Pathogenesis Of Hemolytic Uremic Syndrome Research Articles

Shiga Toxin Activates Complement and Binds Factor H: Evidence for an Active Role of Complement in Hemolytic Uremic Syndrome

Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6-8 and SCRs18-20, but not to SCRs16-17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.

Vacuolisation of human microvascular endothelial cells by enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS), a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis of HUS. We identified an EHEC strain that vacuolates and kills primary human glomerular microvascular endothelial cells (GMVECs) and a human brain microvascular endothelial cell (HBMEC) line. Because the vacuolating effect closely resembles those elicited on other cells by the vacuolating cytotoxin of Helicobacter pylori (VacA), we designated the factor responsible for this effect EHEC vacuolating cytotoxin (EHEC-Vac). EHEC-Vac (a secreted non-serine protease protein) binds to HBMECs rapidly and irreversibly, vacuolates within 30 min after exposure and the effect is maximally apparent at 48 h. Despite the vacuolisation, HBMECs survive for several days before they undergo necrosis. Electron and immunofluorescence microscopy demonstrate that the vacuoles induced by EHEC-Vac originate from lysosomes. Accordingly, they stain with neutral red indicating an acidic microenvironment. Similar to VacA, the EHEC-Vac-mediated vacuolisation is both prevented and reverted by the vacuolar proton pump inhibitor bafilomycin A1, suggesting a similar mechanism of vacuole formation by these toxins. Despite the similarity of phenotypes elicited by EHEC-Vac and VacA, genomic DNA from the EHEC-Vac-producing strain failed to hybridise to a vacA probe, as well as to probes derived from presently known E. coli vacuolating toxins. Through its microvascular endothelium-injuring potential combined with the ability to induce interleukin 6 release from these cells EHEC-Vac might contribute to the pathogenesis of HUS.

Shiga Toxin 1(Stx1) Stimulates Endothelial Cell VWF Secretion through a Ca2+/PKC Signaling Pathway That Does Not Require the Active Toxin Stx1A Subunit

Shiga toxins (Stx) consist of 5 B (binding) subunits that interact with cell surface globotriaosylceramide (Gb3), and a single A subunit that is retrotranslocated into the cytoplasm where it enzymatically inactivates ribosomal RNA. E. coli O157:H7 can express several variants of Shiga toxin (Stx) that cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. The Stx A subunit is required for cytopathic effects on endothelium. In addition, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice, but not in wild-type mice, and this effect requires the presence of von Willebrand factor (VWF). When added to cultured human endothelial cells under conditions of laminar flow, Stx rapidly induces the acute secretion of long strings of VWF that attach to the cell surface and bind platelets with high affinity, which suggests that VWF-induced platelet aggregation might contribute to the pathogenesis of HUS. Whether VWF secretion depends on ribotoxic stress is unknown, and the mechanism of Stx-induced VWF secretion has not been characterized. To address these questions, we investigated VWF secretion by human umbilical vein endothelial cells (HUVECs) treated with Stx1 holotoxin (AB5) or binding subunits (B5). Recombinant Stx1 B5 was expressed in E. coli and purified to homogeneity. The pentameric composition and purity of B5 preparations were demonstrated by gel filtration chromatography and Western blotting. Endotoxin was removed from Stx AB5 and B5 preparations by affinity chromatography. HUVECs were perfused in a parallel plate flow chamber with fluorescently labeled anti-VWF and Stx preparations, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Unexpectedly, we found that Stx1 B5 and Stx1 AB5 were equally potent in stimulating the secretion of VWF strings. String formation was maximal after 5 min and was blocked by soluble analogs of Gb3 or anti-Stx1 B subunit antibodies. Pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min), or a protein kinase C inhibitor (50 nM staurosporine, 30 min) decreased the secretion of VWF strings by 95%, 82%, or 90%, respectively. Treatment with a protein kinase A inhibitor (5 μM H89, 30 min) did not affect VWF string formation. To more directly assess Stx1-induced PLC activation, HUVECs were transfected with a plasmid expressing a PLC-delta PH domain-GFP construct, which binds membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2). When HUVECs were observed by confocal microscopy, both Stx1 AB5 and Stx1 B5 caused a rapid (<60 s) redistribution of fluorescent signal from plasma membrane to cytosol, indicating the acute activation of PLC and hydrolysis of PIP2. In addition, Stx1 AB5 or B5 induced a transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min. Stx1-induced Ca2+ transients were comparable to those induced by 0.1 mM histamine or 1 U/ml thrombin. Stx-induced Ca2+ responses were inhibited by BAPTA-AM or U73122, but not by staurosporine or H89. Treatment with Stx1 AB5 or B5 had no effect on intracellular levels of cAMP. These results indicate that Stx1 B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3, PIP2 hydrolysis by PLC, increased intracellular Ca2+, and activation of PKC. Therefore, Stx1 secreted by enterohemorrhagic E. coli may contribute to the pathogenesis of HUS through at least two mechanisms that affect microvascular endothelium: cell death caused by A subunit-induced ribotoxic stress, and VWF secretion caused by an independent B subunit-induced cell signaling pathway.

