Human blood neutrophils rolling on E-or P-selectin reduced their rolling velocity when intercellular adhesion molecule (ICAM)-1 was available. Similar to mouse neutrophils, this was dependent on P-selectin glycoprotein ligand 1 (PSGL1), ␣ L ␤ 2 integrin, the Src family tyrosine kinase FGR and spleen tyrosine kinase SYK. Blocking phospholipase C or p38 MAP kinase attenuated, but did not abolish the velocity reduction. To test expression of integrin activation epitopes, we adapted an immobilized reporter assay and developed a new homogeneous microfluidics-based reporter antibody binding assay. Rolling on E-or P-selectin induced the extension reporter epitopes KIM127 and NKI-L16, but not the high affinity reporter epitope monoclonal antibody (mAb) 24. This enabled rolling neutrophils to bind to immobilized extension reporter, but not activation reporter antibodies and allowed binding of soluble KIM127 during rolling. We conclude that human neutrophil rolling on E-or P-selectin induces the extended ␣ L ␤ 2 integrin conformation through signaling triggered by PSGL-1 engagement. (Blood. 2010;116(4):617-624) Introduction E-selectin or P-selectin binding to human neutrophils has long been known to induce activation as demonstrated by phosphorylation of p38 MAP kinase. [1][2][3] In Ficoll-isolated human neutrophils, this process leads to arrest from rolling, polarization, and firm adhesion to endothelial monolayers under flow. [4][5][6][7] The proximal signal transduction pathway leading from E-selectin binding to integrin-dependent adhesion is unknown. Whether neutrophil integrins assume the extended or high affinity conformation is also unknown.Mouse neutrophils in whole blood reduce their rolling velocity on E-selectin when intercellular adhesion molecule 1 (ICAM1) is also available. 8 This requires P-selectin glycoprotein ligand 1 (PSGL1), 8,9 a cell surface expressed O-glycan that is a ligand for all 3 selectins. 10 Slow rolling probably involves extension of the integrin LFA-1, which requires the Src family tyrosine kinase Fgr, the immunoreceptor tyrosine-based activation motif (ITAM)-containing adapter molecules 11 and Syk. 8 Binding of isolated human neutrophils to E-selectin increases intracellular calcium levels, 7 which is blocked by phospholipase C inhibition. Indeed, PLC␥2 was recently shown to be activated downstream of Syk after integrin engagement. 12 We found little evidence for LFA-1 activation during mouse neutrophil rolling on P-selectin, but McEver et al reported that rolling on P-selectin can trigger LFA-1 activation. 9 Like other integrins, 13,14 LFA-1 undergoes dramatic conformational changes when activated. 15 Fluorescence resonance energy transfer (FRET) studies show that the cytoplasmic and transmembrane domains of the ␣ L and ␤ 2 subunits of LFA-1 move apart, 16 forcing the extracellular domain of LFA-1 into the extended conformation. 15 Conformational unbending of ␣ 4 ␤ 1 integrin was also shown more directly using FRET between a fluorophore on ␣ 4 subunit and an acceptor in the lipid bila...

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2 S ) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2 S , was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domaincontaining proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.

Acute kidney injury (AKI) is a common clinical problem in critically ill patients and increases in-hospital mortality. Acute loss of renal function (ALRF) reduces leukocyte recruitment into inflamed tissue, but the underlying molecular mechanisms remain unknown. In this study, we investigated the effects of ALRF on the different steps of the leukocyte recruitment cascade by using intravital microscopy, flow chamber experiments, and biochemistry assays. ALRF abolished selectin-induced slow leukocyte rolling on E-selectin/ICAM-1 and P-selectin/ICAM-1 and also reduced transmigration without affecting chemokine-induced arrest. A reduced phosphorylation of spleen tyrosine kinase (Syk), Akt, phospholipase C (PLC) γ2, and p38 MAPK, but not altered expression levels of adhesion molecules on the surface of neutrophils, was responsible for the abolished selectin-mediated slow leukocyte rolling. The results observed in the murine system could be reproduced in flow chamber experiments with human blood. Samples from critically ill patients with sepsis-induced AKI showed a significantly higher rolling velocity on E-selectin/ICAM-1 and P-selectin/ICAM-1 compared to patients with sepsis alone or to healthy volunteers. In conclusion, these data suggest that ALRF inhibits selectin-mediated slow leukocyte rolling by reducing phosphorylation of Syk, Akt, PLCγ2, and p38 MAPK and transmigration.

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When attempting to intubate a trapped car accident victim, video laryngoscopes provide a better view of the glottis, but some delay tracheal intubation significantly. The devices with a tube guide (Airtraq and Ambu Pentax AWS) enable tracheal intubation to be achieved significantly faster and with a lower failure rate than devices without a tube guide. No video laryngoscope outperformed direct laryngoscopy with a Macintosh laryngoscope in this simulation study.

Best quality of CPR was achieved by DACO-CPR because of superior compression frequencies and reduced NFT. In contrast, the full dispatcher-assisted CPR with a longer initial instructing phase (initial NFT) did not result in enhanced CPR quality or an optimised compression depth.

No abstract

Although the minimal material required according to European aviation regulations is provided by all airlines for medical emergencies, there are significant differences in the provision of additional material. The equipment on most airlines is not sufficient for the treatment of specific emergencies according to published medical guidelines (eg, for CPR or acute myocardial infarction).