单克隆抗体S2C4对2型志贺毒素及其亚型毒性的中和作用

纯化的2型志贺毒素(Shiga toxin 2,Stx2)经福尔马林脱毒后免疫BALB/c小鼠制备Stx2单克隆抗体,用体外中和试验对具有中和活性的阳性抗体克隆进行初筛,对所获得的中和抗体的重、轻链同种型及结合特异性进行鉴定,其中和保护作用通过体内、体外中和试验加以验证,最后,中和抗体对Stx2亚型Stx2c和Stx2vha的中和谱用体内中和试验验证．结果显示,12株抗Stx2的阳性抗体克隆中,只有1株具有中和活性,命名为S2C4,其重、轻链同种型为G₁/κ,其靶分子为Stx2的A亚单位,与Stx2的B亚单位或Stx1不结合．在体外中和试验中S2C4可有效中和Stx2对Vero细胞的杀伤作用,同样,S2C4可中和致死量的Stx2及其亚型Stx2c和Stx2vha对小鼠的毒性作用．该抗体有望成为治疗产志贺毒素大肠杆菌感染的候选分子．     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.     

Exosomes released from Shiga toxin 2a–treated human macrophages modulate inflammatory responses and induce cell death in toxin receptor expressing human cells

Shiga toxins (Stxs) produced by Stx‐producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx‐mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte‐derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage‐like differentiated THP‐1 cells treated with Stxs secreted Stx‐associated exosomes (Stx‐Exo) of 90–130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb₃)‐dependent manner. Stx2‐Exo engulfed by Gb₃‐positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK‐2. Stx2‐Exo contained pro‐inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2‐Exo‐mediated human renal cell death. Stx2‐Exo isolated from human primary monocyte–derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx‐containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin‐sensitive cells. Therapeutic interventions targeting Stx‐containing exosomes may prevent or ameliorate Stx‐mediated acute vascular dysfunction.     

Sildenafil reduces polyuria in rats with lithium-induced NDI

Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; α-, β-, and γ-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, γ-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.     

Lithospermic acid B isolated from Salvia miltiorrhiza ameliorates ischemia/reperfusion-induced renal injury in rats

The present study was designed to examine whether lithospermic acid B (LSB) isolated from Salvia miltiorrhiza has an ameliorative effect on renal functional parameters in association with the expression of aquaporin 2 (AQP 2) and Na,K-ATPase in the ischemia-reperfusion induced acute renal failure (ARF) rats. LSB showed strong antioxidant activity against production of reactive oxygen species (ROS), ROS-induced hemolysis, and production of lipid peroxide in a dose-dependent manner. Polyuria caused by down-regulation of renal AQP 2 in the ischemia-reperfusion induced ARF rats was partially restored by administration of LSB (40 mg/kg, i.p.), restoring expression of AQP 2, in renal inner and outer medulla. The expression of Na,K-ATPase alpha1 subunit in outer medulla of the ARF rats was also restored in the ARF rats by administration of LSB, while beta1 subunit level was not altered. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also partially restored in ischemia-ARF rats by administration of LSB. Histological study also showed that renal damages in the ARF rats were abrogated by administration of LSB. Taken together, these data indicate that LSB ameliorates renal defects in rats with ischemia-reperfusion induced ARF, most likely via scavenging of ROS.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage

Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.     

Role of Shiga toxin 2 (Stx2)-binding protein,human serum amyloid P component (HuSAP), in Shiga toxin-producing Escherichia coli infections: assumption from in vitro and in vivo study using HuSAP and anti-Stx2 humanized monoclonal antibody TMA-15

Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections. Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP). Here, we report the role of HuSAP in STEC infections. HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice. By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP. These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP.     

Improved production of holotoxin Stx2 with biological activities by using a single-promoter vector and an auto-induction expression system

The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinant Shiga toxin 2 (rStx2). rStx2 was expressed in the E. coli periplasm in a completely soluble, biologically active form. The final yield of purified rStx2 from each liter of culture in LB medium and ZYM-5052 medium was approximately 2.3 mg and 3.5 mg, respectively. The highest amount of rStx2 accounted for 27.8% of total bacteria protein in ZYM-5052 medium. rStx2A and rStx2B isolated from rStx2 by electroelution were, respectively, identified by N-terminal protein sequencing. Signal peptides with the sequence MKCILFKWVLCLLLGFSSVSYS and MKKMFMAVLFALASVNAMA were identified at the N terminus of rStx2A and rStx2B, respectively. Our rStx2 possessed Vero cell CD₅₀ value about 500 pg and LD₅₀ value approximately 6 ng. rStx2 can be substitute for natural toxin Stx2, which can be used for animal models, drug screening, vaccine research, and so on.     

Evaluation of Shiga toxin 2e‐specific chicken egg yolk immunoglobulin: Production and neutralization activity

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e‐specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti‐Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost‐effective agent to develop for prophylactic foods or diagnosis kits for edema disease.     

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.     

猪水肿病毒素Stx2e的致Vero细胞凋亡作用

摘要：【目的】研究猪水肿病的致病因子志贺毒素2e（Shiga toxin 2e, Stx2e）的致病机理。【方法】以AO/EB荧光染色法、琼脂糖凝胶电泳法和Western blot等方法研究Stx2e对Vero细胞的致凋亡作用及其信号途径。【结果】从细胞形态学和染色质水平证明,Stx2e 能诱导Vero细胞凋亡,并表现出时间和浓度依赖性;同时引起caspase-3表达量明显上调,Bax、caspase-9的表达量没有明显变化。【结论】Stx2e对Vero细胞的致凋亡作用主要通过膜受体通路引起,线粒体信号通路所起的作用较小。     

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

志贺毒素及其预防治疗剂的研究进展

志贺毒素（Shigatoxin,Stx）主要由肠出血性大肠杆菌（EHEC）产生,是其主要的致病毒力因子,可通过引起急性肾衰竭导致死亡。迄今为止尚没有可推荐的治疗方案能够有效地预防或治疗Stx引起的疾病。目前,对于Stx的研究热点主要包括：Stx尚未清楚的致病机理研究,Stx与HUS的相关性研究,以及预防、治疗由Stx引起的疾病的研究。本文就以上几方面对国内外的相关研究进行总结及讨论。     

Reconstitution of active recombinant Shiga toxin (Stx)1 from recombinant Stx1-A and Stx1-B subunits independently produced by E. coli clones

Escherichia coli clones expressing recombinant Shiga toxin (Stx)1-A and recombinant Stx1-B subunits, were established. Culture supernatants of these clones were examined for inhibitory activity on in vitro protein synthesis using luciferase as a reporter enzyme. Culture supernatant of the clone expressing Stx1-A, but not Stx1-B, showed the inhibitory activity. Neither recombinant Stx1-A nor Stx1-B showed Vero cell cytotoxicity. For reconstitution of biologically active toxin, the culture supernatants of the Stx1-A clone and the Stx1-B clone were mixed. The reconstituted recombinant Stx1 showed both Vero cell cytotoxicity and inhibition of in vitro protein synthesis.     

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria

Aims: The Shiga‐like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. Methods and Results: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non‐Stx1‐producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. Conclusions: The anti‐STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. Significance and Impact of the Study: This is the first report regarding the usage of anti‐STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.     

Calpain-mediated AQP2 proteolysis in inner medullary collecting duct

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.     

Decreased expression of AQP2 and AQP4 water channels and Na,K-ATPase in kidney collecting duct in AQP3 null mice

BACKGROUND INFORMATION: Phenotype analysis has demonstrated that AQP3 (aquaporin 3) null mice are polyuric and manifest a urinary concentration defect. In the present study, we report that deletion of AQP3 is also associated with an increased urinary sodium excretion. To investigate further the mechanism of the decreased urinary concentration and significant natriuresis, we examined the segmental and subcellular localization of collecting duct AQPs [AQP2, p-AQP2 (phosphorylated AQP2), AQP3 and AQP4], ENaC (epithelial sodium channel) subunits and Na,K-ATPase by immunoperoxidase and immunofluorescence microscopy in AQP3 null (-/-), heterozygous (+/-) mice, wild-type and unrelated strain of normal mice. RESULTS: The present study confirms that AQP3 null mice exhibit severe polyuria and polydipsia and demonstrated that they exhibit increased urinary sodium excretion. In AQP3 null mice, there is a marked down-regulation of AQP2 and p-AQP2 both in CNT (connecting tubule) and CCD (cortical collecting duct). Moreover, AQP4 is virtually absent from CNT and CCD in AQP3 null mice. Basolateral AQP2 was virtually absent from AQP3 null mice and normal mice in contrast with rat. Thus the above results demonstrate that no basolateral AQPs are expressed in CNT and CCD of AQP3 null mice. However, in the medullary-collecting ducts, there is no difference in the expression levels and subcellular localization of AQP2, p-AQP2 and AQP4 between AQP3 +/- and AQP3 null mice. Moreover, a striking decrease in the immunolabelling of the alpha1 subunit of Na,K-ATPase was observed in CCD in AQP3 null mice, whereas a medullary-collecting duct exhibited normal labelling. Immunolabelling of all the ENaC subunits in the collecting duct was comparable between the two groups. CONCLUSIONS: The results improve the possibility that the severe urinary concentrating defect in AQP3 null mice may in part be caused by the decreased expression of AQP2, p-AQP2 and AQP4 in CNT and CCD, whereas the increased urinary sodium excretion may in part be accounted for by Na,K-ATPase in CCD in AQP3 null mice.     

Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.