Establishment of a method for separating macrophage migrasomes / 细胞与分子免疫学杂志

Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes. Methods Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells. A 0.45 μm filter was employed for reverse filtration and elution to isolate the migrasomes. The morphological characteristics of the migrasomes were then observed using transmission electron microscopy. Western blot analysis was performed to determine the expression of characteristic markers of the migrasomes. The RNA carried by the migrasomes was analysed by using LabChip bioanalyzer. Results Scanning electron microscopy revealed that the migrasomes, with membranous structures, were attached to the tip or bifurcation of the retraction fiber formed in the tail of RAW264.7 cells. Transmission electron microscopy showed that the isolated migrasomes had a typical oval vesicle-like structure with wrinkled membrane surfaces. Western blot analysis confirmed the expression of the characteristic markers phosphatidylinositol glycan anchor biosynthesis class K (PIGK), epidermal growth factor domain-specific O-linked N-acetylglucosamine transferase (EOGT) and tetraspanin 4 (TSPAN4) in the migrasomes, while the EV (extracellular vesicle) markers tumor susceptibility gene 101 (TSG101) and Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X (ALIX) were not detected. Furthermore, the isolated migrasomes were found to be rich in small RNA, which were approximately 25-200 nt in length. Conclusion A method for the extraction of well-structured and high quality migrasomes from macrophages is established.

Results of safety monitoring of BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine in U.S. children aged 5-17 years

ImportanceActive monitoring of health outcomes following COVID-19 vaccination offers early detection of rare outcomes that may not be identified in pre-licensure trials. ObjectiveTo conduct near-real time monitoring of health outcomes following BNT162b2 COVID-19 vaccination in the U.S. pediatric population aged 5-17 years. DesignWe conducted rapid cycle analysis of 20 pre-specified health outcomes, 13 of which underwent sequential testing and 7 of which were monitored descriptively within a cohort of vaccinated individuals. We tested for increased risk of each health outcome following vaccination compared to a historical baseline, while adjusting for repeated looks at the data as well as claims processing delay. SettingThis is a population-based study in three large commercial claims databases conducted under the U.S. FDA public health surveillance mandate. ParticipantsThe study included over 3 million enrollees aged 5-17 years with BNT162b2 COVID-19 vaccination through mid-2022 in three commercial claims databases. We required continuous enrollment in a medical health insurance plan from the start of an outcome-specific clean window to the COVID-19 vaccination. ExposureExposure was defined as receipt of a BNT162b2 COVID-19 vaccine dose. The primary analysis assessed primary series doses together (Dose 1 + Dose 2), and dose-specific secondary analyses were conducted. Follow up time was censored for death, disenrollment, end of risk window, end of study period, or a subsequent vaccine dose. Main Outcome(s) and Measure(s)We monitored 20 pre-specified health outcomes. We performed descriptive monitoring for all outcomes and sequential testing for 13 outcomes. ResultsAmong 13 health outcomes evaluated by sequential testing, 12 did not meet the threshold for a statistical signal in any of the three databases. In our primary analysis, myocarditis/pericarditis signaled following primary series vaccination with BNT162b2 in ages 12-17 years across all three databases. Conclusions and RelevanceConsistent with published literature, our near-real time monitoring identified a signal for only myocarditis/pericarditis following BNT162b2 COVID-19 vaccination in children aged 12-17 years. This method is intended for early detection of safety signals. Our results are reassuring of the safety of the vaccine, and the potential benefits of vaccination outweigh the risks. Key PointsO_ST_ABSQuestionC_ST_ABSDid active monitoring detect potentially elevated risk of health outcomes following BNT162b2 COVID-19 vaccination in the U.S. pediatric population aged 5-17 years? FindingsTwelve of 13 health outcomes did not meet the safety signal threshold following BNT162b2 COVID-19 vaccination in three large commercial claims databases using near real-time monitoring. Myocarditis/pericarditis met the statistical threshold for a signal following primary series vaccination in ages 12-17 years. MeaningResults from near-real time monitoring of health outcomes following BNT162b2 COVID-19 vaccination provide additional reassuring evidence of vaccine safety in the pediatric population. The myocarditis/pericarditis signal is consistent with current evidence and is being further evaluated.