Inhibition of water absorption in human proximal tubular epithelial cells in response to Shiga toxin-2

Postdiarrhea hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children in Argentina. It is well established that Shiga toxin type 2 (Stx2) causes direct damage to glomerular endothelial cells and tubular epithelial cells, leading to a reduction in the water handling capacity of the kidney. In this study, we demonstrate that Stx2 and its B subunit (Stx2B) were able to inhibit water absorption across human renal tubular epithelial cell (HRTEC) monolayers without altering the short circuit current and the (3)H-mannitol permeability. Quantitative evaluation of (14)C-inulin transport across HRTEC monolayers showed a similar transport rate both before and after HRTEC treatment with Stx2 that confirmed the integrity of the paracellular pathway. Furthermore, Stx2 produced significant protein synthesis inhibition of HRTEC at concentrations as low as 0.001 ng/ml and 1 h of incubation, whereas Stx2B did not modify it at concentrations as high as 10,000 ng/ml and 6 h of incubation. Our findings suggest that whereas the action of Stx2 appears to be caused mainly by the inhibition of protein synthesis mediated by the A subunit, the binding of Stx2B subunit to the Gb3 receptor may affect the membrane mechanisms related to water absorption. We speculate that inhibition of water absorption may occur in proximal tubular cells in vivo in response to Stx2 and may contribute to the early event of HUS pathogenesis.

Glycosphingolipids Gb3 and iGb3

The glycosphingolipids globotrihexosylceramide (Gb3, CD77) and isoglobotrihexosylceramide (iGb3) are isomers differing only in one glycosidic bond and have been implicated in several processes of the innate and adaptive immune system.Aims1) To verify the function of Gb3 in the pathogenesis of hemolytic-uremic syndrome as the cellular receptor responsible for cytotoxicity caused by verotoxin (VT) elaborated by Shigella and certain strains of E.coli. 2) To investigate in vivo the previously implicated function of iGb3 as the endogenous lipid ligand responsible for positive selection of invariant natural killer T-cells (iNKT), which have an essential regulatory function in infection, tumor rejection and tolerance.MethodsGeneration of mice deficient in Gb3 and iGb3 synthesizing enzymes and VT injection into Gb3-deficient mice. Analysis of iNKT cell development and function by flow cytometry and by administration of the exogenous agonist alpha-galactosylceramide in iGb3-deficient mice.ResultsFor 1) Gb3-deficient mice were insensitive to otherwise lethal doses of VT, and 2) iGb3-deficient mice showed normal numbers of iNKT cells. Furthermore the function of iNKT cells evolving in iGb3-deficient mice was unaffected.Conclusions1) Gb3 is the cellular receptor mediating verotoxin cytotoxicity in haemolytic-uremic syndrome. 2) In contrast to previous indirect implications, iGb3 cannot be regarded as an endogenous ligand responsible for the positive selection of iNKT cells.

