Flotillins Interact with PSGL-1 in Neutrophils and, upon Stimulation, Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1

Compositional analysis with atomic column spatial resolution by 5th-order aberration-corrected scanning transmission electron microscopy

We show in this article that it is possible to obtain elemental compositional maps and profiles with atomic-column resolution across an InxGa1?xAs multilayer structure from 5th-order aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images. The compositional profiles obtained from the analysis of HAADF-STEM images describe accurately the distribution of In in the studied multilayer in good agreement with Muraki's segregation model [Muraki, K., Fukatsu, S., Shiraki, Y. & Ito, R. (1992). Surface segregation of In atoms during molecular beam epitaxy and its influence on the energy levels in InGaAs/GaAs quantums wells. © Microscopy Society of America 2011.This work was supported by the Spanish MICINN (projects TEC2008-06756-C03-02 and CONSOLIDER INGENIO 2010 CSD2006-0019 and CSD2009-00013), the Junta de Andalucía (PAI research groups TEP-120 and TIC-145; project P08-TEP-03516 and MAT2010-15206), and CAM 2010 project S2009ESP-1503. Work at Oak Ridge National Laboratory was sponsored by the U.S. Department of Energy, Division of Materials Sciences and Engineering (M.V. and S.J.P.).Peer Reviewe

Defining the scope of the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet): A bottom-up and One Health approach

Background: Building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) was proposed to strengthen the European One Health antimicrobial resistance (AMR) surveillance approach. Objectives: To define the combinations of animal species/production types/age categories/bacterial species/specimens/antimicrobials to be monitored in EARS-Vet. Methods: The EARS-Vet scope was defined by consensus between 26 European experts. Decisions were guided by a survey of the combinations that are relevant and feasible to monitor in diseased animals in 13 European countries (bottom-up approach). Experts also considered the One Health approach and the need for EARS-Vet to complement existing European AMR monitoring systems coordinated by the ECDC and the European Food Safety Authority (EFSA). Results: EARS-Vet plans to monitor AMR in six animal species [cattle, swine, chickens (broilers and laying hens), turkeys, cats and dogs], for 11 bacterial species (Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus suis). Relevant antimicrobials for their treatment were selected (e.g. tetracyclines) and complemented with antimicrobials of more specific public health interest (e.g. carbapenems). Molecular data detecting the presence of ESBLs, AmpC cephalosporinases and methicillin resistance shall be collected too. Conclusions: A preliminary EARS-Vet scope was defined, with the potential to fill important AMR monitoring gaps in the animal sector in Europe. It should be reviewed and expanded as the epidemiology of AMR changes, more countries participate and national monitoring capacities improve

AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells

Background and purpose: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs)