Patent Foramen Ovale-associated Stroke and COVID-19 Vaccination

Background: COVID-19 infection has been associated with paradoxical thromboembolism through a patent foramen ovale (PFO) and ischaemic stroke. Such events have not been reported after COVID-19 vaccination. The aim of the present study was to investigate PFO-associated stroke during the mass COVID-19 vaccination in Slovenia. Methods: This prospective study, conducted between 26 December 2020 and 31 March 2022, enrolled consecutive patients (≥18 years) with PFO-associated stroke referred for a percutaneous closure to a single interventional facility in Slovenia. Results: A total of 953,546 people aged between 18 and 70 years received at least one dose of a COVID-19 vaccine approved by the European Medicines Agency. Of the 28 patients presenting with PFO-associated stroke, 12 patients (42.9%) were vaccinated prior to the event, of whom nine were women and three were men, aged between 21 and 70 years. Stroke occurred within 35 days after vaccination in six patients (50%). Clinical presentation included motor dysphasia, paresis, vertigo, ataxia, paraesthesia, headache, diplopia and hemianopia. At hospital discharge, 11 patients (91.6%) had at least one residual ischaemic lesion. Conclusion: A temporal coincidence of COVID-19 vaccination and PFO-associated stroke has been described. A potential cause–effect relationship may only be hypothesised

Effects of LLO and EqtII on permeability of Caco-2 cell monolayer.

<p>Permeability of Caco-2 monolayer to fluorescein and FD3 after treatment with LLO and EqtII. Data represent means of permeability coefficients ± standard error for 2 to 3 independent experiments.</p

Pore formation with LLO or EqtII in Caco-2 cells.

<p><b>A</b> Hemolytic activity of LLO^A318C-L334C in the reduced state (filled symbols) or the oxidized state (open symbols). Average ± S.D, n = 5. <b>B</b> Binding of LLO and LLO^A318C-L334C to multilamellar vesicles. M, molecular weight markers (the bottom band is 50 kDa and the upper is 60 kDa). t, total applied protein; P, protein associated with the pelleted fraction; S, protein remaining in the supernatant after centrifugation. <b>C</b> Time course of the relative drop in TEER (left) after apical application of oxidized (white) or reduced (black) LLO^A318C-L334C or EqtII^V8C-K69C and a relative drop in TEER (right) 3 minutes after apical application of oxidized (dark gray) or reduced (white) LLO^A318C-L334C or EqtII^V8C-K69C. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 3 independent, experiments. <b>D</b> Time course of SYTOX Green staining after application of 125 nM (black), 62.5 nM (dark gray), 31.3 nM (gray) and 15.6 nM (light gray) LLO (left) or 100 nM (black), 50 nM (dark gray) and 25 nM (gray) EqtII (rigt) and control (white squares). <b>E</b> Confocal microscopy of SYTOX Green staining after application of 125 nM LLO or 100 nM EqtII. Bar in panel C represents 30 μM.</p

Effects of LLO and EqtII on tight junction proteins and actin.

<p><b>A</b> Immunostaining and confocal microscopy of claudin-1 (first column), occludin (second column), E-cadherin (third column) and actin and nuclei (fourth column) after treatment with LLO and EqtII. Arrows indicate ruptures of tight junctions. Scale bars represent 20 μm. <b>B</b> Western blot analysis of claudin-1 after 5, 30 and 60 minute exposure to 31.3 nM LLO, 25 nM EqtII, 2.5 mM EDTA and untreated cells (control). M, molecular weight marker.</p

Effects of calcium on TEER drop.

<p><b>A</b> Effects of intra- and extracellular calcium chelation on the action of 125 nM LLO and 100 nM EqtII. For extracellular chelation 2.5 mM EDTA or EGTA was used or the medium was substituted by PBS. For intra- and extracellular chelation BAPTA-AM and PBS (BP) were used. Values for control experiments in DMEM (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130471#pone.0130471.g001" target="_blank">Fig 1</a>) are shown by the dashed line for each protein. <b>B</b> Effects of different concentrations (nM) of ionomycin on TEER. <b>C</b> Time course of relative TEER values after apical application of 4 μM nigericin (filled symbols) and control (open symbols).</p

Cell viability after treatment with LLO and EqtII.

<p><b>A</b> Relative LDH release 3 hours after treatment with LLO (black) or EqtII (gray). <b>B</b> MTT cell viability assay. Relative cell viability 24 hours after treatment with LLO (black) or EqtII (gray). <b>C</b> Light microscopy of Caco-2 cells 24 hours after treatment with LLO (top) or EqtII (bottom). Data represent means of percent of negative controls. Error bars are the standard error of the mean calculated for 2 to 3 independent experiments. Bar in panel C represents 100 μm.</p

Effect of LLO and EqtII on TEER.

<p>Top left: Time course of the relative drop in TEER after apical application of 1 μM (black squares), 250 nM (black circles), 62.5 nM (black triangles) and 15.6 nM (black inverted triangles) or basolateral application of 2 μM (gray squares) LLO and control (white squares). Bottom left: Time course of the drop in TEER after apical application of 100 nM (black squares), 50 nM (black circles) and 25 nM (black triangles) or basolateral application of 1 μM (gray squares) EqtII and control (white circles). Top right: Relative drop in TEER 3 minutes after apical (black; at two-fold increase in concentration from 7.8 nM-1 μM) or basolateral (gray; a four-fold increase from 31.3 nM-2 μM) application of LLO. Bottom right: Relative drop in TEER 3 minutes after apical (black; at two-fold increase in concentration from 12.5–400 nM) or basolateral (gray) application of EqtII. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 4 independent experiments.</p

Regeneration of the Caco-2 monolayer after apical treatment with LLO and EqtII.

<p>Top: Time course of relative TEER values after apical application of 62.5 nM (squares), 31.3 nM (circles), 15.6 nM (triangles) and 7.8 nM (diamonds) LLO. Bottom: Time course of relative TEER values after apical application of 100 nM (squares), 50 nM (circles), 25 nM (triangles) and 12.5 nM (diamonds) EqtII. Data represent means of percent of initial values. Error bars are the standard error of the mean calculated for 2 to 3 independent experiments.</p

Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

<div><p>Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside G_M1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region <i>via</i> caveolin-1–positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.</p></div

Co-localisation of OlyA-mCherry and other membrane markers.

<p>(<b>A–F</b>) Representative images of co-localisation between OlyA-mCherry (red) and other membrane markers (green), as indicated. Scale bar, 5 μm. (<b>G</b>) Quantitative analysis of the co-localisation between OlyA-mCherry and the other membrane markers. Pearson's correlation coefficients were calculated using the JACoP plugin (Image J programme) from optical sections. The degree of co-localisation from the Pearson's value correlation coefficients s were categorised as very strong (0.85 to 1.0), strong (0.49 to 0.84), moderate (0.2 to 0.48), weak/moderate (0.1 to 0.2), weak (−0.26 to 0.09), and very weak (−1 to −0.27). Error bars are the standard errors of the means (n = 5).</p