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Vascular endothelial cells are equipped with numerous specialized granules called Weibel-Palade bodies (WPBs). They contain a cocktail of proteins that can be rapidly secreted (3-5 min) into the vascular lumen after an appropriate stimulus such as thrombin. These proteins are ready without synthesis. Von Willebrand factor (VWF) and P-selectin are the main constituents of WPBs. Upon stimulation, release of ultralarge VWF multimers occurs and assembles into VWF strings on the apical side of endothelium. The VWF A1 domain becomes exposed in a shear-dependent manner recruiting and activating platelets. VWF is able to recruit leukocytes via direct leukocyte binding or via the activated platelets promoting NETosis. Ultralarge VWF strings are ultimately cleaved into smaller pieces by the protease ADAMTS-13 preventing excessive platelet adhesion. Under carefully performed flowing conditions and adequate dose of Shiga toxins, the toxin induces the release of ultralarge VWF multimers from cultured endothelial cells. This basic information allows insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and of STEC-HUS in the diarrhea phase. In TTP, ADAMTS-13 activity is deficient and systemic aggregation of platelets will occur after a second trigger. In STEC-HUS, stimulated release of WPB components in the diarrhea phase of the disease can be presumed to be the first hit in the damage of Gb3 positive endothelial cells., (© 2024. The Author(s).)

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1β). STEC stimulated neutrophils to release elevated levels of IL-1β through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1β secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1β secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1β secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation., Competing Interests: JC is inventor of a patent related to Ecotin. The patent “Immunomodulating and immunostimulating polypeptides for drug-delivery” was filled on February 8, 2019 by the authors’ National Research Council (CONICET) and the University of San Martin (UNSAM) in the United States patent and trademark Office (US2021009371), the European patent office (PCT/IB2019/051026, EP3749364), the China National intellectual property administration (CN111936166), and the National Institute of Intellectual Property from Argentina (AR114683). The filing of the patent did not have any role in experimental design, data collection and analysis, decision to publish, or preparation of this manuscript. The authors have no financial conflicts of interest., (Copyright: © 2023 Sabbione et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear., Methods: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied., Findings: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype., Conclusions: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease., Funding: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Shiga toxin (Stx)-producing Escherichia coli is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS. Mice coinjected with Stx2/LPS exhibited glomerular podocyte and tubular C3 deposits and C3aR overexpression associated with cell damage, which were limited by C3aR antagonist treatment. C3a promoted renal injury by affecting mitochondrial wellness as demonstrated by data showing that C3aR blockade reduced mitochondrial ultrastructural abnormalities and preserved mitochondrial mass and energy production. In cultured podocytes and tubular cells, C3a caused altered mitochondrial fragmentation and distribution, and reduced anti-oxidant SOD2 activity. Stx2 potentiated the responsiveness of renal cells to the detrimental effects of C3a through increased C3aR protein expression. These results indicate that C3aR may represent a novel target in Stx-associated HUS for the preservation of renal cell integrity through the maintenance of mitochondrial function.

The formation of attaching and effacing (A/E) lesions on intestinal epithelium, combined with Shiga toxin production, are hallmarks of enterohemorrhagic Escherichia coli (EHEC) infection that can lead to lethal hemolytic uremic syndrome. Although an animal infection model that fully recapitulates human disease remains elusive, mice orally infected with Citrobacter rodentium(ϕStx2dact), a natural murine pathogen lysogenized with an EHEC-derived Shiga toxin 2-producing bacteriophage, develop intestinal A/E lesions and toxin-dependent systemic disease. This model has facilitated investigation of how: (A) phage gene expression and prophage induction contribute to disease and are potentially triggered by antibiotic treatment; (B) virulence gene expression is altered by microbiota and the colonic metabolomic milieu; and (C) innate immune signaling is affected by Stx. Thus, the model provides a unique tool for accessing diverse aspects of EHEC pathogenesis., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.

Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.

Shiga-toxin (Stx)-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS) is one of the most common causes of acute kidney injury in children. Stx-mediated endothelial injury initiates the cascade leading to thrombotic microangiopathy (TMA), still the exact pathogenesis remains elusive. Interestingly, there is wide variability in clinical presentation and outcome. One explanation for this could be the enhancement of TMA through other factors. We hypothesize that heme, as released during extensive hemolysis, contributes to the etiology of TMA. Plasma levels of heme and its scavenger hemopexin and degrading enzyme heme-oxygenase-1 (HO-1) were measured in 48 STEC-HUS patients. Subsequently, the effect of these disease-specific heme concentrations, in combination with Stx, was assessed on primary human glomerular microvascular endothelial cells (HGMVECs). Significantly elevated plasma heme levels up to 21.2 µM were found in STEC-HUS patients compared to controls and were inversely correlated with low or depleted plasma hemopexin levels (R 2 -0.74). Plasma levels of HO-1 are significantly elevated compared to controls. Interestingly, especially patients with high heme levels (n = 12, heme levels above 75 quartile range) had high plasma HO-1 levels with median of 332.5 (86-720) ng/ml (p = 0.008). Furthermore, heme is internalized leading to a significant increase in reactive oxygen species production and stimulated both nuclear translocation of NF-κB and increased levels of its target gene (tissue factor). In conclusion, we are the first to show elevated heme levels in patients with STEC-HUS. These increased heme levels mediate endothelial injury by promoting oxidative stress and a pro-inflammatory and pro-thrombotic state. Hence, heme may be a contributing and driving factor in the pathogenesis of STEC-HUS and could potentially amplify the cascade leading to TMA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Wijnsma, Veissi, de Wijs, van der Velden, Volokhina, Wagener, van de Kar and van den Heuvel.)

Background: Hemolytic uremic syndrome (HUS), a common subtype of thrombotic microangiopathy (TMA), is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. Shiga toxin-producing Escherichia coli infection is the most common cause of post-diarrheal HUS. Kidney and central nervous system are the primary target organs., Case Presentation: A 64-year-old male presented with HUS following bloody diarrhea. Nephrotic-range proteinuria and hypoalbuminemia were present at the acute stage and renal histology revealed common TMA features. Neurological involvement presented as confusion and impaired cognitive function. Cranial magnetic resonance imaging demonstrated bilateral T2 hyperintensities in the brainstem and insula. The patient received plasma exchange and supportive care. Both the renal and neurological impairments were completely recovered 3 months after the onset., Conclusion: We report an adult patient presenting with nephrotic-range proteinuria and central nervous system involvement at the acute phase of post-diarrheal HUS. The reversibility of the organ damages might predict a favorable outcome.

