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Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)‐producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A‐subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re‐evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS‐pathogenesis and to develop therapeutic approaches.

Introduction: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by clonal hemopoiesis, inflammatory microenvironment and mutations in JAK2, MPL, or CALR genes. Around 10% of patients (pts) do not carry the 3 mutations (Triple negative (TN)). Microparticles (MPs; 0.1-1 μm) are small vesicles deriving from plasma membrane during homeostasis and cell activation of a wide variety of cells with a role in intercellular signalling. Circulating MPs are increased in inflammation and cancer including MPN. However, their role in the pathogenesis of MF is still elusive. Here we compared phenotype, function and microRNAs (miRs) expression of isolated MPs of JAK2V617F mutated and TN pts. Methods: Peripheral blood was collected from 10 MF pts (at diagnosis or out of cytotoxic treatment for at least 3 months) and 10 age/sex-matched healthy donors (HD). MF pts were JAK2V617F mutated (n=5) and TN n=5). MPs were purified from 2 mL of platelet poor plasma by ultracentrifugation and quantified using Nanosight technology. Isolated MPs were characterized by flow cytometry (CytoFLEX, Beckman Coulter) for the expression of platelets (PLT:CD61+CD62P+) and megakaryocytes (MK:CD61+CD62P-) markers. MPs were then analyzed for their ability to modulate the in vitro viability of immunomagnetically isolated CD34+ cells from MF pts and cord blood (CB). MiRs expression of isolated MPs (10e9) from 3 JAK2V617F mutated, 3 TN pts and 3 HD was investigated using TaqMan™ Array Human MicroRNA A Cards (ThermoFisher). Results: The mean number of plasma MPs isolated from MF pts was similar to HD (5*10e10/mL). However, flow cytometry analysis revealed that the mean percentage of PLT-MPs was significantly increased (p

Background: Among stem cell sources for regenerative therapy, human adipose derived stromal/stem cells (hASCs) have attracted a lot of attention over the last decade. hASCs are easily accessible, available in high numbers and their use does not pose ethical concerns. These cells can be obtained with minimal manipulation methods and have been used extensively in the autologous setting. However, the methods for tissue harvesting and grafting remain a crucial issue. This study aims at evaluating the biological features of hASCs obtained with nanofat, a novel liposuction technique, and at comparing them with hASCs obtained with the classical Coleman technique of adipose tissue harvesting. Materials and Methods: The nanofat methodology is a procedure alternative to the classical Coleman technique of fat harvesting and is characterized by the use of a thinner cannula. We analyzed hASCs derived from lipoaspirate samples collected using these two techniques. We assessed the immunophenotype, the rate of cell proliferation, adipogenic, osteogenic and chondrogenic differentiation potential and immunomodulatory properties of nanofat- and Coleman-processed hASCs. Results: Both liposuction procedures enabled isolation and expansion of hASCs with high efficiency. The immunophenotypic characterization showed an antigen profile similar to the pattern expressed by mesenchymal stem cells. hASCs obtained with the two methods displayed multilineage differentiation potential into the adipogenic, osteogenic and chondrogenic commitments. Finally, both nanofat- and Coleman-derived hASCs displayed immunomodulatory properties, as evidenced by their tolerogenic phenotype and their ability to inhibit lymphocyte proliferation in vitro. Conclusions: These results indicate that the stem cell component in the stromal vascular fraction is preserved in lipoaspirates obtained with the nanofat technique.