NFAT-regulated Gene Expression After Tacrolimus

NFAT (Nuclear Factor of Activated T-cells)-Regulated Gene Expression After Tacrolimus -the Basis for Future Tailored Immunosuppression-

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.

Study Overview

• Status: Completed
• Conditions: NFAT Regulated Gene Expression in Tacrolimus Treated Patients

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.

• Study Type: Observational
• Enrollment (Actual): 40

Contacts and Locations

Study Locations

• Austria
  • Graz, Austria, 8036
    • Medical University of Graz, Klin. Abteilung für Transplantationschirurgie

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 14 years to 86 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

As a pilot trial this study comprises of 15 patients who will undergo liver transplantation.

Description

Inclusion Criteria:

• Patients > 18 years of age
• Liver transplantation
• Informed consent

Exclusion Criteria:

• Patients < 18 of years
• Patients taking actively part in a different interventional trial

Study Plan

How is the study designed?

Design Details

• Observational Models: Cohort
• Time Perspectives: Prospective

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
correlation of NFAT-RGE and TAC peak levels	Calculation: cpeak/c0x100	1 year

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
correlation of both NFAT-RGE and TAC (trough and peak) levels with rejection episodes and TAC side effects	Calculation: cpeak/c0x100	1 year

Collaborators and Investigators

• Sponsor: Medical University of Graz

Study record dates

Study Major Dates

• Study Start (Actual): January 18, 2019
• Primary Completion (Actual): November 29, 2021
• Study Completion (Actual): November 29, 2021

Study Registration Dates

• First Submitted: October 17, 2017
• First Submitted That Met QC Criteria: October 17, 2017
• First Posted (Actual): October 20, 2017

Study Record Updates

• Last Update Posted (Actual): February 3, 2022
• Last Update Submitted That Met QC Criteria: February 2, 2022
• Last Verified: February 1, 2022

More Information

Terms related to this study

• Keywords: tacrolimus; gene expression; NFAT
• Other Study ID Numbers: NFAT1

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No