Vafadari, R.

Tacrolimus Inhibits NF-κB Activation in Peripheral Human T Cells (Article)

2013-04-01T00:00:00Z

The calcineurin inhibitor, tacrolimus (TAC), inhibits the protein phosphatase activity of calcineurin, leading to suppression of the nuclear translocation of NFAT and inhibition of T cell activation. Apart from NFAT also the transcription factor NF-κB plays a key functional role in T cell activation. Therefore, blockade of the NF-κB activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-κB activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-κB (p65) phosphorylation was measured by flow cytometry in CD3+ T cells, CD4+ helper T cells and CD8+ cytotoxic T cells in the absence and presence of TAC 10 ng/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 μg/mL (negative control; n = 6). NF-κB transcriptional activity was measured by ELISA and intracellular TNFα protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-κB blockade. Anti-CD3/28-activation induced NF-κB phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p<0.01). Sotrastaurin inhibited NF-κB activation in the respective T cell subsets by 93%, 95% and 86% (p<0.01 vs. no drug), while mycophenolic acid did not affect this activation pathway. Surprisingly, TAC also inhibited NF-κB phosphorylation, by 55% (p<0.01) in CD3+ T cells, by 56% (p<0.01) in CD4+ T cells and by 51% in CD8+ T cells (p<0.01). In addition, TAC suppressed NF-κB DNA binding capacity by 55% (p<0.05) in CD3+ T cells and TNFα protein expression was inhibited in CD3+ T cells, CD4+ T cells and CD8+ T cells by 76%, 71% and 93% resp. (p<0.01 vs. no drug), confirming impaired NF-κB signaling. This study shows the suppressive effect of TAC on NF-κB signaling in peripheral human T cell subsets, measured at three specific positions in the NF-κB activation cascade.

Exploring pharmacodynamics of immunosuppresive agents in transplantation (Doctoral Thesis)

2013-03-01T00:00:00Z

Supported by advancements in technology, surgical techniques and immunosuppressive drugs, solid organ transplantation has become the preferred solution to end stage organ failure. The first solid organ transplantation was performed in 1954 under supervision of Joseph Murray in the Peter Bent Brigham Hospital in Boston. A kidney from a healthy donor was transplanted into his identical twin, who suffered from chronic glomerulonephritis. The transplanted organ functioned immediately and the recipient survived for 25 years while the donor lived for another 56 years. Since the twins were genetically identical, no suppression of the immune system was needed. Transplantation between individuals other than identical twins was made possible a decade later by the use of the combination of azathioprine and corticosteroids, achieving a 1 year allograft1 survival rate of 40 to 50 percent.

Pharmacodynamic analysis of tofacitinib and basiliximab in kidney allograft recipients (Article)

2012-09-15T00:00:00Z

BACKGROUND: The common γ-chain (γc) cytokines signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and play pivotal roles in lymphocyte activation. We investigated the effect of immunosuppressive drugs targeting this pathway, the JAK inhibitor tofacitinib (CP-690,550) and the anti-interleukin (IL)-2R antibody basiliximab, as part of a phase 2 study. METHODS: After whole-blood activation with the γccytokines IL-2, IL-7, and IL-15, STAT5 phosphorylation was determined in T cells of de novo kidney transplantation patients treated with tofacitinib/basiliximab (n=5), calcineurin inhibitor (CNI) (cyclosporine A)/basiliximab (n=4) or CNI (tacrolimus)-based immunosuppression (n=6). The IC50 for phosphorylated STAT (P-STAT) 5 inhibition by tofacitinib was determined in cytokine-activated CD4+and CD8+T cells from healthy individuals (n=4). RESULTS: IC50 was 26, 72, and 37 ng/mL for IL-2, IL-7, and IL-15 activation, in CD4+T cells, respectively; and 35, 61, and 76 ng/mL for IL-2, IL-7, and IL-15 activation, in CD8+T cells, respectively. In kidney transplantation patients, 7 days after starting tofacitinib/basiliximab treatment, cytokine-induced P-STAT5 was inhibited in CD4+T cells (92% for IL-2 activation, 60% for IL-7, and 75% for IL-15), which persisted for the 2-month study period. In contrast, CNI/basiliximab treatment did not affect IL-7-activated or IL-15-activated P-STAT5; only IL-2-activated P-STAT5 was reduced by 77% on day 7 and recovered to pretreatment levels within 2 months. CD8+T cells showed a comparable profile to CD4+T cells. P-STAT5 was not inhibited in CNI-treated control patients. CONCLUSIONS: Tofacitinib therapy strongly inhibits γccytokine-induced JAK/STAT5 activation, whereas basiliximab suppresses IL-2-stimulated activation only. Pharmacodynamic monitoring offers a unique tool to evaluate the biologic effects of immunosuppressive drugs. Copyright

Phosphospecific flow cytometry for pharmacodynamic drug monitoring: Analysis of the JAK-STAT signaling pathway (Article)

2012-09-08T00:00:00Z

Cytokines of the IL-2 receptor family act via activation of the JAK-STAT (janus tyrosine kinase-signal transducer and activator of transcription) signaling pathway. These cytokines are pivotal for the development and function of lymphocyte subsets involved in the immune response after organ transplantation including T, B and natural killer cells. The new small drug molecule and JAK1/3 inhibitor, tofacitinib, is currently being tested in phase II and III clinical trials for rheumatoid arthritis, psoriasis and in organ transplantation. This agent specifically targets the JAK-STAT signaling pathway. Here we discuss phosphospecific flow cytometry as a novel tool to monitor the JAK-STAT signaling pathway in kidney transplant patients and speculate that through the use of this pharmacodynamic tool the efficacy of immunosuppressive drugs can be assessed enabling optimization of the immunosuppressive therapy for individual transplant patients.

Inhibitory effect of tacrolimus on p38 mitogen-activated protein kinase signaling in kidney transplant recipients measured by whole-blood phosphospecific flow cytometry. (Article)

2012-06-27T00:00:00Z

Tacrolimus (TAC), the cornerstone of immunosuppressive therapy after solid organ transplantation, inhibits calcineurin activation. Despite pharmacokinetic monitoring, patients frequently experience toxicity or lack of efficacy, which could be prevented by pharmacodynamic monitoring. In Jurkat T-cell lines, it has been shown that TAC, in addition to calcineurin, inhibits the p38 mitogen-activated protein kinase (MAPK) pathway, which is important in T-cell activation and is therefore a potential drug-specific biomarker. We studied whether TAC inhibits p38 MAPK signaling in primary human T cells and ex vivo in healthy volunteers and kidney transplant recipients. Phorbol-12-myristate-13-acetate/ionomycin-induced MAPK signaling was measured by whole-blood phosphospecific flow cytometry. In vitro, 10-ng/mL TAC inhibited p38 MAPK phosphorylation by a mean of 27% in CD3, 26% in CD4, and 34% in CD8 T cells (P<0.01 compared with baseline). In healthy adults (n=4), 2 hr after a single oral dose of 10-mg TAC, the p38 MAPK activation was inhibited by 35% in CD3, CD4, and CD8 T cells (P<0.05 compared with baseline). In kidney transplant recipients (n=24), TAC predose concentrations (range, 3.2-10.5 ng/mL) were inversely correlated with p38 MAPK activation in CD3, CD4, and CD8 T cells (r=0.51, 0.34, and 0.37, respectively; P<0.01). TAC inhibits activation of the MAPK pathway in a dose-dependent manner in kidney transplant patients and may be a potential marker for immune monitoring.