Immunological and Molecular Characterization of Circulating Exosomes Induced by Lung Transplant Recipients with Covid-19

Exosomes are nano-vesicles of <200 nm released by cells. We have demonstrated the presence of circulating exosomes containing lung self-antigens (SAgs) (Collagen-V, Kα1-Tubulin) and respiratory viral (RVI) antigens in lung transplant recipients (LTxRs) with RVI and rejection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in COVID-19 disease which can result in high morbidity and mortality in LTxRs. The goal of this study is to determine role of exosomes induced after SARS-CoV-2 infection in LTxRs, their origin and immune as well as molecular characteristics. We analyzed exosomes from 20 LTxRs with SARS-CoV-2 infection. Exosomes were isolated from plasma by Exosome Precipitation Kit and characterized by western blot for SARS-CoV-2 Spike (CSP), nucleocapsid protein (CNP), lung SAgs, cardiac SAgs (Myosin, Vimentin), transcription factors CIITA and NFkB, 20S proteasome, endothelial marker Von Willebrand Factor (VWF, VEGFR1 and V-CADHERIN), epithelial marker (beta catenin, MUC1 and FOX A1), angiotensin II type-I receptor (AT1R), macrophage stimulating factor 1 (MST1) and GRANZYME B (GRA-B). Isolated exosomes were also analyzed by transmission electron microscopy (TEM) for the presence of CSP, VWF, lung SAgs and cardiac SAgs. Mice were immunized with isolated exosomes containing CSP and CNP. Immune responses and histopathology of the mice lung tissues were analyzed. Exosomes from SARS-CoV-2 infected LTxRs contained CSP, nucleocapsid, VWF, VEGFR1, V-CADHERIN, beta catenin, MUC1, FOX A1 lung SAgs, cardiac SAgs, transcription factors, MST1, GRA-B and AT1R. TEM of exosomes also revealed the surface expression of spike protein, VWF, Kα1-Tubulin and Vimentin. C57BL/6 mice immunized with exosomes developed antibodies to CSP and CNP. Histopathology of the lungs demonstrated inflammation and lung fibrosis. We demonstrated that SARS-CoV-2 infected LTxRs induced circulating exosomes with CSP, CNP, lung SAgs, epithelial and endothelial markers suggesting that SARS-CoV-2 infection occurs in both endothelial and epithelium of the host. Therefore, we propose that the induced exosomes can activate both endothelium and epithelium leading to cytokine release. Immunized mice developed antibodies specific to CSP and CNP resulting in inflammation in the lung and fibrosis. [ FROM AUTHOR] Copyright of Journal of Heart & Lung Transplantation is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Lung Transplant Recipients with SARS-CoV2 Infection and Bnt162b2 Vaccination Induce Circulating Exosomes with Spike Protein S2 Capable of Inducing Immune Responses to SARS0-CoV2 in Mice

