Escherichia coli lipopolysaccharides produce serotype-specific hypothermic response in biotelemetered rats

We investigated whether LPS-induced hypothermia develops in a serotype-specific manner in biotelemetered conscious rats. Two different Escherichia coli serotypes of LPSs were injected at a dose of 250 mug/kg ip. E. coli O55:B5 LPS elicited an initial hypothermia and subsequent fever, but E. coli O111:B4 LPS caused more potent monophasic hypothermia. Serum tumor necrosis factor (TNF)-alpha levels were dramatically elevated at the initial phase of the hypothermia induced by both LPSs. This elevation tended to subside at the nadir of E. coli O55:B5 LPS-induced response but progressively increased at the nadir of E. coli O111:B4 LPS hypothermia. Serum IL-10 levels were moderately elevated at the initial phase of the hypothermia and persisted at the same level at the nadir of each LPS-induced response. No change was observed at the serum IL-18 levels. A selective cyclooxygenase (COX)-1 enzyme inhibitor, valeryl salicylate (20 mg/kg sc), abolished the hypothermia without any effect on the elevated cytokine levels. Another COX-1-selective inhibitor, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; 1 mg/kg sc) inhibited hypothermic responses as well. Meanwhile, cytokine levels were also reduced by SC-560 treatment. These findings suggest that LPS-induced hypothermia may have serotype-specific characteristics in rats. E. coli O111:B4 LPS has more potent hypothermic activity than E. coli O55:B5 LPS; that may presumably be related to its higher or sustained capability to release antipyretic cytokines, such as TNF-alpha. COX-1 enzyme may be involved in the generation of the hypothermia, regardless of the type of LPS administered.     

Effects of different serotypes of Escherichia coli lipopolysaccharides on body temperature in rats

The effects of Escherichia coli O55:B5, O127:B8, and O111:B4 serotypes' lipopolysaccharides (LPS) on body temperature were investigated in rats. LPSs were injected intraperitoneally at doses of 2, 50, and 250 microg/kg. A multiphasic and no-dose dependent increase in rectal temperature was observed in response to E. coli O55:B5 LPS at all doses, and in response to E. coli O127:B8 LPS at 2 and 50 microg/kg doses. The highest dose of the latter caused a dual change in rectal temperature, in which hypothermia preceded fever. E. coli O111:B4 LPS was either pyrogenic or hypothermic at 2 and 250 microg/kg doses; respectively, whereas a dual response was observed when the 50 microg/kg dose was injected. Although dual responses were observed after administration of all LPSs at 50 microg/kg dose when the body temperature was recorded by biotelemetry, the hypothermia induced by E. coli O55:B5 LPS was significantly smaller. These data suggest that LPSs induce dose and serotype-specific variable changes on body temperature in rats. This variability may be related to the structure of LPSs. The data also indicate that LPS causes hypothermia with or without fever in rats.     

Human C-reactive protein does not protect against acute lipopolysaccharide challenge in mice

The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo. Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS. However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically. Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E. coli O55:B5 or Salmonella typhimurium. Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed. Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls. Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo.     

Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin,NTS-1

G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.     

Genetic analysis of Escherichia coli O111:B4, a strain of medical and biochemical interest.

Procedures have been worked out which allow, for the first time, the genetic analysis of Escherichia coli O111:K58:H2 (O111:B4). The approximate map position of mutant loci was determined by mating with 15 Hfr strains of E. coli K-12. In addition, P1 transduction procedures were used for establishing relative gene order and linkage for any region of the E. coli O111:B4 chromosome. To obtain these, it was necessary to select for a rare P1 lysogen since E. coli O111:B4 is resistant to phage P1. Finally, genetic homology between E. coli strains K-12 and O111:B4 is suggested since they can form stable haploid hybrids, and several loci have similar map positions in the two strains.     

