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Impact of single immunosuppressive drug withdrawal on lymphocyte immunoreactivity

Published:November 25, 2013DOI:https://doi.org/10.1016/j.jss.2013.11.1085

      Abstract

      Background

      Chronic rejection is a major cause of graft loss in kidney transplant recipients. Nonadherence to drug therapy is a well-recognized cause of chronic rejection leading to long-term graft dysfunction and failure for transplant recipients. Immunosuppressive medications with short half-lives that require frequent dosing, such as tacrolimus, complicate transplant regimens and may increase noncompliance. Regimens could be simplified using drugs with long half-lives requiring once-daily administration, such as sirolimus. The impact of missing doses of single agents has not been studied extensively. Erratic compliance or temporary discontinuation of immunosuppressive drugs may have significant implications for chronic rejection.

      Methods

      Our study evaluated the impact of single drug withdrawal of commonly used immunosuppressive agents (sirolimus and tacrolimus) on lymphocyte responses. We analyzed lymphocyte proliferation, cytokine secretion, and adenosine triphosphate generation using a crossover study design with normal healthy patients. Lymphocyte proliferation was assessed using 5-bromo-2-deoxyuridine incorporation, and T cell function was analyzed by examining adenosine triphosphate generation.

      Results

      Our results indicate that sirolimus exerts prolonged suppression of lymphocyte proliferation and decreased interleukin 17A that lasts up to 48 h after drug withdrawal. In comparison, tacrolimus did not have a similar effect on lymphocyte proliferation or interleukin 17A secretion.

      Conclusion

      Future analysis of sirolimus in diverse transplantation populations merits investigation.

      Keywords

      1. Introduction

      Nonadherence with immunosuppressive drug regimens remains a major problem in the long-term management of transplant patients. Presently, the most common regimens include a calcineurin inhibitor (CNI), such as cyclosporine or tacrolimus, which blocks calcineurin signal transduction and prevents activation of the transcription factor nuclear factor of activated T cells. Alternative agents that block the mammalian target of rapamycin (mTOR) pathway, including sirolimus, are available but used less commonly because of the repression of wound healing and dyslipidemia. CNIs have a shorter half-life (approximately 16–27 h for cyclosporine in patients with normal liver function [
      • Naesens M.
      • Kuypers D.R.
      • Sarwal M.
      Calcineurin inhibitor nephrotoxicity.
      ] and 12 h for tacrolimus [
      • Rostaing L.
      • Massari P.
      • Garcia V.D.
      • Mancilla-Urrea E.
      • Nainan G.
      • del Carmen Rial M.
      • et al.
      Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study.
      ]) than sirolimus and are dosed twice a day. In contrast, sirolimus has a longer half-life (62 h [
      • Sommerer C.
      • Schnitzler P.
      • Meuer S.
      • Zeier M.
      • Giese T.
      Pharmacodynamic monitoring of cyclosporin a reveals risk of opportunistic infections and malignancies in renal transplant recipients 65 years and older.
      ]) and is dosed once a day. Thus, the effective management of CNIs requires frequent monitoring of serum drug concentrations and patient adherence.
      A recent cross-sectional study of transplant recipients concluded that there is significantly lower graft survival in patients who are assessed to be nonadherent to transplant medication regimens. As expected, the study also revealed higher late acute rejection rates in this group [
      • De Geest S.
      • Borgermans L.
      • Gemoets H.
      • Abraham I.
      • Vlaminck H.
      • Evers G.
      • et al.
      Incidence, determinants, and consequences of subclinical noncompliance with immunosuppressive therapy in renal transplant recipients.
      ]. A prospective trial indicated that 47% of transplant recipients with acute rejection are identified as nonadherent [
      • Sellares J.
      • de Freitas D.G.
      • Mengel M.
      • Reeve J.
      • Einecke G.
      • Sis B.
      • et al.
      Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.
      ].
      Sommerer et al. [
      • Sommerer C.
      • Schnitzler P.
      • Meuer S.
      • Zeier M.
      • Giese T.
      Pharmacodynamic monitoring of cyclosporin a reveals risk of opportunistic infections and malignancies in renal transplant recipients 65 years and older.
      ] have shown a close relationship between CNI concentrations (cyclosporine or tacrolimus) over the dosing cycle and cytokine transcription, which could promote the development of graft rejection. An analysis of pediatric patients (aged 8–18 y) showed that patients who received a heart, lung, or kidney transplant and had a higher variability in tacrolimus concentrations were at an increased risk of graft loss and late rejection [
      • Pollock-Barziv S.M.
      • Finkelstein Y.
      • Manlhiot C.
      • Dipchand A.I.
      • Hebert D.
      • Ng, V.L.
      • et al.
      Variability in tacrolimus blood levels increases the risk of late rejection and graft loss after solid organ transplantation in older children.
      ]. One interpretation is that nonadherence may be a contributing factor to the variability in tacrolimus concentrations.
      We used a crossover study design of normal healthy patients treated with a single-drug regimen of tacrolimus or sirolimus and analyzed the drug concentration, lymphocyte proliferation, cytokine secretion, and adenosine triphosphate (ATP) generation. In this study, we tested the hypothesis that sirolimus will have greater drug concentrations over the dosing interval and maintain greater immunosuppressive effects. We also postulated that these effects are sustained. Consequently, if a dose of sirolimus is taken irregularly or intermittently, this may be less likely to result in an enhanced immune response that promotes graft rejection. Recently, interleukin 17A (IL-17A) has been implicated to have a role in graft rejection [
      • Snell G.I.
      • Levvey B.J.
      • Zheng L.
      • Bailey M.
      • Orsida B.
      • Williams T.J.
      • et al.
      Interleukin-17 and airway inflammation: a longitudinal airway biopsy study after lung transplantation.
      ]. T helper 17 cells are the most recently identified member of the CD4+ T cell subset and secrete IL-17A [
      • Weaver C.T.
      • Harrington L.E.
      • Mangan P.R.
      • Gavrieli M.
      • Murphy K.M.
      Th17: an effector CD4 T cell lineage with regulatory T cell ties.
      ]. There are several isoforms of IL-17 that include IL-17A to IL-17F, and they are homodimeric peptides between 35 and 52 kD [
      • Crome S.Q.
      • Wang A.Y.
      • Levings M.K.
      Translational mini-review series on Th17 cells: function and regulation of human T helper 17 cells in health and disease.
      ]. In this study, we analyzed the impact of the withdrawal of sirolimus and tacrolimus on immune reactivity and the levels of IL-17A.

