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 W Burke, Jose Nery, David Roth, Violet Esquenazi, Joshua Miller

Research output: Contribution to journalArticle

12 Citations (Scopus)

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
Pages (from-to)227-234
Number of pages8
JournalHuman Immunology
Volume36
Issue number4
DOIs
StatePublished - Jan 1 1993

Fingerprint

Immunosuppressive Agents
Cyclosporine
Interleukin-2
Transplants
Kidney
Polymerase Chain Reaction
Messenger RNA
Physiologic Monitoring
Serum
Mitogens
Thymidine
Healthy Volunteers
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy

Cite this

Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction. / Koutouby, Raghad; Zucker, Carol; Zucker, Keith; Burke, George W; Nery, Jose; Roth, David; Esquenazi, Violet; Miller, Joshua.

In: Human Immunology, Vol. 36, No. 4, 01.01.1993, p. 227-234.

Research output: Contribution to journalArticle

Koutouby, Raghad ; Zucker, Carol ; Zucker, Keith ; Burke, George W ; Nery, Jose ; Roth, David ; Esquenazi, Violet ; Miller, Joshua. / Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction. In: Human Immunology. 1993 ; Vol. 36, No. 4. pp. 227-234.
@article{7fdd81a3faa84388aedf9a6610056d4a,
title = "Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction",
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.",
author = "Raghad Koutouby and Carol Zucker and Keith Zucker and Burke, {George W} and Jose Nery and David Roth and Violet Esquenazi and Joshua Miller",
year = "1993",
month = "1",
day = "1",
doi = "10.1016/0198-8859(93)90129-O",
language = "English",
volume = "36",
pages = "227--234",
journal = "Human Immunology",
issn = "0198-8859",
publisher = "Elsevier Inc.",
number = "4",

}

TY - JOUR

T1 - Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction

AU - Koutouby, Raghad

AU - Zucker, Carol

AU - Zucker, Keith

AU - Burke, George W

AU - Nery, Jose

AU - Roth, David

AU - Esquenazi, Violet

AU - Miller, Joshua

PY - 1993/1/1

Y1 - 1993/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0027318433&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027318433&partnerID=8YFLogxK

U2 - 10.1016/0198-8859(93)90129-O

DO - 10.1016/0198-8859(93)90129-O

M3 - Article

C2 - 8340231

AN - SCOPUS:0027318433

VL - 36

SP - 227

EP - 234

JO - Human Immunology

JF - Human Immunology

SN - 0198-8859

IS - 4

ER -