NFAT-regulated Gene Expression After Tacrolimus
Condition: NFAT Regulated Gene Expression in Tacrolimus Treated Patients
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        As a pilot trial this study comprises of 15 patients who will undergo liver
        transplantation.
      
Biospecimen description: 
      blood samples
    
Keywords: NFAT; gene expression; tacrolimus