity was present in a phylogenetically characterized collection of diarrheagenic E. coli (DEC) strains and to establish whether its expression was associated with their adherence to tissue culture cells. LDC activity was found in most of the pathogenic E. coli strains tested and was absent from Shiga toxin-producing E. coli (STEC) O111 strains (DEC pathotype 8). Analysis of the cad region in these O111 strains indicates that the operon has been rearranged and some of the genes are either missing or disrupted. A similar rearrangement was found in an E. coli O111:H8 strain recently isolated from an outbreak in Texas. Complementation of the LDC-negative strains with the cad operon in trans restored the LDC activity and resulted in a reduction in adherence to tissue culture cells. Initial analysis of the protein profiles on the surface of the O111 strains indicates that the LDC activity has an effect on the expression of the adhesin intimin. Cadaverine had a slight effect on LDC-negative strain adhesion but none on intimin expression. Our data suggest that this pathoadaptive mutation is an important mechanism to control functions potentially implicated in the pathogenesis of these organisms.     

Inhibition of bacterial adhesion of uropathogenic Escherichia coli strains by the urine of patients treated with nitroxoline

The present study evaluates the effects of sub-inhibitory concentrations of nitroxoline, (oxyquinoline derivative) widely used in the treatment of uncomplicated, urinary tract infections, on the adherence of uropathogenic strains of Escherichia coli. These bacterial strains showed mannose sensitive and/or mannose resistant hemagglutinating activity (HA). In the presence of nitroxoline and at sub-MIC concentrations, inhibition of adherence is 90% (MIC/4), 87% (MIC/8), and 70% (MIC/16), whatever HA's are expressed by the E. coli strains. The inhibitory effect on adherence is also observed in the urine after oral administration of 400 mg of nitroxoline. The concentrations of nitroxoline in the urine are determined by microbiological assay (anti-bacterial activity) and by physico-chemical assay (total nitroxoline and free nitroxoline). The percentages of inhibition are related to the concentrations of free and conjugated nitroxoline. For a 1/16 dilution of urine, the inhibitory effect is 70% and 87% respectively 1 h 30 and 2 h 30 after oral administration of nitroxoline. After 5 h, a similar inhibitory effect is observed for a 1/2 dilution of urine. These results justify the performance of a clinical trial on the prophylaxis of recurrent urinary tract infections by nitroxoline.     

Effects of fibronectin and other salivary macromolecules on the adherence of Escherichia coli to buccal epithelial cells.

The effect of saliva and fibronectin (Fn) on the adherence of a type 1 fimbriated strain of Escherichia coli to human buccal epithelial cells was studied. Saliva pretreatment of epithelial cells led to a dose-dependent increase in adherence that was inhibited by alpha-methyl mannoside, which is typical of a type 1 fimbria-mediated event. The molecules responsible for affecting this increased adherence were nondialyzable and were recovered after lyophilization. E. coli adherence was stimulated by individual saliva samples from each of 11 volunteers. Fn inhibited E. coli adherence to saliva-treated buccal cells by more than 60%. Biotinylated E. coli and Fn were reacted with Western blots of whole saliva to identify the receptors that might explain the phenomenon described above. Both E. coli and Fn bound to 57- and 62-kilodalton (kDa) protein bands in Western blots of sodium dodecyl sulfate gels of whole saliva. The binding of E. coli to these bands was inhibited by pretreatment with unlabeled Fn. To study these salivary components, samples of saliva were electrophoresed on sodium dodecyl sulfate gels, strips corresponding to the appropriate molecular weights were cut out, and the proteins were eluted electrophoretically. Material that eluted from strips at 57 and 62 kDa, but not that from a control strip, stimulated E. coli adherence to buccal cells. Alternatively, saliva was fractionated over 100- and 50-kDa cutoff filters. Of the three fractions obtained, only the fraction passing through the 100-kDa filter and retained by the 50-kDa filter stimulated E. coli adherence to buccal cells. This fraction also increased the binding of Fn to buccal cells. These observations suggest the possibility that one or more salivary components bind to the surface of buccal cells and serve as receptors for type 1 fimbriated E. coli. Fn also binds to this isolated material; and it is apparently by these interactions, at least in part, that saliva stimulates and Fn inhibits E. coli adherence. The way in which these interactions may affect bacterial adherence in vivo remains to be elucidated.     

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.     

Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes, and basophils and from polymorphonuclear granulocytes.

We investigated the role of Escherichia coli expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains differed in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature. The various bacterial strains differed in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene B4 generation and reduced w-oxidation of leukotriene B4. Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E. coli.     

Influence of sublethal concentrations of antibiotics on the expression of the mannose-specific ligand of Escherichia coli.

The ability of streptomycin, in subinhibitory concentrations, to differentially suppress the acquisition of the mannose-binding activity of Escherichia coli was demonstrated in several strains, but not one with a ribosomal mutation to high-level streptomycin resistance, rpsL. We also determined that the growth of bacteria in other antibiotics, notably those that interfere with protein synthesis, resulted in diminished mannose-binding activity (as measured by yeast cell agglutination), degree of piliation (as measured by electron microscopy), and adherence to human oral epithelial cells. The aminoglycoside antibiotics streptomycin, gentamicin, and neomycin had the most marked effects relative to their minimum inhibitory concentrations, followed by tetracycline. Both spectinomycin and chloramphenicol had more effect on adherence than on piliation, although spectinomycin had a more pronounced effect on mannose-binding activity than did chloramphenicol. We conclude that antibiotics, at concentrations below their minimum inhibitory concentration, may have profound effects on surface properties of bacteria that may be pertinent for their ability to colonize and infect human mucosal surfaces. The mechanism(s) may vary from one drug to another, but appear to depend on the classic actions of the antibiotics on inhibiting protein synthesis.