Neutrophil extracellular traps induce the epithelial-mesenchymal transition: implications in post-COVID-19 fibrosis (preprint)

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Neutrophils activated with PMA (PMA-Neu), a stimulus known to induce NETs formation, induce both EMT and cell death in the lung epithelial cell line, A549. Notably, NETs isolated from PMA-Neu induce EMT without cell damage. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs. Thus, we tested in an in vitro alveolar model the hypothesis that virus-induced NET may drive EMT. Co-culturing A549 at air-liquid interface with alveolar macrophages, neutrophils and SARS-CoV2, we demonstrated a significant induction of the EMT in A549 together with high concentration of NETs, IL8 and IL1{beta}, best-known inducers of NETosis. Lung tissues of COVID-19 deceased patients showed that epithelial cells are characterized by increased mesenchymal markers. These results show for the first time that NETosis plays a major role in triggering lung fibrosis in COVID-19 patients.

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate (preprint)

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect approximately 50 million people, with over 1 million reported deaths, and a safe and effective vaccine remains urgently needed. Based on previous experience developing vaccines against SARS and MERS, we constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Collectively, these three recombinant protein RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity for their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, and had a lower tendency to form oligomers when compared to the other two proteins and was therefore selected for further vaccine development and evaluation.

Pulmonary Post-Mortem Findings in a Large Series of COVID-19 Cases from Northern Italy (preprint)

Importance: The analysis of lung tissues of patients with COVID-19 may help understand pathogenesis and clinical outcomes in this life-threatening respiratory illness.Objective: To determine the histological patterns in lung tissue of patients with severe COVID-19.Design and Participants: Lungs tissues of 38 cases who died for COVID-19 in two hospital of Northern Italy were systematically analysed. Hematoxylin-eosin staining, immunohistochemistry for the inflammatory infiltrate and cellular components, electron microscopy were performed.Results: The features of the exudative and proliferative phases of Diffuse Alveolar Disease (DAD) were found: capillary congestion, necrosis of pneumocytes, hyaline membrane, interstitial oedema, pneumocyte hyperplasia and reactive atypia, platelet-fibrin thrombi. The inflammatory infiltrate was composed by macrophages in alveolar lumens and lymphocytes mainly in the interstitium. Electron microscopy revealed viral particles within cytoplasmic vacuoles of pneumocytes.Conclusions and Relevance: The predominant pattern of lung lesions in COVID-19 patients is DAD, as described for the other two coronavirus that infect humans, SARS-CoV and MERS-CoV. Hyaline membrane formation and pneumocyte atypical hyperplasia are frequently found. The main relevant finding is the presence of platelet-fibrin thrombi in small arterial vessels; this important observation fits into the clinical context of coagulopathy which dominates in these patients and which is one of the main targets of therapy.Funding Statement: No FundingDeclaration of Interests: No Conflict of InterestEthics Approval Statement: Tissue samples were taken as part of routine autopsies

COVID-19: the key role of pulmonary capillary leakage. An observational cohort study (preprint)

