Isolation and Characterization of Shiga Toxin-Associated Microvesicles.

Microvesicles are shed from cell surfaces during infectious or inflammatory conditions and may contribute to the pathogenesis of disease. During Shiga toxin-producing Escherichia coli (STEC) infection, microvesicles are released from blood cells. These microvesicles play a part in inflammation, thrombosis, hemolysis, and the transfer of the main virulence factor of STEC strains, Shiga toxin, to target organ cells.

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor.

Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles.

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model.

Blockade of the kallikrein-kinin system reduces endothelial complement activation in vascular inflammation.

Background: The complement and kallikrein-kinin systems (KKS) are activated during vascular inflammation. The aim of this study was to investigate if blockade of the KKS can affect complement activation on the endothelium during inflammation.

Methods: Complement deposition on endothelial microvesicles was assayed in vasculitis patient plasma samples and controls.

Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases.

Extracellular vesicles are cell-derived membrane particles ranging from 30 to 5,000 nm in size, including exosomes, microvesicles, and apoptotic bodies. They are released under physiological conditions, but also upon cellular activation, senescence, and apoptosis. They play an important role in intercellular communication.

Extracellular vesicles in renal disease.

Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information.

C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles.

The kinin system is activated during vasculitis and may contribute to chronic inflammation. C1-inhibitor is the main inhibitor of the kinin system. In this study, we investigated the presence of the kinin B1 receptor on endothelial microvesicles and its contribution to the inflammatory process.

Microvesicle transfer of kinin B1-receptors is a novel inflammatory mechanism in vasculitis.

During vasculitis, activation of the kinin system induces inflammation, whereby the kinin B1-receptor is expressed and activated after ligand binding. Additionally, activated blood cells release microvesicles into the circulation. Here we determined whether leukocyte-derived microvesicles bear B1-kinin receptors during vasculitis, and if microvesicles transfer functional B1-receptors to recipient cells, thus promoting inflammation.

Complement Interactions with Blood Cells, Endothelial Cells and Microvesicles in Thrombotic and Inflammatory Conditions.

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.

Enterohemorrhagic Escherichia coli Pathogenesis and the Host Response.

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic bacterial strain capable of causing watery or bloody diarrhea, the latter termed hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). HUS is defined as the simultaneous development of non-immune hemolytic anemia, thrombocytopenia, and acute renal failure. The mechanism by which EHEC bacteria colonize and cause severe colitis, followed by renal failure with activated blood cells, as well as neurological symptoms, involves the interaction of bacterial virulence factors and specific pathogen-associated molecular patterns with host cells as well as the host response.

A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry.

The combined role of galactose-deficient IgA1 and streptococcal IgA-binding M Protein in inducing IL-6 and C3 secretion from human mesangial cells: implications for IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial cell proliferation and extracellular matrix expansion associated with immune deposits consisting of galactose-deficient polymeric IgA1 and C3. We have previously shown that IgA-binding regions of streptococcal M proteins colocalize with IgA in mesangial immune deposits in patients with IgAN. In the present study, the IgA-binding M4 protein from group A Streptococcus was found to bind to galactose-deficient polymeric IgA1 with higher affinity than to other forms of IgA1, as shown by surface plasmon resonance and solid-phase immunoassay.

Complement activation associated with ADAMTS13 deficiency in human and murine thrombotic microangiopathy.

This study addressed the contribution of ADAMTS13 deficiency to complement activation in thrombotic thrombocytopenic purpura (TTP). Renal tissue and blood samples were available from 12 TTP patients. C3 and C5b-9 deposition were demonstrated in the renal cortex of two TTP patients, by immunofluorescence and immunohistochemistry, respectively.

A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis.

Phenotypic expression of ADAMTS13 in glomerular endothelial cells.

Background: ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13⁺/⁺) and deficient (Adamts13⁻/⁻) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells.

Methodology/principal Findings: Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice.

Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is commonly associated with Shiga toxin (Stx)-producing Escherichia coli O157:H7 infection. This study examined patient samples for complement activation on leukocyte-platelet complexes and microparticles, as well as donor samples for Stx and lipopolysaccharide (O157LPS)-induced complement activation on platelet-leukocyte complexes and microparticles. Results, analyzed by flow cytometry, showed that whole blood from a child with HUS had surface-bound C3 on 30% of platelet-monocyte complexes compared with 14% after recovery and 12% in pediatric controls.

Shiga toxin and lipopolysaccharide induce platelet-leukocyte aggregates and tissue factor release, a thrombotic mechanism in hemolytic uremic syndrome.

Background: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF.

Methodology/principal Findings: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood.

A novel mutation in the complement regulator clusterin in recurrent hemolytic uremic syndrome.

A novel heterozygous mutation in the clusterin gene, nucleotide position A1298C (glutamine>proline Q433P), was detected in exon 7 of a child with recurrent hemolytic uremic syndrome (HUS). The same mutation was found in the child's two siblings and mother but not in 120 controls. In addition, a previously described heterozygous mutation was detected in the gene encoding membrane cofactor protein (MCP) causing a 6 base-pair deletion 811-816delGACAGT in exon 6.

Factor H dysfunction in patients with atypical hemolytic uremic syndrome contributes to complement deposition on platelets and their activation.

Atypical hemolytic uremic syndrome (aHUS) may be associated with mutations in the C-terminal of factor H (FH). FH binds to platelets via the C-terminal as previously shown using a construct consisting of short consensus repeats (SCRs) 15 to 20. A total of 4 FH mutations, in SCR15 (C870R) and SCR20 (V1168E, E1198K, and E1198Stop) in patients with aHUS, were studied regarding their ability to allow complement activation on platelet surfaces.

Platelet activation in hemolytic uremic syndrome.

Platelet consumption in platelet-fibrin aggregates leading to thrombocytopenia and small vessel obstruction are major features of the hemolytic uremic syndrome (HUS). Although thrombocytopenia has been correlated to poor prognosis, the mechanisms by which thrombocytopenia develops in HUS have not been completely elucidated. However, plausible explanations have been platelet contact with thrombogenic surfaces and/or direct contact with an aggregating agent.

Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.

This study presents evidence that human platelets bind lipopolysaccharide (LPS) from enterohemorrhagic Escherichia coli (EHEC) through a complex of toll-like receptor 4 (TLR4) and CD62, leading to their activation. TLR4 colocalized with CD62 on the platelet membrane, and the TLR4 specificity of LPS binding to platelets was confirmed using C57BL/10ScN mice lacking Tlr4. Only platelets from TLR4 wild-type mice bound O157LPS in vitro.