Stx)-producing Escherichia Coli Infection Research Articles

Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS).

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.

The Antibiotic Polymyxin B Impairs the Interactions between Shiga Toxins and Human Neutrophils.

Hemolytic uremic syndrome (HUS) is the life-threatenig sequela of intestinal infections by Shiga toxin (Stx)-producing Escherichia coli (STEC) in children. Human neutrophils specifically bind Stx through TLR4, the receptor of LPS. The binding could be considered protective (Stx sequestration) or harmful (toxin delivery to target organs). The amount of Stx on neutrophils is in equilibrium with the amount of Stx present in the gut, and it is also related to renal and neurologic symptoms. The TLR4-mediated interaction of LPS with innate immune cells is hampered by the well-known antibiotic polymyxin B. In this study, we show that the same antibiotic impairs the binding of Stx to neutrophils, also blocking their functional effects (release of CXCL8, formation of neutrophil/platelet aggregates) involved in HUS pathogenesis. Controls for contaminating LPS in Stx-induced neutrophil responses inhibited by polymyxin B were performed. Stx interact with human neutrophils through their A chain, since these leukocytes do not express globotriaosylceramide, the specific receptor for Stx B chains. Consistently, polymyxin B blocked the enzymatic activity of Stx1, Stx2, Stx1 A chain, and the analogous plant protein gelonin, whereas the antibiotic did not show any protective effect on Stx-induced cytotoxicity in globotriaosylceramide-expressing Raji cells. Antibiotic administration is not recommended in human STEC infections during the prodromal intestinal phase, and the toxicity of polymyxin B could further discourage its therapeutic use. However, nontoxic, nonbactericidal polymyxin derivatives have been developed and might be used in animal models of STEC infection to study their efficacy in preventing the onset of HUS during the systemic blood phase of Stx.

A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins).

Bovine macrophages sense Escherichia coli Shiga toxin 1.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections in cattle are asymptomatic; however, Stx impairs the initiation of an adaptive immune response by targeting bovine peripheral and intraepithelial lymphocytes. As presumptive bovine mucosal macrophages (Mø) are also sensitive to Stx, STEC may even exert immune modulatory effects by acting on steps preceding lymphocyte activation at the Mø level. We therefore studied the expression of the Stx receptor (CD77), cellular phenotype and functions after incubation of primary bovine monocyte-derived Mø with purified Stx1. A significant portion of bovine Mø expressed CD77 on their surface, with the recombinant B-subunit of Stx1 binding to >50% of the cells. Stx1 down-regulated significantly surface expression of CD14, CD172a and co-stimulatory molecules CD80 and CD86 within 4 h of incubation, while MHC-II expression remained unaffected. Furthermore, incubation of Mø with Stx1 increased significantly numbers of transcripts for IL-4, IL-6, IL-10, IFN-γ, TNF-α, IL-8 and GRO-α but not for IL-12, TGF-β, MCP-1 and RANTES. In the course of bovine STEC infections, Stx1 appears to induce in Mø a mixed response pattern reminiscent of regulatory Mø, which may amplify the direct suppressive effect of the toxin on lymphocytes.

Protective efficacy and pharmacokinetics of human/mouse chimeric anti-Stx1 and anti-Stx2 antibodies in mice.

In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.

New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A serious sequela of disease, the hemolytic uremic syndrome (HUS) may arise in up to 25% of patients. The development of HUS after STEC infection is linked to the presence of Stx. STEC strains may produce one or more Stxs, and the Stxs come in two major immunological groups, Stx1 and Stx2. A multitude of possible therapeutics designed to inhibit the actions of the Stxs have been developed over the past 30 years. Such therapeutics are important because antibiotic treatment of STEC infections is contraindicated due to an increased potential for development of HUS. The reason for the increased risk of HUS after antibiotic treatment is likely because certain antibiotics induce expression of the Stxs, which are generally associated with lysogenic bacteriophages. There are a few potential therapeutics that either try to kill STEC without inducing Stx expression or target gene expression within STEC. However, the vast majority of the treatments under development are designed to limit Stx receptor generation or to prevent toxin binding, trafficking, processing, or activity within the cell. The potential therapies described in this review include some that have only been tested in vitro and several that demonstrate efficacy in animals. The therapeutics that are currently the furthest along in development (completed phase I and II trials) are monoclonal antibodies directed against Stx1 and Stx2.