Hemolytic uremic syndrome (HUS) is a severe renal disease that is often preceded by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). The exact mechanism of Stx-mediated inflammation on human glomerular microvascular endothelial cells (HGMVECs) during HUS is still not well understood. In this study, we investigated the effect of Stx1 on the gene expression of proteins involved in leucocyte-mediated and complement-mediated inflammation. Our results showed that Stx1 enhances the mRNA and protein expression of heparan sulfate proteoglycan (HSPG) syndecan-4 in HGMVECs pre-stimulated with tumor necrosis factor α (TNFα). CD44 was upregulated on mRNA but not on protein level; no effect on the mRNA expression of other tested HSPGs glypican-1 and betaglycan was observed. Furthermore, Stx1 upregulated the mRNA, cell surface expression, and supernatant levels of the intercellular adhesion molecule-1 (ICAM-1) in HGMVECs. Interestingly, no effect on the protein levels of alternative pathway (AP) components was observed, although C3 mRNA was upregulated. All observed effects were much stronger in HGMVECs than in human umbilical endothelial cells (HUVECs), a common model cell type used in endothelial studies. Our results provide new insights into the role of Stx1 in the pathogenesis of HUS. Possibilities to target the overexpression of syndecan-4 and ICAM-1 for STEC-HUS therapy should be investigated in future studies.

Shiga toxins (Stxs), syn. Vero(cyto)toxins, are potent bacterial exotoxins and the principal virulence factor of enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin-producing E. coli (STEC). EHEC strains, e.g., strains of serovars O157:H7 and O104:H4, may cause individual cases as well as large outbreaks of life-threatening diseases in humans. Stxs primarily exert a ribotoxic activity in the eukaryotic target cells of the mammalian host resulting in rapid protein synthesis inhibition and cell death. Damage of endothelial cells in the kidneys and the central nervous system by Stxs is central in the pathogenesis of hemolytic uremic syndrome (HUS) in humans and edema disease in pigs. Probably even more important, the toxins also are capable of modulating a plethora of essential cellular functions, which eventually disturb intercellular communication. The review aims at providing a comprehensive overview of the current knowledge of the time course and the consecutive steps of Stx/cell interactions at the molecular level. Intervention measures deduced from an in-depth understanding of this molecular interplay may foster our basic understanding of cellular biology and microbial pathogenesis and pave the way to the creation of host-directed active compounds to mitigate the pathological conditions of STEC infections in the mammalian body.

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., Competing Interests: G.A. reports grants from Progetto Organizzazione non Lucrativa di Utilità Sociale–Alice Associazione per la Lotta alla Sindrome Emolitico Uremica, during the conduct of the study. M.B. reports grants from Progetto Organizzazione non Lucrativa di Utilità Sociale–Alice Associazione per la Lotta alla Sindrome Emolitico Uremica, during the conduct of the study. X.H. reports grants from USDA-ARS National Program NP108, CRIS project 2030–42000–049–00D, during the conduct of the study., (Georg Thieme Verlag KG Stuttgart · New York.)

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8., Competing Interests: The authors have declared that no competing interests exist.

Glomerulonephritis (GN) refers to a group of renal diseases affecting the glomeruli due to the damage mediated by immunological mechanisms. A large proportion of the disease manifestations are caused by disturbances in the complement system. They can be due to genetic errors, autoimmunity, microbes or abnormal immunoglobulins, like modified IgA or paraproteins. The common denominator in most of the problems is an overactive or misdirected alternative pathway complement activation. An assessment of kidney function, amount of proteinuria and hematuria are crucial elements to evaluate, when glomerulonephritis is suspected. However, the cornerstones of the diagnoses are renal biopsy and careful examination of the complement abnormality. Differential diagnostics between the various forms of GN is not possible based on clinical features, as they may vary greatly. This review describes the known mechanisms of complement dysfunction leading to different forms of primary GN (like IgA glomerulonephritis, dense deposit disease, C3 glomerulonephritis, post-infectious GN, membranous GN) and differences to atypical hemolytic uremic syndrome. It also covers the basic elements of etiology-directed therapy and prognosis of the most common forms of GN. Common principles in the management of GN include treatment of hypertension and reduction of proteinuria, some require immunomodulating treatment. Complement inhibition is an emerging treatment option. A thorough understanding of the basic disease mechanism and a careful follow-up are needed for optimal therapy., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The inflammatory response of the host to Shiga toxin and/or lipopolysaccharide (LPS) of Escherichia coli (E. coli) is included in (HUS). The TLR4-LPS complex is internalized and TLR4 induced inflammatory signaling is stopped by targeting the complex for degradation. Rab7b, a small guanosine triphosphatase (GTPase) expressed in monocytes, regulates the later stages of the endocytic pathway., Objective: we studied the Rab7b participation on the TLR4 endocytic pathway and its effect on monocyte cytokine production along the acute course of pediatric Shiga toxin-associated HUS., Methods and Results: Monocytes were identified according to their positivity in CD14 expression. Surface TLR4 expression in monocytes from 18 HUS patients significantly increased by day 1 to 6, showing the highest increase on day 4 compared to monocytes of 10 healthy children. Significant higher surface TLR4 expression was accompanied by increased proinflammatory intracellular cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In contrast, after these time points, surface TLR4 expression and intracellular TNF-α levels, returned to near control levels after 10 days. Furthermore, confocal immunofluorescence microscopy proved colocalization of increased intracellular TLR4/Rab7b determined by Pearson's coefficient in monocytes from HUS patients from day 1 on the highest colocalization of both proteins by day 4. Decreased TLR4/Rab7b colocalization was shown 10 days after HUS onset., Conclusion: The colocalization of TLR4 and Rab7b allows us to suggest Rab7b participation in the control of the TLR4 endocytic pathway in HUS patient monocytes. A consequential fall in cytokine production throughout the early follow up of HUS is demonstrated., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Shiga toxin 1 (STx1) and 2 (STx2), produced by Shiga toxin-producing Escherichia coli , cause lethal untreatable disease. The toxins invade cells via retrograde trafficking. Direct early endosome-to-Golgi transport allows the toxins to evade degradative late endosomes. Blocking toxin trafficking, particularly at the early endosome-to-Golgi step, is appealing, but transport mechanisms of the more disease-relevant STx2 are unclear. Using data from a genome-wide siRNA screen, we discovered that disruption of the fusion of late endosomes, but not autophagosomes, with lysosomes blocked the early endosome-to-Golgi transport of STx2. A subsequent screen of clinically approved lysosome-targeting drugs identified tamoxifen (TAM) to be a potent inhibitor of the trafficking and toxicity of STx1 and STx2 in cells. The protective effect was independent of estrogen receptors but dependent on the weak base property of TAM, which allowed TAM to increase endolysosomal pH and alter endosomal dynamics. Importantly, TAM treatment enhanced survival of mice injected with a lethal dose of STx1 or STx2. Thus, it may be possible to repurpose TAM for treating Shiga toxin-producing E. coli infections., (© 2019 Selyunin et al.)

Peptide-based biomaterials are a promising class of antimicrobial agents that work by physically damaging bacterial cell membranes rather than targeting intracellular factors, resulting in less susceptibility to drug resistance. Herein we report the synthesis of cationic, star-shaped polypeptides with 3 to 8 arms and their evaluation as antimicrobial agents against different types of bacteria. The effects of the arm number and side chain group on their antimicrobial activities were systematically investigated. Compared to their linear counterparts, these star-shaped polypeptides exhibited potent antibacterial activity (which may involve adhesion and disruption processes). The increase of the arm number can efficiently increase the antibacterial activities up until 8 arms, which did not exhibit further improvement of antibacterial activities. Poly(l-lysine) (PLL) modified with an indole group (PLL-g-indo) exhibited the best antibacterial activity among all grafted copolypeptides and improved cytotoxic selectivity towards pathogens over mammalian cells without compromising their hemolytic activities. In vivo studies showed that the star-shaped PLL-g-indo can effectively suppress Enterohaemorrhagic E. coli (EHEC) infection and attenuate the clinical symptoms in mice, suggesting that they are promising antimicrobial agents.

Objectives: Interleukin (IL)-33 plays an important role in host defense, immune regulation, and inflammation. This study assessed IL-33's role in the pathogenesis of severe hemolytic uremic syndrome (HUS) induced by enterohemorrhagic Escherichia coli (EHEC). We also investigated the clinical significance of IL-33 and soluble ST2 (soluble form of IL-33 receptor) serum levels in patients with EHEC-induced HUS., Methods: The role of IL-33 in Shiga toxin (STx)-2-induced endothelial injury was studied in human umbilical vein endothelial cells (HUVECs) in vitro. Blood samples were obtained from 21 HUS patients and 15 healthy controls (HC). The IL-33 and sST2 serum levels were quantified using an enzyme-linked immunosorbent assay. The results were compared to HUS' clinical features., Results: Cytotoxic assays indicated that IL-33 enhanced STx2 toxicity in HUVECs. Serum IL-33 levels in most HUS patients were below the lowest detection limit. On the other hand, serum sST2 levels in patients during the HUS phase were significantly higher than those in HC and showed a correlation with disease severity. Serum sST2 levels in patients with encephalopathy were significantly higher than those in patients without it. A serum sST2 level > 63.2 pg/mL was associated with a high risk of encephalopathy. Serum sST2 levels significantly correlated with serum levels of inflammatory cytokines related to the development of HUS., Conclusions: Our results indicate that IL-33 contributes to the severity of EHEC-induced HUS. Serum sST2 level in HUS patients correlated with disease activity, which suggests its potential role as a marker for disease activity and development of encephalopathy in patients with EHEC-induced HUS.

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB 5 toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases., (Copyright © 2018. Published by Elsevier GmbH.)

Hemolytic uremic syndrome is a rare complication of invasive pneumococcal infection (pnHUS). Its pathogenesis is poorly understood, and treatment remains controversial. The emerging role of complement in various forms of HUS warrants a new look at this "old" disease. We performed a retrospective analysis of clinical and laboratory features of three sequential cases of pnHUS since 2008 associated with pneumonia/pleural empyema, two due to Streptococcus pneumoniae serotype 19 A. Profound depletion of complement C3 (and less of C4) was observed in two patients. One patient was Coombs test positive. Her red blood cells (RBCs) strongly agglutinated with blood group compatible donor serum at 0 °C, but not at 37 °C. All three patients were treated with hemodialysis, concentrated RBCs, and platelets. Patient 2 received frozen plasma for hepatic failure with coagulation factor depletion. Intravenous immunoglobulin infusion, intended to neutralize pneumococcal neuraminidase in patient 3, was associated with rapid normalization of platelets and cessation of hemolysis. Two patients recovered without sequelae or disease recurrence. Patient 2 died within 2½ days of admission due to complicating Pseudomonas aeruginosa sepsis and multiorgan failure. Our observations suggest that pnHUS can be associated with dramatic, transient complement consumption early in the course of the disease, probably via the alternative pathway. A critical review of the literature and the reported cases argue against the postulated pathological role of preformed antibodies against the neuraminidase-exposed Thomsen-Friedenreich neoantigen (T antigen) in pnHUS. The improved understanding of complement regulation and bacterial strategies of complement evasion allows to propose a testable, new pathogenetic model of pnHUS. This model shifts emphasis from the action of natural anti-T antibodies toward impaired Complement Factor H (CFH) binding and function on desialylated membranes. Removal of neuraminic acid residues converts (protected) self to non-self surfaces that supports membrane attack complex (MAC) assembly. Complement activation is potentially exacerbated by decreased CFH availability following tight CFH binding to pneumococcal evasion proteins and/or by the presence of genetic variants of complement regulator proteins. Detailed clinical and experimental investigations are warranted to better understand the role of unregulated complement activation in pnHUS. Instead of avoidance of plasma, a new, integrated model is evolving, which may include short-term therapeutic complement blockade, particularly where genetic or functional APC dysregulation is suspected, in addition to bacterial elimination and, potentially, neuraminidase neutralization., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Hemolytic-uremic syndrome (HUS) is a form of thrombotic microangiopathy, in the course of which some patients may develop chronic kidney disease (CKD). From a clinical point of view, it is important to search for markers that allow for early identification of patients at risk of a poor prognosis., Objectives: The study evaluated the serum and urine levels of liver-type fatty acid binding protein (L-FABP) and interleukin 6 (IL-6)., Material and Methods: The study was conducted in 29 children with a history of HUS. The relationship between L-FABP and IL-6 and anthropometric measurements, the value of estimated glomerular filtration rate (eGFR) and albuminuria were additionally evaluated., Results: In children after HUS, L-FABP and IL-6 concentration in both serum and urine was significantly higher in comparison to the control group. No differences in L-FABP and IL-6 concentration in serum and urine depending on the type of HUS and gender were noted. Correlation between L-FABP and IL-6 in serum and urine with eGFR and urine albumin-creatinine ratio (ACR) in the total group of patients after HUS was not detected. In the group of children after 6 month observation after HUS, a negative correlation of L-FABP concentration with eGFR was found., Conclusions: The results indicate that the higher concentration of L-FABP in serum and urine of children with a history of HUS can be the result of protracted injury initiated during the acute phase of the disease. Lack of correlation of L-FABP concentration with the ACR may be associated with a short (less than 6 months) observation after acute renal failure or merely temporary renal tubular damage in the acute phase of the disease. In contrast, higher levels of IL-6 in serum and urine in children after HUS compared to healthy children and the negative correlation of L-FABP concentration and eGFR in children after 6 month observation after HUS may confirm their participation in CKD. Thus, L-FABP and IL-6 seem to be good biomarkers of chronic kidney damage in survivors of the acute phase of HUS.

