Journal: bioRxiv

Article Title: Therapeutic poxviruses induce the secretion of immunostimulating and anti-tumoral extracellular vesicles

doi: 10.1101/2025.09.19.677320

Figure Lengend Snippet: A) Workflow for assessing the presence of cellular proteins (CD63, ICAM1 and CD86) and virus-encoded therapeutic payloads (IL-12, CD40L and OVA peptide SIINFEKL presented on MHCI) on EVs secreted by mouse dendritic cells (DC2.4) and isolated by ultracentrifugation (UC) or size exclusion chromatography (SEC; EVs-rich fractions (EVs SEC, fractions 1-4) and soluble proteins-rich fractions (Prots SEC, fractions 5-10)) following 100nm filtration. Payloads were quantified by electrochemiluminescence immunoassay (B-G) on EVs isolated from non-infected cells (mock) or from cells infected with empty or armed MVA virus (encoding IL12, CD40L and OVA peptide SIINFEKL payloads). B) Quantification of CD63 on EVs. EVs mock Vs EVs empty virus p=0,0007; EVs mock Vs EVs armed virus UC p= 0,0001; EVs mock Vs EVs armed virus SEC p< 0,0001; EVs mock Vs Soluble proteins armed virus SEC p< 0,0001; One-way Anova; N=5 independent experiments. C) Quantification of ICAM1 on EVs. EVs mock Vs EVs empty virus: p=0,0276; EVs mock Vs EVs armed virus UC p= 0,0012; EVs mock Vs EVs armed virus SEC p= 0,0425; EVs mock Vs Soluble proteins armed virus SEC p= 0,83; One-way Anova; N=4 independent experiments. D) Quantification of CD86 on EVs. EVs mock Vs EVs empty virus: p=0,19; EVs mock Vs EVs armed virus UC p>0,99; EVs mock Vs EVs armed virus SEC p= 0,73; EVs mock Vs Soluble proteins armed virus SEC p= 0,99; Kruskal-Wallis; N=4 independent experiments. E) Quantification of CD40L on EVs. EVs mock Vs EVs empty virus: p>0,99; EVs mock Vs EVs armed virus UC p<0,0001; EVs mock Vs EVs armed virus SEC p= 0,011; EVs mock Vs Soluble proteins armed virus SEC p>0,99; EVs armed virus SEC Vs Soluble proteins armed virus SEC p=0,0125; One-way Anova; N=4 independent experiments. F) Quantification of MHCI bound OVA peptide (SIINFEKL). EVs mock Vs EVs empty virus: p>0,99; EVs mock Vs EVs armed virus UC p=0,023; EVs mock Vs EVs armed virus SEC p= 0,0341; EVs mock Vs Soluble proteins armed virus SEC p>0,99; Kruskal-Wallis; N=4 independent experiments. E) Quantification of IL-12. EVs mock Vs EVs empty virus p=0,045; EVs mock Vs EVs armed virus UC p>0,99; EVs mock Vs Soluble proteins armed virus SEC p=0,0004; EVs armed virus SEC Vs Soluble proteins armed virus SEC p=0,045; Kruskal-Wallis test. N=5 independent experiments. * p<0,05; ** p<0,01; *** p<0,005; **** p<0,001; ***** p<0,0001; ns: non-significant.

Article Snippet: The following detection antibodies were used: CD63 Antibody, anti-mouse Biotin (clone REA563, Miltenyi Biotec); CD54 (ICAM-1) Antibody, anti-mouse, Biotin (clone YN1/1.7.4, #130-104-213, Miltenyi Biotec); CD86 Antibody, anti-mouse, Biotin (clone PO3.3, #130-101-944, Miltenyi Biotec); CD154 (CD40L) Antibody, anti-mouse, Biotin (clone MR1, #130-101-900, Miltenyi Biotec); H-2Kb/SIINFEKL Antibody, anti-mouse Biotin (clone 25-D1.16, Miltenyi Biotec).

