Christine Lorkowski

Differential impact of the CYP3A5*1 and CYP3A5*3 alleles on pre-dose concentrations of two tacrolimus formulations.

Objectives We investigated the pharmacokinetic and pharmacogenetic implications of conversion from a twice-daily (P-Tac) to a once-daily (A-Tac) tacrolimus (Tac) formulation. Methods We analyzed Tac levels in a cohort of 41 renal transplant patients with a stable graft function over a period of 1 year before and after conversion. Results After conversion, the patients had, on average, significantly lower Tac trough and dose-normalized trough levels (14%, P=0.0004 and 23%, P=0.001, respectively) despite similar doses. CYP3A5*3/*3 patients (n=27) required significantly lower Tac doses with both the formulations to reach Tac target levels (P-Tac 39%, P=0.011; A-Tac 36%, P=0.003) compared with *1/*3 patients (n=13). Interestingly, after the conversion, mean Tac trough levels and dose-normalized trough level remained almost constant in *1/*3 patients, but decreased significantly in *3/*3 patients (16%, P=0.001 and 25%, P=0.006). Conclusion This study provides further evidence that the CYP3A5*1/*3 polymorphism significantly impacts Tac pharmacokinetics. Moreover, we show for the first time a pharmacogenetic effect on two different Tac formulations, as Tac trough levels of *3/*3 patients declined significantly after conversion to identical A-Tac doses.

Effects of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function and activation

Humoral rejection processes may lead to allograft injury and subsequent dysfunction. Today, only one B‐cell‐specific agent is in clinical use and the effects of standard and new immunosuppressant substances on B‐cell activation and function are not fully clarified. The impact of sotrastaurin, mycophenolic acid and everolimus on human B‐lymphocyte function was assessed by analysing proliferation, apoptosis, CD80/CD86 expression and immunoglobulin and IL‐10 production in primary stimulated B cells. In addition, B‐cell co‐cultures with pre‐activated T cells were performed to evaluate the effect of the different immunosuppressive agents on T‐cell‐dependent immunoglobulin production. Sotrastaurin did not inhibit B‐cell proliferation, CD80/CD86 expression, and IgG production and had only minor effects on IgM levels at the highest concentration administered. In contrast, mycophenolic acid and everolimus had strong effects on all B‐cell functions in a dose‐dependent manner. All immunosuppressive agents caused decreased immunoglobulin levels in T‐cell‐dependent B‐cell cultures. The data provided here suggest that mycophenolic acid and everolimus, but not sotrastaurin, are potent inhibitors of human B‐lymphocyte function and activation.

Effects of the new immunosuppressive agent AEB071 on human immune cells

UNLABELLED BACKGROUND. The novel immunosuppressive agent AEB071 is currently being evaluated for its capability to prevent rejection after kidney transplantation as a potential adjunct to calcineurin inhibitor-based regimen. AEB071 is a selective protein kinase C inhibitor and has been shown to be well tolerated in humans. We here present extensive in vitro studies that contribute to the understanding of AEB071 effects on human lymphocyte, natural killer (NK) cell and dendritic cell (DC) action. METHODS The impact of AEB071 on several T-cell activation and costimulatory markers was assessed. Furthermore, assays were performed to study the effect on T-cell proliferation and intracellular cytokine production. Additionally, the effect of AEB071 on DC maturation and their capacity to stimulate allogeneic T-cells was examined. Also, an evaluation of AEB071 effects on the lytic activity of human NK cells was performed. RESULTS We were able to show that T-cell proliferation and cytokine production rates are significantly reduced after AEB071 administration. Also, mitogen-induced T-cell activation characterized by expression levels of surface markers could be significantly inhibited. In contrast, the T-cell stimulatory capacity of AEB071-treated mature monocyte-derived DC (Mo-DC) is not reduced, and AEB071 administration does not prevent lipopolysaccharide (LPS)-induced Mo-DC maturation. It could be demonstrated that AEB071 significantly inhibited the cytotoxic activity of NK cells. CONCLUSIONS The promising immunosuppressive agent AEB071 has a strong impact on T-cell activation, proliferation and cytokine production as well as NK cell activity, but not DC maturation in vitro, and therefore, seems to function T-cell and NK cell specific via protein kinase C (PKC) inhibition.

Identification of T Cell-Mediated Vascular Rejection After Kidney Transplantation by the Combined Measurement of 5 Specific MicroRNAs in Blood.

