NFAT-regulated Gene Expression After Tacrolimus

NFAT Regulated Gene Expression in Tacrolimus Treated Patients

NFAT, gene expression, tacrolimus

Aim of the study is measurement of NFAT-RGE (IL-2 (interleukin-2), IFN-γ (interferon-gamma), GM-CSF (granulocyte monocyte colony stimulating factor)) after tacrolimus (TAC) in de-novo immunosuppressed patients after liver transplantation (LT), to test the hypothesis that in de-novo TAC patients receiving mycophenolate mofetil (MMF) and steroids after LT there is an inverse correlation of NFAT-RGE and TAC peak levels at 1.5 hours after TAC intake.

The trial will be conducted as a prospective, longitudinal study. The study is a single-centre study performed at the Medical University of Graz, Department of Surgery, Division of Transplant Surgery and the Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz. All the patients on the LT wailting list at the Division of Transplant Surgery, Medical University of Graz are screened according to both inclusion and exclusion criteria. The study period is 1 year. One NFAT-RGE baseline measurement is performed directly before LT; NFAT-RGE-measurements after LT are performed at clearly defined timepoints. Study population. As a pilot trial this study comprises of 15 patients who will undergo LT Objectives: * To test the hypothesis that in de-novo TAC patients receiving mycophenolate mofetil (MMF) and steroids after LT there is an inverse correlation of NFAT-RGE and TAC peak levels at 1.5 hours after TAC intake * To correlate both NFAT-RGE and TAC (trough and peak) levels with rejection episodes and TAC side effects Approach: NFAT-RGE is determined in 15 patients just before LT, and after LT after 1 day, 1 week, 2 weeks, and after 1, 6 and 12 months. Methods. Heparinized peripheral blood is stimulated with 1 ml of complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 100 ng/ml phorbol 12-myristate 13-acetate (PMA) and 5 mcg/ml ionomycin (Sigma-Aldrich Corp., St.Louis, MO, USA) for 3 hours at 37°C. Following ex vivo immune activation, after red cell lysis with ACK buffer (0.15 M NH4CL, 1.0 mM KHCO3), leukocytes are lysed with 400 mcl of MagNA-Pure lysis buffer supplemented with an additional 1% (W/v) of dithiothritol (RAS, Mannheim, Germany), and the sample is frozen at -70°C. After thawing, mRNA is isolated with the RNA Blood mini Kit (Quiagen) device using the mRNA standard protocol for cells. RNA is reverse transcribed using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, life technologies). The 3 NFAT-regulated genes (IFN-γ, IL-2, GM-CSF) were identified as suitable genes for this essay from previous studies \[18, 26\]. Target mRNA sequences of IFN- γ, IL-2, GM-CSF and 3 reference genes are amplified using commercially available PrimePCR ddPCR Gene Expression Probe Assays (Biorad) by digital PCR (Biorad). The RGE after TAC intake is calculated as cpeak/c0x100, where c0 is the adjusted number of transcripts at the TAC predose level and cpeak is the number of transcripts 1.5 (c1.5) hours after drug intake.

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