Cytokines play an important role in the pathogenesis of the severe complications of Shiga toxin-producing Escherichia coli (STEC) infection, such as hemolytic uremic syndrome (HUS) and acute encephalopathy. A 3-year-old boy with acute encephalopathy associated with STEC O-157 HUS showed increased levels of IL-6 and IL-10, which normalized after methylprednisolone pulse therapy, and additionally exhibited a transient increase of glutamine on MR spectroscopy. This finding suggests that excitotoxicity, in addition to hypercytokinemia, may play an important role in the pathogenesis of HUS encephalopathy., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Shiga toxin is the main virulence factor of enterohemorrhagic Escherichia coli , a non-invasive pathogen that releases virulence factors in the intestine, causing hemorrhagic colitis and, in severe cases, hemolytic uremic syndrome (HUS). HUS manifests with acute renal failure, hemolytic anemia and thrombocytopenia. Shiga toxin induces endothelial cell damage leading to platelet deposition in thrombi within the microvasculature and the development of thrombotic microangiopathy, mostly affecting the kidney. Red blood cells are destroyed in the occlusive capillary lesions. This review focuses on the importance of microvesicles shed from blood cells and their participation in the prothrombotic lesion, in hemolysis and in the transfer of toxin from the circulation into the kidney. Shiga toxin binds to blood cells and may undergo endocytosis and be released within microvesicles. Microvesicles normally contribute to intracellular communication and remove unwanted components from cells. Many microvesicles are prothrombotic as they are tissue factor- and phosphatidylserine-positive. Shiga toxin induces complement-mediated hemolysis and the release of complement-coated red blood cell-derived microvesicles. Toxin was demonstrated within blood cell-derived microvesicles that transported it to renal cells, where microvesicles were taken up and released their contents. Microvesicles are thereby involved in all cardinal aspects of Shiga toxin-associated HUS, thrombosis, hemolysis and renal failure., Competing Interests: The authors declare no conflict of interest.

Ribotoxic Shiga toxins are the primary cause of hemolytic uremic syndrome (HUS) in patients infected with Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC), a pathogen class responsible for epidemic outbreaks of gastrointestinal disease around the globe. HUS is a leading cause of pediatric renal failure in otherwise healthy children, resulting in a mortality rate of 10% and a chronic morbidity rate near 25%. There are currently no available therapeutics to prevent or treat HUS in STEC patients despite decades of work elucidating the mechanisms of Shiga toxicity in sensitive cells. The preclinical development of toxin-targeted HUS therapies has been hindered by the sporadic, geographically dispersed nature of STEC outbreaks with HUS cases and the limited financial incentive for the commercial development of therapies for an acute disease with an inconsistent patient population. The following review considers potential therapeutic targeting of the downstream cellular impacts of Shiga toxicity, which include the unfolded protein response (UPR) and the ribotoxic stress response (RSR). Outcomes of the UPR and RSR are relevant to other diseases with large global incidence and prevalence rates, thus reducing barriers to the development of commercial drugs that could improve STEC and HUS patient outcomes., Competing Interests: The authors declare no conflict of interest.

We identified the mucus-activatable Shiga toxin genotype stx2d in the most common hemolytic uremic syndrome-associated Escherichia coli serotype, O157:H7. stx2d was detected in a strain isolated from a 2-year-old boy with bloody diarrhea in Spain, and whole-genome sequencing was used to confirm and fully characterize the strain.

Background: Deficiency of complement factor H-related plasma proteins and complement factor H autoantibody-positive hemolytic uremic syndrome (DEAP-HUS), which is characterized by the deficiency of complement-factor H-related (CFHR) plasma proteins and the subsequent formation of autoantibodies against complement factor H (CFH), has been reported to have an adverse outcome in one third of patients. Therapy options include prompt removal of antibodies by plasma exchange and immunosuppressive therapy. Recently, restoration of complement control using the monoclonal antibody eculizumab has been shown to be effective as first- and as second-line therapy in cases of therapy resistance or severe side effects of the applied therapy., Diagnosis/treatment: Here, we report a 6-year-old girl with DEAP-HUS and first-line therapy with eculizumab under immunosuppressive therapy with glucocorticoids and mycophenolate mofetil (MMF). This therapy led to a prompt and sustained clinical recovery, to a stable reduction of complement activation, and to a rapid decline in autoantibody titer. A second increase in the autoantibody titer was successfully treated with methylprednisolone and the child remained in remission. After 8.3 months of sustained complement control and 4.5 months of stable antibody suppression, eculizumab was successfully discontinued without any sign of relapse., Conclusions: To our knowledge, this is the first reported case of a child with DEAP-HUS treated with the combination of eculizumab and immunosuppression as first-line therapy avoiding any HUS- or therapy-related complications and resulting in prompt clinical recovery. Importantly, clinical remission is maintained after discontinuation of eculizumab under stable immunosuppression.

