Journal: Frontiers in Immunology

Article Title: Molecular determinants of STEC-HUS: from complement activation to microvascular thrombosis

doi: 10.3389/fimmu.2026.1749811

Figure Lengend Snippet: Prothrombotic effects of STEC-HUS serum are dependent on WPB exocytosis from endothelial cells. P-selectin (A) and vWF (B) expression on unstimulated HMEC-1 exposed to serum from patients with acute STEC-HUS (n = 5 for P-selectin experiments; n = 6 for vWF experiments), in the presence or in the absence of different complement inhibitors (sCR1, 150 µg/mL; factor B inhibitor, iptacopan,10 µM; eculizumab 100 µg/mL) or to a pool of control sera (normal human serum, NHS), run in parallel. Results are shown as pixel 2 /high-power field (HPF) of stained surface area. Data are mean ± SD. Circles represent single patients’ data. The addition of either sCR1, or iptacopan or eculizumab to patient’s serum significantly decrease both P-selectin and vWF expression induced by patient’s serum alone. *P < 0.05 vs STEC-HUS serum alone (ANOVA, followed by Tukey’s multiple comparisons test for data of P-selectin expression and by Holm-Šídák’s multiple comparisons test for data of vWF expression). (C) Endothelial surface area covered by thrombi on ADP-activated HMEC-1 exposed to serum from STEC-HUS patients collected during the acute phase of the disease and then perfused with whole blood. Before the experiments, HMEC-1 were left for 16 hours with medium added or not with the RalA inhibitor BQU57 (10 µM). Data are expressed as mean ± SD of percentages of serum-induced thrombus formation in respect to a pool of control sera (normal human serum, NHS), run in parallel in each experiment and set as 100% (n = 3 independent experiments). Circles indicate single patients’ data. Horizontal dashed lines indicate upper and lower limits of the normal range . *P < 0.05 (paired Student’s t test). (D) Representative confocal microscopy images (original magnification X200) of experiments of thrombus formation (green staining) relative to Figure 6C . Scale bar: 100 µm.

Article Snippet: Cells were washed again and treated with the following specific antibodies: FITC-conjugated rabbit anti-human C3c-complement (Dako, that recognizes C3c, part of C3 and C3b, 1:300 final dilution in Dapi 1 μg/mL); or rabbit anti-human complement C5b-9 complex (Calbiochem, 1:200 final dilution in PBS1X) followed by FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:50 final dilution in 1 μg/mL Dapi); or goat anti-human C4 (Abcam, 1:100 final dilution in PBS1X) followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:200 final dilution in Dapi 1 μg/mL); or FITC-conjugated anti-human IgG (Sigma Aldrich, 1:32 final dilution in 1 μg/mL Dapi); or mouse anti-human P-selectin (R&D System, 20 μg/mL final concentration in PBS1X), followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:60 final dilution in 1 μg/mL Dapi); or rabbit anti-human vWF (Dako, 10 μg/mL final concentration in PBS1X), followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:50 final dilution in 1 μg/mL Dapi).

Techniques: Expressing, Control, Staining, Confocal Microscopy

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: A , Schematic workflow for in-cell ELISA. HUVEC were cultured for 6 hours with serum from either healthy controls (collected pre-pandemic) (n=38) or patients hospitalized with COVID-19 (n=118). Cells were then fixed and surface expression of E-selectin ( B ), VCAM-1 ( C ), or ICAM-1 ( D ) was quantified. Median values are indicated by horizontal lines. Groups were analyzed by Mann-Whitney test; ****p<0.0001. E , Beyond the 118 COVID-19 patients tested in panel D, surface expression of ICAM-1 was tested in the context of an additional 126 unique patient samples. Patients requiring mechanical ventilation (n=101) are compared to hospitalized patients who were not mechanically ventilated (n=143); p<0.01 by Mann Whitney test. F , Serum from healthy controls (n=38) and COVID-19 patients (n=102) were assessed for soluble E-selectin. COVID-19 samples were compared to controls by Mann-Whitney test; ****p<0.0001. G , Soluble E-selectin was compared to HUVEC E-selectin expression as presented in panel B. Correlation was determined by Spearman’s method. H , HUVEC were cultured for 6 hours with plasma from healthy controls (n=36), intensive care unit patients with non-COVID sepsis (n=100), or patients hospitalized with COVID-19 (n=72). Cells were then fixed and surface expression of ICAM-1 was quantified. Groups were analyzed by one-way ANOVA with correction for multiple comparisons by Holm-Sidak’s method; *p<0.05 and ***p<0.001.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: In-Cell ELISA, Cell Culture, Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: HUVEC were cultured for 6 hours with serum from either healthy controls (collected pre-pandemic) (n=38) or patients hospitalized with COVID-19 (n=118). Cells were then fixed and surface expression of E-selectin ( A ), VCAM-1 ( B ), or ICAM-1 ( C ) was quantified. Median values are indicated by horizontal lines. Data were presented as raw absorbance (optical density 650 nm). Groups were analyzed by Mann-Whitney test; ****p<0.0001.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Cell Culture, Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: Soluble E-selectin in COVID-19 serum was compared to laboratory and clinical data when available on the same day as the serum collection. Spearman’s correlations are presented for C-reactive protein (n=83) ( A ), D-dimer (n=72) ( B ), calprotectin (n=102) ( C ), oxygenation efficiency (n=99) (pulse oximetry/fraction of inspired oxygen, D ).

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques:

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: A , Serum was pooled from 3 patients with positive aCL IgG or 5 patients with positive aPS/PT IgG. HUVEC monolayers were then treated with 2.5% COVID or control serum for 6 hours. Calcein-AM-labeled neutrophils were then added as described in Methods. Scale bar=200 microns. Mean ± standard deviation is presented for n=3 independent experiments; *p<0.05 by one-way ANOVA corrected by Dunnett’s test. B-D , IgG was depleted from each of the aforementioned pools. Activation of HUVEC was determined after culture for 6 hours as defined by surface expression of E-selectin ( B ), VCAM-1 ( C ), or ICAM-1 ( D ). The experiment was repeated on 3 different days, and bars represent mean and standard deviation. Groups were compared by 2-sided paired t-test; *p<0.05 and **p<0.01. E , IgG was purified from the pooled samples referenced in A-C, and then supplemented (100 μg/ml) into control serum that had been depleted of IgG. Activation of HUVEC was determined after culture for 6 hours as defined by surface expression of ICAM-1. Groups were compared by one-way ANOVA with correction for multiple comparisons by Tukey’s test; **p<0.01.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Labeling, Standard Deviation, Activation Assay, Expressing, Purification