      2. Materials and methods

      2.1 Patients

      Patients were recruited from the general population in San Diego (n = 5) in accordance with the University of California, San Diego Human Research Protection Program. Inclusion criteria for study patients were age 18–65 y, body mass index 30 or less, and normotensive with no current or recent infections. Exclusion criteria included a previous history or existing health problems, such as diabetes, hypertension, heart disease, kidney disease, recurrent infections, neoplasm (or history of such), chronic obstructive pulmonary disease, peripheral vascular disease (no palpable foot pulses), and/or prescription, or over the counter medications except for occasional acetaminophen or non–steroidal anti-inflammatory drugs. A signed informed consent form was obtained from each participant before any study procedures. Patients with proteinuria >300 gm/dL, fasting cholesterol >300 mg/dL, fasting triglycerides >400 mg/dL, platelets <100,000/mm3, and/or white blood cell count <2.5 mm3 were excluded.
      After the initial recruitment phase, a complete chemistry panel, including liver function tests, complete peripheral blood count with manual normal white cell differential, lipid panel, hemoglobin A1c (HgbA1C), and urinalysis were obtained to rule out any occult abnormalities. Pregnancy tests were done for female patients to rule out pregnancy and patients were counseled to use an effective form of contraception during the entire trial through the 30-d safety follow-up visit.
      After recruitment, patients underwent a baseline 2-h oral glucose tolerance test (OGTT) and received a single test dose administration of the assigned medication, tacrolimus or sirolimus, to determine the optimal dose.

      2.2 Pharmocokinetics models

      Population and individual patient pharmacokinetic modeling was performed using NONMEM (version 6; Icon US, Ellicott City, MD). A two-compartment model (subroutine advan4 trans1) with first-order absorption pharmacokinetics was calculated using the tacrolimus dose and the blood concentration values (blood concentration at t = 0, 2, 12, 24, and 48 h) obtained from five healthy patients. For sirolimus, a two-compartment model was calculated using the sirolimus dose and the blood concentration values (blood concentration at t = 0, 2, 24, 48, and 96 h) obtained from the same five healthy patients. Maximum likelihood estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (Vd/F).