Background MicroRNAs (miRNAs, miR) hold important roles in the posttranscriptional regulation of gene expression. Their function has been correlated with kidney disease, and they might represent a new class of biomarkers for frequent evaluation of renal graft status. We analyzed their potential in identifying severe T cell–mediated vascular rejection (TCMVR) (Banff 4-II/III) in kidney transplanted patients. Methods Microarray experiments and semiquantitative real-time reverse transcription polymerase chain reaction were performed with total RNA isolated from blood cells of kidney graft recipients. Initial microarray analysis revealed 23 differentially expressed miRNAs distinguishing patients with TCMVR from patients with stable grafts. From these, we validated and further determined the expression of 6 differentially expressed miRNAs and 2 control miRNAs in 161 samples from patients with T cell–mediated rejection (Banff 3-Borderline, Banff 4-I/II/III), Banff-2 antibody-mediated rejection, Banff-5 interstitial fibrosis/tubular atrophy, in samples from stable patients and in samples from patients with urinary tract infection using real-time reverse transcription polymerase chain reaction. Results Expression levels of all 6 candidate miRNAs were significantly downregulated in blood of TCMVR patients compared to the other groups and displayed high sensitivities and specificities for diagnosing TCMVR. The combination of 5 miRNAs, identified by an unbiased multivariate logistic regression followed by cross-validation, enhanced the sensitivity and specificity for the diagnosis of TCMVR after renal transplantation. Conclusions The combined measurement of miRNA-15B, miRNA-16, miRNA-103A, miRNA-106A, and miRNA-107 may help to better identify TCMVR after renal transplantation in a precise and clinically applicable way.

Free microRNA levels in plasma distinguish T-cell mediated rejection from stable graft function after kidney transplantation.

The potential diagnostic value of circulating free miRNAs in plasma compared to miRNA expression in blood cells for rejection processes after kidney transplantation is largely unknown, but offers the potential for better and timely diagnosis of acute rejection. Free microRNA expression of specific blood cell markers was measured in 160 plasma samples from kidney transplant patients under standard immunosuppressive therapy (steroids±mycophenolic acid±calcineurin inhibitor) with stable graft function, urinary tract infection, interstitial fibrosis and tubular atrophy, antibody-mediated rejection (ABMR), Borderline (Banff3), tubulo-interstitial (Banff4-I) and vascular rejection (Banff4-II/III) applying RT-PCR. The expression levels of specific microRNAs miR-15B, miR-103A and miR-106A discriminated patients with stable graft function significantly (p-values 0.001996, 0.0054 and 0.0019 resp.) from patients with T-cell mediated rejection (TCMR) and from patients with urinary tract infection (p-values 0.0001, <0.0001 and 0.0001, resp.). A combined measurement of several microRNAs after multivariate logistic regression improved the diagnostic value supported by subsequent cross-validation. In conclusion, the measurement of circulating microRNAs in plasma from patients with renal transplants distinguishes TCMR and urinary tract infection from stable graft function. In contrast to miRNA expression measurement in blood cells it does not allow a discrimination from ABMR or interstitial fibrosis and tubular atrophy.

Combined standard and novel immunosuppressive substances affect B-lymphocyte function☆

A considerable fraction of renal transplanted patients is susceptible to humoral rejection. Today well-established therapy regimens are available to control antibody-mediated rejection in the short term. Nevertheless, donor-specific antibodies persist and graft function deteriorates over time. This might be due to insufficient maintenance immunosuppression - which always consists of two to three drugs with different mechanisms of action. Since T- and B-cell functions always depend on each other in the alloimmune response it is of interest to analyze the effects of combined standard and new immunosuppressive substances with T-cell inhibitory properties on B-cell function. The effectiveness of complementary administrations of sotrastaurin, mycophenolic acid and everolimus on the activation and function of human primary B-lymphocytes was tested. Everolimus and mycophenolic acid alone and in combination proved to be highly effective in suppressing B-cell activation, whereas the proteinkinase C inhibitor sotrastaurin had an unexpected and reverse impact on various B-cell functions when applied in combination with the mammalian target of rapamycin and the inosine monophosphate dehydrogenase inhibitor.

