PMC:7172841 / 26462-34599 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7172841","sourcedb":"PMC","sourceid":"7172841","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7172841","text":"STAR★Methods\n\nKey Resources Table\nREAGENT or RESOURCE SOURCE IDENTIFIER\nAntibodies\nCD14-FITC (RMO52 clone) Beckman Coulter Cat#IM0645U; RRID: AB_130992\nCD45-PC5 (J33 clone) Beckman Coulter Cat#IM2653U; RRID: AB_10641226\nHLA-DR-RD1 (9-49 clone) Beckman Coulter Cat#6604366; RRID: AB_2832962\nIgG1-PE isotype control (679.1Mc7 clone) Beckman Coulter Cat#A07796; RRID: AB_2832963\nIgG1-FITC isotype control (679.1Mc7 clone) Beckman Coulter Cat#A07795; RRID: AB_2832964\nCD3-FITC/CD4-PE Antibody Cocktail (UCHT1/13b8.2 clones) Beckman Coulter Cat#A07733; RRID: AB_2832965\nCD3-FITC/CD8-PE Antibody Cocktail Beckman Coulter Cat#A07734; RRID: AB_2832966\nCD3-FITC/CD(16+56)-PE Antibody Cocktail (UCHT1/3G8/N901 (NKH-1)) Beckman Coulter Cat#A07735; RRID: AB_1575965\nCD19-FITC/CD5-PE Antibody Cocktail Beckman Coulter Cat#IM1346U; RRID: AB_2832967\nQuantibrite Anti-HLA-DR/Anti-Monocyte (L243/MϕP9) BD Biosciences Cat#340827; RRID: AB_400137\nMouse Anti-Human IL-6-PE (AS12 clone) BD Biosciences Cat#340527; RRID: AB_400442\nChemicals, Peptides, and Recombinant Proteins\nVersaLyse Lysing Solution Beckman Coulter A09777\nFixative Solution Beckman Coulter A07800\neBioscience™ Brefeldin A Solution (1000x) Invitrogen 00-4506-51\nIntraPrep Permeabilization Reagent Beckman Coulter A07802\nRPMI 1640 W/ Stable Glutamine W/ 25 MM HEPES Biowest L0496\nLymphosep, Lymphocyte Separation Media Biowest L0560\nPBS Dulbecco’s Phosphate Buffered Saline w/o Magnesium, w/o Calcium Biowest L0615\nFBS Superior; standardized Fetal Bovine Serum, EU-approved Biochrom S0615\nGentamycin Sulfate BioChemica PanReac AppliChem A1492\nPenicillin G Potassium Salt BioChemica PanReac AppliChem A1837\nLipopolysaccharides from Escherichia coli O55:B5 Sigma-Aldrich L2880\nCritical Commercial Assays\nHuman IL-1β uncoated ELISA Invitrogen 88-7261\nHuman TNF-α uncoated ELISA Invitrogen 88-7346\nHuman IL-6 ELISA Invitrogen 88-7066\nHuman IL-17A (homodimer) uncoated ELISA Invitrogen 88-7176\nHuman Ferritin ELISA ORGENTEC Diagnostika GmbH ORG 5FE\nHuman IFN-γ ELISA Diaclone 950.000.192\nSoftware and Algorithms\nGraphPad Prism Graphpad Software https://www.graphpad.com\nSPSS IBM https://www.ibm.com/analytics/spss-statistics-software\n\nResource Availability\n\nLead Contact\nFurther information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Evangelos J. Giamarellos-Bourboulis (egiamarel@med.uoa.gr).\n\nMaterials Availability\nThis study did not generate new unique reagents.\n\nData and Code Availability\nData of this study are available after communication with the Lead Contact.\n\nExperimental Model and Subject Details\nBlood was sampled from patients with CAP and sepsis and CAP by SARS-CoV-2 of either gender within the first 24 h of hospital admission (Table 1). CAP was defined as the presence of new infiltrate in chest X-ray in a patient without any contact with any healthcare facility the last 90 days; sepsis was defined by the Sepsis-3 criteria (Singer et al., 2016). COVID was diagnosed for patients with CAP confirmed by chest X-ray or chest computed tomography and positive molecular testing of respiratory secretions. For patients who required MV, blood sampling was performed within the first 24 h from MV and results were used for this analysis. Exclusion criteria were infection by the human immunodeficiency virus; neutropenia; and any previous intake of immunosuppressive medication (corticosteroids, anti-cytokine biologicals, and biological response modifiers). SRF was defined as severe decrease of the respiratory ratio necessitating intubation and mechanical ventilation. The studies were conducted under the 78/17 approval of the National Ethics Committee of Greece; the 23/12.08.2019 approval of the Ethics Committee of Sotiria Athens General Hospital; and the 26.02.2019 approval of the Ethics Committee of ATTIKON University General Hospital. Written informed consent was provided by patients or by first-degree relatives in case of patients unable to consent.\nAll data of patients with bacterial CAP screened for participation in the randomized clinical trial with the acronym PROVIDE (ClinicalTrials.gov NCT03332225) were used. All patients admitted for CAP by SARS-CoV-2 from March 3 until March 30, 2020 participated in the study. No randomization to experimental groups was needed according to the study design.\n\nMethod Details\nWhite blood cells were incubated for 15 min in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France) and Quantibrite HLA-DR/anti-monocyte PerCP-Cy5 (Becton Dickinson, Cockeysville Md). White blood cells were also incubated for 15 min with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525 nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667 nm, Beckman Coulter). Fluorospheres (Beckman Coulter) were used for the determination of absolute counts. Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Isotypic IgG controls stained also with anti-CD45 were used for each patient. Results were expressed as absolute molecules of HLA-DR on CD14/CD14+CD45+ cells, percentages, and MFI.\nIn separate experiments, whole blood was treated with 10 μg/mL brefeldin A (Invitrogen, Carlsbad, CA, USA) for 60 min at 37°C in 5% CO2 followed by incubation in the dark for 15 min at room temperature with anti-CD4 FITC, anti-CD14 FITC (Beckman Coulter) and anti-CD45 PC5 followed by red blood cells lysis. Stained cells were then washed with Dulbecco’s Phosphate Buffered Saline and permeabilized with the IntraPrep Parmeabilization Reagent kit (Beckman Coulter). Anti-IL-6 PE (Beckman Coulter) was added and analyzed after flow cytometry.\nPeripheral blood mononuclear cells (PBMCs) were isolated after gradient centrifugation over Ficoll (Biochrom, Berlin, Germany) for 20 min at 1,400 g. After three washings in ice-cold PBS pH 7.2, PBMCs were counted in a Neubauer plate with trypan blue exclusion of dead cells. They were then diluted in RPMI 1640 enriched with 2mM of L-glutamine, 500 μg/mL of gentamicin, 100 U/mL of penicillin G, 10 mM of pyruvate, 10% fetal bovine serum (Biochrom) and suspended in wells of a 96-well plate. The final volume per well was 200 μL with a density of 2 × 106 cells/mL. PBMCs were exposed in duplicate for 24 h or 5 days at 37°C in 5% CO2 to different stimuli: 10 ng/mL of Escherichia coli O55:B5 lipopolysaccharide (LPS, Sigma, St. Louis, USA); 5 × 105 colony forming units of heat-killed Candida albicans; 25% of patient plasma; or 10 μg/mL of Tocilizumab (Roche, Athens, Greece). After incubation, cells were removed and analyzed for flow cytometry. Concentrations of TNFα, IL-1β, IL-6, IL-17A and IFNγ were measured in cell supernatants and/or serum in duplicate by an enzyme immunoassay (Invitrogen, Carlsbad, California, USA). The lowest detections limits were as follows: for TNF-α, 40 pg/mL; for IL-1β, 10 pg/mL; for IL-6, 10 pg/mL; for IL-17A, 10 pg/mL; and for IFN-γ, 12.5 pg/mL. Concentrations of ferritin were measured in serum by an enzyme-immunoassay (ORGENTEC Diagnostika GmbH, Mainz, Germany); the lower limit of detection was 75 ng/mL.\nSix patients with immune dysregulation were treated with single intravenous infusion of 8 mg/kg of Tocilizumab (RoAcremra, Roche) based on the National Health Care off-label provision for COVID-19. Absolute lymphocyte blood counts were compared before and after Tocilizumab infusion.\n\nQuantification and Statistical Analysis\nQualitative data were presented as percentages and confidence intervals (CIs) and compared by the Fisher exact test. Qualitative data were presented as mean and standard deviation and compared by the Student’ s “t-test”; they were presented as mean and standard error and compared by the Mann-Whitney U test for small groups. Paired comparisons were done by the Wilcoxon’s rank-signed test. Non-parametric correlations were done according to Spearman. 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