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Objective To establish a detection system of ultra high performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) for everolimus concentration in whole blood of liver transplant recipients. Methods The proteins of samples were precipitated with methanol and zinc sulfate, and everolimus-D4 was used as the internal standard. Phenomenex Kinetex PFP column was used. The mobile phase A was water (containing 2 mmol/L ammonium formate and 0.1% formic acid), and the mobile phase B was methanol (containing 2 mmol/L ammonium formate and 0.1% formic acid). The gradient elution was performed with the flow rate of 1 mL/min, the column temperature of 50 ℃ and the injection volume of 1 μL. The multi - reaction monitoring mode was used to quantitatively analyze with electrospray positive ionization. The UPLC-MS/MS detection system required only 100 μL of whole blood, and could achieve a sufficient lower limit of quantification without complicated sample preparation. The total running time was within 4.5 min. Linear regression (1/x 2 ) analysis was performed using peak area of everolimus / peak area of everolimus-D4 (y) and concentration of everolimus / concentration of everolimus-D4 (x) to calculate the calibration function and analyze its accuracy and linear relationship. UPLC-MS/MS was used to detect the trough blood concentration of everolimus in blood samples of 5 recipients after liver transplantation. Results The accuracy of quality control was within 15%, and the linear relationship of everolimus was good in the blood concentration range of 1-100 ng /mL (R² >0.990). Trough blood concentration of everolimus measured in blood samples of 5 liver transplant recipients ranged from 3.77 to 9.27 ng/mL. Conclusions The detection system of UPLC-MS/MS in this study is suitable for monitoring the concentration of everolimus in whole blood of liver transplant recipients because of its high accuracy, simple sample processing method and short detection time. Objective To establish a detection system of ultra high performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) for everolimus concentration in whole blood of liver transplant recipients. Methods The proteins of samples were precipitated with methanol and zinc sulfate, and everolimus-D4 was used as the internal standard. Phenomenex Kinetex PFP column was used. The mobile phase A was water (containing 2 mmol/L ammonium formate and 0.1% formic acid), and the mobile phase B was methanol (containing 2 mmol/L ammonium formate and 0.1% formic acid). The gradient elution was performed with the flow rate of 1 mL/min, the column temperature of 50 ℃ and the injection volume of 1 μL. The multi - reaction monitoring mode was used to quantitatively analyze with electrospray positive ionization. The UPLC-MS/MS detection system required only 100 μL of whole blood, and could achieve a sufficient lower limit of quantification without complicated sample preparation. The total running time was within 4.5 min. Linear regression (1/x 2 ) analysis was performed using peak area of everolimus / peak area of everolimus-D4 (y) and concentration of everolimus / concentration of everolimus-D4 (x) to calculate the calibration function and analyze its accuracy and linear relationship. UPLC-MS/MS was used to detect the trough blood concentration of everolimus in blood samples of 5 recipients after liver transplantation. Results The accuracy of quality control was within 15%, and the linear relationship of everolimus was good in the blood concentration range of 1-100 ng /mL (R² >0.990). Trough blood concentration of everolimus measured in blood samples of 5 liver transplant recipients ranged from 3.77 to 9.27 ng/mL. Conclusions The detection system of UPLC-MS/MS in this study is suitable for monitoring the concentration of everolimus in whole blood of liver transplant recipients because of its high accuracy, simple sample processing method and short detection time. [ABSTRACT FROM AUTHOR]