The selective biomarker IL-8 identifies IFTA after kidney transplantation in blood cells☆

Cellular and antibody-mediated rejection processes and also interstitial fibrosis/tubular atrophy (IFTA) lead to allograft dysfunction and loss. The search for accurate, specific and non-invasive diagnostic tools is still ongoing and essential for successful treatment of renal transplanted patients. Molecular markers in blood cells and serum may serve as diagnostic tools but studies with high patient numbers and differential groups are rare. We validated the potential value of several markers on mRNA level in blood cells and serum protein level in 166 samples from kidney transplanted patients under standard immunosuppressive therapy (steroids±mycophenolic acid±calcineurin inhibitor) with stable graft function, urinary tract infection (UTI), IFTA, antibody-mediated rejection (ABMR), and T-cell-mediated rejection (TCMR) applying RT-PCR and ELISA. The mRNA expression of RANTES, granulysin, granzyme-B, IP-10, Mic-A and Interferon-γ in blood cells did not distinguish specifically between the different pathologies. We furthermore discovered that the mRNA expression of the chemokine IL-8 is significantly lower in samples from IFTA patients than in samples from patients with stable graft function (p<0.001), ABMR (p<0.001), Borderline (BL) TCMR (p<0.001), tubulo-interstitial TCMR (p<0.001) and vascular TCMR (p<0.01), but not with UTI. Serum protein concentrations of granzyme-B, Interferon-γ and IL-8 did not differ between the patient groups, RANTES concentration was significantly different when comparing UTI and ABMR (p<0.01), whereas granulysin, Mic-A and IP-10 measurement differentiated ongoing rejection or IFTA processes from stable graft function but not from each other. The measurement of IL-8 mRNA in blood cells distinguishes clearly between IFTA and other complication after kidney transplantation and could easily be used as diagnostic tool in the clinic.

MicroRNA regulation in blood cells of renal transplanted patients with interstitial fibrosis/tubular atrophy and antibody-mediated rejection

Interstitial fibrosis/tubular atrophy (IFTA) is associated with reduced allograft survival, whereas antibody-mediated rejection (ABMR) is the major cause for renal allograft failure. To identify specific microRNAs and their regulation involved in these processes, total RNA from blood cells of 16 kidney transplanted (KTx) patients with ABMR, stable graft function (SGF) and with T-cell mediated rejection (TCMR) was isolated. MicroRNA expression was determined by high-throughput sequencing. Differentially expressed candidate microRNAs were analyzed with RT-PCR in patients with SGF (n = 53), urinary tract infection (UTI) (n = 17), borderline rejection (BL) (n = 19), TCMR (n = 40), ABMR (n = 22) and IFTA (n = 30). From the 301 detected microRNAs, 64 were significantly regulated between the three cohorts. Selected candidate microRNAs miR-223-3p, miR-424-3p and miR-145-5p distinguished TCMR and ABMR from SGF, but not from other pathologies. Most importantly, miR-145-5p expression in IFTA patients was significantly downregulated and displayed a high diagnostic accuracy compared to SGF alone (AUC = 0.891) and compared to SGF, UTI, BL, TCMR and ABMR patients combined (AUC = 0.835), which was verified by cross-validation. The identification of miR-145-5p as IFTA specific marker in blood constitutes the basis for evaluating this potentially diagnostic microRNA as biomarker in studies including high numbers of patients and different pathologies and also the further analysis of fibrosis causing etiologies after kidney transplantation.

The Regulation of IFN Type I Pathway Related Genes RSAD2 and ETV7 Specifically Indicate Antibody-Mediated Rejection After Kidney Transplantation

Antibody‐mediated rejection (ABMR) after kidney transplantation (KTx) remains the crucial obstacle to successful long‐term graft function. The identification of gene signatures involved in ABMR could grant the basis for better prevention and treatment strategies.

Conversion to Belatacept based regimen does not change T-cell phenotype and function in renal transplantation.

Belatacept offers a new option for renal allograft recipients who are suffering from side effects of calcineurin inhibitors or mTOR inhibitors,which may result in renal and extrarenal benefits.We prospectively performed flow cytometric immunophenotyping with a T-cell panel. In total we were able to fully investigate the immunophenotypic change in 8 patients before and after conversion from calcineurin inhibitor (n = 5) or mTOR inhibitor (n=2) to Belatacept or additional administration (n=1). Cells were analysed pre conversion, 1 month, 3 months, 6 months and 12 months after first Belatacept administration. The percentage of central memory, naïve, effector memory and terminally differentiated effector memory CD4+ and CD4− T-cells was determined. CD28, CD25 and CD69 expression on CD4+ and CD4− T-cells was measured ex vivo and also after 3 days of mitogen stimulation. Intracellular cytokines IFNgamma and IL-2 were measured after polyclonal cellular stimulation. The expression of activation markers and intracellular cytokines as well as the percentage of T-cell subsets did not change significantly during the observation period compared to the time-point pre conversion. Therefore the conversion of calcineurin inhibitor or mTOR inhibitor to Belatacept seems to have no obvious impact on the immunophenotype of T-cells in patients after kidney transplantation.