Treatment and prevention of enterohemorrhagic Escherichia coli infection and hemolytic uremic syndrome

Over a quarter century after the discovery of verocytotoxin and the first report by Karmali and colleagues of cases of postdiarrheal hemolytic uremic syndrome (HUS) caused by verotoxigenic Escherichia coli (VTEC), otherwise known as Shiga-toxigenic E. coli (STEC), successful treatment of these infections has remained elusive. This is because the pathological insult producing the clinical picture of HUS occurs early in the disease process and curtails quickly, making treatment intervention a largely vain hope. Nevertheless, understanding of the pathogenesis of HUS has expanded and, as a result, we can expect a future breakthrough in the treatment of this life-threatening condition. This review examines the pathogenesis of HUS and explores targets for treatment, including the reasons why certain therapies have failed and why future therapies could be successful. This review also examines the status of vaccine development in prevention of VTEC/STEC disease.

Interactions of Shiga-like toxin with human peripheral blood monocytes

The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx.

Involvement of the fractalkine pathway in the pathogenesis of childhood hemolytic uremic syndrome

AbstractThrombotic microangiopathy and acute renal failure are cardinal features of postdiarrheal hemolytic uremic syndrome (HUS). These conditions are related to endothelial and epithelial cell damage induced by Shiga toxin (Stx) through the interaction with its globotriaosyl ceramide receptor. However, inflammatory processes contribute to the pathogenesis of HUS by sensitizing cells to Stx fractalkine (FKN), a CX3C transmembrane chemokine expressed on epithelial and endothelial cells upon activation, is involved in the selective migration and adhesion of specific leukocyte subsets to tissues. Here, we demonstrated a selective depletion of circulating mononuclear leukocytes expressing the receptor for FKN (CX3CR1) in patients with HUS. We found a unique phenotype in children with HUS distinct from that seen in healthy, uremic, or infected controls, in which monocytes lost CX3CR1, down-modulated CD62L, and increased CD16. In addition, the CD56dim natural killer (NK) subpopulation was decreased, leading to an altered peripheral CD56dim/CD56bright ratio from 10.0 to 4.5. It is noteworthy that a negative correlation existed between the percentage of circulating CX3CR1+ leukocytes and the severity of renal failure. Finally, CX3CR1+ leukocytes were observed in renal biopsies from patients with HUS. We suggest that the interaction of CX3CR1+ cells with FKN present on activated endothelial cells may contribute to renal injury in HUS.

Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and α-Sarcin

Treatment of human endothelial cells with Shiga toxin 1 and 2 leads to the upregulation of genes encoding proinflammatory molecules involved in the pathogenesis of hemolytic-uremic syndrome. The paradoxical effect of inhibitors of mRNA translation, such as Shiga toxins, that at the same time induce protein expression was investigated by studying the relationship between their enzymatic activity (abstraction of adenine from nucleic acids) and the induction of interleukin-8 and granulocyte-macrophage colony-stimulating factor in human endothelial cells. As a positive control, the fungal toxin alpha-sarcin, acting on the same rRNA sequence targeted by Shiga toxins with a different mechanism (RNase activity), was used. The three toxins caused ribosomal lesions that, in turn, induced the activation of p38 stress kinase with kinetics that paralleled the inhibition of translation. Alpha-sarcin was devoid of activity on DNA. Shiga toxin 2 targeted nuclear DNA with more rapid kinetics than did Shiga toxin 1. Since the fungal ribotoxin was fully effective in the induction of proinflammatory proteins, we conclude that damage to ribosomes is indispensable and sufficient to activate protein expression via induction of the stress-kinase cascade. However, gene upregulation events induced by Shiga toxin 2 were much more efficient than those triggered by Shiga toxin 1, although the two toxins impaired translation to the same extent and had overlapping time courses of stress kinase activation. Regulations independent of the ribotoxic stress were assumed to operate in intoxicated cells. We hypothesized that the two bacterial toxins recognize different DNA sequences inducing different regulating effects on gene expression.

