Advia 2021i

Full blood count and hemoglobin was assessed every 24 h through a hematocytometer (Siemens Advia 2021i, Erlangen, Germany). At the same time points, serum iron was measured using a colorimetric assay (Sekisui Diagnostics, Lexington, MA). Total iron-binding capacity (TIBC) was measured using a colorimetric assay (Pointe Scientific). Transferrin levels were calculated using the formula TIBC (μmol/L)/25.1. Transferrin saturation was calculated with the formula serum iron/(25.1 × total transferrin) × 100%. Ferritin (LsBio), hepcidin (LsBio), and interleukin 6 (IL-6) levels (Millipore Iberica, S.A., Madrid, Spain) were measured by enzyme-linked immunosorbent assay kits. Lung tissue was homogenized and cultured on agar plates at 37 °C with 5% CO₂. The number of CFUs was counted the next day.

During the surgical procedure, animals (10-week-old female and male Wistar rats, Charles River Germany) were kept under continuous 2% isoflurane anesthesia and the skin was cleaned and disinfected by Betaseptic^®. A midline incision was made from the base of the neck posteriorly over the thorax (2–2.5 cm). The thorax was opened by cutting through the sternal manubrium and pectoral muscles, the connective tissue was torn/snipped and the thymus was pulled out with the aid of a slim cotton-tipped applicator and artery forceps. To prevent pneumothorax, the chest was gently compressed between thumb and forefingers before the cavity was closed and sutured. In sham-operated animals, the same procedure was applied without removal of the thymus. Animals were allowed to recover for at least 2 weeks after thymectomy (or sham-operation) before start of compound treatment.
MLT-943 was applied at 20 mg/kg/day p.o., from the 2nd week post thymectomy onward and for 9 weeks. Leukocyte populations in EDTA-anticoagulated blood were measured using an ADVIA 2021i (Siemens Medical Solutions Diagnostics).