Lipopolysaccharides and lipoteichoic acids stimulate rat mast cells to cysteinyl leukotriene synthesis

We have investigated the ability of lipopolysaccharides (LPS) and lipoteichoic acids (LTA) to induce rat peritoneal mast cells to degranulation and histamine release, and to cysteinyl leukotriene (LT) generation. We have stated that LPS Salmonella Enteritidis, LPS Escherichia coli O111:B4 and LPS E. coli O55:B5 did not activate rat mast cells to degranulation and histamine release. However, LPSs induced LT synthesis and secretion; the strongest stimulant to generation of LT was LPS E. coli O55:B5 (concentration of LT in supernatant was 830.5 +/-15.2 pg/ml). We have also observed that LTA Staphylococcus aureus and LTA Bacillus subtilis stimulated rat mast cells to degranulation and histamine secretion, even though the percentage of the releases histamine was relatively low (10.0 +/- 1.4 and 10.4 +/- 5.4 at antigen concentration, respectively). At the same time, LTA of both of the bacterial species strongly activate LT generation by mast cells (concentrations of LT in supernatants were 777.9 +/- 11.2 pg/ml and 734.0 +/- 38.3 pg/ml, respectively, at the antigen concentration 50 ng/ml). Our results have shown that LPS oraz LTA activate rat mast cells to secretion of proinflammatory mediators.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

特异性卵黄抗体(IgY)对小鼠大肠杆菌败血症的保护作用

目的:研究特异性IgY对小鼠大肠杆菌败血症的保护作用.方法:以灭活的E.coli O111免疫产蛋母鸡,抗体经水稀释及盐析分离纯化.ELISA法检测大肠杆菌特异性IgY对大肠杆菌及LPS的结合活性.腹腔注射E.coli O111(1011cfu/mL)建立小鼠败血症模型,攻毒剂量为0.1mL/10g体重.小鼠随机分为5组,分别给药保护:空白组(生理盐水)、阴性对照组(非特异性IgY,20mg/mL)、阳性对照组(头孢哌酮20mg/mL)、高剂量组(特异性IgY,40mg/mL),低剂量组(特异性IgY,20mg/mL).给药剂量为:攻毒前,0.15mL/10g体重,每天一次,共两天;攻毒后,0.25mL/10g体重,每天一次,共七天.观察小鼠临床表现、体重变化、白细胞(WBC)和血小板(PLT)数变化及各组小鼠的死亡率.结果:特异性IgY与E.coli O111和LPS均有体外结合活性.大肠杆菌攻毒后,小鼠体重下降,各组小鼠外周血中WBC和PLT数均有不同程度的下降.特异性IgY保护组各项指标较快恢复到正常水平,其他组恢复缓慢.各组小鼠七天内的死亡率分别为:空白组与阴性对照组都为100%;阳性对照组60%;低剂量IgY组30%;高剂量IgY组10%.结论:特异性IgY对小鼠大肠杆菌败血症有保护作用.     

In vitro selection of Escherichia coli O157:H7-specific RNA aptamer

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes life-threatening symptoms in humans worldwide. To rapidly and properly identify the pathogen and avoid its toxic effects, ligands which can directly and specifically bind to the virulent E. coli O157:H7 serotype should be identified. In this study, a RNA aptamer-based ligand which can specifically distinguish the pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX method. To this end, an RNA library was first incubated with the E. coli K12 strain, and the RNAs binding to the strain were discarded. The precluded RNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. This was evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer that distinguishes between the virulent serotype and the nonpathogenic strain specifically bound to an O157:H7-specific lipopolysaccharide which includes the O antigen. This novel O157:H7-specific aptamer could be of potential application as a diagnostic ligand against the pathogen-related food borne illness.     

Harnessing aptamers for electrochemical detection of endotoxin

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K(d)) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.     

An aptamer beacon responsive to botulinum toxins

Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs.     

脂多糖保守表位模拟肽的筛选与鉴定

用针对脂多糖保守表位的单抗2B4对噬菌体随机12肽库进行亲和筛选,通过噬菌体ELISA实验及脂多糖(LPS)竞争抑制实验鉴定阳性克隆.经三轮筛选后,与抗体结合的噬菌体得到明显富集,噬菌体ELISA结果显示,阳性率达80%.将其中12个阳性噬菌体克隆做鼠伤寒杆菌和大肠杆菌LPS竞争抑制实验,抑制作用非常明显,有良好的剂量依赖关系,证明这12个克隆与LPS具相似表位.DNA测序并推导噬菌体展示肽的氨基酸序列为,GPPQWFFSQPQL（5/12,41.7%）,LPQYFWNTATTA（3/12,25%）,FPQNHWNVPWAT（2/12,16.6%）,HSQSFWNAPLAM和AHPWTHGYFPPL（1/12,8.3%）.实验结果表明,用2B4抗体筛选到的噬菌体短肽克隆可模拟保守表位,即脂多糖的模拟肽（位）.     