Purpose: Exosomes are small vesicles which are released by cells into body fluids. We have demonstrated the presence of circulating exosomes with viral antigens in lung transplant recipients (LTxRs) diagnosed with respiratory viral infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection results Covid-19 disease and SARS-CoV2 infection of LTxRs can be severe with poor clinical outcomes. The goal of this single center study is to determine the development of antibody responses specific to SARS-CoV2 in LTxRs, characterize the immune and molecular markers in the circulating exosomes induced and its role in eliciting immunity. Method(s): To determine that antibody responses and induction of circulating exosomes we enrolled LTxRs with SARS-CoV2 infection (n=50), following 2 doses of vaccination (n=100). Exosomes were isolated from plasma by exosome precipitation kit followed by 0.2 micron filtration and size determination by NanoSight300. Exosomes were subjected to transmission electron microscopy for spike (CSP) and nucleocapsid (CNP) antigens. Exosomes were also characterized by western blot for immune and molecular markers (NFkB, CIITA, 20S proteasome, beta catenin and VWF). C57BL/6 mice were immunized with circulating exosomes isolated from LTxRs with infection. Result(s): 78% of SARS-CoV2 infected LTxRs developed antibodies to CSP and CNP as opposed to normal infected individuals. In contrast, only 55% vaccinated LTxRs developed antibodies to SARS-CoV2 spike. Exosomes from SARS-CoV2 infected and vaccinated individuals contained CSP S2, CNP and immune and molecular markers. Transmission electron microscopy also revealed the presence of CSP and CNP on exosomes. C57BL/6 mice immunized with exosomes carrying CSP developed antibodies to SARS-CoV2 spike antigens. Severe inflammation and lung lesions were also demonstrated in the lungs of mice immunized with exosomes carrying CSP. Conclusion(s): In conclusion, we demonstrated that SARS-CoV2 infected and vaccinated LTxRs induced circulating exosomes with SARS-CoV2 CSP. In addition, exosomes contained important immune activating molecules suggesting that the exosomes induced by SARS-CoV2 may have a physiological role in inducing immune responses. Immunization of mice with exosomes from SARS-CoV2 infected and vaccinated LTxRs not only induced SARS-CoV2 spike specific antibody but also resulted in inflammation and lung lesions in the immunized animals.

Is the Swansea Hip Interrogation Fracture Tool Reliable When Used in Our Local Hospital Population?

Introduction: The Swansea Hip interrogation Fracture Tool (SHiFT) was suggested combining the Clinical Frailty score and Nottingham hip fracture scores, and then using this combined score to aid clinical decision making during COVID 19 pandemic. The tool suggested three groups with suggested treatment decisions;scores 2 to 8 have surgery within 36 hours, scores 9 to 12 have surgery potentially delayed up to 7 days, scores 12 plus to be non-operative management. Swansea hospital treats approximately 500 to 600 hip fractures annually. Musgrove Park Hospital treats approximately 450 hip fractures annually therefore the aim of your study was to assess the reliability of this tool in our local population. Methods: A retrospective review of patients admitted with a hip fracture between January 2018 and December 2019. The date of discharge, the date of death if applicable, the clinical frailty score and Nottingham hip score, and the SHiFT score were recorded. The original study assessed mortality at 4 months therefore similar local data was collected. The local results were compared to the original SHiFT outcomes. Results: The original SHiFT study had 124 patients with an annual mortality rate of 26%. Our study had 103 patients with mortality rate of 26%. Our SHiFT scores ranged from 2 to 16, 25.2% had a score between 2 and 8, 50.5% had a score between 9 and 12, and 24.3% had a score of 13 to 16. The original SHiFT study scores range from 6 to 15 with respective percentage in each group of 34%, 56%, 10%. In our study 3.8% of patients with score between 2 and 8 died within 4 months, 9.6% of patients with scores 9 to 12, and 36% with scores 13 to 16. The percentage of deaths at 4 months within the same groups in the original study was 2%, 34%, and 58%. Conclusion: Whilst we recognise that clinical decision-making regarding resource allocation is difficult especially during a pandemic, we would not recommend using a tool such as SHiFT as this was not a reliable tool. Whilst our local population had a higher percentage of frailer patients with comorbidity this did not translate into higher mortality. Therefore, using this tool would have resulted in numerous patients being triage for inappropriate treatment decisions. We recognise that the main limitation in this study as well as the original study is the small patient numbers.