Hemolytic uremic syndrome (HUS) is a type of thrombotic microangiopathy, in the course of which some patients may develop chronic kidney disease (CKD). It is clinically important to investigate the markers of a poor prognosis. The levels of angiotensinogen (AGT) and interleukin-18 (IL-18) in serum and urine were evaluated. Study was conducted in 29 children with a history of HUS. Serum and urine AGT concentration was significantly higher in children after HUS as compared to the control group. No differences depending on the type of HUS and gender were noted. The serum concentration of IL-18 in children after HUS was significantly lower, whereas in urine did not differ significantly between the sick and healthy children. A negative correlation between the concentration of AGT in serum and albuminuria in patients after HUS was detected. The results indicate that the concentration of AGT in serum and urine in children after HUS increases, which may indicate the activation of the intrarenal renin-angiotensin-aldosterone system. The statement, that AGT may be a good biomarker of CKD after acute kidney injury due to HUS requires prospective studies with follow-up from the acute phase of the disease on a larger group of patients. Reduced IL-18 serum concentration in children after HUS with no difference in its urine concentration may indicate a loss of the protective effects of this cytokine on renal function due to previously occurred HUS.

Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans.

Escherichia coli O157:H7 is a subtype of Shiga toxin-producing E. coli that is associated with haemorrhagic colitis and haemolytic uremic syndrome (HUS). Studies of populations in endemic areas have reported that the presence of specific antibodies against the O157 lipopolysaccharide (LPS) is associated with a lower incidence of diarrhoea and HUS. Phage display and IgG anti-O157 LPS antibodies were used in the present study to select peptide mimotopes of O157 LPS expressed in protein III of the M13 phage. Synthetic peptides (SP) were designed using the derived amino acid sequences obtained from DNA nucleotides of 63 selected phagotopes. The LxP/YP/SxL motif was identified in five of the phagotope amino acid sequences. Antibody responses against the phagotopes and their corresponding SPs were evaluated. SP12, one of the designed SP, induced the production of antibodies against the homologous peptide (1:800) and O157 LPS (1:200). The specificity of anti-SP12 antiserum was confirmed by analyzing its response to SP3, an SP with a different amino acid sequence than that of SP12, as well as against an E. coli LPS different from O157. Competitive studies with SP12 and O157 LPS showed a significant decrease in anti-SP12 and anti-LPS O157 antiserum responses against SP12 and O157 LPS, respectively. Eighteen (82%) of the 22 human serum samples with positive reactivity against E coli O157 LPS reacted with SP12 SP (cut-off >0.4). These results support the idea that SP12 is an immunogenic mimotope of O157 LPS., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The alternative pathway (AP) of complement can recognize nonself structures by only two molecules, C3b and factor H. The AP deposits C3b covalently on nonself structures via an amplification system. The actual discrimination is performed by factor H, which has binding sites for polyanions (sialic acids, glycosaminoglycans, phospholipids). This robust recognition of 'self' protects our own intact viable cells and tissues, while activating structures are recognized by default. Foreign targets are opsonized for phagocytosis or killed. Mutations in factor H predispose to severe diseases. In hemolytic uremic syndrome, they promote complement attack against blood cells and vascular endothelial cells and lead, for example, to kidney and brain damage. Even pathogens can exploit factor H. In fact, the ability to bind factor H discriminates most pathogenic microbes from nonpathogenic ones., (© 2016 Federation of European Biochemical Societies.)

Background: Over the last forty years, our laboratory has accumulated a collection of over 1000 cultured fibroblast lines derived from patients from around the world referred with signs of inborn errors of cobalamin or folate metabolism, including several hundred with complementation-confirmed diagnoses. By accurately classifying patient disorders into classes representing blocks affecting specific reactions, we have provide the basis for rational assessment of phenotypic heterogeneity, and development of methods for diagnosis, treatment and prognosis. These resources have been valuable in identification of causal genes for known inborn errors. Since 2000, we and our collaborators identified the genes for the cblA (MMAA), cblB (MMAB), cblC (MMACHC), cblD (MMADHC), and cblF (LMBRD1) disorders., Results: Whole exome sequencing of DNA from a patient with severe combined immunodeficiency (SCID), megaloblastic anemia and hemolytic uremic syndrome identified mutations in the MTHFD1 gene, which encodes a trifunctional enzyme involved in interconversion of folate coenzyme derivatives. This disorder demonstrates the importance de novo pyrimidine synthesis in the etiology of SCID. Mutations in the ABCD4 gene have been identified in four patients with accumulation of unbound cobalamin in lysosomes; this gene encodes a lysosomal membrane protein that plays a role in the transport of cobalamin across this membrane. Mutations in the HCFC1 gene on the X chromosome have been identified in several male patients that had received a diagnosis of cblC on the basis of complementation studies in cultured fibroblasts. HCFC1 encodes a transcription factor that regulates expression of a number of genes, including MMACHC, the gene that is mutated in patients with the cblC disorder. These studies demonstrate that with the advent of affordable whole exome sequencing, it has been possible to identify genes for novel inborn errors of cobalamin metabolism, often working from a small number of affected patients., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: Endothelial dysfunction plays a pivotal role in the pathogenesis of postenteropathic hemolytic uremic syndrome (HUS), most commonly caused by Shiga toxin (Stx)-producing strains of Escherichia coli., Methods: To identify new treatment targets, we performed a metabolomic high-throughput screening to analyze the effect of Stx2a, the major Stx type associated with HUS, on human renal glomerular endothelial cells (HRGEC) and umbilical vein endothelial cells (HUVEC). Cells were treated either with sensitizing tumor necrosis factor α (TNF-α) or Stx2a, a sequence of both or remained untreated., Results: We identified 341 metabolites by combined liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry. Both cell lines exhibited distinct metabolic reaction profiles but shared elevated levels of free fatty acids. Stx2a predominantly altered the nicotinamide adenine dinucleotide (NAD) cofactor pathway and the inflammation-modulating eicosanoid pathway, which are associated with lipid metabolism. In HRGEC, Stx2a strongly diminished NAD derivatives, leading to depletion of the energy substrate acetyl coenzyme A and the antioxidant glutathione. HUVEC responded to TNF-α and Stx2a by increasing production of the counteracting eicosanoids prostaglandin I2, E1, E2, and A2, while in HRGEC only more prostaglandin I2 was detected., Conclusions: We conclude that disruption of energy metabolism and depletion of glutathione contributes to Stx-induced injury of the renal endothelium and that the inflammatory response to Stx is highly cell-type specific., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated., Objectives: We investigated the effects of EspP on clot formation and lysis in human blood., Methods: Human whole blood and plasma were incubated with EspP(WT )at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured., Results and Conclusions: Human whole blood or plasma incubated with EspP(WT) was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspP(WT) had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS.

Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation.

Haemolytic uraemic syndrome (HUS) is the major complication of Escherichia coli gastrointestinal infections that are Shiga toxin (Stx) producing. Monocytes contribute to HUS evolution by producing cytokines that sensitize endothelial cells to Stx action and migration to the injured kidney. As CC chemokine receptors (CCRs) are involved in monocyte recruitment to injured tissue, we analysed the contribution of these receptors to the pathogenesis of HUS. We analysed CCR1, CCR2 and CCR5 expression in peripheral monocytes from HUS patients during the acute period, with healthy children as controls. We observed an increased expression of CCRs per cell in monocytes from HUS patients, accompanied by an increase in the absolute number of monocytes CCR1+, CCR2+ and CCR5+. It is interesting that prospective analysis confirmed that CCR1 expression positively correlated with HUS severity. The evaluation of chemokine levels in plasma showed that regulated on activation of normal T-cell-expressed and -secreted (RANTES) protein was reduced in plasma from patients with severe HUS, and this decrease correlated with thrombocytopenia. Finally, the expression of the higher CCRs was accompanied by a loss of functionality which could be due to a mechanism for desensitization to compensate for altered receptor expression. The increase in CCR expression correlates with HUS severity, suggesting that the dysregulation of these receptors might contribute to an increased risk of renal damage. Activated monocytes could be recruited by chemokines and then receptors could be dysregulated. The dysregulation of CCRs and their ligands observed during the acute period suggests that a chemokine pathway would participate in HUS development.

Background: Recombinant human soluble thrombomodulin (rhTM) is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C. In order to clarify the efficacy of rhTM for the treatment of typical hemolytic uremic syndrome (t-HUS), we examined changes in renal damage in t-HUS mice treated with rhTM or vehicle alone., Methods: We used severe and moderate t-HUS mice injected with shiga toxin (Stx) and lipopolysaccharide (LPS). The severe t-HUS mice were divided into two subgroups [an rhTM subgroup (Group A) and a saline subgroup (Group B)] along with the moderate t-HUS mice [an rhTM subgroup (Group C) and a saline subgroup (Group D)]. Groups E and F were healthy mice treated with rhTM or saline, respectively., Results: All mice in Group B died at 80-90 h post-administration of Stx2 and LPS whereas all mice in Group A remained alive. Loss of body weight, serum creatinine level, endothelial injury and mesangiolysis scores at 24 h after administration in the t-HUS mice treated with rhTM were lower than those in t-HUS mice treated with saline. The levels of hemoglobin at 6 h and platelet counts at 24 h after administration in Group A were higher than those in Group B. Serum interleukin (IL)-6, IL-1β and tumor necrotic factor (TNF)-α levels at 24 h after administration in Group A were lower than those in Group B. Serum C5b-9 levels at 24 h after the administration and serum fibrinogen degradation product (FDP) at 72 h after the administration of Stx2 and LPS were lower in Group A than in Group B., Conclusions: These results indicate that rhTM might afford an efficacious treatment for t-HUS model mice via the inhibition of further thrombin formation and amelioration of hypercoagulant status., (© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

The molecular linkage between ultralarge (UL) von Willebrand factor (VWF) multimers and the alternative complement pathway (AP) has recently been described. Endothelial cell (EC)-secreted and anchored ULVWF multimers (in long stringlike structures) function as both hyperadhesive sites that initiate platelet adhesion and aggregation and activating surfaces for the AP. In vitro, the active form of C3, C3b binds to the EC-anchored ULVWF multimeric strings and initiates the assembly on the strings of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b). In vivo, activation of the AP via this mechanism proceeds all the way to generation of terminal complement complexes (C5b-9)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The complement system is activated in the vasculature during thrombotic and inflammatory conditions. Activation may be associated with chronic inflammation on the endothelial surface leading to complement deposition. Complement mutations allow uninhibited complement activation to occur on platelets, neutrophils, monocytes, and aggregates thereof, as well as on red blood cells and endothelial cells. Furthermore, complement activation on the cells leads to the shedding of cell derived-microvesicles that may express complement and tissue factor thus promoting inflammation and thrombosis. Complement deposition on red blood cells triggers hemolysis and the release of red blood cell-derived microvesicles that are prothrombotic. Microvesicles are small membrane vesicles ranging from 0.1 to 1 μm, shed by cells during activation, injury and/or apoptosis that express components of the parent cell. Microvesicles are released during inflammatory and vascular conditions. The repertoire of inflammatory markers on endothelial cell-derived microvesicles shed during inflammation is large and includes complement. These circulating microvesicles may reflect the ongoing inflammatory process but may also contribute to its propagation. This overview will describe complement activation on blood and endothelial cells and the release of microvesicles from these cells during hemolytic uremic syndrome, thrombotic thrombocytopenic purpura and vasculitis, clinical conditions associated with enhanced thrombosis and inflammation.