      2.3 Study arms and samples

      The drug treatment phase lasted 3 d for tacrolimus and sirolimus with the last dose taken on the morning of the fourth day. Peripheral whole blood samples were collected at the baseline visit before starting medications. For tacrolimus, concentrations were measured at timed intervals of 2 h and 48 h after the final dose. Sirolimus concentrations were measured at 24, 48 and 96 h after the final dose because of the long half-life of sirolimus. Tacrolimus and sirolimus levels were measured from the whole blood using a microparticle enzyme immunoassay (Cylex, Columbia, MD).

      2.4 Mitogen stimulation

      Peripheral blood mononuclear cells were analyzed using a mitogen stimulation assay (Cylex, Columbia, MD). This assay quantitates the level of intracellular ATP. A mitogen stimulant (phytohemagglutinin) was added to the whole blood samples and the cells were incubated at 37°C for 16–18 h. Control samples were incubated without phytohemagglutinin. The CD4+ T cells were purified with magnetic beads coated with anti-CD4 monoclonal antibody. The purified CD4+ T cells were lysed to release the intracellular ATP, which was read using a luminometer. (http://www.cylex.net/pdf/ImmuKnow_Insert-cx.pdf). The assay results were normalized for area under the curve (AUC) for drug exposure. The results in the two study arms were analyzed using paired t-test to assess the significant difference set at P value <0.05.

      2.5 Levels of IL-17A

      IL-17A levels were assessed using peripheral blood CD4+ T cells stimulated with anti-CD3/CD28. A commercially available enzyme-linked immunosorbent assay kit was used to quantify the cytokines (per protocol provided by manufacturer eBioscience, San Diego, CA ). A paired t-test analysis was used to assess for significance.

      2.6 Lymphocyte proliferation

      Lymphocyte proliferation was determined by incorporating BrdU (5-bromo-2-deoxyuridine) during DNA synthesis. The peripheral blood samples were processed with (Ficoll-Paque, Pittsburgh, PA) to obtain mononuclear cells. The CD4+ T cells were isolated with an indirect magnetic labeling system and activated using (Dynabeads, Grand Island, NY) coated with human anti-CD3/CD28 antibodies. The cells were cultured in a 96-well plate at 37°C for 48 h. BrdU was added to the cultures for the last 6 h of incubation. The absorbance was measured at 370 nm using an enzyme-linked immunosorbent assay plate reader.

      2.7 Urine protein/creatinine ratios

      Urine samples were collected before the start of the study, during administration of the drug, and 1 wk after the completion of drug administration. These samples were evaluated for urine protein/creatinine ratios.

      2.8 Statistical methods

      Paired t-test analysis was used to evaluate the significant differences between the groups. Analysis of variance was performed on the OGTT to determine if there was a significant change in glucose handling during the drug administration. Data were stored and analyzed in (Microsoft Excel, Redman, WA).