A Poly- N -Acetylglucosamine−Shiga Toxin Broad-Spectrum Conjugate Vaccine for Shiga Toxin-Producing Escherichia coli

ABSTRACTMany pathogens produce the β-(1−6)-linked poly-N-acetylglucosamine (PNAG) surface polysaccharide that is being developed as a broadly protective antimicrobial vaccine. However, it is unknown whether systemically injected PNAG vaccines or antibodies would provide protective immunity against pathogens confined to the gastrointestinal tract such as Shiga toxin (Stx)-producing Escherichia coli (STEC), an important group of gastrointestinal (GI) pathogens for which effective immunotherapeutics are lacking. To ascertain whether systemic IgG antibody to PNAG impacts this infectious situation, a vaccine consisting of a synthetic nonamer of nonacetylated PNAG, 9GlcNH2, conjugated to the Shiga toxin 1b subunit (9GlcNH2-Stx1b) was produced. Rabbit antibodies raised to the conjugate vaccine were tested for bacterial killing and toxin neutralization in vitro and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including E. coli O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups.

English

Hemolytic-uremic syndrome (HUS), mainly caused by Shiga toxin (Stx) producingEscherichia coli (STEC) such as E. coli O157:H7 and STEC/EAggEC, is a serious complication predominantly leading to renal failure and even death. We have previously reported that a monoclonal antibody effectively neutralizes Stx2 in vitro and in vivo toxicity models. As a therapeutic agent against HUS, the mouse origin of this antibody can trigger human anti-murine antibody (HAMA) reactions thereby restricting its clinical application. In order to reduce its immunogenicity for use in humans, in this study, a mouse/human chimeric antibody designated rS2C4-IgG1 was developed in baculovirus/insect cell expression system. Analysis of antigen-binding and competitive binding revealed that rS2C4-IgG1 possessed specificity and affinity similar to that of S2C4. Results from cytotoxicity assays and mouse toxicity model analysis showed that rS2C4-IgG1offers neutralizing activity comparable to its parent MAb in vitro and in vivo. Therefore, the chimeric rS2C4-IgG1 had great potential for use in the treatment of STEC infection.   Key words: Shiga toxin 2, HUS, STEC, chimeric rS2C4-IgG1.

Shiga Toxin 1 Induces on Lipopolysaccharide-Treated Astrocytes the Release of Tumor Necrosis Factor-alpha that Alter Brain-Like Endothelium Integrity

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.

Serotype, Shiga Toxin (Stx) Type, and Antimicrobial Resistance of Stx-Producing Escherichia coli Isolated from Humans in Shizuoka Prefecture, Japan (2003^|^ndash;2007)

The serotype, Shiga toxin (Stx) type, and antimicrobial resistance patterns of 138 Stx-producing Escherichia coli (STEC) strains isolated from humans between 2003 and 2007 in Shizuoka Prefecture, Japan were characterized. The predominant O serogroups of the STEC isolates were O157, O26, and O111. Antimicrobial susceptibility testing of the STEC isolates showed that 31 of the 138 isolates (22.5%) were resistant to antibiotics. Compared to the results reported in the previous studies, a higher rate of STEC O157 isolates were susceptible to all the antimicrobial agents used in this study. However, antimicrobial susceptibility data from this study showed that antimicrobial resistance patterns have increased by 6 compared to the survey performed by Masuda et al. between 1987 and 2002 (Jpn. J. Food Microbiol., 21, 44-51, 2004). This indicates that STEC isolates have evolved to show a variety of antimicrobial resistance patterns. It is important to consider the population of isolates showing decreased susceptibility to clinically relevant drugs such as ciprofloxacin (CPFX) and fosfomycin (FOM). All the 3 STEC isolates resistant to nalidixic acid showed low susceptibility to CPFX (MIC, 0.25-0.5 μg/ml). In addition, a decreased susceptibility to FOM was clearly observed in the E. coli O26 isolates. Our findings also showed that 1 STEC O26 strain could possibly be a chromosomal AmpC β-lactamase hyperproducer. These results suggest that antimicrobial therapy may be less effective in patients with non-O157 STEC infections than in those with STEC O157 infections.

An Epidemiologic Surveillance of Shiga-like Toxin-producing Escherichia coli Infection in Argentinean Children

Shiga-like toxin (Stx)-producing Escherichia coli (STEC) infection is an ongoing health issue that can lead to serious complications, including hemolytic uremic syndrome (HUS) and death. This study assessed demographic and epidemiologic information of STEC infection among Argentinean children. A prospective surveillance of 2435 screened children (age, 0.5-15 years) presenting with watery diarrhea and/or bloody diarrhea was undertaken to evaluate the clinical course of STEC infection. Prevalence of STEC infection was 4.1% among subjects presenting with watery diarrhea for ≤ 5 days' duration, bloody diarrhea for ≤ 36 hours' duration, or both. Incidence of STEC infection was significantly higher in the subjects with bloody diarrhea. Ninety-three STEC+ children underwent further evaluation, of whom 8 (8.6%) developed HUS. White blood cells, particularly neutrophils, were abnormally elevated at screening in 5 of 8 HUS subjects. Quantifiable serum Stx-2 values were noted within 24 to 48 hours after the onset of bloody diarrhea in 3 HUS subjects using a validated chemiluminescence assay, with levels quickly dissipating by HUS onset. Results suggest that young STEC-positive children with bloody diarrhea and exhibiting neutrophilic leukocytosis in the early course of their diarrhea are at risk for HUS progression. The observation of measurable concentrations of Stx-2 levels in the early post-bloody-diarrhea period and rapid dissipation at the time of HUS onset requires further evaluation.

Similarity of Shiga Toxin–producingEscherichia coliO104:H4 Strains from Italy and Germany

Similarity of Shiga Toxin–producing<i>Escherichia coli</i>O104:H4 Strains from Italy and Germany

Recent Progress of Shiga Toxin Neutralizer for Treatment of Infections by Shiga Toxin-Producing Escherichia coli

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.

An Orally Applicable Shiga Toxin Neutralizer Functions in the Intestine To Inhibit the Intracellular Transport of the Toxin

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells.

Safety and Pharmacokinetics of Chimeric Anti-Shiga Toxin 1 and Anti-Shiga Toxin 2 Monoclonal Antibodies in Healthy Volunteers

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic-uremic syndrome (HUS). The rates of STEC infection and complications, including death, are highest among young children and elderly individuals. There are no causal therapies. Because Stx is the primary pathological agent leading to organ injury in patients with STEC disease, therapeutic antibodies are being developed to neutralize systemically absorbed toxin during the early phase of the infection. Two phase I, single-dose, open-label, nonrandomized studies were conducted to evaluate the safety and pharmacokinetics of the chimeric monoclonal antibodies (antitoxins) against Stx 1 and 2 (calphaStx1 and calphaStx2, respectively). In the first study, 16 volunteers received 1 or 3 mg/kg of body weight of calphaStx1 or calphaStx2 as a single, short (1-h) intravenous infusion (n = 4 per group). In a second study, 10 volunteers received a 1-h infusion of calphaStx1 and calphaStx2 combined at 1 or 3 mg/kg (n = 5 per group). Treatment-emergent adverse events were mild, resolved spontaneously, and were generally unrelated to the antibody infusion. No serious adverse events were observed. Human antichimeric antibodies were detected in a single blood sample collected on day 57. Antibody clearance was slightly greater for calphaStx1 (0.38 +/- 0.16 ml/h/kg [mean +/- standard deviation]) than for calphaStx2 (0.20 +/- 0.07 ml/h/kg) (P = 0.0013, t test). The low clearance is consistent with the long elimination half-lives of calphaStx1 (190.4 +/- 140.2 h) and calphaStx2 (260.6 +/- 112.4 h; P = 0.151). The small volume of distribution (0.08 +/- 0.05 liter/kg, combined data) indicates that the antibodies are retained within the circulation. The conclusion is that calphaStx1 and calphaStx2, given as individual or combined short intravenous infusions, are well tolerated. These results form the basis for future safety and efficacy trials with patients with STEC infections to ameliorate or prevent HUS and other complications.

Shiga toxin-producing Escherichia coli O157 in beef and chicken burgers, and chicken carcasses in Buenos Aires, Argentina

We describe the isolation and characterization of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 from cooked and uncooked beef and chicken burgers and from chicken carcasses collected during sampling procedures in 2001 and 2002 in Buenos Aires City, Argentina. Of the 24 STEC O157:H7 strains isolated, 20 were recovered from 19 (6.8%) out of 279 samples of beef and chicken burgers, and 4 strains from 4 (10.3%) out of 39 chicken carcasses. The samples were analyzed following the USDA/FSIS 2002 method. The prevalent stx genotype was stx 2 and stx 2c (12 strains, 50%). All strains were characterized as eae and ehxA-positive. By XbaI-PFGE, the strains yielded 10 different patterns. Eighteen out of 24 strains were grouped in four clusters: #1 (4 strains, AREXHX01.0043), #2 (4 strains, AREXHX01.0022), #3 (8 strains, AREXHX01.0139), and #4 (2 strains, AREXHX01.0200). Identical strains by phage typing, stx genotyping and PFGE were detected in uncooked and cooked beef and chicken burgers in different restaurants, which had been collected on the same or different sampling dates. These findings help to underline the importance of STEC O157 detection in meat products, to improve active surveillance, and to define control strategies in order to prevent new cases of STEC infection.