Objective: To screen Hemolytic Uremic Syndrome (HUS) related differentially expressed genes using the microarray data of neonatal microvascular endothelial cells from human skin treated with or without Shiga toxin, and study the the mechanism of HUS from multiple angles., Materials and Methods: The microarray dataset GSE32710 was download from gene expression database GEO (Gene Expression Omnibus), which included a total of 12 samples, 6 samples were treated with Shiga toxin while the others were normal without any treatments. Then the raw chip data were preprocessed by R package, and T-test was utilized for differentially expressed genes screening. The selected differentially expressed genes were subjected to GO functional and KEGG pathway analysis. Following that, human protein interaction network, synergetic effects among the differentially expressed genes and regulation of post-transcriptional miRNA were integrated so as to construct a molecular interaction network associated with HUS and excavate the sub-function modules., Results: Trough differential expression screening, 195 of HUS related marker genes were obtained, and 294 of gene pairs with significant co-expression were achieved. Molecular interaction network associated with HUS excavated 302 miRNAs and 117 differentially expressed genes, among which miRNA-30c and miRNA-30d may play important roles during the development of HUS., Conclusions: In this study, we used a method that explained the biological mechanism of HUS systematically from gene transcription level and different levels of biological information such as protein interaction, post-transcriptional regulation of gene expression as well as synergy effects of gene expression, which may provide new therapeutic targets for HUS.

Frequencies of human leucocyte antigens (HLA) were determined in 287 classic hairy cell leukaemia (HCL) patients. With respect to both population (n = 287) and allele (2n = 574) frequency respectively, the most common HLA class I and II antigens expressed were HLA-A*02 (49·1% and 28·6%), HLA-B*07 (21·3% and 11·1%), HLA-C*07 (46·7 and 28·2%), HLA-DQB1*03 (62·7% and 37·3%), HLA-DRB1*11 (30·0% and 16·0%) and HLA-DRB4*01 (45·3% and 29·6%). In comparing 6-14 databases of control Caucasians to 267 Caucasian HCL patients, only HLA-DRB1*11 was consistently over-represented in HCL, 31·1% of patients vs. 17-19·9% of controls (P = 0·0055 to <0·0001) and 16·5% of alleles vs. 6·5-12·3% of control alleles (P = 0·022 to <0·0001). HLA-DRB1*11 is a known risk factor for acquired thrombotic microangiopathy. Anti-CD22 recombinant immunotoxin BL22 in HCL was associated with a 12% incidence of completely reversible grade 3-4 haemolytic uraemic syndrome (HUS), mainly during the second or third retreatment cycle. Of 49 HCL patients receiving ≥2 cycles of BL22, 7 (14%) had HUS and HLA-DRB1*11 was expressed in 71% of 7 with HUS compared with only 21% of 42 without (P = 0·015). These data suggest that DBR1*11 may be a marker for increased susceptibility to HCL and, among HCL patients, could be a risk factor for BL22-induced HUS., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Shiga toxin (Stx)-producing Escherichia coli is the offending agent of postdiarrhea-associated hemolytic uremic syndrome (HUS), a disorder of glomerular ischemic damage and widespread microvascular thrombosis. We previously documented that Stx induces glomerular complement activation, generating C3a responsible for microvascular thrombosis in experimental HUS. Here, we show that the presence of C3 deposits on podocytes is associated with podocyte damage and loss in HUS mice generated by the coinjection of Stx2 and LPS. Because podocyte adhesion to the glomerular basement membrane is mediated by integrins, the relevance of integrin-linked kinase (ILK) signals in podocyte dysfunction was evaluated. Podocyte expression of ILK increased after the injection of Stx2/LPS and preceded the upregulation of Snail and downregulation of nephrin and α-actinin-4. Factor B deficiency or pretreatment with an inhibitory antibody to factor B protected mice against Stx2/LPS-induced podocyte dysregulation. Similarly, pretreatment with a C3a receptor antagonist limited podocyte loss and changes in ILK, Snail, and α-actinin-4 expression. In cultured podocytes, treatment with C3a reduced α-actinin-4 expression and promoted ILK-dependent nuclear expression of Snail and cell motility. These results suggest that Stx-induced activation of the alternative pathway of complement and generation of C3a promotes ILK signaling, leading to podocyte dysfunction and loss in Stx-HUS., (Copyright © 2014 by the American Society of Nephrology.)

Background: Hemolytic uremic syndrome (HUS) is a systemic and potentially fatal complication of gastroenteritis secondary to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infection characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal damage. Shiga toxin (Stx), the toxin principle in HUS, is produced locally within the gut following EHEC colonization and is disseminated via the vasculature. Clinical development of HUS currently has no effective treatment and is a leading cause of renal failure in children. Novel post-exposure therapies are currently needed for HUS; therefore, the purpose of this study was to investigate the efficacy of a Stx receptor mimic probiotic in a porcine model of HUS. Edema disease, an infection of swine caused by host adapted Shiga toxin-producing Escherichia coli (STEC) and mediated by Shiga toxin 2e (Stx2e), shares many pathogenic similarities to HUS. In this study, three-week old piglets were inoculated with STEC and 24 hours later treated twice daily with a probiotic expressing an oligosaccharide receptor mimic for Stx2e to determine if the probiotic could reduce intestinal toxin levels., Methods: Piglets were orally inoculated with 10(10) CFU of STEC strain S1191 eight days after weaning. Beginning day 1 post-inoculation, piglets were treated orally twice daily with 5 × 10(11) CFU of either the receptor mimic probiotic or a sham probiotic for 10 days. Intestinal Stx2e levels were assessed daily via Vero cell assay. The efficacy of the probiotic at reducing intestinal Stx2e, vascular lesions, and clinical disease was evaluated with repeated measures ANOVA and Fisher's exact test as appropriate., Results: The probiotic significantly reduced intestinal Stx2e, as reflected by decreased fecal toxin titers on days 3-8 post-inoculation (p < 0.01). Despite this reduction in intestinal toxin levels, however, the probiotic failed to reduce the incidence of vascular necrosis in target organs and had no effect on clinical disease., Conclusions: The data suggest that post-exposure treatment with a Stx-binding probiotic is effective in reducing intestinal toxin burden. Future studies could target this approach for possible development of post-exposure interventions.