      3. Results and discussion

      3.1 Results

      Normal patients were enrolled and all participated in the two study arms: sirolimus and tacrolimus administration (Table 1). Drug effects on lymphocyte activation were determined by mitogen responses, proliferation, and cytokine production. These patients had no underlying medical conditions and were not on any prescribed medications. There were no significant differences in blood pressure, baseline creatinine, or HgbA1c. The median age of the patients was 49. The systolic blood pressures of study patients ranged from 101–124 mm Hg. Average HgbA1c was approximately 5.4%. There were no dropouts and no observed complications during the study or at 1 mo safety follow-up visit.
      Table 1Characteristics of patients. Baseline demographics and other characteristics of the study patients.
      PatientAgeSexRaceBMIBPCreatinineUr pr/crHgb A1 CT Chol
      133MHispanic29.2113/660.900.045.6185
      249MCaucasian28.6120/780.880.045.5226
      350MCaucasian29.4124/811.020.035.6240
      454MCaucasian24.8105/720.720.065.5188
      519FHispanic26.3101/600.710.045.2191
      BMI = body mass index; BP = blood pressure; T Chol = total cholesterol; Ur pr/cr = urine total protein/creatinine spot ratio.
      Characteristics of patients: no significant difference was detected in baseline blood pressure, HgbA1C, creatinine, and total cholesterol (T chol). The baseline urine total protein/creatinine ratios ranged from 0.03–0.06 mg/dL. None of the participants had preexisting medical conditions or were taking prescription medications.
      Pharmacokinetic parameters for sirolimus and tacrolimus were analyzed (Table 2, Table 3). For the sirolimus group, the mean AUC(0–24) (ng*h/mL) was 0.462 with a standard deviation of 0.125. The mean half-life for sirolimus was 63.7 h with a standard deviation of 7 h. For the tacrolimus group, the mean AUC was 0.220 with a standard deviation of 0.07. The mean half-life for tacrolimus was 40 h with a standard deviation of 9 h. The drug concentrations for tacrolimus were within the recommended clinical therapeutic range for all patients (Fig. 1A). The drug concentrations for sirolimus were also within clinically relevant therapeutic ranges (Fig. 1B).
      Table 2Pharmacokinetic parameters of sirolimus concentration.
      IDDOSE (mg)V2 (L)V3 (L)KA (h-1)K (h-l)K23 (h-1)K32 (h-1)CL (L/h)T1/2 (h)AUC(0–24) (ng*h/mL)
      15.0215.681008.600.380.080.270.0617.2059.600.29
      26.0100.11837.710.320.111.250.1510.8564.100.55
      35.042.60618.260.180.190.750.058.1868.700.61
      43.088.92472.820.320.080.200.047.0571.900.43
      54.071.98582.650.490.130.880.119.3554.300.43
      Mean4.6103.86704.010.340.120.670.0810.5263.700.46
      Std1.166.16215.740.120.050.440.053.997.000.13
      CL = clearance; k = elimination rate constant; KA = numeric value to express the degree to which a compound or drug dissociates in aqueous solution (e.g. blood in this study); k23 = distribution rate constant (central to peripheral); k32 = distribution rate constant (peripheral to central); V2 = central compartment; V3 = peripheral compartment; t1/2 = half-life.
      Pharmacokinetic parameters of sirolimus concentration: Blood samples were obtained for analysis of sirolimus concentration. A two-compartment model was calculated using the sirolimus dose and blood concentration values (blood concentration at t = 0, 2, 24, 48, and 96 h). Maximum likelihood estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (Vd/F).
      Table 3Pharmacokinetic parameters of tacrolimus in concentration.
      IDDOSE (mg)V2 (L)V3 (L)KA (h-1)K (h-l)K23 (h-1)K32 (h-1)CL (L/h)t1/2 (h)AUC(0–12) (ng*h/mL)
      14.009.44500.100.301.302.100.0412.3046.000.33
      210.00126.801194.000.110.350.230.0343.7648.000.23
      32.0019.60602.640.210.701.380.0513.7346.500.15
      43.0065.59547.120.660.290.820.1018.9228.900.16
      53.5014.48468.760.580.993.080.1014.3930.400.24
      Mean4.5047.18662.520.370.731.520.0620.6240.000.22
      Std3.2049.83301.370.240.431.110.0313.179.400.07
      CL = clearance; k = elimination rate constant; KA = numeric value to express the degree to which a compound or drug dissociates in aqueous solution (e.g. blood in this study); k23 = distribution rate constant (central to peripheral); k32 = distribution rate constant (peripheral to central); t1/2 = half-life; V2 = central compartment; V3 = peripheral compartment.
