Journal: bioRxiv

Article Title: Microengineered transplantation of human solid tumors for in vitro studies of CAR T immunotherapy

doi: 10.1101/2025.04.25.650709

Figure Lengend Snippet: a, Conceptual diagram illustrating pairwise mapping of directional ligand-receptor interactions between different cell types in the engineered tumor model. b,c, Chord diagrams of cell pair-specific ligand-receptor interactions with adjusted p-values < 0.05 and total mean expression > 0.35. d, Visualization of interactions between CAR T cells (CAR Ts) and endothelial cells (Endos) mediated by CXCR3/DPP4-CXCL10/CXCL11. e, Violin plots comparing the expression of these mediators across all cell types. f, Experimental timeline for CAR T cell infusion and drug treatment. g, Representative confocal micrographs of single tumors treated with CAR T cells and different concentrations of LAF237. Blood vessels are not shown in these images. Scale bars, 250 μm. h, Representative images of CAR T cell-infused tumors at Day 26. Scale bars, 200 µm. i, Quantification and comparison of tumor area (top) and CAR T cell-occupied hydrogel area (bottom). Shaded in grey is a period of CAR T cell infusion and daily LAF237 treatment. Data are presented as mean ± SEM (n ≥ 3). j,k, Quantification of CXCL10 and CXCL11 levels ( j ) and the activity of DPP4 ( k ) in device effluent. Shaded in grey is a period of CAR T cell infusion and daily LAF237 treatment. Data are presented as mean ± SEM in ( j ) and as mean ± SD in ( k ) (n ≥ 3). CAR T cells derived from three healthy donors were tested in this study.

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies against CXCL10 (MAB266-100, R&D systems).

Techniques: Expressing, Comparison, Activity Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil-fibroblast crosstalk drives immunofibrosis in Crohn’s disease through IFNα pathway

doi: 10.3389/fimmu.2024.1447608

Figure Lengend Snippet: UC and CD intestinal tissues are characterized by differential spatial distribution of neutrophils, attracted in the intestinal tissue by fibroblast-derived IL-8. (A) Neutrophil Elastase IHC staining (brown cells) and (B) Masson’s trichrome (cyan fibers) indicating the presence of neutrophils and fibrotic areas respectively, in serial cross sections obtained from the same intestinal biopsies. Thickness between the serial cross sections in (A, B) was 4 μm. (C) IL-8 levels in supernatants of PIFs measured by a bead-based flow cytometric assay. (D) IL-8 mRNA assessed by RT-qPCR and (E) IL-8 protein levels in PIFs immunostaining (blue: DAPI, green: IL-8, red: Vimentin). (F) IL-8 expression assessed by immunostaining in intestinal tissue fibroblasts (blue: DAPI, green: IL-8, red: Vimentin). Dotted frames indicate the zoomed-in areas, which are provided to assess the co-expression of IL-8 and Vimentin. White arrowheads show double positive IL-8/Vimentin cells observed in UC and CD patients. (G) Chemotactic capacity of the PIFs’ supernatant on HI neutrophils, before and after the neutralization of IL-8, assessed by a transwell migration assay. (A, B, E, F) One representative example out of four independent experiments, performed in different subjects of each group, is shown. (A, B) Optical microscopy, magnification: 100x, (E, F) Confocal microscopy, magnification: 400x, Scale Bar: 10μm. Nonparametric Kruskal-Wallis followed by Dunn’s multiple comparisons test was performed in (C, D) , n=4 , *p<0.05, ns, not significant. (G) Bayesian unpaired t-tests, followed by the Benjamini-Hochberg correction, were used to compare the migratory capacity of HI PIFs supernatants to UC and CD. For comparisons between PIFs supernatants that were treated with IL-8 neutralizing antibody (anti-IL-8), and supernatants treated with IgG isotype control (Iso IgG), Bayesian paired t-tests were performed, n=4 , *p<0.05, ***p<0.001, ns, not significant. Data are expressed as mean ± SEM. CD, Crohn’s disease; DMEM, Dulbecco’s Modified Eagle Medium; HI, healthy individuals; IHC, immunohistochemistry; PIFs, primary intestinal fibroblasts; UC, ulcerative colitis;.

Article Snippet: In brief, NETs were captured with a human anti-MPO antibody (1:500 dilution; HM2164; clone 6G3-mouse IgG1; Hycult Biotech; Uden, Netherlands), and an anti-double-stranded DNA antibody was used for DNA detection (Cell Death Detection ELISA Kit; 11544675001; Merck; Kenilworth, New Jersey, USA).

