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Neutrophilic granulocytes (NG) are the main drivers of pathological inflammation in COVID-19. Objective. To specify the mechanisms of immunopathogenesis of COVID-19 based on a comparative immunological study of the number and phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets with an assessment of their effector functions against changing profile of NG-associated cytokines IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma. Patients and methods. In patients with moderate-to-severe and severe COVID-19, we determined IL-1beta, TNFalpha, IL-6, IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma (ELISA), the phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets, NF-kappaB-NG (CYTOMICS FC500), phagocytically active NG (%), neutrophil extracellular traps (NETs), NG in apoptosis, and the activity of NADPH oxidase. Results. In COVID-19 against the background of IFNalpha and IFNgamma production blockade and high levels of NG-associated IL-8, IL-18, IL-17A, VEGF-A, a reduction in the number of mature and functionally active CD16brightSD62LbrightCD11bbrightCD63-NG subsets was revealed, as well as an increase in the number of CD16dimSD62LdimSD11bbrightCD63-NG subsets with an immunosuppressive phenotype and CD16brightSD62LbrightSD11bbrightCD63bright-NG subsets with high cytotoxic activity and ability to form NETs, a decrease in the percentage of phagocytically active NG and an increase in the activity of NADPH oxidase, NETs, and NG in apoptosis. Conclusion. IFNalpha deficiency provokes a hyperergic response of NG-associated cytokines, which leads to the formation of uncontrolled immune inflammation involving NG subsets with an immunosuppressive and cytotoxic phenotype, exacerbating the course of COVID-19. The use of recombinant IFNalpha-2b with antioxidants (Viferon) in the early stages of the disease can help to restore immune homeostasis, normalize the level of NG-associated cytokines, reduce NERTs, and achieve good clinical efficacy.Copyright © 2022, Dynasty Publishing House. All rights reserved.
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IDDF2023-ABS-0045 Figure 1 IDDF2023-ABS-0045 Figure 2 IDDF2023-ABS-0045 Figure 3 IDDF2023-ABS-0045 Figure 4
ABSTRACT
Neutrophilic granulocytes (NG) are the main drivers of pathological inflammation in COVID-19. Objective. To specify the mechanisms of immunopathogenesis of COVID-19 based on a comparative immunological study of the number and phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets with an assessment of their effector functions against changing profile of NG-associated cytokines IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma. Patients and methods. In patients with moderate-to-severe and severe COVID-19, we determined IL-1beta, TNFalpha, IL-6, IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma (ELISA), the phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets, NF-kappaB-NG (CYTOMICS FC500), phagocytically active NG (%), neutrophil extracellular traps (NETs), NG in apoptosis, and the activity of NADPH oxidase. Results. In COVID-19 against the background of IFNalpha and IFNgamma production blockade and high levels of NG-associated IL-8, IL-18, IL-17A, VEGF-A, a reduction in the number of mature and functionally active CD16brightSD62LbrightCD11bbrightCD63-NG subsets was revealed, as well as an increase in the number of CD16dimSD62LdimSD11bbrightCD63-NG subsets with an immunosuppressive phenotype and CD16brightSD62LbrightSD11bbrightCD63bright-NG subsets with high cytotoxic activity and ability to form NETs, a decrease in the percentage of phagocytically active NG and an increase in the activity of NADPH oxidase, NETs, and NG in apoptosis. Conclusion. IFNalpha deficiency provokes a hyperergic response of NG-associated cytokines, which leads to the formation of uncontrolled immune inflammation involving NG subsets with an immunosuppressive and cytotoxic phenotype, exacerbating the course of COVID-19. The use of recombinant IFNalpha-2b with antioxidants (Viferon) in the early stages of the disease can help to restore immune homeostasis, normalize the level of NG-associated cytokines, reduce NERTs, and achieve good clinical efficacy.Copyright © 2022, Dynasty Publishing House. All rights reserved.
