Platelet Imaging

The knowledge gained through imaging platelets has formed the backbone of our understanding of their biology in health and disease. Early investigators relied on conventional light microscopy with limited resolution and were primarily able to identify the presence and basic morphology of platelets. The advent of high resolution technologies, in particular, electron microscopy, accelerated our understanding of the dynamics of platelet ultrastructure dramatically. Further refinements and improvements in our ability to localize and reliably identify platelet structures have included the use of immune-labeling techniques, correlative-fluorescence light and electron microscopy, and super-resolution microscopies. More recently, the expanded development and application of intravital microscopy in animal models has enhanced our knowledge of platelet functions and thrombus formation in vivo, as these experimental systems most closely replicate native biological environments. Emerging improvements in our ability to characterize platelets at the ultrastructural and organelle levels include the use of platelet cryogenic electron tomography with quantitative, unbiased imaging analysis, and the ability to genetically label platelet features with electron dense markers for analysis by electron microscopy

Association of SARS-CoV-2 nucleocapsid viral antigen and the receptor for advanced glycation end products with development of severe disease in patients presenting to the emergency department with COVID-19

IntroductionThere remains a need to better identify patients at highest risk for developing severe Coronavirus Disease 2019 (COVID-19) as additional waves of the pandemic continue to impact hospital systems. We sought to characterize the association of receptor for advanced glycation end products (RAGE), SARS-CoV-2 nucleocapsid viral antigen, and a panel of thromboinflammatory biomarkers with development of severe disease in patients presenting to the emergency department with symptomatic COVID-19.MethodsBlood samples were collected on arrival from 77 patients with symptomatic COVID-19, and plasma levels of thromboinflammatory biomarkers were measured.ResultsDifferences in biomarkers between those who did and did not develop severe disease or death 7 days after presentation were analyzed. After adjustment for multiple comparisons, RAGE, SARS-CoV-2 nucleocapsid viral antigen, interleukin (IL)-6, IL-10 and tumor necrosis factor receptor (TNFR)-1 were significantly elevated in the group who developed severe disease (all p<0.05). In a multivariable regression model, RAGE and SARS-CoV-2 nucleocapsid viral antigen remained significant risk factors for development of severe disease (both p<0.05), and each had sensitivity and specificity >80% on cut-point analysis.DiscussionElevated RAGE and SARS-CoV-2 nucleocapsid viral antigen on emergency department presentation are strongly associated with development of severe disease at 7 days. These findings are of clinical relevance for patient prognostication and triage as hospital systems continue to be overwhelmed. Further studies are warranted to determine the feasibility and utility of point-of care measurements of these biomarkers in the emergency department setting to improve patient prognostication and triage

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

Outcomes after ultramassive transfusion in the modern era: An Eastern Association for the Surgery of Trauma multicenter study

BACKGROUND: Despite the widespread institution of modern massive transfusion protocols with balanced blood product ratios, survival for patients with traumatic hemorrhage receiving ultramassive transfusion (UMT) (defined as ≥20 U of packed red blood cells [RBCs]) in 24 hours) remains low and resource consumption remains high. Therefore, we aimed to identify factors associated with mortality in trauma patients receiving UMT in the modern resuscitation era. METHODS: An Eastern Association for the Surgery of Trauma multicenter retrospective study of 461 trauma patients from 17 trauma centers who received ≥20 U of RBCs in 24 hours was performed (2014-2019). Multivariable logistic regression and Classification and Regression Tree analysis were used to identify clinical characteristics associated with mortality. RESULTS: The 461 patients were young (median age, 35 years), male (82%), severely injured (median Injury Severity Score, 33), in shock (median shock index, 1.2; base excess, -9), and transfused a median of 29 U of RBCs, 22 U of fresh frozen plasma (FFP), and 24 U of platelets (PLT). Mortality was 46% at 24 hours and 65% at discharge. Transfusion of RBC/FFP ≥1.5:1 or RBC/PLT ≥1.5:1 was significantly associated with mortality, most pronounced for the 18% of patients who received both RBC/PLT and RBC/FFP ≥1.5:1 (odds ratios, 3.11 and 2.81 for mortality at 24 hours and discharge; both p \u3c 0.01). Classification and Regression Tree identified that age older than 50 years, low initial Glasgow Coma Scale, thrombocytopenia, and resuscitative thoracotomy were associated with low likelihood of survival (14-26%), while absence of these factors was associated with the highest survival (71%). CONCLUSION: Despite modern massive transfusion protocols, one half of trauma patients receiving UMT are transfused with either RBC/FFP or RBC/PLT in unbalanced ratios ≥1.5:1, with increased associated mortality. Maintaining focus on balanced ratios during UMT is critical, and consideration of advanced age, poor initial mental status, thrombocytopenia, and resuscitative thoracotomy can aid in prognostication. LEVEL OF EVIDENCE: Prognostic, level III

Activation of the α7 nAChR Reduces Acid-Induced Acute Lung Injury in Mice and Rats

New evidence indicates that neural mechanisms can down-regulate acute inflammation. In these studies, we tested the potential role of the α7 nicotinic acetylcholine receptor (α7 nAChR) in a rodent model of acid-induced acute lung injury. We first determined that the α7 nAChR was expressed by alveolar macrophages and lung epithelial cells. Then, using an acid-induced acute lung injury mouse model, we found that nicotine, choline, and PNU-282987 (a specific α7 nAChR agonist) decreased excess lung water and lung vascular permeability, and reduced protein concentration in the bronchoalveolar lavage (BAL). Deficiency of α7 nAChR resulted in a 2-fold increase in excess lung water and lung vascular permeability. The reduction of proinflammatory cytokines (macrophage inflammatory protein-2 and TNF-α) in the BAL with nicotine probably resulted from the suppression of NF-κB activation in alveolar macrophages. The beneficial effect of nicotine was also tested in rat model of acid-induced acute lung injury in which BAL protein and receptor for advanced glycation end products (RAGE), a marker of type I cell injury, were reduced by nicotine treatment. These results indicate that activation of α7 nAChR may provide a new therapeutic pathway for the treatment of acute lung injury