Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum.

The pathogenesis of Escherichia coli-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing E. coli (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela. Stx2a, associated to circulating cells (early toxemia) or extracellular vesicles (late toxemia) in blood, is considered the main pathogenic factor in the development of eHUS. Recently, it was found that the functional properties of Stx2a (binding to circulating cells and complement components) change according to modifications of the structure of the toxin, i.e., after a single cleavage of the A subunit resulting in two fragments, A1 and A2, linked by a disulfide bridge. Herein, we describe a method to be used for the detection of the cleaved form of Stx2a in the serum of STEC-infected or eHUS patients. The method is based on the detection of the boosted inhibitory activity of the cleaved toxin, upon treatment with reducing agents, on a rabbit cell-free translation system reconstituted with human ribosomes. The method overcomes the technical problem caused by the presence of inhibitors of translation in human serum that have been stalled by the addition of RNAase blockers and by treatment with immobilized protein G. This method, allowing the detection of Stx2a at concentrations similar to those found by ELISA in the blood of STEC-infected patients, could be a useful tool to study the contribution of the cleaved form of Stx2a in the pathogenesis of eHUS.

Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing Escherichia coli (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at g-forces corresponding to 1 µm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS.

An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum.

Shiga toxins (Stx) released by Stx-producing E. coli (STEC) are virulence factors that are most closely 3associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.

Extrarenal Manifestations in Shigatoxin-associated Haemolytic Uremic Syndrome.

BACKGROUND: Shigatoxin-associated haemolytic uremic syndrome (STEC-HUS) is the most frequent cause of acute kidney injury in children worldwide. Extrarenal manifestations are the main determinants for both, short- and long-term prognosis of patients with STEC-HUS. PATIENTS: 46 patients treated over the last 10 years for STEC-HUS in a single center. METHODS: This retrospective study analysed the incidence and outcome of extrarenal manifestations in our cohort of children with STEC-HUS. Risk factors for extrarenal involvement and adverse outcome were assessed by detailed chart review. RESULTS: Eleven extrarenal manifestations occurred in 9/46 patients comprising 8 neurological, 2 gastro-intestinal, and 1 cardiovascular complication. One patient died from cerebral bleeding. Liver transplantation was required in a girl 18 months after HUS due to secondary sclerosing cholangitis. PATIENTS with extrarenal manifestations were significantly younger and presented with higher leucocyte counts and higher alanine aminotransferase levels at admission. Renal replacement therapy was necessary for a longer period than in patients without extrarenal complications. CONCLUSION: Extrarenal manifestations occurred in about 20% of our patients with STEC-HUS. The identification of risk-factors will help to provide a better management of these patients which might also include novel treatment strategies like complement inhibition.

Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum.

Shiga-like toxins (verotoxins) are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The (15)N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with (15)NH4Cl. Trypsin digestion of the (15)N-labeled protein yields a set of (15)N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h.

Serum Shiga toxin 2 values in patients during acute phase of diarrhoea-associated haemolytic uraemic syndrome.

AIM: Shiga toxins are delivered via systemic circulation and are considered to be the cause of diarrhoea-associated haemolytic uraemic syndrome (HUS), as they injure endothelial cells, particularly in the glomeruli. This study measured Shiga toxin 2 (Stx2) in the serum of children affected in by HUS due to Stx2 producing Escherichia coli. METHODS: The concentration of free Stx2 was measured in the serum of 16 children, collected immediately after admission to the clinic in the acute phase of HUS, using a sandwich enzyme-linked immunosorbent assay. The family members of two children were also investigated, with the relative toxicity of Stx2 assessed by a Vero cell-based fluorescent assay. RESULTS: Stx2 was found in the serum of eight of the 16 children who were investigated. It was also detected in four of the six family members not showing symptomatic HUS, with an extremely high level in two. CONCLUSION: An absent or rather low concentration of Stx2 was found in the serum of children admitted to the clinic with diarrhoea-associated HUS. The high concentration of Stx2 in family members without HUS, but mostly with watery diarrhoea and raised functional activity, was in line with the concept of early injury by Stx2.

Inhibition of verotoxin (VT) 2 absorption into systemic blood from intestine by repeated administration of bovine immune colostral antibody against VT2 in mice.

BACKGROUND/PURPOSE: Whether absorption of verotoxin (VT) 2 from the intestine in mice is inhibited by administration bovine immune colostral antibody against VT2 was investigated. METHODS: Three-week-old mice were administered VT2 solution at 477.8 ng/mL or 955.6 ng/mL, and bovine immune colostral antibody against VT2 was then administered three times. Whey without antibody against VT2 was administered to control mice. Serum levels of VT2 were measured by fluorescence enzyme immunoassay. RESULTS: Serum levels of VT2 in mice administered VT2 solution at 477.8 ng/mL and bovine immune colostral antibody against VT2 scarcely changed. By contrast, serum levels of VT2 in control mice increased and peaked 12 hours after administration. Peak values were 15.4 ± 5.04 ng/mL. Furthermore, serum levels of VT2 at 12 hours and 16 hours in control mice were significantly higher than in mice administered bovine colostral antibody against VT2. Serum levels of VT2 in mice administered antibody at 955.6 ng/mL showed no significant differences between repeated administration of bovine immune colostral antibody and controls. CONCLUSION: These results suggest that absorption of VT2 from the intestine was inhibited by repeated administration of bovine immune colostral antibody against VT2 at early stages of Escherichia coli O157:H7 infection, whereas VT2 in the intestine remained at low levels.

Síndrome urémico hemolítico con compromiso renal leve debido a una cepa de Escherichia coli productora de toxina Shiga [Shiga-toxin-producing Escherichia coli (STEC)] 0145 / Hemolytic uremic syndrome with mild renal involvement due to Shiga toxin-producing Escherichia coli (STEC) 0145 strain

El síndrome urémico hemolítico (SUH) es una afección caracterizada por la presencia de la tríada clásica: anemia hemolítica microangiopática, trombocitopenia y compromiso renal agudo. Los casos de SUH sin insuficiencia renal pueden confundirse con otras enfermedades hematológicas. Presentamos un caso de SUH pediátrico causado por una cepa de Escherichia coli productora de toxina Shiga Shiga-toxin-producing Escherichia coli (STEC) O145 con el genotipo stx2, ehxA, eae subtipo ?1. El niño no requirió diálisis durante la etapa aguda del SUH, evolucionó favorablemente y no tuvo recurrencias hasta el último control; además, mantuvo cifras normales de presión arterial y función renal normal. Esto puede deberse a varios factores: características de la cepa STEC infectante y susceptibilidad del hospedero al daño renal, entre otros. Este hallazgo destaca la participación regional de STEC no-O157 en enfermedades de la infancia y la importancia de realizar una vigilancia activa de todas las formas de SUH

Hemolytic uremic syndrome (HUS) is a disorder characterized by the presence of the classic triad: microangiopathic hemolytic anemia, thrombocytopenia and acute renal injury. HUS without acute renal failure can be confused with other hematologic diseases. An infantile HUS caused by a Shiga-toxin-producing Escherichia coli (STEC) O145 strain carrying genotype stx2, ehxA, eae subtype ?1 is herein reported. The infant did not require dialysis during the acute stage of HUS, evolved favorably, maintained normal blood pressure and normal renal function and had no recurrence until the last control. This could be due to several factors, such as the characteristics of infecting STEC strain and a reduction in host susceptibility to renal injury. This report highlights the regional participation of non-O157 STEC in childhood diseases and the importance of performing active surveillance for all forms of HUS

Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies.

Shiga toxin-producing Escherichia coli (STEC) food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS). Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs). We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t(1/2) α to be 3 min and the clearance phase or t(1/2) ß to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

Shiga toxin type 2 (Stx2), a potential agent of bioterrorism, has a short distribution and a long elimination half-life, and induces kidney and thymus lesions in rats.

Shiga toxin type 2, a major virulence factor produced by the Shiga toxin-producing Escherichia coli, is a potential toxin agent of bioterrorism. In this study, iodine-125 (125I) was used as an indicator to describe the in vivo Stx2 biodistribution profile. The rats were injected intravenously (i.v.) with 125I-Stx2 at three doses of 5.1-127.5 µg/kg body weight. Stx2 had a short distribution half-life (t (1/2)α, less than 6 min) and a long elimination half-life in rat. The toxicokinetics of Stx2 in rats was dose dependent and nonlinear. Stx2 concentrations in various tissues were detected at 5-min, 0.5-h, and 72-h postinjection. High radioactivity was found in the lungs, kidneys, nasal turbinates, and sometimes in the eyes, which has never been reported in previous studies. In a preliminary assessment, lesions were found in the kidney and thymus.

Ingested Shiga toxin 2 (Stx2) causes histopathological changes in kidney, spleen, and thymus tissues and mortality in mice.

The Shiga toxin (Stx)-producing bacterial strain, Escherichia coli O157:H7, colonizes the distal small intestine and the colon, initiating serious illness, including hemolytic-uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Although intravenous administration of purified Stx to primates has been able to reproduce the features of HUS, it has not been conclusively established as to whether ingestion of Stx alone without the bacterium poses a potential health risk. To help answer this question, in this study, we fed Shiga toxin 2 (Stx2) directly into the stomachs of mice via gavage. Our data show that ingestion of Stx2 at a concentration of 50 µg/mouse induces weight loss and kills the mice at 3-5 days post-gavage. Additional studies revealed that the toxin retains activity at low pH, that its activity is neutralized by treatment with toxin-specific antibody, and that about 1% of the fed toxin is absorbed into the blood circulation. Lethality by intraperitoneal (IP) injection of Stx2 occurred at much lower doses than by ingestion. Detailed histopathological evaluation of stained tissues by light microscopy revealed severe histopathological changes in kidneys, spleen, and thymus but not in the pancreas, lymph nodes, heart, lungs, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon. The pathological changes in the kidney appeared similar to those seen in humans with HUS. The cited data suggest that (a) most but not all of the toxin is inactivated in the digestive tract, (b) part of the oral-ingested toxin is absorbed from the digestive tract into the circulation, (c) enough active toxin reaches susceptible organs to induce damage, and (d) Stx2 in the absence of toxin-producing bacteria can be harmful to mice. The results are clinically relevant for food safety because we also found that heat treatments (pasteurization) that destroy bacteria did not inactivate the heat-resistant toxin produced and secreted by the bacteria.