Lack of specific binding of Shiga-like toxin (verocytotoxin) and non-specific interaction of Shiga-like toxin 2 antibody with human polymorphonuclear leucocytes

After gastrointestinal infection with Shiga-like toxin (Stx) producing Escherichia coli, the toxin is transported from the intestine to the renal microvascular endothelium. This is the main target for Stx in humans. Previous studies indicated that polymorphonuclear leucocytes (PMN) could serve as carriers for Stx in the systemic circulation. As at a later stage we could not confirm these data, we performed new studies. The binding of Stx1 to PMN was determined in vitro (isolated human PMN and whole blood) and in vivo (injection in mice). The specificity of binding of an antibody against Stx2 to PMN from patients with haemolytic uraemic syndrome (HUS) was determined. This was compared with binding to PMN from healthy controls, and patients after haemodialysis (HD) or on peritoneal dialysis (PD). Furthermore, PMN were incubated with Stx to study possible activation. No specific binding of Stx1 to PMN could be detected. After intravenous injection of the toxin in mice, it was not associated with PMN. The binding of an antibody against Stx2 to PMN was detected in both patients with HUS and patients after HD, but not in patients on PD. Stx was not able to activate PMN. PMN are not acting as transporter for Stx in the pathogenesis of HUS. The interaction of a Stx antibody with PMN from HUS patients is not specific as it can also be observed in patients after HD (possibly due to activation of the PMN). Therefore, binding of Stx antibody to PMN is not reliable as a diagnostic tool for HUS.

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene ( stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic–uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)- hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.

Increased Tissue Factor Bearing Microparticles in Hemolytic Uremic Syndrome.

Microparticles (MP) are circulating cellular fragments that are increased in thrombotic conditions including TTP, antiphospholipid syndrome, and HIT. Tissue factor (TF)-bearing MP are thought to be procoagulant in these conditions. Hemolytic Uremic Syndrome (HUS) is a thrombotic disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and renal failure secondary to glomerular thrombotic microangiopathy. In children, HUS most often occurs after infection with E. coli O157H7. It is hypothesized that E. coli verotoxin injures renal and other endothelium leading to activation of hemostasis, thrombotic microangiopathy and HUS. We studied 56 children at the time of presentation with bloody diarrhea associated with E. coli O157H7 infection (typically day 4 of illness) prior to developing HUS, and 5 healthy children as controls. Subsequently 14 children (Pre-HUS) went on to develop HUS while 42 (Uncomplicated) resolved their illness without developing HUS. We also studied 15 children that presented with already developed HUS (HUS group). We analyzed the total number of MPs, percent of TF-bearing microparticles and their cellular derivation using a 5-color flow cytometry assay. Endothelial-derived MP are identified as positive for CD144 (Ve-Cad)-PE, monocyte-derived MP are positive for CD14-PE-Cy7, and platelet derived MP are positive for CD41a-PerCP. MPs were collected from plasma by ultracentrifugation (100,000g). MPs were defined based on size (0.5–1 um) and strong annexin V binding. The MP distribution in the control group was similar to reports by others: 204 ±98 ×103 MPs/mL of plasma (55% platelet-, 5% endothelial- and 15% monocyte-derived), only 2.6% of MP express TF. The MP distribution in Uncomplicated was 996 ±766 ×103 MPs/mL (36% plat-, 11% endo- and 15% mono-derived) and 5.4% express TF; while pre-HUS showed 1197±1108 ×103 MPs/mL (21% plat-, 12% endo- and 11% mono-derived) and 8.2% express TF. The MP distribution in the HUS group was 1183±949 ×103 MPs/mL (45% plat-, 15% endo- and 19% mono-derived) and 10.9% express TF. Compared to controls, children infected with E. coli O157H7 showed a higher number of total MPs (p<0.02, Mann Whitney) and a higher number of tissue factor bearing MP (p<0.004). Compared to the Uncomplicated group, the pre-HUS group showed fewer platelet MPs (p<0.04). Progression from Pre-HUS to HUS is characterized by increased number of TF-bearing MP distributed in all cellular populations (p<0.01). In a prior study the pre-HUS group showed increased hemostatic activation as indicated by higher levels of F1.2 and D-dimer. We conclude that E. coli 0157:H7 infection results in increased microparticle production versus controls, and increased tissue factor positive microparticles. Development of HUS is associated with further increases in tissue factor positive microparticles, which correlates with the increased hemostatic activation observed in previous studies, suggesting a pathologic role for the release TF-bearing MP in HUS. Interestingly, Pre-HUS patients show reduced numbers of platelet derived microparticles versus uncomplicated patients that resolve without developing HUS, but when HUS develops the number of TF-bearing MP increases. Whether this reduction of platelet-derived microparticles preceding HUS development results from increased binding of MP to endothelium or reduced production of MP in pre-HUS are ongoing studies that may shed some light into the pathogenesis of HUS.

