Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction

Raghad Koutouby, Carol Zucker, Keith Zucker, George Burke, Jose Nery, David Roth, Violet Esquenazi, Joshua Miller

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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. Although there was no difference in mean proliferation responses or in mean whole blood CsA levels between groups B and C, mean IL-2 mRNA levels were 38% and 68% of the group A levels, respectively, indicating that standard CsA blood level alone may not reflect the immunosuppressive effect of the drug. Therefore, it is proposed that this, a rapid, sensitive, and quantitative PCR analysis of IL-2 mRNA should be useful in longitudinal monitoring of patients who may not be appropriately suppressed at "therapeutic" CsA blood levels.

Original languageEnglish (US)
Pages (from-to)227-234
Number of pages8
JournalHuman Immunology
Volume36
Issue number4
DOIs
StatePublished - Apr 1993

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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