il 2 dy202  (R&D Systems)

Journal: Journal of Cardiovascular Pharmacology

Article Title: Evidence of Cardiotoxic Immune Activation by Triple Immune Checkpoint Blockade: A Translational Alert for Clinical Surveillance in Patients With Cancer

doi: 10.1097/FJC.0000000000001798

Figure Lengend Snippet: Effects of nivolumab–relatlimab combined with ipilimumab or atezolizumab on secretion of cytokines in the supernatant of cocultures of hPBMCs with HFC cells. HFC cells were cocultured with lymphocytes (effector–target ratio 5:1) and treated for 48 hours at 37°C with the combination of nivolumab + relatlimab (light gray bars), ipilimumab or atezolizumab (dark gray bars) used alone, or in combinations (black bars) at the indicated concentration. IL-2 (A) and granzyme B (B) levels (pg/mL) were obtained by using the duoset ELISA kit from R & D Systems performed on cell supernatants. Cells untreated or treated with an unrelated hIgG (with bars) were used as negative controls. Error bars depict means ± SD. P -values for the combinations relative to respective single agents are *** P < 0.001; ** P < 0.01.

Article Snippet: Supernatants were collected after 48 hours of coculture and analyzed for IL-2 (DY202) and granzyme B (DY2906) concentrations using DuoSet ELISA kits (R&D Systems, Minneapolis, MN) endowed with a sensitivity of 50 pg/mL.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Pharmacology

Article Title: Evidence of Cardiotoxic Immune Activation by Triple Immune Checkpoint Blockade: A Translational Alert for Clinical Surveillance in Patients With Cancer

doi: 10.1097/FJC.0000000000001798

Figure Lengend Snippet: Proposed mechanism of immune checkpoint inhibitor (ICI)-driven disruption of immune tolerance and cardiomyocyte injury. Under physiologic conditions, regulatory T cells (Tregs) and inhibitory checkpoint pathways—including cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1)—cooperate to maintain peripheral tolerance and protect cardiac tissue from autoreactive immune responses. ICI therapy blocks these inhibitory signals at multiple levels: (I) anti-CTLA-4 antibodies prevent CTLA-4 engagement on both activated T cells and Tregs, thereby dampening Treg-mediated suppression and enhancing effector T-cell activation; (ii) anti-LAG-3 antibodies abrogate an additional inhibitory checkpoint on activated T cells, further potentiating T-cell cytotoxicity; and (iii) antibodies targeting PD-1 or PD-L1 disrupt the PD-1/PD-L1 axis, which normally delivers inhibitory signals on engagement of PD-1 on activated T cells with PD-L1 expressed on cardiomyocytes. Cumulative blockade of these checkpoints removes critical layers of peripheral tolerance and unleashes effector T cells that recognize cardiac antigens as targets, leading to immune attack against cardiomyocytes. This loss of tolerance contributes to proinflammatory cytokine secretion (eg, IL-2, IL-1β, IL-6, IL-8, CCL2, IL-17), enhanced cytotoxic mediator release such as granzyme B, and activation of the cardiomyocyte NLRP3–MyD88–NF-κB axis, ultimately driving myocardial inflammation and injury. The figure summarizes the central paradigm supported by our coculture experiments, wherein triplet checkpoint blockade (anti-CTLA-4 + anti-PD-1 + anti-LAG-3 or anti-PD-L1) produced the strongest disruption of immune tolerance and the most pronounced cardiotoxic effects.

Article Snippet: Supernatants were collected after 48 hours of coculture and analyzed for IL-2 (DY202) and granzyme B (DY2906) concentrations using DuoSet ELISA kits (R&D Systems, Minneapolis, MN) endowed with a sensitivity of 50 pg/mL.

Techniques: Disruption, Activation Assay, Produced

Journal: British Journal of Biomedical Science

Article Title: Formulation and In Vitro Characterisation of Withaferin A-Loaded Liposomal Gels for the Topical Management of Chronic Inflammatory Skin Conditions

doi: 10.3389/bjbs.2025.14847

Figure Lengend Snippet: Withaferin A (WA) Reduces TNF-α-Induced Inflammation and Matrix Metalloproteinase levels in HUVEC and HDFa’s. (A) IL-6 secretion in HUVECs and HDFa’s and treatment with Withaferin A-loaded liposomes (either 1 mg/mL noted as WA-L or 5 mg/mL noted as WA-H), resulted in a concentration-dependent reduction of IL-6 levels. (B) MMP-9 secretion in HDFa’s was significantly elevated by TNF-α stimulation however treatment with Withaferin A-loaded liposomal gel reduced MMP-9 levels. Cells were stimulated with 5 ng/mL TNF-α for 3 h and treated with Withaferin A-loaded liposomal gels (WA-L and WA-H corresponding to a Withaferin A loading to 1 and 5 mg/mL) for 24 h followed by appropriate ELISA analysis. Data are presented as mean ± standard deviation, n = 3 independent experiments. Statistical significance was assessed by One-way ANOVA: **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To assess the anti-inflammatory and matrix remodelling regulatory potential of Withaferin A, levels of IL-6 and MMP9 were quantified in cell culture supernatants using the Human DuoSet enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, USA, DY202 and DY911 respectively).

Techniques: Liposomes, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation