U plex biomarker group 1

Serum levels of cytokines (TNF-α, IL-17A) were measured in patients by electrochemiluminescence before (guselkumab; n=12, adalimumab; n=11) and 6 months after the treatment with guselkumab (n=5) or adalimumab (n=7). Serum samples were isolated immediately after taking blood from patients and stored in a −80°C freezer; serum samples were not thawed until cytokine measurement. The MESO SCALE DISCOVERY S-PLEX Human IL-17A kit (#K15067L-1, Meso Scale Diagnostics, LLC, Rockville, MD, USA) was used to measure IL-17A (fg/mL), and the U-PLEX Biomarker Group 1 (#K151C3S-1, Meso Scale Diagnostics, LLC, Rockville, MD, USA) was used to measure TNF-α (pg/mL).

Inflammatory cytokines/chemokines were quantified in whole-lung homogenates using a Meso Scale multiplex cytokine array following the manufacturer’s instructions (U-PLEX Biomarker Group 1, Meso Scale Diagnostics). HIF-1α was quantified using ELISA (R&D Systems) and confirmed by Western blot using the ProteinSimple Wes microcapillary system (Bio-Techne) following the manufacturer’s instructions using an anti–HIF-1α Ab (D1S7W, catalog no. 36169S, Cell Signaling Technology). ACE2 was quantified in A549 cell lysates (using anti–ACE-2 Ab, catalog no. 4355S, Cell Signaling Technology) using a ProteinSimple Wes microcapillary Western blot. Total protein levels were determined using the total protein detection module for chemiluminescence assays for Simple Western according to the manufacturer’s instructions (Bio-Techne). The target protein signal was normalized to total protein levels.

In cohorts 1 and 2, serum cytokine (IFN-γ, IL-6, TNF-α, IL-17A, IL-21, IL-22, and IL-23) concentrations were measured. Similarly, these concentrations were also measured in the healthy control (HC) group, matched for age and sex. The concentrations of cytokines at the initiation of bDMARD therapy were measured after a 1-year observation period in all patients in both cohorts. The MESO SCALE DISCOVERY S-PLEX Human IL-17A kit (#K15067L-1, Meso Scale Diagnostics, LLC, Rockville, MD, USA) was used to measure IL-17A (fg/ml), and the U-PLEX Biomarker Group 1 (#K151C3S-1, Meso Scale Diagnostics, LLC, Rockville, MD, USA) was used to measure IFN-γ (pg/ml), IL-6 (pg/ml), TNF-α (pg/ml), IL-21 (pg/ml), IL-22 (pg/ml), and IL-23 (pg/ml) [10 (link)].