Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction.
We have developed a rapid (24 hours) quantitative PCR assay to measure the direct effect of cyclosporine on IL-2 mRNA production by activated PBMC cultures from renal transplant patients. The PBMCs were purified from normal laboratory volunteers (group A, n = 26), CsA-treated renal transplant patients with good renal function, tested between 3 and 8 weeks (group B, n = 14) or between 2 and 8 years (group C, n = 15) after surgery, and stimulated with PHA in media supplemented with either patient serum or pooled commercially obtained AB serum. The mRNA was then isolated and, using semiquantitative PCR or quantitative PCR with a competitive inhibitor, the relative levels or exact levels of IL-2 mRNA (in attomoles) could be measured. A 3-day confirmatory lymphoproliferation assay of [3H]thymidine incorporation was also performed on the samples. Kinetic analysis of the data from group A showed that the peak level of IL-2 transcription into mRNA occurred at 6 hours after mitogen stimulation. Increasing in vitro concentrations of CsA in this group resulted in lower IL-2 mRNA levels and a shift in the peak time to 12-24 hours. In the transplant recipients, there was no correlation between individual CsA blood levels and proliferation responses. However, some correlation was found between CsA blood levels and IL-2 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Koutouby
R
R
Department of Pediatrics, University of Miami School of Medicine, Florida 33101.
Zucker
C
C
Zucker
K
K
Burke
G
G
Nery
J
J
Roth
D
D
Esquenazi
V
V
Miller
J
J
D001483:Base Sequence; D016572:Cyclosporine / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000187:drug effects; D006084:Graft Rejection / Q000517:prevention & control; D006801:Humans; D007165:Immunosuppression Therapy; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology*; D016030:Kidney Transplantation / Q000276:immunology*; D007700:Kinetics; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D015166:Monitoring, Immunologic; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*
D007376:Interleukin-2; D012333:RNA, Messenger; D016572:Cyclosporine