Objective: To review the clinical features and pathophysiologic mechanisms of the thrombotic microangiopathies (TMAs) including acquired and congenital thrombotic thrombocytopenic purpura (TTP), Shiga toxin-induced and atypical (non-Shiga toxin-induced) hemolytic uremic syndrome (HUS), and the TMAs associated with pregnancy, drugs, and organ transplantation., Methods: PubMed Medline was used to identify articles published from 2000 to July 2013 using the following key words: thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, Shiga toxin, ADAMTS13, and eculizumab. Articles in languages other than English, papers available in abstract form only, and nearly all single case reports were excluded. Small series, reports from registries and study groups, reviews, guidelines, and articles concerning pathophysiology and therapy were preferentially considered., Results: Impaired post-secretion processing of unusually large von Willebrand multimers due to deficiency of ADAMTS13 (IgG antibodies or congenital), dysregulation of the alternative complement pathway (mutations and/or specific antibodies), and endothelial injury are pathophysiologic mechanisms involved in the TMAs. Acquired and congenital TTP are due primarily to severe ADAMTS13 deficiency, atypical HUS is commonly associated with complement dysregulation, and Shiga toxin, drugs, immune complexes, and others likely damage endothelium. However, there is considerable mechanistic overlap, and the TMAs often have multifactorial causation. Plasma procedures, complement pathway inhibition, immunosuppression, and general supportive care are the principal therapies., Conclusions: The TMAs are very important conditions because of their associated organ damage and mortality rates. Prompt recognition and categorization by both clinical presentation and pathophysiologic mechanisms should become routine as they are crucial to an optimal treatment plan. Treatment advances have substantially reduced the morbidity of these disorders. Investigational therapies are promising., (© 2013 Published by Elsevier Inc.)

Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy characterized by uncontrolled complement activation against endothelial and blood cells. Mutations in the C-terminal target recognition domains 19-20 of complement regulator factor H (FH) are strongly associated with aHUS, but the mechanisms triggering disease onset have remained unresolved. Here we report that several aHUS-related mutations alter the binding of FH19-20 to proteins where lysines have reacted with malondialdehyde (MDA). Although FH19-20 did not interact with MDA-modified hexylamine, lysine-containing peptides, or a proteolytically degraded protein, it bound to MDA-modified polylysine. This suggests that FH19-20 recognizes only clustered MDA adducts. Binding of MDA-modified BSA to FH19-20 was ionic by nature, depended on positive residues of FH19-20, and competed with the polyanions heparin and DNA. This could not be explained with the mainly neutral adducts known to form in MDA modification. When positive charges of lysines were eliminated by acetic anhydride instead of MDA, the acetylated BSA started to bind FH19-20. Together, these results indicate that negative charges on the modified proteins dominate the interaction with FH19-20. This is beneficial for the physiological function of FH because by binding to the negative charges of the modified target, FH could prevent excess complement activation initiated by naturally occurring antibodies recognizing MDA epitopes with multiple different structures. We propose that oxidative stress leading to formation of MDA adducts is a common feature for triggers of aHUS and that failure of FH in protecting MDA-modified surfaces from complement activation is involved in the pathogenesis of the disease.

The atypical hemolytic uremic syndrome (aHUS) is a paradigm of a disease, caused by overactivation of the alternative complement pathway secondary to a not well-understood trigger event. About 60 % of the patients present genetic or acquired abnormalities in the proteins of the alternative complement pathway. In 40 % of the cases the affected protein is the complement regulator Factor H (FH)-30 % due to mutations and 10 % because of anti-FH autoantibodies. Here we describe the detailed protocol for a rapid test to analyse the functional defect associated with genetic or acquired FH-related abnormalities. It can be applied for the characterization of the underlying complement defect in aHUS, based on spontaneous lysis of non-sensitized sheep erythrocytes in contact with patients' plasma or serum.

The haemolytic uraemic syndrome (HUS) is the most frequent cause of acute renal failure in childhood. We investigated L-arginine/NO pathway in 12 children with typical HUS and 12 age-matched healthy control subjects. Nitrite and nitrate, the major NO metabolites in plasma and urine, asymmetric dimethylarginine (ADMA) in plasma and urine, and dimethylamine (DMA) in urine were determined by GC-MS and GC-MS/MS techniques. Urinary measurements were corrected for creatinine excretion. Plasma nitrate was significantly higher in HUS patients compared to healthy controls (P = 0.021), whereas urine nitrate was borderline lower in HUS patients compared to healthy controls (P = 0.24). ADMA plasma concentrations were insignificantly lower, but urine ADMA levels were significantly lower in the HUS patients (P = 0.019). Urinary DMA was not significantly elevated. In HUS patients, nitrate (R = 0.91) but not nitrite, L-arginine, or ADMA concentrations in plasma correlated with free haemoglobin concentration. Our results suggest that both NO production and ADMA synthesis are decreased in children with typical HUS. We hypothesize that in the circulation of children with HUS a vicious circle between the L-arginine/NO pathway and free haemoglobin-mediated oxidative stress exists. Disruption of this vicious circle by drugs that release NO and/or sulphydryl groups-containing drugs may offer new therapeutic options in HUS.

Hemolytic uremic syndrome (HUS) in children is usually caused by Shiga-like toxin-producing Escherichia coli, but approximately 5% of cases are caused by invasive pneumococcal infection (P-HUS). Reported herein is the case of a 9-month-old HUS patient with pneumococcal meningitis who needed hemodialysis for 12 days. Decreased sialylation was characterized in both transferrin N-glycans and IgA1 O-glycans, analyzed in the acute phase on mass spectrometry, consistent with S. pneumonia-produced sialidases hydrolyzing both α2,3- and α2,6-linked sialic acids. The method will complement the T-antigen activation test and help to understand the molecular pathology related to P-HUS., (© 2013 The Authors. Pediatrics International © 2013 Japan Pediatric Society.)

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr(1605)-Met(1606) peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.