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Background: The incidence and the outcomes of pulmonary embolism (PE) missed during emergency department (ED) workup are largely unknown. Objectives: To describe the frequency, demographics, and outcomes of patients with delayed diagnosis of PE. Methods: We retrospectively compared patients diagnosed with PE during ED workup (early diagnosis) with patients diagnosed with PE thereafter (delayed diagnosis). Electronic health records (EHR) of 123,560 consecutive patients who attended a tertiary hospital ED were screened. Data were matched with radiology and pathology results from the EHR. Results: Of 1,119 patients presenting to the ED with early workup for PE, PE was diagnosed in 182 patients (80.5%) as early diagnosis. Delayed diagnosis was established in 44 cases (19.5%) using radiology and/or autopsy data. Median age of patients with early diagnosis was significantly lower as compared to delayed diagnosis (67 vs. 77.5 years). Main symptoms were dyspnea (109 patients [59.9%] in early, 20 patients [45.5%] in delayed diagnosis), chest pain (90 patients [49.5%] in early, 8 patients [18.2%] in delayed diagnosis), and nonspecific complaints (16 patients [8.8%] in early, 13 patients [29.5%] in delayed diagnosis). In-hospital mortality was 1.6% in early diagnosis and 43.2% in delayed diagnosis. Conclusions: Delayed diagnosis of PE carries a worse prognosis than early diagnosis. This discrepancy may arise from either delayed therapy, confounding variables (e.g., older age), or both. Possible reasons for delayed diagnoses are nonspecific presentations and symptoms overlapping with preexisting conditions.

Background End-of-life decisions (EOLD) represent potentially highly consequential decisions often made in acute situations, such as 'do not attempt resuscitation' (DNAR) choices at emergency presentation. Aim We investigated DNAR decisions in an emergency department (ED) to assess prevalence, associated patient characteristics, potential medical and economic consequences and estimate contributions of patients and physicians to DNAR decisions. Design Single-centre retrospective observation, including ED patients with subsequent hospitalization between 2012 and 2016. Primary outcome was a DNAR decision and associated patient characteristics. Secondary outcomes were mortality, admission to intensive care unit and use of resources. Methods Associations between DNAR and patient characteristics were analysed using logistic mixed effects models, results were reported as odds ratios (OR). Median odds ratios (MOR) were used to estimate patient and physician contributions to variability in DNAR. Results Patients of 10 458 were attended by 315 physicians. DNAR was the choice in 23.3% of patients. Patients' characteristics highly associated with DNAR were age (OR = 4.0, 95% CI = 3.6-4.3) and non-trauma presentation (OR = 2.3, 95% CI = 1.9-2.9). In-hospital mortality was significantly higher (OR = 5.4, CI = 4.0-7.3), and use of resources was significantly lower (OR = 0.7, CI = 0.6-0.8) in patients choosing DNAR. There was a significant effect on DNAR by both patient (MOR = 1.8) and physician (MOR = 2.0). Conclusions DNAR choices are common in emergency patients and closely associated with age and non-trauma presentation. Mortality was significantly higher, and use of resources significantly lower in DNAR patients. Evidence of a physician effect raises questions about the choice autonomy of emergency patients in the process of EOLD.

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-β1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti–TGF-β1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-β1–mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti–TGF-β1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-β1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell–derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-β1–dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.

Ectosomes are vesicles shed directly from the cell surface. Human polymorphonuclear neutrophils release ectosomes (PMN-Ect) upon their activation. PMN-Ect expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, and down-modulate the inflammatory response of human macrophages and dendritic cells exposed to TLR-2 and -4 ligands. This down-modulation is mediated by PS via the engagement and activation of the Mer receptor tyrosine kinase (MerTK). In the present study, we demonstrate that exposure of macrophages to PMN-Ect activates directly 2 additional pathways, an immediate Ca(2+) flux and a rapid release of TGF-beta1. As expected, the Ca(2+) flux was necessary for the activation of TLR-2 pathway with the release of cytokines. However, MerTK blockade with antibodies did not modify the Ca(2+) flux, indicating an independent activation of Ca(2+) by PMN-Ect. Striking was that the rapid release of TGF-beta1 was independent of the MerTK pathway and did not require a Ca(2+) flux. TGF-beta1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-beta1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-beta1 by macrophages was seen only for PMN-Ect and not for PS-liposomes or erythrocyte ectosomes, which express PS. However, blocking the PS of PMN-Ect inhibited TGF-beta1 release, suggesting that PS expression was necessary although not sufficient for this release. Interestingly, the effects of PMN-Ect pre-exposure were lasting for 24h with the macrophages being less receptive to TLR-2 activation and TGF-beta1 stores remaining low. In sum, PMN-Ect induce several signaling pathways in resting and stimulated macrophages, which include independently the MerTK pathway, Ca(2+) flux and the release of stored TGF-beta1, and each might influence the immunomodulatory effects of macrophages.

Microparticles (MP) shed by platelets (PLT) during storage have procoagulant activities, but little is known about their properties to modify inflammation or immunity. In this study, we studied the capacity of MP present in PLT concentrates to alter the function of macrophages and dendritic cells (DC). The size of the purified MP was between 100 and 1000 nm, and they expressed phosphatidylserine; surface proteins of PLT (CD61, CD36, CD47), including complement inhibitors (CD55, CD59), but not CD63; and proteins acquired from plasma (C1q, C3 fragments, factor H). These characteristics suggest that the MP shed by PLT are formed by budding from the cell surface, corresponding to ectosomes. The purified PLT ectosomes (PLT-Ect) reduced the release of TNF-α and IL-10 by macrophages activated with LPS or zymosan A. In addition, PLT-Ect induced the immediate release of TGF-β from macrophages, a release that was not modified by LPS or zymosan A. Macrophages had a reduced TNF-α release even 24 h after their exposure to PLT-Ect, suggesting that PLT-Ect induced a modification of the differentiation of macrophages. Similarly, the conventional 6-d differentiation of monocytes to immature DC by IL-4 and GM-CSF was modified by the presence of PLT-Ect during the first 2 d. Immature DC expressed less HLA-DP DQ DR and CD80 and lost part of their phagocytic activity, and their LPS-induced maturation was downmodulated when exposed to PLT-Ect. These data indicate that PLT-Ect shed by stored PLT have intrinsic properties that modify macrophage and DC differentiation toward less reactive states.

At the earliest stage of activation, human polymorphonuclear neutrophils release vesicles derived directly from the cell surface. These vesicles, called ectosomes (PMN-Ect), expose phosphatidylserine in the outer membrane leaflet. They inhibit the inflammatory response of human monocyte-derived macrophages and dendritic cells to zymosan A (ZymA) and LPS and induce TGF- 1 release, suggesting a reprogramming toward a tolerogenic phenotype. The receptors and signaling pathways involved have not yet been defined. Here, we demonstrate that PMN-Ect interfered with ZymA activation of macrophages via inhibition of NF B p65 phosphorylation and NF B translocation. The MerTK (Mer receptor tyrosine kinase) and PI3K/Akt pathways played a key role in this immunomodulatory effect as shown using specific MerTK-blocking antibodies and PI3K inhibitors LY294002 and wortmannin. As a result, PMN-Ect reduced the transcription of many proinflammatory genes in ZymA-activated macrophages. In sum, PMN-Ect interacted with the macrophages by activation of the MerTK pathway responsible for down-modulation of the proinflammatory signals generated by ZymA.