Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs activate the NF-κB/AP-1 pathways in human reporter macrophages via TLR2 signaling. A dTHP1-XBlue cells were treated with Ef -EVs (100 − 50,000 EVs/cell). LPS (100 ng/mL) was used as a positive control. B dTHP1-XBlue cells were treated with EVs derived from clinical E. faecalis bloodstream isolates (1000-10,000 EVs/cell). LPS (100 ng/mL) and Pam 3 CSK 4 (100 ng/mL) were used as positive controls. C-E) dTHP1-XBlue cells were pretreated with increasing concentrations (from 0.1 to 5 µg/mL) of anti-hTLR2-IgA mAb (dark pink) or human IgA2 control mAb (light pink) for 1 h. Subsequently, cells were treated with either (C) EVs (7000 EVs/cell), (D) Pam 3 CSK 4 (TLR2 ligand, 1 ng/mL), or (E) LPS (TLR4 ligand, 1 ng/mL) in the presence of antibodies for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) in (C, blue), Pam 3 CSK 4 (1 ng/mL) in (D, dark blue), or LPS (1 ng/mL) in (E, red) were used as positive activating controls while cells treated with cell culture medium supplemented alone ( C - E , light green) were used as negative controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive controls. Data are shown as means ± SD of three independent experiments ( N = 3, n ≥ 3) and analyzed either by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test in (A and B) or by two-way analysis of variance (ANOVA) followed by Šídák multiple comparison post hoc test in ( C - E )

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Positive Control, Derivative Assay, Control, Cell Culture, Activation Assay, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 controls EV-induced immune activation but does not function as an endocytic receptor for EV uptake. dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with DiI-labeled Ef -EVs (7000 EVs/cell) in the presence of antibodies for 4 and 24 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium supplemented with DiI-labeled Ef -EVs (7000 EVs/cell) were used as positive activating controls. For EV internalization assays, mean fluorescence intensity (B and C, upper panel) and EV-positive cells (B and C, lower panel) were quantified after 4 ( A and B ) and 24 h ( A and C ) of EV treatment by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. NF-κB/AP-1 activation in D was measured after 24 h of EV incubation as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are presented as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Activation Assay, Control, Labeling, Incubation, Cell Culture, Fluorescence, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 engagement by Pam 3 CSK 4 -SUVs triggers inflammatory activation but does not enhance SUV internalization. A - C ) dTHP1-XBlue cells were treated with Pam 3 CSK 4 -SUVs (6 µM) containing increasing amounts of Pam 3 CSK 4 (from 0 to 0.4 mol% total SUV lipid composition) for 18 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.4 mol% total SUV lipid composition) were used as positive activating controls. D - F ) dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) in the presence of antibodies for 18 h. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) were used as positive activating controls. For both experiments, cells incubated in cell culture medium alone were used as negative controls. Mean fluorescence intensity (B and E, upper panel) and SUV-positive cells (B and E, lower panel) were quantified after 18 h of treatment by measuring fluorescence intensity associated with SUVs on the APC channel. In C and F, NF-κB/AP-1 activation was measured after 18 h of SUV treatment as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are shown as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Activation Assay, Incubation, Cell Culture, Control, Fluorescence, Activity Assay, Comparison

Journal: Scientific Reports

Article Title: SARS-CoV-2-specific B cell responses in non-draining lymph nodes and antibody functionalities in immunized end-stage renal disease patients

doi: 10.1038/s41598-025-27815-y

Figure Lengend Snippet: The B cell isotypes in the LN and PB compartment, the horizontal black lines indicate the average percentage in both compartments ( a ) IgM in switched B cells, lower in LNs than in PB at 8% (IQR: 5% − 11%) or 13% (IQR: 11% − 26%) ( p = 0.0001), respectively; ( b ) IgM in S-binding B cells, no difference between the LNs and PB at 29% (IQR: 16% − 51%) or 15% (IQR 6% − 33%) ( p = 0.058), respectively; ( c ) IgA in switched B cells, no difference between LNs and PB at 36% (IQR 27% − 43%) or 37% (IQR 32% − 43%) ( p = 0.207), respectively; ( d ) IgA in S-binding B cells, in LNs higher than in PB at 22% (IQR 11% − 32%) or 8% (IQR 5% − 15%) ( p = 0.0002), respectively; ( e ) IgG in switched B cells, in LNs higher than in PB at 49% (IQR 42% − 53%) or 42% (IQR 32% − 48%) ( p = 0.0110), respectively; ( f ) IgG in S-binding B cells, in LNs lower than in PB at 53% (IQR: 44% − 67%) or 73% (IQR 61% − 80%) ( p = 0.0001), respectively. PRM (primary), two vaccinations; BST (booster three), three vaccinations; BSF (booster four), four vaccinations; INFX (infection), prior infection; HYB (hybrid), immunity in which the infection was prior to the last of two vaccinations.

Article Snippet: The following surface mAbs were used: CD19 BV785, CD20 APC-Fire750, CD27 BB700, IgD PE-CF594, CD24 BV650, CD38 BUV563, IgM BUV395, IgG BUV496 (all from BD Biosciences), CXCR3 R718 (eBioscience), IgG1 PE, IgG2 PE, IgG2 FITC, IgG3 FITC (all from SouthernBiotech), IgA PE-Vio770, IgA2 FITC (both from Miltenyi Biotec), and IgA1 PE (Abcam).

Techniques: Binding Assay, Infection