human fibronectin duoset elisa  (R&D Systems)

Journal: ACR Open Rheumatology

Article Title: Lymphocyte Activation Gene 3 Regulation of Profibrotic Cytokines and Type I Collagen Production in Patients With Systemic Sclerosis

doi: 10.1002/acr2.70120

Figure Lengend Snippet: Impact of agonistic LAG‐3 antibody on proinflammatory cytokine secretion by PBMCs. (A) dcSSc (n = 8) PBMCs were prestimulated with CD3/CD28 for 24 hours in monocultures or (B) cocultured with allogeneic dcSSc fibroblasts. The cultures were treated with either an LAG‐3 Q22 agonistic antibody (LAG‐3 Ag) or an isotype control (LAG‐3 Isotype) for 48 hours. In monocultures, the agonistic LAG‐3 Ag significantly reduced the levels of IFNγ, IL‐1β, IL‐4, and TNFα. In cocultures, IFNγ, IL‐12p70, IL‐13, IL‐2, IL‐4, and TNFα were decreased significantly. In both setups, IL‐10 levels were significantly increased by the addition of agonistic LAG‐3 isotype–treated samples. Data are presented as mean with SD Q23 (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFNγ, interferon γ; IL, interleukin; LAG‐3, lymphocyte Q24 activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell; TNFα, tumor necrosis factor α (1425 pg/mL vs 1847 pg/mL; P = 0.001); compared to the control IgG, there was still a notable increase in IL‐10 production (8.33 pg/mL vs 6.10 pg/mL; P = 0.01). No significant changes were observed in the production of IL‐1β or IL‐6 (Figure ). Agonistic LAG‐3 antibodies suppress type I IFN production and reduce fibroblast matrix production. PBMCs from patients with dcSSc (n = 8) were stimulated with CD3/CD28 for 24 hours and cultured with or without allogeneic dcSSc fibroblasts for 48 hours. The cultures were then treated with either the LAG‐3 Ag or the isotype control (LAG‐3 Isotype). (C, D) The LAG‐3 Ag significantly reduced the production of bioactive IFNα/β in monocultures and cocultures, as measured in HEK‐Blue cells, compared to the LAG‐3 isotype control. (E) In cocultures, levels of type 1 procollagen and (F) fibronectin were significantly reduced by LAG‐3 Ag. Data are presented as mean with SD (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFN, interferon; LAG‐3, lymphocyte activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell.

Article Snippet: Quantification of sLAG‐3 (LAG‐3 Human Enzyme‐Linked Immunosorbent Assay [ELISA] kit # BMS2211; Invitrogen), type 1 procollagenα (Human Pro‐Collagen I alpha 1 DuoSet ELISA Cat.DY6220‐05; R&D Systems), and fibronectin (Human Fibronectin DuoSet ELISA Cat. DY1918‐05; R&D Systems) in the plasma and supernatants was performed according to the manufacturer's protocol.

Techniques: Control, Activation Assay, Cell Culture

Journal: ACS applied materials & interfaces

Article Title: Nanotopography Influences Host-Pathogen Quorum Sensing and Facilitates Selection of Bioactive Metabolites in Mesenchymal Stromal Cells and Pseudomonas aeruginosa Co-Cultures.

doi: 10.1021/acsami.4c09291

Figure Lengend Snippet: Figure 1. Characterization of functional active coatings on Ti nanotopographies. (a) Representative SEM images of flat control, nanospike (NS), and nanonetwork (NN) together with surface height and surface area measurements (table); scale bar 2 μm. (b) Chemical structure of PEA. (c) Titanium, carbon, and oxygen spectra of PEA coated flat, NS, and NN taken by X-ray photoelectron spectroscopy (XPS) surfaces. Each color corresponds to the number on the PEA chemical structure. (d) 1 × 1 μm AFM micrographs from the flat, NS, and NN surfaces before coating (top row) and after coating with PEA for 90 s at 100 W using plasma polymerization, followed by fibronectin (FN) for 1 h (bottom row). (e) Roughness (Rq) and (f) contact angle (3 μL sessile water drop) measurements for flat, NS, and NN with PEA coating and PEA+FN coating. ELISA was used to quantify the amount of (g) FN and (h) BMP2 adsorbed onto flat, NS, and NN samples after one h of coating. (i) The percentage of BMP2 released into solution after PEA+FN coating, quantified at days 1, 3, 5, 7, 9, 12, and 14. (e−h) Average represented as bars with individual values and standard deviation. Comparison of differences was tested using a Kruskal−Wallis test with a p-value <0.05 (*) considered significant, and <0.001 (**) highly significant. Together these results show that the PEA+FN+BMP2 coating could be used on Ti flat, NS, and NN nanotopographies to test with bacteria, hMSCs, and co-cultures.