Asymptomatic healthy slaughterhouse workers in South Korea carrying Shiga toxin-producingEscherichia coli

A total of 1602 stool samples from healthy employees in a slaughter company were screened by PCR for Shiga toxin (Stx)-producing Escherichia coli (STEC). The PCR product of Stx-encoding genes was detected in 90 (5.6%) of 1602 stool samples. Among the 90 stx-positive workers, the Residual Products Handlers and Slaughterers had rates of 8.0% and 6.0%--higher than Inspectors, Grading Testers and Livestock Hygiene Controllers at 3.3%, 2.0% and 3.5%, respectively. Forty-nine (54.4%) were shown to have stx2; 25 (27.7%) carried stx1 and 16 (17.7%) had both stx1 and 2. Distribution of the stx PCR-positive workers by age revealed an increase in STEC infection with age (P<0.05). Phenotypic and genotypic traits of nine STEC strains isolated from eight slaughter plant workers were characterized. A variety of serotypes, five O serogroups (O8, O54, O59, O103 and O153) and two H serogroups (H7 and H32) were found, but none of the strains belonged to the serogroup O157. Eight Vero cell cytotoxicity assay-positive strains were isolated from the workers and these workers were asymptomatic and healthy. The results of the study show that slaughter plant workers are at high risk of STEC infection.

Gnotobiotic Piglet Infection Model for Evaluating the Safe Use of Antibiotics againstEscherichia coliO157:H7 Infection

Shiga toxin (Stx)-producing Escherichia coli (STEC), especially O157:H7, cause bloody diarrhea, and in 3%-15% of individuals the infection leads to hemolytic uremic syndrome (HUS) or other complications. Use of antibiotics to treat STEC infections is controversial. Here, we describe the use of piglets to evaluate the efficacy and mechanism of action of antibiotics in these infections. The effects of 2 antibiotics on STEC toxin production and their mechanisms of action were first determined by enzyme-linked immunosorbent assay and subsequently evaluated clinically in the gnotobiotic piglet infection model. In vitro treatment of clinical and isogenic strains with ciprofloxacin increased the production of Stx2 via phage induction but not the production of Stx1. Azithromycin caused no significant increase in toxin production. After treatment with ciprofloxacin, infected piglets had diarrhea and the severe fatal neurological symptoms associated with Stx2 intoxication. Characteristic petechial hemorrhages in the cerebellum were more severe in ciprofloxacin-treated animals than in control animals. In contrast, azithromycin-treated piglets survived the infection and had little or no brain hemorrhaging. The increased in vitro toxin production caused by ciprofloxacin was strongly correlated with death and an increased rate of cerebellar hemorrhage, in contrast to the effect of azithromycin. The piglet is a suitable model for determining the effectiveness and safety of antibiotics available to treat patients.

Neuronal apoptosis and inflammatory responses in the central nervous system of a rabbit treated with Shiga toxin-2

BackgroundShiga toxins (Stxs) are the major agents responsible for hemorrhagic colitis and hemolytic-uremic syndrome (HUS) during infections caused by Stx-producing Escherichia coli (STEC) such as serotype O157:H7. Central nervous system (CNS) involvement is an important determinant of mortality in diarrhea associated-HUS. It has been suggested that vascular endothelial injuries caused by Stxs play a crucial role in the development of the disease. The current study investigates the relationship between the cytotoxic effects of Stxs and inflammatory responses in a rabbit brain treated with Stx2.MethodsIn a rabbit model treated with purified Stx2 or PBS(-), we examined the expression of the Stx receptor globotriaosylceramide (Gb3)/CD77 in the CNS and microglial activation using immunohistochemistry. The relationship between inflammatory responses and neuronal cell death was analyzed by the following methods: real time quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) to determine the expression levels of pro-inflammatory cytokines, and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method to detect apoptotic changes.ResultsGb3/CD77 expression was detected in endothelial cells but not in neurons or glial cells. In the spinal cord gray matter, significant levels of Gb3/CD77 expression were observed. Severe endothelial injury and microvascular thrombosis resulted in extensive necrotic infarction, which led to acute neuronal damage. Conversely, in the brain, Stx receptor expression was much lower. The observed neuropathology was less severe. However, neuronal apoptosis was observed at the onset of neurological symptoms, and the number of apoptotic cells significantly increased in the brain at a later stage, several days after onset. Microglial activation was observed, and tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the CNS parenchyma was significantly up-regulated. There was significant overexpression of TNF-α transcripts in the brain.ConclusionThis study indicates that Stx2 may not directly damage neural cells, but rather inflammatory responses occur in the brain parenchyma in response to primary injury by Stx2 in vascular endothelial cells expressing Gb3/CD77. These findings suggest that neuroinflammation may play a critical role in neurodegenerative processes during STEC infection and that anti-inflammatory intervention may have therapeutic potential.

Emerging Shiga Toxin-Producing Escherichia coli Serotypes in Europe: O100:H- and O127:H40

Novel and as yet rare non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) serotypes are emerging in Europe. Two different sorbitol-fermenting STECs, O100:H- carrying the virulence gene stx2 and O127:H40 carrying stx1 and eae genes (found in two related subjects), were isolated from patients' stool samples. Non-O157 STEC infections in humans are currently under-diagnosed. This report highlights the need for, and importance of, screening for Shiga toxins or serotypes other than just O157.