Techniques: Virus, Isolation, Size-exclusion Chromatography, Filtration, Electrochemiluminescence, Infection

Journal: Frontiers in Immunology

Article Title: Extracellular vesicles derived from antigen-presenting cells pulsed with foot and mouth virus vaccine-antigens act as carriers of viral proteins and stimulate B cell response

doi: 10.3389/fimmu.2024.1440667

Figure Lengend Snippet: EVs-FMDVi express FMDV proteins, classical EVs markers, and immunoregulatory molecules. EVs were coupled with aldehyde/sulfate latex beads of 4 µm diameter and incubated with different fluorochrome-conjugated monoclonal antibodies anti-CD9, anti-CD81, CD63, MHC-II, CD11c, CD86 as detailed in materials and methods. For viral protein detection on EVs, FITC-labeled IgG purified from an FMDV-immune bovine serum was used, and FITC-labeled IgG obtained from a healthy unimmunized bovine serum was used as a corresponding control (Control). Comparison of percentages for different marker proteins and FMDV viral proteins for each EV population. The mean value ± SEM of the Median Fluorescence Intensity (MFI) for each of the markers evaluated is also shown. For statistical analysis, the Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used for the analysis of MFI, and one-factor ANOVA followed by Bonferroni’s multiple comparisons test for the analysis of percentages. Only significant differences are indicated in the graphs (p<0.05 *; p<0.01**; p<0.001 ***). The data correspond to 15 independent experiments.

Article Snippet: After being saturated with 100 mM glycine, EVs were incubated with the following monoclonal antibodies: 0.02µg/100µl anti-MHC-II-PE (Biolegend-clone: M5/114.15.2), 0.2µg/100µl anti-CD11c-APC (Biolegend-clone: N418), 1.5 µg/100µl anti-CD86-FITC (Miltenyi-clone: PO3.3), 1 µg/100µl anti-CD9-biotin (eBioscience-clone: MEM61), 1 µg/100µl anti-CD81-biotin (eBioscience clone: Eat2) or 1 µg/100µl polyclonal antibody anti-CD63-biotin (MyBioSource).

Techniques: Incubation, Labeling, Purification, Control, Comparison, Marker, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Allergological work-up outcome.

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Example of a positive BAT-FC in patient 6. Gating strategy for basophils: ( A ) doublet exclusion, FSC-H vs. FSC-A; basophils were identified as low SSC-A and CD123 + ( B ) and CD123 + HLA-DR neg cells ( C ). ( D ) The univariate histogram shows a positive BAT determined by an increased percentage of CD63 expression on the patient’s basophils with the highest concentration of oxaliplatin (250 µg/mL). ( E ) The univariate histogram (green) shows a positive BAT determined by increased mean fluorescence intensity (MFI) on the patient’s basophils at 250 µg/mL oxaliplatin; the gray histogram corresponds to the unstimulated cells. ( F ) Table showing the percentage of CD63+, MFI, and SI of CD203c in patient 6. Data were analyzed using FACSDiva software (verion 8.0.2).

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Expressing, Concentration Assay, Fluorescence, Software

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Examples of positive sIgE-microarray, BAT-microarray, and immunofluorescence detection of CD63 upon drug stimulation in patient 6, allergic to oxaliplatin, and patient 21, allergic to carboplatin. Cells were visualized under at ×100 magnification using a Olympus IX70 fluorescent microscope.

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Microarray, Immunofluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: BAT-microarray immunoassay. The y -axis shows the average CD63 expression represented as average z -scores. The x -axis shows the drug concentration: oxaliplatin ( A ) and carboplatin ( B ) 1:100 molar ratio. The standardized fluorescence intensity represented as the average z -score was considered positive if it exceeded three (dotted line).

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Microarray, Expressing, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Matrimeres are systemic nanoscale mediators of tissue integrity and function

