Blood collection

At all three visits, phlebotomists drew two 3 ml serum tubes, one 5 ml EDTA-containing tube and one 3 ml Li-Heparin tube. The blood in serum tubes was allowed to clot at room temperature for 30 minutes. All tubes were centrifuged to separate plasma or serum. EDTA-plasma was divided over four 0.5 ml tubes, which were stored at -80 degrees until analysis. Serum and Li-Heparin tubes were transported to an external laboratory (Hospital Gelderse Vallei, Ede, The Netherlands) for same-day analysis. Results of kidney function (estimated glomerular filtration rate, eGFR) were obtained on the same day to immediately exclude participants when eGFR dropped below <30 ml/min /1.73m². Levels of vitamin D (by Liquid chromatography-mass spectrometry), IGF-1 (by Luminescence-enhanced immuno-enzymatic assay) and albumin (by bromocersol purple method) were measured at the laboratory of Hospital Gelderse Vallei.

At the first and last visit, additional blood was collected in 9 ml citrate tubes for immunological assessments. These tubes were transported to an external laboratory (Sanquin, Amsterdam, The Netherlands), where peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque separation, washed and counted. PBMC composition (T-cells, B-cells, monocytes and natural killer (NK) cells) was assessed by flow cytometry. PBMCs were stimulated (in triplicate) with a mix of Tetanus Toxoid, Tubulin PPD and Candida Albicans to trigger a memory reaction of T-cells. Production of interferon-gamma (IFNγ) and interleukin-13 (IL-13) was measured in supernatants by enzyme-linked immunosorbent assays.