Background: Natural killer (NK) cells are enriched in the liver and are the main regulators in liver transplantation regarding rejection or tolerance, viral infection, or tumor recurrence. Immunosuppression consists of a triple drug standard regimen comprising tacrolimus (TAC) and corticosteroids (CS) with either mycophenolate mofetil (MMF) or sirolimus (SIR)/everolimus (EVE). The aim of this study was to evaluate the impact of trough levels of these regimens under clinical conditions and exposure on human NK-cell activity and function in order to better understand the antiviral and anti-tumor effects of mammalian target of rapamycin inhibitor (mTORI)., Methods: Peripheral blood mononuclear cells (PBMCs) were collected from liver transplant recipients and healthy controls. Number and phenotypes of NK cells in vivo were analyzed by flow cytometry. In this study we simulated the immunosuppressive microenvironment in vitro . PBMCs were cultured at the clinically effective plasma concentration of drugs for 3 d to detect the effect of immunosuppressants on NK cells. Drug type and concentration: single drug [EVE, 5 ng/mL; SIR, 5 ng/mL; TAC, 5 ng/mL; cyclosporine A (CSA), 125 ng/mL; MMF, 15 µg/mL; CS, 0.5 µg/mL] and combined immunosuppressants (Group 1: TAC, 5 ng/mL + MMF, 15 µg/mL + CS, 0.5 µg/mL; Group 2: TAC, 5 ng/mL + SIR, 5 ng/mL + CS, 0.5 µg/mL; Group 3: TAC, 5 ng/mL + EVE, 5 ng/mL + CS, 0.5 µg/mL). In addition, NK cells were sorted from PBMCs and treated under the above conditions to detect NK cell killing function and RNA transcription characteristics., Results: CS significantly impaired the cytolytic activity of NK cells, followed by MMF and SIR/EVE. CS and TAC/CSA significantly decreased the secretion of IFN-γ and CD107a. NK cell function in liver transplant recipients was most pronouncedly inhibited by a triple immunosuppressive regimen, with CS playing the most prominent role compared with the other drugs. The MMF-containing regimen demonstrated a significant increase in the expression of suppressive genes, especially of the Siglec7/9 family. The SIR group had stronger NK cell activity compared with that of the MMF group, although liver transplantation patients have lower NK cell activity and function., Conclusions: Despite an overall comparable immunosuppressive efficiency in terms of prevention of acute rejection, a mTORIs-including regimen might be considered as having less impact on NK cell function., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://hbsn.amegroups.com/article/view/10.21037/hbsn-22-438/coif). All authors reported that the project was funded by B.N. research start-up fund (2018KYQ003RC2017001), National Natural Science Foundation of China (#8202290021, #92169118, #91942310), National Key Research and Development Program of China (#2021YFC2300600), Anhui Provincial Natural Science Foundation (#2008085J35), and the Fundamental Research Funds for the Central Universities (WK9110000055). B.N. serves as an unpaid editorial board member of Hepatobiliary Surgery and Nutrition. The authors have no other conflicts of interest to declare., (2023 Hepatobiliary Surgery and Nutrition. All rights reserved.)

Abstract: The frequent occurrence of cutaneous squamous cell carcinomas (SCCs) containing weakly tumorigenic human papillomaviruses (HPVs) following iatrogenic immunosuppression for organ transplantation remains incompletely understood. Here we address this problem in the light of recent insights into (1) the association of low-risk β-HPVs with skin SCCs in the rare genetic syndromes of epidermodysplasia verruciformis and xeroderma pigmentosum, (2) the frequent recovery of post-transplant tumor control on substituting calcineurin-inhibitory with mTOR-inhibitory immunosuppression, (3) the unexpectedly favorable prognosis of node-positive SCCs containing high-risk α-HPVs originating in the activated immune niche of the oropharynx, (4) the rapid occurrence of HPV-negative SCCs in ultraviolet (UV)-damaged skin of melanoma patients receiving Raf-inhibitory drugs, and (5) the selective ability of β-HPV E6 oncoproteins to inhibit Notch tumor-suppressive signaling in cutaneous and mesenchymal tissues. The crosstalk so implied between oncogenic UV-induced mutations, defective host immunity, and β-HPV-dependent stromal-epithelial signaling suggests that immunosuppressants such as calcineurin inhibitors intensify mitogenic signalling in TP53-mutant keratinocytes while also abrogating immune-dependent Notch-mediated tumor repression. This emerging interplay between solar damage, viral homeostasis and immune control makes it timely to reappraise strategies for managing skin SCCs in transplant patients. [Copyright &y& Elsevier]