Integrative analyses identify genes and their functional consequences underlying COVID-19 hospitalization

Recent emergence of SARS-Cov-2 has resulted in unprecedented spread of COVID-19 exhibiting wide variability in individuals’ symptoms. Despite rapid progress in characterizing the role of host genetics in SARS-Cov-2 infection, there is limited understanding of the role of host genetic variation and the molecular mechanisms including the knowledge of genes and pathways that contribute to COVID-19. Previous research to understand the mechanisms underlying severe COVID-19 outcomes have focused on lung- and brain-related pathologies. Here, we integrated a genome-wide association study of COVID-19 hospitalization (7,885 cases and 961,804 controls from COVID-19 Host Genetics Initiative) with mRNA expression, splicing, and protein levels (n = 18,502). We identified 27 genes related to inflammation and coagulation pathways whose genetically predicted expression was associated with COVID-19 hospitalization. These genes converge on cytokine-cytokine and the JAK-STAT signaling pathways. We functionally characterized the 27 genes using phenome- and laboratory-wide association scans in Vanderbilt Biobank (BioVU;n = 85,460) and identified coagulation-related clinical symptoms, immunologic, and blood-cell-related biomarkers. We replicated these findings in the African-American cohort here in BioVU and found concordant results. This study highlights putative causal genes impacting COVID-19 severity and symptomology through the host inflammatory response.

The relationship between symptoms and functional physiological outcomes in survivors of severe COVID-19 pneumonia

P136 Table 1Results of correlation analysis Correlation analysis 4MGS 1STSreps SpO2% desaturation Results r p-value r p-value r p-value Pre-COVID mMRC dyspnoea score 0(0–1) -0.267** <0.001 -0.285** <0.001 -0.108 0.094 Post-COVID mMRC dyspnoea score 1(0–2) -0.442** <0.001 -0.457** <0.001 -0.143* 0.025 NRS breathlessness 3(0–5) -0.287** <0.001 -0.406** <0.001 -0.490 0.445 NRS fatigue 3(0–5) -0.315** <0.001 -0.379** <0.001 -0.190* 0.003 NRS cough 0(0–2) -0.660 0.292 -0.153* 0.017 0.083 0.194 NRS pain 1(0–4) -0.278** <0.001 -0.346** <0.001 -0.188* 0.003 NRS sleep difficulty 2(0–5) -0.246** <0.001 -0.386** <0.001 -0.122 0.057 Data are presented as median (interquartile range) or frequency (proportion%;95% confidence interval). SpO2% desaturation = SpO2% desaturation from baseline during 1 minute sit to stand test;1STSreps = repetitions per minute during 1 minute sit to stand test;4MGS = 4 metre gait speed;mMRC = modified Medical Research Council;NRS = 0 – 10 numerical rating scale;r = Spearman correlation coefficient. *indicates statistical significance at 0.05 level. **indicates statistical significance at 0.001 level.ConclusionRespiratory symptoms were not strong predictors of 4-metre gait speed and 1-minute sit-to-stand test performance. These data highlight the importance of face-to-face testing to objectively assess functional limitation in patients recovering from severe COVID pneumonia.

Toward a critical pedagogy of crisis

Crises in our society - climate, covid-19 and mass migration - seem to define not only the experience of learning but also the experience of living and even surviving that in turn have implications for adult learning. We explore the concept of experience and examine whether it plays a role in addressing the need for transformative learning. Our allies in this task are Oskar Negt from the Frankfurt School tradition, L. A. Paul from a philosophical tradition and Rene Arcilla. Negt is useful for rethinking the role of experience in pedagogy. Paul helps identify the not-knowing aspect of our current experience and our inability to imagine how decisions translate into one's way of living and being in the world. Arcilla emphasises the importance of keeping conversations going. Jack Mezirow's transformation theory (relying on Habermas) informs the understanding of adult learning and how we can transform our way of being and living while facing experiences of crises and disorientation.

