Ignite Creation Date:
2024-05-06 @ 10:38 AM
Last Modification Date:
2024-10-26 @ 12:33 PM
Study NCT ID:
NCT03315858
Status:
COMPLETED
Last Update Posted:
2022-02-03
First Post:
2017-10-17
Brief Title:
NFAT-regulated Gene Expression After Tacrolimus
Sponsor:
Medical University of Graz
Organization:
Medical University of Graz
Study Overview
Official Title:
NFAT Nuclear Factor of Activated T-cells-Regulated Gene Expression After Tacrolimus -the Basis for Future Tailored Immunosuppression-
Status:
COMPLETED
Status Verified Date:
2022-02
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief 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 15 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 15 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 ngml phorbol 12-myristate 13-acetate PMA and 5 mcgml ionomycin Sigma-Aldrich Corp StLouis MO USA for 3 hours at 37C Following ex vivo immune activation after red cell lysis with ACK buffer 015 M NH4CL 10 mM KHCO3 leukocytes are lysed with 400 mcl of MagNA-Pure lysis buffer supplemented with an additional 1 Wv of dithiothritol RAS Mannheim Germany and the sample is frozen at -70C 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 cpeakc0x100 where c0 is the adjusted number of transcripts at the TAC predose level and cpeak is the number of transcripts 15 c15 hours after drug intake
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 15 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 ngml phorbol 12-myristate 13-acetate PMA and 5 mcgml ionomycin Sigma-Aldrich Corp StLouis MO USA for 3 hours at 37C Following ex vivo immune activation after red cell lysis with ACK buffer 015 M NH4CL 10 mM KHCO3 leukocytes are lysed with 400 mcl of MagNA-Pure lysis buffer supplemented with an additional 1 Wv of dithiothritol RAS Mannheim Germany and the sample is frozen at -70C 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 cpeakc0x100 where c0 is the adjusted number of transcripts at the TAC predose level and cpeak is the number of transcripts 15 c15 hours after drug intake
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None