Summary: Despite the increase in long-term survival, liver transplant recipients still exhibit higher morbidity and mortality than the general population. This is in part attributed to the lifelong administration of immunosuppression and its associated side effects. Several studies reported in the last decades have evaluated the impact of immunosuppression minimization in liver transplant recipients, but results have been inconsistent due to the heterogeneity of study designs and insufficient sample sizes. On the other hand, complete immunosuppression withdrawal has proven to be feasible in approximately 20% of carefully selected liver transplant recipients, especially in older patients and those with longer duration after transplantation. The long-term risks and clinical benefits of this strategy, however, also need to be clarified. As a consequence, and despite the general perception that a large proportion of liver recipients are over-immunosuppressed, it is currently not possible to derive evidence-based guidelines on how to manage long-term immunosuppression to improve clinical outcomes. Large clinical trials of drug minimization and/or withdrawal focused on clinically-relevant long-term outcomes are required. Development of personalized medicine tools and a deeper understanding of the pathogenesis of idiopathic inflammatory graft lesions will be pre-requisites to achieve these goals. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell-death 1 (PDCD1) are two genes encoding coinhibitory immunoreceptors that are involved in regulation transplant rejection and tolerance induction. Thus, CTLA-4 and PDCD1 may be good candidate genes to evaluate in liver transplant rejection. In this retrospective study, we investigated whether four functional single nucleotide polymorphisms (SNP) of the CTLA-4 gene and PDCD1 gene were associated with susceptibility to liver transplant rejection. The SNPs −1772T>C (rs733618), −1661A>G (rs4553808) of the CTLA-4 gene, and the SNPs 7146G>A (rs11568821), 7209C>T (rs41386349) of the PDCD1 gene were genotyped by polymerase chain reaction allele specific restriction enzyme analysis (PCR-ASRA) in 100 liver recipients with acute rejection, 104 liver transplant recipients without acute rejection and 100 healthy control individuals. For the selected SNPs we did not detect any significant difference in genotypic and allelic frequencies between liver transplant recipients with and without acute rejection. In conclusion, our results suggest that the tested SNPs may not be associated with susceptibility to acute liver transplant rejection in a Caucasian population. [Copyright &y& Elsevier]

Abstract: Introduction: Renal transplant recipients are at increased risk of venous thromboembolic events, which is in part caused by their treatment with maintenance immunosuppressive drugs. Because we observed an increased incidence of venous thromboembolic events in renal transplant recipients treated with the mTOR inhibitor (mTORi) everolimus, we aimed to identify prothrombotic mechanisms of this immunosuppressive drug. Materials and Methods: In a single center study, nested in a multi-center randomized controlled trial, we measured parameters of coagulation, anti-coagulation and fibrinolysis in renal transplant recipients, receiving the mTORi everolimus (n=16, mTOR group) and compared them to a similar patient group, receiving a calcineurin inhibitor and/or mycophenolate sodium (n=20, non-mTOR group). All patients were at least 6months following transplantation with a stable transplant function. Results: The use of an mTORi was associated with significantly higher levels of von Willebrand factor, prothrombin fragment 1+2, thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 as compared to a non-mTORi based immunosuppressive regimen. Conclusions: Treatment with an mTORi leads to increased endothelial activation, thrombin formation and impaired fibrinolysis in renal transplant recipients. This suggests an increased risk of thrombotic events in renal transplant recipients treated with mTOR inhibitors. A prospective study to establish the precise risk of thrombotic events in these patients is urgently needed. [Copyright &y& Elsevier]

Abstract: This study aimed to investigate the association between longitudinal exposure to mycophenolic acid (MPA) and acute rejection (AR) risk in the first year after renal transplantation, and to propose MPA exposure targets conditionally to this association. A joint model, adjusted for monitoring strategy (fixed-dose versus concentration-controlled) and recipient age, was developed; it combined a mixed-effects model to describe the whole pattern of MPA exposure (i.e. area under the concentration–time curve (AUC)) and a survival model. MPA AUC thresholds were determined using time-dependent receiver-operating characteristics (ROC) curves. Data from 490 adult renal-transplant recipients, representative of the general population of adult renal-transplant patients (i.e. including patients considered at low immunological risk-enrolled in the OPERA trial as well as second renal transplant and patients co-treated by either cyclosporine or tacrolimus), were analyzed. A significant association was found between the longitudinal exposure to MPA (MPA AUCs= f(t)) and AR (p =0.0081), and validated by bootstrapping. A significant positive correlation was observed between time post-transplantation and ROC thresholds which increased in average from 35mgh/L in the first days to 41mgh/L beyond six months post-transplantation (p <0.001). Using a new modeling approach which recognizes the repeated measures in a same patient, this study supports the association between MPA exposure and AR. [Copyright &y& Elsevier]

