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IntroductionThe mechanisms of the SARS-CoV-2-triggered complex alterations in immune cell activation and production of cytokines in lung tissue remain poorly understood, in part because of the limited use of adequate tissue models that simulate the structure and cell composition of the lung in vivo. We developed a novel ex vivo model of SARS-CoV-2 infection of lung explants, that maintains the intact tissue composition and the viral load for up to 7–10 days. Using this model, we studied cytokine production during SARS-CoV-2 infection.Materials and methodsLung tissue was monitored for viability and cell composition using flow cytometry and histological analysis. SARS-CoV-2 infection was verified immunohistochemically, viral loads in tissue and culture medium were monitored by qPCR. A panel of 41 cytokines was measured in culture medium using xMAP technology.ResultsThe explant lung tissue was viable and maintained viral infection that influenced the cytokine production. Elevated concentrations of G-CSF, GM-CSF, GRO-a, IFN-g, IL-6, IL-8, IP-10, MCP-3, MIP-1a, PDGF-AA, and VEGF, and decreased IL-1RA concentration were observed in infected tissue compared to non-infected tissue.DiscussionOur results generally reflect the data obtained in COVID-19 patients. GRO-a, IFN-g, IL-6, IL-8, MCP-1, MCP-3, and RANTES correlated with the viral load, forming a distinct pro-inflammatory cluster. Thus, our lung ex vivo model faithfully reproduces some aspects of cytokine alterations in COVID-19 patients at an early disease stage, making the investigation of SARS-CoV-2 infection mechanisms more accessible and providing a potential platform for antiviral drug testing.

Abstract Aims Insulin‐like growth factor binding protein‐4 (IGFBP‐4) fragments have been shown to predict the risk of major adverse cardiovascular events, including segment‐elevation myocardial infarction, in patients with acute coronary syndrome. We evaluated the prognostic value of the carboxy‐terminal fragment of IGFBP‐4 (CT‐IGFBP‐4) for all‐cause mortality in emergency room patients with acute heart failure (AHF). Methods and results CT‐IGFBP‐4, N‐terminal pro brain natriuretic peptide (NT‐proBNP), and C‐reactive protein (CRP) were measured at admission from the lithium‐heparin plasma of 156 patients with AHF. All‐cause mortality was recorded for 1 year. Receiver operator characteristic (ROC) curves, Kaplan–Meier, and Cox proportional hazard ratio analyses were performed to evaluate the prognostic value of the various clinical variables, CT‐IGFBP‐4, NT‐proBNP, CRP, and their combinations. During 1 year of follow‐up, 52 (33.3%) patients died. CT‐IGFBP‐4 only weakly correlated with NT‐proBNP (Pearson correlation coefficient r = 0.16, P = 0.044) and did not correlate with CRP (r = 0.08, P = 0.35), emphasizing the different nature of these biomarkers. The receiver operator characteristic area under the curve (ROC AUC) of CT‐IGFBP‐4 for the prediction of all‐cause mortality (0.727) was significantly higher than that of NT‐proBNP (0.680, P = 0.045) and CRP (0.669, P = 0.016). The combination of CT‐IGFBP‐4, NT‐proBNP, and CRP predicted mortality significantly better (ROC AUC = 0.788) than any of the biomarkers alone (P

KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at pCa>8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC. [ABSTRACT FROM AUTHOR]