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Background: Intestinal infections with Shiga toxin-producing Escherichia coli (STEC) in children can lead to the hemolytic uremic syndrome (HUS). Shiga toxins (Stx) released in the gut by bacteria enter the blood stream and target the kidney causing endothelial injury. Free toxins have never been detected in the blood of HUS patients, but they have been found on the surface of polymorphonuclear leukocytes (PMN)., Methods: With respect to their clinical features, the clinical relevance of the amounts of serum Stx (cytotoxicity assay with human endothelial cells) and PMN-bound Stx (cytofluorimetric assay) in 46 patients with STEC-associated HUS was evaluated., Results: Stx-positive PMN were found in 60% of patients, whereas negligible amounts of free Stx were detected in the sera. Patients with high amounts of Stx on PMN showed preserved or slightly impaired renal function (incomplete form of HUS), whereas cases with low amounts of Stx usually presented evidence of acute renal failure., Conclusions: These observations suggest that the extent of renal damage in children with STEC-associated HUS could depend on the concentration of Stx present on their PMN and presumably delivered by them to the kidney. As previously shown by experimental models from our laboratory, high amounts of Stx could induce a reduced release of cytokines by the renal endothelium, with a consequent lower degree of inflammation. Conversely, low toxin amounts can trigger the cytokine cascade, provoking inflammation, thereby leading to tissue damage.

The endothelial damage induced by Stx represents the main pathogenic event in the HUS associated with STEC infections in humans. Stx, released in the gut by bacteria, enter the bloodstream and are targeted to renal endothelia. The role of PMN as a toxin carrier has been the object of controversy. In this paper, we confirm the binding of Stx1 to PMN, also showing its degranulating effects on full-loaded leukocytes, and support the carrier role of PMN by using a two-chamber transmigration device, in which PMN, loaded in vitro with different amounts of Stx1, transmigrated through confluent monolayers of endothelial cells, mimicking the toxin-induced renal endothelial injury. Stx1 was transferred during PMN transmigration, impairing protein synthesis and triggering production of proinflammatory cytokines in endothelial cells. PMN, carrying low toxin amounts, induced the release of high levels of cytokines in viable endothelial cells, whereas cytokine production was blocked in cells challenged with PMN fully loaded with Stx as a result of an almost total impairment of translation and of the activation of the apoptotic program. In agreement with previous unexplained observations in animal models, the results obtained with our experimental setting suggest that a self-amplifying circle triggered by low doses of toxin may lead to the production of proinflammatory mediators of renal damage in HUS.

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

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

Two dendritic fullerene (DF) monoadducts, 2 and 3, which can carry up to 9 and 18 negative charges, respectively, were examined with respect to electrostatic complexation with Cytochrome c (Cytc). To facilitate comprehensive photophysical investigations, the zinc analogue of Cytc (ZnCytc) was prepared according to a novel, modified procedure. The association of ZnCytc and DF, and consequential photoinduced electron transfer within ZnCytc-DF from the photoexcited protein to the fullerene, was proven by fluorescence spectroscopy and transient absorption spectroscopy. These findings were also supported by circular dichroism as well as by extensive molecular dynamics (MD) simulations.