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Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo a rapid transcriptional program, resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation, and structural tissue damage occur. Because CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and ribonucleic acid (RNA)-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector because they are highly customizable, and airway PMNs have previously been shown by our group to process and use their cargo efficiently [1]. Furthermore, our prior work on CF airway PMNs [2] led to identification of the long noncoding RNA MALAT1, the transcription factor Ehf, and the histone deacetylase/long-chain fatty deacylase HDAC11 as potential targets to modulate CF airway PMN dysfunction. Method(s): H441 human club epithelial cells were chosen for EV production because they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive cytomegalovirus or U6 promoter with an additional puromycin selection cassette. EVs were generated in serumdepleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy and nanoparticle tracking analysis and cargo content by western blot (protein) or qualitative reverse transcription polymerase chain reaction (RNA). Enhanced green fluorescent protein and messenger ribonucleic acid (mRNA) were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry, and bacterial (PA01 and Staphylococcus aureus 8325-4) killing and viral (influenza Avirus [IAV] H1N1/PR/8/34;SARS-CoV-2/Washington) clearance assays were conducted. Result(s): To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C'D box motif recognized by the RNA-binding protein L7Ae was included in the 3' untranslated region of the expressed RNA, and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing small interfering RNAs against MALAT1 or HDAC11 showed greater ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARSCoV- 2. PMNs expressing Ehf showed greater clearance of bacteria and viruses. Conclusion(s): Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial and viral clearance by airway-recruited PMNs. Expression of Ehf in airway PMNs may be a pathogen-agnostic approach to enhancing clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases and for use of EVs as a promising method for cargo delivery to these cells. It is our expectation that, by treating the immune compartment of CF airway disease, pathogentherapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance into the cell types of interest.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved
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Background: Use of ultrasound contrast agent spread increasingly in worldwide. Although few side effects are published, number of increase anaphylactic reaction is reported. A recent cautionary remark was published about polyethylene glycol (PEG) as culprit for some ultrasound contrast agent hypersensitivity (Krantz et al. JACIP. Avril 2020). A 22 years-old woman was referred for a severe anaphylaxis after a second injection of Sulphur Hexafluoride (SonoVue, Bracco, Milan, Italy). The investigation was performed to explore hepatic multinods. She already had presented with uncomfortable feeling, generalized pruritus and nausea few minutes after the first injection of Sonovue, 10 months earlier. Three months before, she has recieved also a first injection of SPIKEVAX without any reaction. As Sonovue and SPIKEVAX contain PEG, we looked for a hypersensitivity to PEG and RNAm COVID-19 vaccine. Method(s): Skin tests and basophil activation test (BAT) with expression of CD63 were performed for SonoVue, PEG 3350 and 4000 and COMIRNATY. Result(s): Skin tests were negative for all products except the undiluted IDT to COMIRNATY. BAT to PEG and SonoVue were negative but positive to COMIRNATY. Despite a fractioned administration of COMIRNATY, the patient presented with an acute urticaria few minutes after the last injection Conclusion(s): The relationship between PEG, RNAm vaccine, and hypersensitivity to SonoVue is once again highlighted by this new report. (Soni et al. J Am Soc Echocardiogr, Oct 2021).
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Background: There is an urgent need to identify novel biomarkers for early detection of severe cases of COVID-19. Extracellular Vesicles (EVs) contain cargoes derived from the cell of origin that include proteins, lipids, and nucleic acids and have been used as biomarkers in other diseases. Aim: To identify EVs-derived biomarkers in broncho-alveolar lavages (BAL) and plasma for the early detection of severe COVID-19. Methods: We included patients hospitalized without and with severe COVID-19 and non-hospitalized mild cases. EVs were isolated from BAL and plasma, and characterized by western blot, electron microscopy and mass spectrometry-based proteomics. EVs derived proteins in both BAL and plasma from severe cases but not from mild or controls will be selected to generate a mathematical model to distinguish severe from mild and non-COVID-19. An independent validation cohort will be used to evaluate sensitivity and specificity. Results: We isolated EVs from BAL and plasma of non-COVID-19 patients and optimized the protocol for mass spectrometry. The EVs isolated were expressing the common EVs markers such as CD63, stomatin and TSG101 and electron microscopy confirmed the enrichment of EVs after isolation. Proteomic analysis showed a great overlap in the EVs proteins detected between different patients. We found 80% of the proteins were EVs derived confirming the EVs isolation protocol. Conclusions: Isolated EVs from plasma and BAL showed characteristic markers, microscopy patterns and proteomic analysis compatible with EV origin. We are currently isolating EVs from COVID- 19 patients and would identified proteins that allow us to predict COVID-19 severity.
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Background: The management and mechanisms involved in allergies to mRNA anti-SARS Cov2 vaccines remain debated. The availability of mRNA vaccines limits their use for Basophil Activation Test (BAT). Cryopreservation of mRNA vaccines from leftover doses or vials may help standardize this assay better. Methods: Individuals with respectively negative (n = 10) and positive (n = 10) skin testing for mRNA vaccines were included in this study. CD63 upregulation in basophils was compared using fresh leftover or cryopreserved mRNA vaccines from Pfizer BioNTech (BNT162b2) and Moderna (mRNA-1273). Spike protein expression was used as a surrogate marker for the evaluation of mRNA vaccine's function in vitro. Results: CD63 upregulation in basophils was significantly higher in patients with positive intradermal skin testing (IDR) with both mRNA vaccines. A significant correlation was found comparing (1) fresh BNT162b2 and mRNA-1273 vaccines, (2) cryopreserved and fresh BNT162b2 vaccines, (3) cryopreserved and fresh mRNA-1273 vaccines, and (4) cryopreserved BNT162b2 and mRNA-1273 vaccines. Importantly, CD63 upregulation in basophils was independent of the capacity of the mRNA vaccines to induce spike expression. Conclusions: BAT performed with mRNA vaccines can be used as IDR surrogates to identify COVID-19 mRNA vaccine-sensitized individuals. These mRNA vaccines can be safely cryopreserved for BAT assays independently of their in vitro function. The current findings will allow broader use of mRNA vaccines for diagnostic tests and research purposes.
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The basophil activation test (BAT) is a flow cytometric assay that evaluates the percentage of activation or degranulation of peripheral blood basophils, after “in vitro” exposure to specific allergens. In sensitized patients, the stimulation of peripheral blood basophils with a specific allergen induces or up-regulates the expression of molecules, such as CD63 and CD203c, which represent, markers of degranulation and activation of basophils, respectively. The validity of the BAT requires a negative control (sterile saline) and a positive control (anti-IgE molecules). Several studies have demonstrated the role of the BAT in supporting the diagnosis of drug, food and hymenoptera venom allergy. The BAT has shown a low sensitivity but good specificity in diagnosing allergy to drugs such as NSAIDs, beta-lactam antibiotics, quinolones and muscle relaxants. In food allergy, the sensitivity and specificity of the BAT depends on the food;in the case of peanut allergy the sensitivity reaches 96% while the specificity the 100%. In addition, the BAT is an useful tool to monitor the natural resolution of allergies and the clinical effects induced by either immunotherapy or anti-IgE treatment. Finally, the BAT has been utilized to study the pathogenetic mechanisms underlying several IgE-mediated diseases. For example, in patients affected by severe bronchial asthma, the BAT has demonstrated the ability of Staphylococcus aureus enterotoxins to induce the activation of basophils supporting the role of these enterotoxins as “triggers” of the inflammatory cascade in bronchial asthma. In patients with cystic fibrosis the BAT can be used to dis-criminate allergic bronchopulmonary aspergillosis from Aspergillus colonization. More recently, the BAT has been demonstrated as a potential diagnostic tool to evaluate allergy to the polyethylene glycol (PEG) present in the anti-SARS-CoV-2 BNT162b2 mRNA vaccine.