Comparative analysis of the abilities of Shiga toxins 1 and 2 to bind to and influence neutrophil apoptosis.

Hemolytic-uremic syndrome (HUS), the life-threatening complication following infection by the intestinal pathogen Escherichia coli O157:H7, is due to the ability of the pathogen to produce toxins in the Shiga toxin (Stx) family. Activated neutrophils are observed in HUS patients, yet it is unclear whether Stx exerts a direct effect on neutrophils or whether the toxin acts indirectly. The effect of Stx1 and Stx2 on human neutrophils was examined. Neither Stx1 nor Stx2 altered the rate of neutrophil apoptosis. Minimal binding of either toxin to neutrophils was observed, and the toxin was easily eluted from the cells. Stx1 and Stx2 were found to circulate in the plasma of mice following intravenous injection, and both toxins were cleared rapidly from the blood. Together these results suggest that neither Stx1 nor Stx2 interacts directly with neutrophils.

Human serum amyloid P component protects against Escherichia coli O157:H7 Shiga toxin 2 in vivo: therapeutic implications for hemolytic-uremic syndrome.

Shiga toxin (Stx) 2 causes hemolytic-uremic syndrome (HUS), an intractable and often fatal complication of enterohemorrhagic Escherichia coli O157:H7 infection. Here, we show that serum amyloid P component (SAP), a normal human plasma protein, specifically protects mice against the lethal toxicity of Stx2, both when injected into wild-type mice and when expressed transgenically; in the presence of human SAP, there was greatly reduced in vivo localization of Stx2 to the kidneys, suggesting a possible mechanism of protection. In humans, circulating SAP concentrations did not differ between patients with suspected enterohemorrhagic E. coli infection with antibodies to E. coli O157:H7 lipopolysaccharide and those without antibodies or between patients with HUS and those without it. However, the potent protection conferred by human SAP in the mouse model suggests that infusion of supplemental SAP may be a useful novel therapeutic approach to the treatment of this devastating condition.

Binding of shiga toxin 2e to porcine erythrocytes in vivo and in vitro.

Shiga toxin 2e (Stx2e), produced by host-adapted Shiga toxin-producing Escherichia coli (STEC) strains, causes edema disease in weaned pigs. Edema disease is manifested as vascular necrosis, edema, neurologic signs, and death. In this study we sought to determine the correlation between the presence of Stx2e in the blood of STEC-inoculated pigs and the disease outcome. Eleven of 15 (73%) pigs with clinical and 5 of 35 (14%) pigs with subclinical edema disease had detectable levels of Stx2e in the red-blood-cell (RBC) fraction of their blood but not in serum or plasma. The presence of Stx2e in the RBC fraction was strongly associated with the development of clinical disease (relative risk, 5.8; P < 0.0001). Subclinical pigs with Stx2e in their blood developed more-extensive vascular lesions than pigs without detectable Stx2e in their blood (average proportions of necrotic arterioles, 63 and 27.5%, respectively; P = 0.001). Variations in RBC-bound Stx2e levels could in part reflect variations in the binding capacity of RBCs. As an initial step toward addressing this possibility, assays were conducted to determine if pigs vary in the Stx2e binding capacity of their RBCs. An in vitro study of noninoculated pigs demonstrated two phenotypes based on the capacity of the RBCs to bind Stx2e. While RBCs from most of the pigs consistently bound high levels of Stx2e (high-binding phenotype), consistently low Stx2e binding was detected in RBCs from a few pigs (low-binding phenotype). The low- and high-binding phenotypes of individual pigs remained consistent throughout repeated samplings over 2 months.

Evidence of persisting serum antibodies to Escherichia coli O157 lipopolysaccharide and Verocytotoxin in members of rural communities in England.

The techniques of enzyme-linked immunosorbent assay (ELISA) and immunoblotting were used to examine a total of 1667 sera, from apparently healthy members of rural communities in England, for antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157 and Verocytotoxins (VT). Twenty-nine sera from 22 individuals were shown to have antibodies specific for E. coli O157 LPS. Some of these lived on livestock farms and had occupational contact with cattle, suggesting that personnel working with farm animals may produce serum antibodies to the O157 LPS antigens. Fifteen people had IgG class antibodies to O157 LPS, suggesting long-term exposure to E. coli O157 and five people had serum antibodies on more than one occasion showing evidence of persistent antibodies to O157 LPS. Thirteen sera from 12 of 22 individuals also contained antibodies to VT1, VT2 or both toxins. Ten sera contained antibodies to VT1 and VT2, three sera contained antibodies to VT2 only.