Relevance of neutrophils in the murine model of haemolytic uraemic syndrome: mechanisms involved in Shiga toxin type 2-induced neutrophilia

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the haemolytic uraemic syndrome (HUS). However, the contribution of the inflammatory response in the pathogenesis of the disease has also been well established. Neutrophils (PMN) represent a central component of inflammation during infections, and patients with high peripheral PMN counts at presentation have a poor prognosis. The mouse model of HUS, by intravenous injection of pure Stx type 2 (Stx2), reproduces human neutrophilia and allows the study of early events in the course of Stx2-induced pathophysiological mechanisms. The aim of this study was to address the contribution of PMN on Stx2 toxicity in a murine model of HUS, by evaluating the survival and renal damage in mice in which the granulocytic population was depleted. We found that the absence of PMN reduced Stx2-induced lethal effects and renal damage. We also investigated the mechanisms underlying Stx2-induced neutrophilia, studying the influence of Stx2 on myelopoyesis, on the emergence of cells from the bone marrow and on the in vivo migration into tissues. Stx2 administration led to an accelerated release of bone marrow cells, which egress at an earlier stage of maturation, together with an increase in the proliferation of myeloid progenitors. Moreover, Stx2-treated mice exhibited a lower migratory capacity to a local inflammatory site. In conclusion, PMN are essential in the pathogenesis of HUS and neutrophilia is not merely an epiphenomenon, but contributes to Stx2-damaging mechanism by potentiating Stx2 toxicity.

New Aspects in the Pathogenesis of Enteropathic Hemolytic Uremic Syndrome

Shiga toxin (Stx) subtyping suggests that the clinical outcome of infections caused by Shiga toxin-producing ESCHERICHIA COLI (STEC) depends, in large part, on the STX genotype of the infecting strain. Whereas the presence of the STX2 or STX2C genotype is associated with the ability of STEC to cause the hemolytic uremic syndrome (HUS), strains possessing STX2D or STX2E have been isolated from patients with less severe disease. In addition to the type of Stx, the level of Stx production might be critical for the pathogenicity of STEC. Control of Stx expression appears to be at the level of transcription. Injury to microvascular endothelial cells is the key event underlying the pathogenesis of HUS. We could show that in addition to Stx, STEC also produces other putative virulence factors, such as cytolethal distending toxin, which can contribute to the endothelial injury by interference with the cell cycle, which results in inhibition of cell proliferation and finally cell death.

Shiga toxin 2 and lipopolysaccharide cause monocytic THP-1 cells to release factors which activate platelet function

Platelet and monocyte activation may contribute to hemolytic anemia, thrombocytopenia and renal failure associated with the hemolytic uremic syndrome (HUS) caused by Escherichia coli O157:H7. Since Shiga toxins (Stxs) and lipopolysaccharide (LPS) from this bacterium are implicated in the pathogenesis of HUS, we examined whether stimulation of THP-1 human monocytic cells by Shiga toxin 2 (Stx2) and LPS can lead to the activation of platelet function. We now show that Stx2 causedTHP-1 cells to release the chemokines IL-8, MDC, and RANTES and that the presence of LPS further stimulated this release. IL-8 was produced in greatest amount and was an effective co-agonist for inducing platelet aggregation. Primary human monocytes also released large amounts of IL-8 in response to LPS and Stx2. Factors released byTHP-1 cells exposed to Stx2 and LPS activated platelet function as evidenced by increased aggregation, serotonin secretion, P-selectin exposure and by the formation of stable platelet-monocyte aggregates. Our data therefore show that monocytes exposed to E.coli-derived Stx2 and LPS release factors which activate platelet function.

Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium

Microvascular endothelial damage underlies the pathological changes in haemorrhagic colitis and the haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) are presently the best characterised EHEC virulence factors that can cause the microvascular endothelium injury. Stxs are released by EHEC in the intestine, absorbed across the gut epithelium into the circulation, and transported to small vessel endothelial cells. Then, they presumably injure the host cell by inhibiting protein synthesis, stimulating prothrombotic messages, or inducing apoptosis. The net result is a multi-organ thrombotic process. Moreover, Stxs stimulate a variety of non-endothelial cells to produce and secrete inflammatory mediators (cytokines, chemokines, adhesion molecules) which could potentiate the effects of Stxs on endothelial cells. The association of HUS with Stx-negative E. coli strains stimulated intensive research on putative non-Stx virulence factors, which might also contribute to the pathogenesis of HUS and haemorrhagic colitis. Based on current data, cytolethal distending toxin, EHEC haemolysin, and subtilase cytotoxin might be such candidates.

Decrease of thrombomodulin contributes to the procoagulant state of endothelium in haemolytic uraemic syndrome

Sirs, The observation that Shiga-like toxin 2 (stx2) appears to reduce the surface expression of thrombomodulin (TM) on human glomerular endothelial cells is intriguing, and might be relevant in the pathogenesis of haemolytic uraemic syndrome (HUS) if it is confirmed [1]. Fern ndez et al. [1] pre-stimulated the cells with tumour necrosis factor-a (TNF) to increase surface expression of the stx receptor, Gb3, and thus increase the sensitivity of the cells to the pleomorphic effects of the toxin. These effects include protein synthesis inhibition, stress responses and cell death, depending on the dose of toxin, the position of the cells in the cell cycle and the specific responses of the cell type. Surface-bound TM was quantified using an in situ cell ELISA method. For the groups of cells pre-stimulated with TNF there is a broad correlation between the reduction in measured TM and the reduction in protein synthesis. However, when a different protein synthesis inhibitor, cycloheximide, was used alone at a concentration that gave a modest reduction in protein synthesis, there was no reduction in TM. The authors concluded that stx2 specifically downregulates the expression of TM. However, this cannot be deduced from the data as they are presented. What the reader does not know is whether the same number of endothelial cells remains viable under the conditions of the experiment. Cell killing by different protein synthesis inhibitors is not consistently proportional to the level of inhibition of protein synthesis achieved. If there was more endothelial detachment or cell death with stx2, the apparent reduction in TM might simply represent the smaller number of endothelial cells left at the end of the incubation period. What one actually wants to know is whether the TM expression per cell is reduced. The authors could very easily go some way towards addressing this by vital staining and counting the cells at the end of the experiment to show that the monolayer of endothelial cells was in fact intact. It might be even better to use fluorescence-activated cell sorting, or to test for and quantify cell necrosis and apoptosis. Without this, the suggested down-regulation of TM by stx2 and its role in the pathogenesis of HUS remain somewhat speculative.

Active Site-Inactivated Recombinant Factor VIIa Blocks Shiga Toxin-Induced Upregulation of Tissue Factor Activity on Human Glomerular Endothelial Cells and Human Proximal Tubular Epithelial Cells.

Post-diarrheal hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children, is most often caused by Shiga toxin (Stx)-producing Escherichia coli infection. The most widely recognized tissue damage in HUS occurs within the kidney, most prominently with local thrombosis and platelet-fibrin accumulation, suggesting activation of the coagulation cascade. We have shown that human proximal tubular epithelial cells (HK-2 cells) exposed to Stx-1 augment their constitutive surface tissue factor (TF) activity by 3.2-fold, and that Stx-1 treatment following TNF-alpha exposure enhances TF activity by 2.7-fold on human glomerular endothelial cells (HGECs). We investigated the possibility of modulating this cellular response to Stx-1 by blocking the TF pathway with active site-inactivated recombinant factor VIIa (irFVIIa), which retains the ability to bind TF, but is enzymatically inactive, thereby inhibiting thrombin formation. Moreover, its action is limited to exposed, functional TF and does not significantly prolong the bleeding time in vivo. Monolayers of both HK-2 cells and TNF-alpha-activated HGECs were incubated at 37°C with Stx-1. Functional TF was determined chromogenically as the production rate of factor Xa. The use of 60 nM irFVIIa completely abrogated the enhanced activity of TF caused by 4 hours incubation with 10 pM Stx-1 of TNF-alpha-activated HGECs (N = 4; Figure 1). On HK-2 cells, irFVIIa (60 – 200 nM) also effectively reversed the observed TF upregulation after exposure to 1 nM Stx-1 for 22 hours as well as blocked the constitutive TF expression which is seen with this cell type (N = 3; Figure 2). The TF pathway may prove to be of major importance in the pathogenesis of HUS, and direct targeting of this pathway with irFVIIa might provide a novel therapeutic approach to this disease. [Display omitted] [Display omitted]

