However, buy BAY 73-4506 to date, the expression level of CD30 on the cell surface of CD4 and CD8 lymphocyte subsets in patients with SLE and its role in the pathogenesis are not known. We have focused our study in the determination of CD30 expression on CD3 T lymphocytes and CD4/CD8

subsets from SLE patients mainly with lupus nephritis. The intracellular level of the cytokines, IL-4, interferon γ (IFNγ), IL-10, and transforming growth factor β (TGFβ), were also investigated in the CD3 T cell population to analyse their relationship with the CD30 expression. Ten healthy volunteers from the blood bank and twenty-one patients with SLE from the Nephrology Section of our Hospital were included in this research. All of them gave their informed consent, as well as patients with SLE fulfilled the American College of Rheumatology revised criteria [16]. Eighteen patients were women (18/21) and three were men (3/21), with a mean age of 43.67 ± 13.81 (mean ± SD) years. The mean age of healthy controls (7 women and 3 men) was 38 ± 12 years. Ten of 21 patients (10/21) presented positivity for antibodies to double-stranded DNA (anti-dsDNA). The mean for the serum levels of C3 and C4 complement factors was 98.57 ± 24.75 mg/dl (normal range: 83–175 mg/dl)

and 16.86 ± 7.78 mg/dl (normal range: 15–45 mg/dl), respectively. Disease activity was assessed by SLE-Disease Activity Index (SLEDAI): seventeen patients had inactive SLE, and four

patients presented active SLE with SLEDAI >4 [17]. According to the WHO classification, five patients did not present lupus nephritis, and the remaining ones had a different Ibrutinib in vivo grade of renal alteration: (1) 12 with class IV, (2) 2 with class V, (3) 1 with class III and (4) 1 with class II [18]. The patients with nephritis were treated with mycophenolate mofetil (n = 12) and cyclophosphamide (n = 4), and the patients without renal alteration were treated with a low dose of prednisone and/or hydroxychloroquine. The cells were isolated from heparinized venous blood by density-gradient centrifugation (Ficoll-Hypaque, Sigma-Aldrich, St. Louis, MO, USA). Afterwards, mononuclear cells were washed twice in phosphate-buffered saline (PBS) and resuspended in 1.5 ml of RPMI-1640 cell culture Bcl-w medium (Gibco, Scotland, UK) supplemented with streptomycin (100 IU/ml) and penicillin (100 IU/ml). For basal staining conditions, 0.5 ml of diluted lymphocytes obtained immediately after cell isolation remained as non-stimulated. Lymphocyte cells at a concentration of 1 × 106/ml (1 ml per tube) were stimulated for 24 h with 50 ng/ml of phorbol myristate acetate (PMA) (Sigma-Aldrich, Steinheim, Germany) and 1 μm of ionomycin (Sigma-Aldrich, Steinheim, Germany) in 5% CO2 at 37 °C. A protein transport inhibitor (BD GolgiPlug™, Becton Dickinson) was added to the last 5 h of incubation time for the intracellular cytokine staining protocol.