Lung transplant recipients with severe acute respiratory syndrome-coronavirus 2 infection and asymptomatic carriers waiting for lung transplantation induce circulating exosomes with severe acute respiratory syndrome-coronavirus 2 spike protein s2

Purpose: Exosomes are vesicles released by cells into body fluids. We demonstrated increased circulating exosomes with lung self-antigens (Collagen V, Kα1 Tubulin) and viral antigens in lung transplant recipients (LTxRs) undergoing rejection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), an important risk factor for LTxRs in immunosuppression and patients with diseased lungs waiting for transplantation. Our goal is to determine that exosomes from LTxRs with SARSCoV2 infection carry SARS-CoV2 spike protein. Methods: We analyzed 30 patients waiting for LTx with no clinical symptoms and 7 LTxRs with SARS-CoV2 infection and symptoms. Exosomes were isolated from plasma by precipitation kit, 0.2 micron filtration and size determination by Nanosight300. Eluted protein from gel was analyzed by mass spectrometry and peptides were aligned with SARS-CoV2. Transmission electron microscopy of exosomes was performed for spike and nucleocapsid antigen. Exosomes were also characterized by western blot for immune and molecular markers. Serum cytokines were analyzed using 25 Plex on Luminex. Results: Exosomes from symptomatic (7/7) and 7/30 (23.3%) asymptomatic LTxRs contained SARS-CoV2 spike protein S2 and increased levels of SARS-CoV2 RNA. SARS-CoV2 spike protein in exosomes was confirmed by mass spectroscopy. Transmission electron microscopy from symptomatic and asymptomatic LTxRs revealed spike protein and nucleocapsid antigen on exosomes. Exosomes contained macrophage stimulating factor 1, GRAnzyme B and angiotensin type II receptor 1 proteins. Increased levels of CXCL10 in sera were detected in SARS-CoV2 positive symptomatic patients, agreeing with the reports that CXCL10 levels correlate with disease severity. Conclusions: SARS-CoV2 infected LTxRs symptomatic and asymptomatic induced circulating exosomes having spike protein, nucleic acid and antigens related to viral entry (angiotensin type II receptor), infection (macrophage stimulating factor 1) and cytotoxic molecule (GRAnzyme B) suggesting that the exosomes induced by SARS-CoV2 will have functional consequences. Exosomes also contained increased levels of viral RNA and antigens suggesting that circulating exosomes may provide a noninvasive tool for detection of SARS-CoV2 infection.

Implementing fully automated kinase inhibitor characterization using a robotic system

About 50 kinase inhibitors have been approved by the FDA for different indications so far. On the way from target validation to approval, the biochemical characterisation of a novel kinase inhibitor is a challenging, yet absolutely critical task. High resolution, kinetic molecular profiling can enable better data-driven decision making early on in the drug discovery process, not only saving time and resources, but also leading to superior molecular design. Arctoris developed a robotics-enabled process for fully automated kinase inhibitor characterisation, providing an unparalleled depth of data capture, going beyond the current state-of-the-art of biochemical assay setup. We validated our technology platform establishing assays against four members of the Janus Kinase family (JAK1, JAK2, JAK3, TYK2), profiling a set of JAK inhibitors. Of note, several JAK inhibitors with prior FDA approval for other indications entered clinical trials for COVID-19 treatment, making this target class particularly relevant for an in-depth study. Reagent validation, assay development, calibration, and optimization were expedited through systematic multifactorial experimental design, high density assay plate formats and versatile automated liquid handling. The Arctoris Ulysses platform affords 9 orders of magnitude range in liquid volume handling, with picolitre precision and contact-free digital dispensing for true, non-serial, independent experimentation. Fully automated protocols were optimized, validated, versioned, and explicitly encoded. Robust potency measurements of all inhibitors were established against each of the JAK targets, revealing molecules with distinct isoform selectivity. Designing and selecting molecules with specific activity profiles enables the fine tuning of pharmacology and the avoidance of unwanted off-target toxicity. Our unique platform, assay design, and deep expertise enabled the identification of molecules within the JAK inhibitor set that exhibit a range of kinetic properties. Our mechanistic analyses can help to elucidate the mode of inhibition (competitive, allosteric, synergistic etc.) as well as provide information pertaining to the kinetic selectivity that may be present but missed by focusing solely on potency.