doi: 10.1101/2024.03.25.586585

Figure Lengend Snippet: (A) The nanostructure subpopulations from mouse D1 MSCs. (i) Percentage of subpopulations pulled down by anti-mouse CD63 (αCD63), anti-mouse FN (αFN), or both, compared to biotin-only control (ctrl.) determined by the gel-based pull-down assay. (ii) Percentage of the triton-X (TX) sensitive fraction before (ctrl.) and after depletion (dep.) with FN or CD63 antibody. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 via one-way ANOVA with Tukey’s multiple comparisons test. (iii) Quantification of nanoscale subpopulations in terms of CD63, FN, and TX sensitivity. (Left) Proportional Venn diagram. (Right) Percentage of subpopulations. n = 3 experiments. (B) Morphological profiling of small nanostructures from mouse D1 MSCs after negative selection with FN or CD63 antibody-coated magnetic beads. (i) T-distributed stochastic neighbor embedding (t-SNE) plot based on 12 morphological parameters of small nanostructures from 9-11 TEM images of crude (ctrl., n = 1581), FN-depleted ( n = 895), and CD63-depleted ( n = 679) samples pooled from 3 different batches. Cluster numbers are assigned by the Leiden clustering algorithm. The color scheme is based on the relative representation of each nanostructure cluster by CD63-depleted (CD63 lo , hence FN hi , red) vs. FN-depleted (FN lo , hence CD63 hi , blue) samples. (ii) Fraction of clusters represented by nanostructures in each group. (Above) Visualization of cluster fractions under the t-SNE plot. (Below) Representative TEM images from each group with an overlay from the color scheme in (i). Scale bar = 300 nm. (C) Quantification of morphological parameters for each nanostructure cluster after negative selection. (Above) Representative nanostructures. Scale bar = 200 nm. (Below) Circular equivalent diameter (CED) and solidity. n = 47-198 for each group. (D) Confirmation of nanostructures after positive selection with FN or CD63 antibody and visualization of single vesicles or particles. (i) Representative images showing vesicular CD63 + and non-vesicular FN + nanostructures. Scale bar = 50 nm. (ii) CED and solidity. n = 24 for CD63 + and n = 38 for FN + nanostructures. The error bars denote s.d.

Article Snippet: Streptavidin-coated magnetic beads ( Dynabeads Biotin Binder; #11047, Thermo) were washed 3 times with PBS followed by incubation with biotin anti-mouse CD63 antibody (Clone REA563; Miltenyi Biotec) or biotin anti-mouse fibronectin (FN) antibody (#IRBAMSFBNGFBL; Innovative Research).

Techniques: Control, Pull Down Assay, Selection, Magnetic Beads

Journal: bioRxiv

Article Title: Matrimeres are systemic nanoscale mediators of tissue integrity and function

doi: 10.1101/2024.03.25.586585

Figure Lengend Snippet: (A) FN + NVEPs are present in mouse blood plasma. (i) Representative TEM images showing the distinct morphology of FN + NVEPs vs. CD63 + EVs in plasma after positive selection with the respective antibodies. Scale bar = 50 nm. (ii) Concentration of nanostructures. (iii) Hydrodynamic sizes of nanostructures. The average data points were fitted to log-normal distribution curves. (iv) Percentage of FN + matrimeres. (v) Concentration of FN + NVEPs. For (ii)-(v), mice were also injected intraperitoneally ( i.p. ) with lipopolysaccharide (LPS, 10 mg/kg), and blood plasma were analyzed after 8 h. n = 3 mice, * p < 0.05, *** p < 0.001 via unpaired T-test. (B) Mass spectrometry analysis of FN + NVEPs from MSCs vs. plasma. (i) Proteome coverage comparison; label-free protein quantitation based on log-transformed intensity. (ii) Fn1 sequence coverage comparison. Red denotes detected regions; histograms above denote peptide overlap density; blue and yellow denote the phosphorylation and oxidation sites detected, respectively. Pooled from n = 2 biological replicates. (C) Effects of fractionated small nanostructures from mouse D1 MSCs on restoration of endothelial barrier function after LPS treatment in vitro . The HUVEC monolayer plated on TEER electrodes was treated with LPS (1 μM) for 6 h, followed by washout and addition of 1.5 x 10 8 small nanostructures per well after gel-based depletion (dep.) with FN and/or CD63 antibody at t = 6.5 h. (i) TEER kinetics over 24 h. The y-axis starts from 500 Ω.cm 2 . A dotted vertical line indicates the time when the samples were added. The data points from t = 6.5 h to 24 h were fitted to one-phase association curves. (ii) Percentage of TEER recovery after LPS treatment from t = 6.5 h to 24 h. n = 3 experiments. (D) Effects of inhibiting RGD and FAK on the recovery of TEER by the CD63-depleted fraction after LPS treatment. DMSO, cilengitide (200 nM) or PF-573228 (100 nM) was added simultaneously with CD63 - nanostructures. (i) TEER kinetics. (ii) Percentage of TEER recovery. (iii) Half-time ( t 1/2 ) of TEER recovery. n = 3 experiments. (E) Effects of fractionated small nanostructures from MSCs on restoration of lung endothelial barrier function after acute lung injury in vivo . (i) Overview of strategy to determine the efficacy of fractionated nanostructures from MSCs in a mouse model of LPS-induced lung injury. Mice were treated with LPS (10 mg/kg) for 4 h, followed by intratracheal ( i.t. ) administration of mouse D1 MSC-secreted nanostructures after antibody (ab)-based depletion (dep.). (ii) Lung edema by quantifying lung tissue wet-dry ratio. (iii) Lung vascular permeability by quantifying Evans blue albumin (EBA) accumulation. The doses are indicated in the unit of 10 8 per 20g mouse. n = 4 mice for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 via one-way ANOVA with Tukey’s multiple comparisons test for (C) and (D) , and via Welch’s one-way ANOVA with Dunnett’s T3 post-test for (E) . The error bars denote s.d.