Hemolytic uremic syndrome associated with Clostridium difficile colitis

Sirs, The most common cause of diarrheal hemolytic uremic syndrome (HUS) in children in North America is Escherichia coli type O 157 H 7 infection [1]. While there are some reports of other organisms being isolated from fecal samples of children with diarrheal HUS, such as viruses [2], salmonella [3], and campylobacter [4], a causal role for these agents in the pathogenesis of HUS has not been convincingly demonstrated. Serological studies in some children thought to have nonE. coli -related HUS have shown a specific immune response to the E. coli O 157 H 7 lipopolysaccharide, indicating a coinfection with the verotoxin-producing E. coli [5]. We recently encountered an 11-year-old child with Clostridium difficile colitis who developed HUS. This young man was admitted with a 5-day history of severe cramping abdominal pain, emesis, and bloody diarrhea. Within 2 days of admission, his hemoglobin and platelet counts dropped, he developed oliguric renal failure, and had evidence of shistocytes on his peripheral blood smear. There was no laboratory evidence of disseminated intravascular coagulation. A stool culture (using the sorbitol MacConkey agar for E. coli detection) on admission (5 days into his illness) was positive only for C. difficile, with no detection of E. coli O 157 H7. Due to this atypical finding, a renal biopsy was performed and confirmed the diagnosis of HUS. A subsequent stool test for shiga toxin (by ELISA) was negative. This child was managed conservatively with fluid restriction, the use of diuretics and anti-hypertensive medications, and was able to avoid dialysis. HUS has been previously reported in association with C. difficile colitis and pseudomembranous colitis [6]. However, in these children fecal samples were not evaluated for the presence of shiga-like toxin, which can be produced by non-O 157 H7 strains of E. coli and result in HUS. Although we did not evaluate our patient for the presence of antibodies to E. coli, the sensitivity of the combined stool studies for detecting HUS-causing E. coli (ELISA and culture) is reported to be very high [7], making E. coli an unlikely co-pathogen. We would definitely recommend adding C. difficile to the list of proven causes of diarrheal HUS in children, and suggest that testing for this pathogen be considered, at least in children with HUS and negative stool studies for E. coli.

Flow cytometry detection of Shiga toxins in the blood from children with hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is the main cause of acute renal failure in early childhood. Most cases are due to intestinal infections from Escherichia coli strains (STEC) which produce by Shiga toxin (Stxs). Stx1 and Stx2 produced by STEC in the gut are absorbed into the circulation and, after binding on polymorphonuclear leukocytes (PMNs), are targeted to renal endothelium. The aim of the present work was the development of a method to detect Stxs bound on circulating PMNs and to diagnose STEC infections in patients with HUS. White blood cells isolated after erythrocytic lysis were incubated with anti-Stxs mouse monoclonal antibodies in the presence of human serum to saturate Fc receptors on PMNs. After incubation with fluorescein isothiocyanate-goat anti-mouse immunoglobulin G, flow cytometric analysis was used to demonstrate the cell-bound fluorescence. The method was quick (3 h), sensitive (femtomoles), and capable of detecting both Stxs. The presence of Stxs was detected on PMNs from six patients with HUS: four patients had serologic or microbiological evidence of STEC infection, whereas the other two patients had no evidence of STEC infection when employing the standard diagnostic methods. The method described is rapid, simple, and based on commercially available reagents, and it might be more sensitive than the standard methods for diagnosis of STEC infection. It also allows the detection of Stxs in blood, a key step to monitor the pathogenesis of HUS.