Article Snippet: FN and BMP2 levels in the supernatants were calculated using enzyme-linked immunosorbent assay (ELISA) duo-set Human Fibronectin (DY1918, R&D systems), and Human BMP2 (DY355, R&D systems), respectively, following instructions from the manufacturer.

Techniques: Functional Assay, Control, Spectroscopy, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison, Bacteria

Journal: Cell Reports Medicine

Article Title: Spatial transcriptomic validation of a biomimetic model of fibrosis enables re-evaluation of a therapeutic antibody targeting LOXL2

doi: 10.1016/j.xcrm.2024.101695

Figure Lengend Snippet: The anti-LOXL2 antibody AB0023 does not inhibit collagen-cross linking, tissue stiffness, or catalytic amine oxidase activity in a disease-relevant model of fibrosis (A) LOXL2 protein levels were assayed in the serum of patients with IPF ( n = 12) and control subjects ( n = 13). (B) Expression of LOXL2 within spatially resolved regions of control and IPF lung tissue. Data from Eyres et al. (C) Representative images of mRNA expression of LOXL2 (green chromagen) and PLOD2 (red chromagen) within a fibroblast focus (∗) of IPF lung tissue and the 3D spheroid model using RNAscope RNA in situ hybridization. Scale bars are 20 μm. Arrows indicating cells with co-expression of LOXL2 and PLOD2 . (D–I) Lung fibroblasts from patients with IPF ( n = 3 donors across 2 independent experiments) were used in the 3D spheroid model in the presence of AB0023 or an isotype control antibody at the same concentrations, as well as with PXS-S2A or its vehicle control (0.1% DMSO). (D) Total mature trivalent (PYD + DPD) collagen cross-links determined by ELISA ( n = 3). (E) Tissue stiffness measured from parallel-plate compression testing determined by Young’s modulus and represented as a proportion of control ( n = 6). (F) Total collagen content determined by hydroxyproline assay. (G) LOXL2 catalytic amine oxidase activity within the conditioned media was assessed using an activity-based probe ( n = 3). (H) VEGFA within cell-conditioned media determined by ELISA. (I) Fibronectin within cell-conditioned media determined by ELISA. Data are mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Unpaired two-tailed t test (A) (t = 2.651, degrees of freedom = 23), Wilcoxon test with Benjamini-Hochberg multiple test correction (B), and ANOVA with Šídák’s multiple comparisons test (D–H) were used to evaluate statistical significance (F values: (D) 7.508, (E) 2.862, (F) 0.8966, (G) 33.06, (H) 34.1, and (I) 45.03. Degrees of freedom: (D) 12, (E) 30, (F) 12, (G) 12, (H) 12, and (I) 12). Error bars are standard deviation.

Article Snippet: VEGFA and Fibronectin concentrations were assayed in conditioned media from 3D spheroid models using Human VEGF DuoSet ELISA and Human Fibronectin DuoSet ELISA kits (RnD systems).

Techniques: Activity Assay, Control, Expressing, RNAscope, RNA In Situ Hybridization, Enzyme-linked Immunosorbent Assay, Hydroxyproline Assay, Two Tailed Test, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Spatial transcriptomic validation of a biomimetic model of fibrosis enables re-evaluation of a therapeutic antibody targeting LOXL2

doi: 10.1016/j.xcrm.2024.101695

Figure Lengend Snippet:

Article Snippet: VEGFA and Fibronectin concentrations were assayed in conditioned media from 3D spheroid models using Human VEGF DuoSet ELISA and Human Fibronectin DuoSet ELISA kits (RnD systems).

Techniques: Control, Recombinant, Luminex, RNAscope, Enzyme-linked Immunosorbent Assay, Gene Expression, Software