      Pharmacokinetic parameters of tacrolimus concentration: Blood samples were obtained for the analysis of tacrolimus concentration. A two-compartment model with first-order absorption pharmacokinetics was calculated using the tacrolimus dose and the blood concentration values (blood concentration at t = 0, 2, 12, 24, and 48 h).
      Figure thumbnail gr1
      Fig. 1(A) Tacrolimus concentrations: participants received three and a half days of tacrolimus administration and doses were based on pharmacokinetic modeling for each individual patient. Serum samples were obtained from participants on day 4, 2 h after the last morning dose of tacrolimus (2 h). A second serum sample was obtained at 48 h after the last dose of tacrolimus (48 h). Whole blood samples were analyzed using a microparticle enzyme immunoassay. (B) sirolimus serum concentrations: participants received 4 d of sirolimus administration and doses were based on pharmacokinetic modeling for each individual patient. Serum samples were obtained from participants on day 4, 2 h after the last morning dose of sirolimus (2 h). Other samples were obtained at 48 and 96 h after the last dose of sirolimus. Whole blood samples were analyzed using a microparticle enzyme immunoassay.
      The effects of single-drug immunosuppression on lymphocyte proliferation were determined by BrdU incorporation (Fig. 2). The BrdU assay at baseline, before drug administration, was not significantly different for both the study arms (Fig. 2A). At 48 h after the last dose of the drug was administered, lymphocyte proliferation was reassessed. The BrdU was normalized for drug exposure using AUC of drug level. The sirolimus group continued to exhibit decreased immune responses (Fig. 2B). In contrast, the tacrolimus group exhibited rebound immune reactivity, which was significantly higher than with sirolimus. Analysis of T cell activation, based on mitogen responses, suggests similar trend as with the BrdU analysis (Fig. 2C).
      Figure thumbnail gr2
      Fig. 2(A) BrdU levels at baseline: samples were processed for lymphocyte proliferation using incorporation of BrdU during DNA synthesis. Blood samples were obtained from participants before the first dose of drug for both the study arms (tacrolimus or sirolimus). (B) lymphocyte proliferation. Samples were obtained at 48 h after the last dose of drug administration (three and a half days of bid dosing in the tacrolimus arm with the last dose on day 4 and 4 d of daily dosing for sirolimus arm with the last dose on day 4). Lymphocyte proliferation was analyzed using incorporation of BrdU during DNA synthesis. The results are normalized for AUC for drug exposure. (C) Cylex levels at 48 h: Peripheral blood lymphocytes were analyzed for phytohemagglutinin mitogen stimulation by the Cylex assay. Samples were obtained at the same time as samples for lymphocyte proliferation. These samples were obtained before the start of drug administration and 48 h after the last dose. Additional samples were obtained for the sirolimus arm at 96 h after the last dose.
      T cell function was also assessed by measuring cytokine production (Fig. 3). Peripheral blood T cells were stimulated with anti-CD3/CD28, and the cytokine IL-17A was analyzed. A significant decrease in IL-17A production in the group receiving sirolimus was detected. The IL-17A levels in the sirolimus arm were decreased at 24 h but not at 48 h. The tacrolimus group did not reveal a decrease in IL-17A either at 2 or 24 h after the last dose.
      Figure thumbnail gr3
      Fig. 3(A) In the two study arms, patients were given sirolimus orally and serum IL-17A (a proinflammatory cytokine) levels were examined at 0 and 24 h after administration (P < 0.04). (B) in the two study arms, patients were given sirolimus orally and serum levels of IL-17A were measured at 0 and 48 h after the last administration. (c) in the two study arms, patients were given tacrolimus orally and serum levels of IL-17A were evaluated at 0 and 2 h after the last administration. (D) in the two study arms, patients were given tacrolimus orally and serum levels of IL17A were measured at baseline (0 h) and 48 h after the last drug dose.
      Urine total protein/creatinine ratios were determined at baseline before drug administration, during the trial, and at 1 wk after the completion of the trial. No significant change in the protein/creatinine ratios at these time points was noted (not shown). Drug effects on glucose tolerance were also analyzed. The OGTTs were compared using analysis of variance, and no significant difference was detected between tacrolimus and sirolimus groups (not shown).

