Xn 350

Following treatment, samples of the plasma perfusate were assayed for markers of clot degradation. Hemoglobin and D-dimer concentrations were measured as described previously to assess hemolysis and fibrinolysis, respectively [33 (link)]. Briefly, hemoglobin was measured via Drabkin’s reagent assay (Sigma-Aldrich, St. Louis, MO, USA). Aminocaproic acid (100 μg/mL) was added to aliquots used to assess fibrinolysis directly after treatment in order to halt rt-PA activity. A latex immune-turbidimetric assay (STA R Max, Stago, Ansières sur seine, France) was used to quantify D-dimer in fibrinogen equivalent units. An assay (Diagnostica Stago Inc., USA) was used to determine total fibrin degradation products, including those non-specific to rt-PA activity. The assay for non-specific fibrin degradation products was semi-quantitative, and a range of concentrations were reported: 0–10 μg/mL, 10–20 μg/mL, or 20–40 μg/mL. Separate aliquots of the perfusate were assayed to determine the concentration of intact red blood cells and intact platelets shed from the clot during treatment using a hematology analyzer (XN-350, Sysmex, Lincolnshire, IL, USA).

PBMCs were isolated from healthy controls using density centrifugation as described previously. CD4⁺ T-cells were isolated from the PBMC fraction using the EasySep™ CD4⁺ T-cell isolation kit (Stemcell Technologies) as per the manufacturer’s instructions. The T-cells were stained with 2µM CellTrace Violet (Invitrogen). A 96-well plate (Eppendorf) was coated with anti-CD3 (1:1000, Clone OKT3, Invitrogen) for 90min. The coating solution was removed prior to use. Wells without coating served as negative controls.
Monocytes from patients or in vitro polarization, as described above, were counted (XN-350, Sysmex) and resuspended in RPMI-1640 supplemented with 10% foetal calf serum, 2mM L-glutamine and PenStrep. Next, monocytes and T-cells at a 1:10 ratio (monocytes:T-cells) were added to the coated plate in a total volume of 200µl. The cells were incubated for 72h, 37°C, at 5% CO₂. The cells were detached through gentle pipetting, centrifuged, and stained with anti-CD3 (clone UCHT1, alexa fluor 700, 1:200), anti-CD25 (clone M-A251, PerCP Cy5.51:200), anti-HLA-DR (clone G46-6, APC-H71:200) and anti-CTLA-4 (clone BNI3, PE, 1:50), all from BD, for 25min, RT. Finally, the cells were washed once with PBS and analysed using flow cytometry (CytoFLEX).

Finger prick blood samples were subjected to routine blood tests. A routine analyzer (XN-350, SYSMEX, Hyogo, Kobe, Japan) was used for detection. The LYM count, monocyte (MON) count, PLT count and mean platelet volume (MPV) were recorded. Additionally, other hematological parameters were calculated: the LMR is the ratio of lymphocytes to monocytes, the NLR is the ratio of neutrophils to lymphocytes, the MPV/PLT is the MPV divided by the PLT count, and the LYM*PLT is the lymphocyte count multiplied by the platelet count.

Platelet and neutrophil counts were performed in EDTA-anticoagulated whole blood on a Sysmex XN-350 (Sysmex Europe, Norderstedt, Germany) automated cell counter and adjusted for weight change. Urea concentration was measured in mouse plasma using the Quantichrom Urea Assay Kit (BioAssay Systems). Arginase 1 concentration in mouse plasma was measured using a Mouse Arginase 1 ELISA Kit (Abcam, Amsterdam, Netherlands). Arginase activity was measured using the same assay as in human plasma. LDH activity was measured in mouse plasma using Lactate Dehydrogenase Activity Assay Kit (Sigma-Aldrich). Plasma A1M levels were measured using the Mouse alpha-1-microglobulin ELISA Kit (Novus Biologicals, Centennial, CO). The Glomax Discovery System was used for detection.

At time of inclusion, a rheumatologist performed a physical examination and clinical data regarding date of RA debut, smoking habits, radiographic changes, presence of subcutaneous nodules, treatment, and previous vaccinations were collected using a structured protocol. Disease activity score (DAS28) [21 (link)] was used to assess disease activity in RA. Serum levels of rheumatoid factor (RF) and anticitrullinated protein antibodies (ACPA) were analyzed as routine clinical samples at the Department of Clinical Immunology and Transfusion Medicine, Region Skåne, Lund. C-reactive protein (CRP) in plasma, erythrocyte sedimentation rate (ESR), and white blood cell (WBC) count in blood were analyzed as routine clinical samples at the Department of Clinical Chemistry, Region Skåne, Lund. WBC count in blood was also determined with Sysmex XN-350 (Sysmex Europe GmbH).