PAP染色法在微生态学中应用的研究——Ⅰ.细菌染色方法的建立

在微生态学中应用过氧化物酶—抗过氧化物酶(PAP)染色法鉴定细菌的研究还未见报道。本文报告了用埃希氏大肠杆菌(O_(111)B_4)腹腔感染小鼠,取其多种脏器制石蜡切片,建立PAP染色程序。确定了第一抗体(兔抗埃希氏大肠杆菌O_(111)B_4型血清)最佳染色滴度为1:800～3200。观察到埃希氏大肠杆菌(O_(111)B_4)定位于组织器官上的状态。在细菌鉴定上PAP染色法较其它方法更具有优点。     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

S-form lipopolysaccharide (LPS), but not lipid A or R-chemo-type LPS, induces interleukin-6 production in vitamin D3-differentiated THP-1 cells

Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A. We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity. Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E. coli-type lipid A (compound 506), E. coli Re mutant LPS (ReLPS), or alkali-treated LPS. The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406). An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production. These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells.     

Stimulation of adhesion molecules on vascular endothelium by capsular polysaccharide, lipopolysaccharide and components of lipopolysaccharide from Bacteroides thetaiotaomicron

The aim of this study was to assay the influence of capsular polysaccharide (CPS), lipopolysaccharide (LPS) and components of B. thetaiotaomicron lipopolysaccharide--polysaccharide part (PS) and lipid part (lipid A) on the expression of adhesion molecules associated with inflammation (ICAM-1, VCAM-1, E-selectin) on the surface of vascular endothelial cells. Capsular polysaccharide was isolated by the method of Poxton and Ip (1981). Lipopolysaccharides were extracted using the hot phenol-water method (Westphal and Jann, 1965). Components of LPS were prepared by mild acid hydrolysis of lipopolysaccharide. Experiments with bacterial compounds at concentrations 10, 1, 0.1 and 0.01 (mg/ml) were performed on HMEC-1 cell line (human dermal microvascular endothelial cells). Immunoenzymatic ELISA test with mouse monoclonal antibodies against human: ICAM-1, VCAM-1 and E-selectin was applied to determine adhesion molecules. Resting HMEC-1 and E. coli O55:B5 LPS were used as controls in each experiment. Lipopolysaccharides were the strongest stimulants of endothelial adhesion molecules. Capsular polysaccharide caused the expression of three adhesion molecules, but only at the highest concentration (10 mg/ml). The stimulatory activities of LPS lipid components were much higher than the activities of polysaccharide parts. PS preparations did not reveal the property of adhesion molecule stimulation or their activities were weak. The activity of B. thetaiotaomicron cell-surface antigens in the process of adhesion molecule stimulation on vascular endothelium was lower than the activity of E. coli LPS.     

Adhesion of human T lymphocytes and granulocytes to endothelial cells stimulated by cell-surface antigens of Bacteroides thetaiotaomicron

The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 micrograms/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA (in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS.     

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.     

Adhesion of human granulocytes and T lymphocytes to vascular endothelial cells after stimulation with Bacteroides fragilis endotoxin and enterotoxin]

The aim of presented study was to estimates the number of human granulocytes and T lymphocytes adhering to 1 mm2 of vascular endothelial cell culture stimulated by Bacteroides fragilis endotoxins (LPS) and enterotoxin (BFT). HMEC-1 cells were activated with bacterial preparations at the concentration of 10 (micrograms/ml for 4 and 24 hours. Granulocytes and T lymphocytes were isolated from peripheral blood of healthy blood donors. The adhesion tests of granulocytes and adhesion tests of resting and activated with PMA (at the concentration of 10 ng/ml) T lymphocytes to the non-stimulated and stimulated by B. fragilis compounds (LPS and BFT) vascular endothelium were performed. The number of viable leukocytes, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The results of experiments indicate that granulocytes and T lymphocytes (resting and after activation with PMA even in greater number) adhere to the endothelial cells stimulated by B. fragilis endotoxins and enterotoxin. B. fragilis toxins are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS. B. fragilis LPS and BFT preparations stimulate endothelial cells to the adhesion of granulocytes in similar manner, whereas the activation of vascular endothelium to the adhesion of T lymphocytes is differentiated.