Background: COVID-19 induces progressive hypoxemic respiratory failure and acute respiratory distress syndrome, mostly due to a dysregulated inflammatory response. Since the first observations of COVID-19 patients, significant hypoalbuminemia was detected. This study aimed to investigate the hypothesis that hypoalbuminemia in COVID-19 patients is due to pulmonary capillary leakage and to test its correlation with indicators of respiratory function. Methods: 174 COVID-19 patients, 92 admitted to the Intermediate Medicine ward (IMW), and 82 to the Intensive Care Unit (ICU) at Luigi Sacco Hospital in Milan were included in this study. Findings: Serum albumin concentration was decreased in the whole cohort, with ICU patients displaying lower values than IMW patients [20 (18-23) vs 28 (24-33) g/L, p<0.0001]. Lower albumin values were found in patients belonging to a more compromised group (lower PaO2 to FiO2 ratio and worst chest X-ray findings). In a subset of 26 patients, analysis of bronchoalveolar lavage fluid (BALF) highlighted high protein concentrations, which were correlated to Interleukin-8 and Interleukin-10 BALF concentration. The length of hospitalisation [20 (15-29) vs 8 (5-14) days, p<0.0001] and death rate (52.4% vs 21.7%, p<0.0001) were higher in ICU than in IMW patients, while a strict relation between hypoalbuminemia and 30 day-survival was detected in the whole cohort. Electron microscopy examinations of eight out of ten autopsy lung tissues showed diffuse loosening of interendothelial junctional complex. Interpretation: The degree of hypoalbuminemia can be considered as a useful severity marker in hospitalised COVID-19 patients. Pulmonary capillary leak syndrome secondary to the hyperinflammatory state plays a key role in the pathogenesis of COVID-19 respiratory dysfunction and should be regarded as a therapeutic target.

Liver and COVID 19 Infection: A Very Preliminary Lesson Learnt from Histological Post-mortem Findings in 48 patients (preprint)

COVID-19 breakout in Italy has caused a huge number of severely ill patients with a serious increase in mortality. Although lungs seem to be the main target of the infection very few information are available about liver involvement in COVID-19 infection, that could possibly evocate a systemic disease targeting a lot of organs. Since now there are no reports of large series of histological evaluation of liver morphology in this setting. Knowledge of histological liver findings connected to clinical data is crucial in management of this disease.Post-mortem wedge liver biopsies from 48 patients died for COVID-19 infection were available from two main hospitals located in northern Italy, Lombardy; all sample were obtained during autopsies. No patient has a significant clinical complain of liver disease or signs of liver failure before and during hospitalization; for each of them laboratory data focused on liver were available. All liver samples showed minimal inflammation features; on the other side, many histological pictures compatible with vascular alterations were observed, characterized by portal vein braches number increase associated with lumen massive dilatation, partial or complete recent luminal thrombosis of portal and sinusoidal vessels, fibrosis of portal tract, focally severely enlarged and fibrotic. Our preliminary results concerning histological liver involvement in COVID-19 infection confirm the clinical impression that liver failure is not a main concern and this organ is not the target of significant inflammatory damage; histopatological findings are highly suggestive for marked alteration of intrahepatic blood vessel network secondary to systemic alterations induced by virus that could target, besides lung parenchyma, cardiovascular system, coagulation cascade or endothelial layer of blood vessels.

Pulmonary post-mortem findings in a large series of COVID-19 cases from Northern Italy (preprint)

Importance. The analysis of lung tissues of patients with COVID-19 may help understand pathogenesis and clinical outcomes in this life-threatening respiratory illness. Objective. To determine the histological patterns in lung tissue of patients with severe COVID-19. Design and participants. Lungs tissues of 38 cases who died for COVID-19 in two hospital of Northern Italy were systematically analysed. Hematoxylin-eosin staining, immunohistochemistry for the inflammatory infiltrate and cellular components, electron microscopy were performed. Results. The features of the exudative and proliferative phases of Diffuse Alveolar Disease (DAD) were found: capillary congestion, necrosis of pneumocytes, hyaline membrane, interstitial oedema, pneumocyte hyperplasia and reactive atypia, platelet-fibrin thrombi. The inflammatory infiltrate was composed by macrophages in alveolar lumens and lymphocytes mainly in the interstitium. Electron microscopy revealed viral particles within cytoplasmic vacuoles of pneumocytes. Conclusions and relevance. The predominant pattern of lung lesions in COVID-19 patients is DAD, as described for the other two coronavirus that infect humans, SARS-CoV and MERS-CoV. Hyaline membrane formation and pneumocyte atypical hyperplasia are frequently found. The main relevant finding is the presence of platelet-fibrin thrombi in small arterial vessels; this important observation fits into the clinical context of coagulopathy which dominates in these patients and which is one of the main targets of therapy.