Investigations.  Blood samples were obtained from patients while they were fasting for measurement of levels of glucose, insulin, lipids, urea, uric acid, creatinine, aminotransferases, thyroid stimulating hormone (TSH) and cortisol profile. An oral glucose tolerance test was then performed with the administration of 1.75 g of glucose per kilogram of body weight (maximal dose – Belnacasan datasheet 75 g). Ambulatory blood pressure monitoring (ABPM).  Blood pressure was measured three times using mercury sphygmomanometer with appropriate cuff size according to the

American Heart Association guidelines. Additionally, all subjects’ blood pressure was monitored for 24 h with the use of ABPM monitor and analysed after completion in the appropriate software. Flow cytometry.  Mononuclear cells were isolated from peripheral blood by centrifugation over Histopaque (Sigma). A flow cytometric analysis of T cell subpopulations was performed using the following markers: anti-CD3 (phycoerythrin-cyanin 5 PECy5 conjugated, UCHT1 clone), anti-CD4 (phycoerythrin-cyanin 7 PECy7 conjugated, SFCI12T4D11 clone), anti-CD25 (phycoerythrin-Texas Red ECD conjugated, AG-014699 clinical trial B1.49.9 clone), anti-CD127 (=IL-7R, fluorescein isothiocyanate FITC

conjugated, eBioRDR5 clone) and FoxP3 (phycoerythrin PE conjugated, 259D/C7 clone) purchased from Beckman Coulter (Brea, CA, USA), Beckton Dickinson (San Jose, CA, USA) and eBioscience (San Diego, CA, USA). Respective isotype control antibodies were used. Intracellular staining 17-DMAG (Alvespimycin) HCl was performed according to the manufacturer’s instructions (Fix/Perm Buffer from Beckton Dickinson). The samples were analysed by five-colour flow cytometer Beckman Cytomics FC 500 MPL using CXP software ver 2.0 (Beckman Coulter). A minimum of 105 events were acquired for each analysis. The percentages of positive cells were calculated. To determine absolute cell counts, a small volume of blood was analysed for complete blood count (CBC) with differential. The

absolute counts were determined by multiplying the frequency of positive cells obtained in cytometric analysis by the number of lymphocytes [G/l] as determined by CBC. The following subpopulations were noted: CD4+,CD4+CD25high,CD4+CD127low/−,CD4+CD25highCD127low/−, CD4+CD25highFoxP3+. Cell separation.  T regulatory cells were isolated from mononuclear cells according to the producer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolation of CD4+CD25+CD127dim/− regulatory T cells was performed in a two-step procedure. First, non-CD4+ and CD127high cells were indirectly magnetically labelled with a cocktail of biotin-conjugated antibodies and Anti-Biotin MicroBeads. The labelled cells were subsequently depleted by separation over a MACS® Column. In the second step, CD4+CD25+CD127dim/− regulatory T cells were directly labelled with CD25 MicroBeads and isolated by positive selection from the pre-enriched CD4+ T cell fraction.