Article Snippet: Streptavidin-coated magnetic beads ( Dynabeads Biotin Binder; #11047, Thermo) were washed 3 times with PBS followed by incubation with biotin anti-mouse CD63 antibody (Clone REA563; Miltenyi Biotec) or biotin anti-mouse fibronectin (FN) antibody (#IRBAMSFBNGFBL; Innovative Research).

Techniques: Clinical Proteomics, Selection, Concentration Assay, Injection, Mass Spectrometry, Comparison, Protein Quantitation, Transformation Assay, Sequencing, Phospho-proteomics, In Vitro, In Vivo, Permeability

Journal: bioRxiv

Article Title: Matrimeres are systemic nanoscale mediators of tissue integrity and function

doi: 10.1101/2024.03.25.586585

Figure Lengend Snippet: (A) Evaluation of DNA as a structural component of FN + matrimeres. (i) Total amount of DNA in mouse D1 MSC-secreted nanostructures without depletion (crude) or after gel-based depletion (dep.) with FN or CD63 antibody. (ii) Sensitivity of (left) FN + matrimeres and (right) CD63 + EVs to DNase I treatment ex vivo . The crude pellet from MSCs was treated without (ctrl.) or with DNase I (50 U/ml) for 1 h at 37°C, followed by washout and pull-down assay to determine the number of FN + matrimeres or CD63 + EVs normalized by the number of MSCs. **** p <0.0001, ns: not significant via unpaired T-test. n = 4 experiments. (B) Effects of DNase I on the efficacy of the CD63-depleted fraction (1.5 x 10 8 per 20g mouse) in LPS-induced lung injury in vivo . (i) Lung tissue wet-dry ratio. (iii) EBA accumulation in lung tissue. n = 4 mice for each group. (C) Effects of pharmacological agents on FN + matrimere production from MSCs. (i) Overview of the tested drugs and their known targets. Mouse D1 MSCs were cultured in the presence of DMSO, BMS-P5 (1 μM), BafA1 (200 nM), CA-C1 (1 μM), or ML-SA5 (20 μM) for 1 day, followed by quantification of FN + matrimeres secreted per cell. (ii) Number of small nanostructures per cell. (iii) Percentage of FN + matrimeres. (iv) Number of FN + matrimeres per cell. n = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 via one-way ANOVA with Tukey’s multiple comparisons test for (A), i and (D) ii-iv, and via Welch’s one-way ANOVA with Dunnett’s T3 post-test for (B). The error bars denote s.d.

Article Snippet: Streptavidin-coated magnetic beads ( Dynabeads Biotin Binder; #11047, Thermo) were washed 3 times with PBS followed by incubation with biotin anti-mouse CD63 antibody (Clone REA563; Miltenyi Biotec) or biotin anti-mouse fibronectin (FN) antibody (#IRBAMSFBNGFBL; Innovative Research).

Techniques: Ex Vivo, Pull Down Assay, In Vivo, Cell Culture