Techniques: Derivative Assay, Immunohistochemistry, Flow Cytometry, Quantitative RT-PCR, Immunostaining, Expressing, Neutralization, Transwell Migration Assay, Microscopy, Confocal Microscopy, Control, Modification

Journal: PLoS ONE

Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

doi: 10.1371/journal.pone.0250265

Figure Lengend Snippet: A) Supernatants of control- and PMA-treated, freshly isolated human neutrophils were diluted at 1:50, 100, 200, 400, 800 and 1600 (corresponding to relative concentrations of 32, 16, 8, 4, 2 and 1) and assessed by the initial MPO-DNA complex ELISA protocol. Shown data correspond to one representative experiment of calibrator batch #2. B) and C) Microwells were coated with MPO antibody, IgG2b isotype control or were left uncoated. B) Supernatant of freshly isolated and PMA-stimulated human neutrophils at dilutions corresponding to relative concentrations from 32 to 1 or C) plasma samples from two human donors at 1:2 dilution were evaluated with the initial ELISA protocol. Note that data points of isotype control and uncoated wells overlap in 1B.

Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

doi: 10.1371/journal.pone.0250265

Figure Lengend Snippet: For assessment of A)—C) serially diluted calibrator and D) 1:2 diluted plasma, the following modifications of the initial MPO-DNA complex ELISA protocol were applied: Microwells were coated with A) 1 - 5 μg/ml rabbit anti-human MPO polyclonal antibody (no. 07-496-I, EMD Millipore), B) 4 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam) or C) and D) 1 μg/ml mouse anti-ds DNA monoclonal antibody (no. ab27156, Abcam), 1 μg/ml mouse IgG2a kappa monoclonal isotype control (no. 010-001-332, Rockland Immunochemicals) or were left uncoated. For MPO-DNA complex detection A) peroxidase-conjugated mouse anti-DNA monoclonal antibody (clone MCA-33, as described for the initial ELISA protocol), B) 0.031 – 2 μg/ml HRP-labeled mouse anti-human MPO monoclonal antibody (no. NBP2-41406H, Bio-Techne) or C) and D) 0.5 μg/ml biotinylated mouse anti-human MPO monoclonal antibody (no. HM2164BT, HyCult Biotech) followed by 0.5 μg/ml streptavidin-HRP were applied.

Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, Labeling

Journal: PLoS ONE

Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

doi: 10.1371/journal.pone.0250265

Figure Lengend Snippet: A) Microwells were coated with MPO antibody, various isotype controls or were left uncoated. Calibrator or 1:2 diluted plasma samples were analyzed according to the initial MPO-DNA complex ELISA protocol. The calibrator with a relative concentration of “0” equals blank. B) MPO antibody (Bio-Rad) and C) isotype control (IgG2b, Bio-Techne) were processed to Fab and Fc fragments. Western blot analysis after reducing SDS-PAGE (12.5% gel) of unprocessed antibodies (lane 2), papain cleaved unpurified antibodies (lane 3), purified Fab fragments (fractions 1–3, lanes 4–6) as well as Fc fragments (fractions 1–3, lanes 7–9) are depicted. Molecular weight marker (Kaleidoscope™ Prestained Standard) was applied in lane 1 and is given in kDa. Intact immunoglobulin G heavy chains (IgG H ) in lane 2 and the respective Fc and Fab fragment chains in lanes 3–9 are indicated with blue, black and green arrowheads, respectively. Please note that the immunoglobulin G light chain (IgG L ) remains unaltered by the fragmentation process and has a molecular weight close to the fragmented Fab heavy chain. Hence, the two protein bands could not be resolved and are both indicated by the green arrowhead. D) and E) MPO antibody and isotype control (for both: Fab fragment, fraction 1) were coated onto microwells and incubated with D) serially diluted calibrator or E) 1:2 diluted plasma samples. The further ELISA procedure was performed as indicated for the initial MPO-DNA complex ELISA protocol.

Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, SDS Page, Purification, Molecular Weight, Marker, Incubation

Journal: PLoS ONE

Article Title: ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

doi: 10.1371/journal.pone.0250265

Figure Lengend Snippet: A) and C) Serially diluted calibrator or B) and D) plasma samples diluted at 1:10, 1:30 and 1:100 (with sample diluent of the ABTS ELISA Buffer Kit) were supplemented A) and B) without (w/o) or with increasing doses (10 μg/ml, 100 μg/ml, 500 μg/ml) of protein A-purified mouse IgG from normal mouse serum or C) and D) without (w/o) or with graded doses (0.1%, 1%, 10%) of TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according the initial protocol in microwells coated with MPO antibody, isotype control or left uncoated. Note that data points of uncoated microwells overlap in 8A and C. E) Plasma was diluted 1:100 without (w/o) or with supplementation of 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was performed according to the initial protocol in microwells coated with MPO antibody or isotype control. F) and G) Plasma of two donors at dilutions of 1:100, 1:200 and 1:400 was supplemented with 10% TRU Block Ready reagent. The further procedure of the MPO-DNA complex ELISA was based on the initial protocol with microwells coated with MPO antibody or isotype control or left uncoated.

Article Snippet: Alternative detection antibodies and other reagents: horseradish peroxidase (HRP)-labeled mouse anti-human MPO monoclonal antibody (clone MPO421-8B2, IgG1, RRID:AB_2827763, no. NBP2-41406H, Bio-Techne), biotinylated mouse anti-human MPO monoclonal antibody (clone 266-6K1, IgG1, RRID:AB_10234434, no. HM2164BT, HyCult Biotech, Uden, The Netherlands) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay