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. After in vivo injection, O157LPS bound to platelets from wild-type mice, which had lower platelet counts than did mice lacking TLR4. Mouse experiments confirmed that O157LPS binding to TLR4 is the primary event leading to platelet activation, as shown by CD40L expression, and that CD62 further contributes to this process. Activation of human platelets by EHEC-LPS was demonstrated by expression of the activated GPIIb/IIIa receptor, CD40L, and fibrinogen binding. In perfusion experiments, platelet activation on endothelial cells was TLR4 and CD62 dependent. O157LPS was detected on platelets from 12 of 14 children with EHECassociated hemolytic uremic syndrome (HUS) and on platelets from 2 children before the development of HUS but not on platelets of EHEC-infected children in whom HUS did not develop (n ‫؍‬ 3). These data suggest that O157LPS on platelets might contribute to platelet consumption in HUS. (Blood. 2006;108:167-176)

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BackgroundAggregates 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 FindingsThe 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. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF–expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery.Conclusions/SignificanceBy triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

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. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

Atypical hemolytic uremic syndrome (aHUS) may be associated with mutations in the C-terminal of factor H (FH).

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Å resolution shows how the 995‐residue polypeptide forms a unique proteolytic chamber of more than 10 000 Å3 in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C‐terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N‐terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge‐bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.

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A set of prominent designers embarked on a research journey to explore aesthetics in movement-based design. Here we unpack one of the design sensitivities unique to our practice: a strong first person perspective-where the movements, somatics and aesthetic sensibilities of the designer, design researcher and user are at the forefront. We present an annotated portfolio of design exemplars and a brief introduction to some of the design methods and theory we use, together substantiating and explaining the first-person perspective. At the same time, we show how this felt dimension, despite its subjective nature, is what provides rigor and structure to our design research. Our aim is to assist researchers in soma-based design and designers wanting to consider the multiple facets when designing for the aesthetics of movement. The applications span a large field of designs, including slow introspective, contemplative interactions, arts, dance, health applications, games, work applications and many others.