      3.2 Discussion

      Current immunosuppressive regimens for transplant recipients involve multiple therapeutic agents, including a combination of steroids, CNIs, mTor inhibitors, mycophenolic acid, and occasionally azathioprine [
      • Weber T.
      • Abendroth D.
      • Schelzig H.
      Rapamycin rescue therapy in patients after kidney transplantation: first clinical experience.
      ,
      • Halloran P.F.
      Immunosuppressive drugs for kidney transplantation.
      ,
      • Denton M.D.
      • Magee C.C.
      • Sayegh M.H.
      Immunosuppressive strategies in transplantation.
      ]. Multidrug regimens have proven to be the most effective in preventing graft rejection [
      • Weber T.
      • Abendroth D.
      • Schelzig H.
      Rapamycin rescue therapy in patients after kidney transplantation: first clinical experience.
      ,
      • Halloran P.F.
      Immunosuppressive drugs for kidney transplantation.
      ,
      • Denton M.D.
      • Magee C.C.
      • Sayegh M.H.
      Immunosuppressive strategies in transplantation.
      ]. Thus, most clinical studies analyze the effect of a cocktail of immunosuppressive agents.
      To determine the immunosuppressive effects of a single agent (tacrolimus or sirolimus), we investigated the immune efficacy of each drug in a crossover study in normal patients. Limitations of this study include a small number of participants and short duration of drug response. Nevertheless, using a crossover study design we detected a significant difference in lymphocyte proliferation and cytokine secretion in an effort to analyze single-drug effects.
      Sirolimus (an mTOR inhibitor) and tacrolimus (CNI) are the two successful cornerstone drugs used to prevent graft rejection. The side effects of these drugs include high blood pressure, rash, decreased blood platelets, or, in the case of tacrolimus, nephrotoxicity or viral infections (cytomegalovirus), and hypertension. CNIs are used in the first month after transplantation with a reduction of CNI use for short and long-term treatment [
      • Naesens M.
      • Kuypers D.R.
      • Sarwal M.
      Calcineurin inhibitor nephrotoxicity.
      ,
      • Rostaing L.
      • Massari P.
      • Garcia V.D.
      • Mancilla-Urrea E.
      • Nainan G.
      • del Carmen Rial M.
      • et al.
      Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study.
      ,
      • Cataneo-Davila A.
      • Zuniga-Varga J.
      • Correa-Rotter R.
      • Alberu J.
      Renal function outcomes in kidney transplant recipients after conversion to everolimus-based immunosuppression regimen with CNI reduction or elimination.
      ,
      • Ireland R.
      Transplantation: Does switching to a CNI-free regimen reduce cancer risk?.
      ]. Nephrotoxicity is a major problem accompanying CNI use that leads to acute and chronic renal failure. Sirolimus has been used as a rescue drug after unsuccessful CNI use.
      For both sirolimus and CNIs, an additional consideration may be noncompliance of medication [
      • Prendergast M.B.
      • Gaston, R.S.
      Optimizing medication adherence: an ongoing opportunity to improve outcomes after kidney transplantation.
      ]. Interestingly, Greenstein and Siegar [
      • Greenstein S.
      • Siegal B.
      Odds probabilities of compliance and noncompliance in patients with a functioning renal transplant: a multicenter study.
      ] examined questionnaires from 1547 renal transplant recipients and found that patients' reasons for noncompliance included the belief that the transplant procedure had improved their health, not the medication [
      • Greenstein S.
      • Siegal B.
      Odds probabilities of compliance and noncompliance in patients with a functioning renal transplant: a multicenter study.
      ].
      We found a significant difference in lymphocyte proliferation between sirolimus and tacrolimus treatment. Results from the BrdU and Cylex assays indicate that sirolimus, in the absence of other immunosuppressants, exhibits reduced lymphocyte responsiveness with a trend toward continued suppression at 48 h. In contrast, a similar effect is not observed with tacrolimus. This difference may be related to the longer half-life of sirolimus. No differences in proteinuria and OGTTs were detected.
      We also observed that the half-life of tacrolimus is longer in healthy volunteers than reported in renal transplant recipients. The half-life for sirolimus is similar in both the groups. The prolonged half-life of tacrolimus compared with previous reports may be because of concomitant medications or co-morbidities in transplant populations. In this study, most of the samples were obtained at the end of the dosing interval so that the absorption rate constant (ka) is fixed for some individual patients using literature values. The influence of covariants such as weight, age, gender, and hematocrit were not assessed because of the limited sample size.
      Prior studies regarding the impact of tacrolimus or sirolimus are variable. In in vitro studies, tacrolimus and anti-CD3/anti-CD28 activated T cells increased IL-2 messenger RNA (eight of 11 healthy patients) [
      • Hartel C.
      • Schumacher N.
      • Fricke L.
      • Ebel B.
      • Kirchner H.
      • Muller-Steinhardt M.
      Sensitivity of whole-blood T lymphocytes in individual patients to tacrolimus (FK 506): impact of interleukin-2 mRNA expression as surrogate measure of immunosuppressive effect.
      ]. Kidney transplant patients (n = 8) who had received tacrolimus or cyclosporine before transplant exhibited unaffected IL-2 mRNA concentrations (four of 8 samples). Whole blood samples incubated with anti-CD3/anti-CD28 monoclonal antibodies with tacrolimus exhibited an increase in IL-2 mRNA [
      • El-Safa E.A.
      • Fredericks S.
      • MacPhee I.
      • Holt D.W.
      • Johnston A.
      Paradoxical response to tacrolimus assessed by interleukin-2 gene expression.
      ].
      CNIs are the mainstay in immunosuppressive protocol in transplantation. In this study, we did not observe inhibition of CD4+ T cell proliferation and activation based on mitogen response or cytokine production. These results may be because of the short duration of drug exposure or to the limited effects of single-drug treatment. Our data suggest that tacrolimus may require longer treatment periods to induce immunosuppression. Long-term treatment periods may necessitate additional therapeutic modalities, such as induction with antibodies or high dose steroids during the post-transplant period. In contrast, sirolimus rapidly induced immunosuppressive effects. Future studies investigating the comparative effectiveness of sirolimus in a diverse patient group (e.g., adherent and partially adherent) are warranted.

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