With advancements in immunosuppressive strategies and availability of better immunosuppressive agents, survival rate following liver transplantation has improved significantly in the recent times. Besides improvements in surgical techniques, the most important factor that has contributed to this better outcome is the progress made in the field of immunosuppression. Over the last several years, the trend has changed to tailored immunosuppression with the aim of achieving optimal graft function while avoiding its undesirable side effects. Induction agents are no longer used routinely and the aim is to provide minimal immunosuppression in the maintenance phase. The present review discusses the various types of immunosuppressive agents, their mechanism of action, clinical utility, advantages and disadvantages, and their side effects in short and long-term. It also discusses about tailoring immunosuppression in presence of various situations such as renal dysfunction, metabolic syndrome, hepatitis C recurrence, cytomegalovirus infections and so on. The issue of chronic kidney disease and the available renal sparing immunosuppressive strategies has been particularly stressed upon. Finally, it discusses about the practical aspects of various immunosuppression regimens including drug monitoring. [Copyright &y& Elsevier]

Chronic kidney disease (CKD) after solid organ transplant is a common clinical presentation, affecting 10% to 20% of liver, heart, and lung transplant recipients and accounting for approximately 5% of the kidney transplant waiting list. The causes of CKD are different for different types of transplants and are not all, or even predominantly, due to calcineurin inhibitor toxicity, with significant heterogeneity particularly in liver transplant recipients. Many solid organ transplant recipients with advanced CKD benefit from kidney transplantation but have a higher rate of death while waitlisted and higher mortality after transplant than the general kidney failure population. Recent organ allocation policies and proposals have attempted to address the appropriate identification and prioritization of candidates in need of a kidney transplant, either simultaneous with or after nonkidney transplant. Future research should focus on predictive factors for individuals identified as being at high risk for progression to kidney failure and death and on strategies to preserve kidney function and minimize the CKD burden in this unique patient population., (Copyright © 2021 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Theoretically, an early protocol biopsy (PB) serves to detect subclinical rejection (SCR), allowing early treatment and prevention of acute rejection (AR) and chronic graft injuries. In this retrospective study, we investigated the incidence of biopsy-proven AR (BPAR) and the usefulness of a 3-month PB in detecting SCR in kidney transplant (KT) and simultaneous pancreas−kidney transplant (SPKT) recipients who received triple immunosuppression and basiliximab. Methods: Between January 2007 and September 2009, 116 patients received transplantation (KT = 112, SPKT = 4). In August 2008, we changed our PB policy and started to collect PB after 3 months instead of a pre-discharge biopsy performed 1 month after transplantation. Here we compare the incidence of SCR (defined as Banff grade Ia or higher) between the pre-discharge PB group and the 3-month PB group. PB was obtained from 41 patients before discharge (pre-discharge PB group), and from 49 patients 3 months after transplantation (3-month PB group). Results: Among all recipients, 21 patients were diagnosed with BPAR (estimated incidence of BPAR 20.1%); including 13 (62.0%) diagnosed from 31 to 180 postoperative days (POD), and only 3 (14.3%) within 30 POD. The incidence of BPAR was not different between the two groups (19.5 and 20.8%, respectively); however, 4 of 8 recipients in the 3-month PB group were diagnosed with SCR, compared to none in the pre-discharge PB group ( P < 0.05). Conclusion: Since the use of triple immunosuppression and basiliximab delayed the onset of AR, we recommend that in order to detect SCR, PB should be obtained 3 months postoperatively. [ABSTRACT FROM AUTHOR]

In response to recently published KDIGO (Kidney Disease: Improving Global Outcomes) guidelines for the care of kidney transplant recipients (KTRs), the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative (KDOQI) organized a working group of transplant nephrologists and surgeons to review these guidelines and comment on their relevance and applicability for US KTRs. The following commentaries on the KDIGO guidelines represent the consensus of our work group. The KDIGO transplant guidelines concentrated on aspects of transplant care most important to this population in the posttransplant period, such as immurlosuppression, infection, malignancy, and cardiovascular care. Our KDOQI work group concurred with many of the KDIGO recommendations except in some important areas related to immunosuppression, in which decisions in the United States are largely made by transplant centers and are dependent in part on the specific patient population served. Most, but not all, KDIGO guidelines are relevant to US patients. However, implementation of many may remain a major challenge because of issues of limitation in resources needed to assist in the tasks of educating, counseling, and implementing and maintaining lifestyle changes. Although very few of the guidelines are based on evidence that is strong enough to justify their being used as the basis of policy or performance measures, they offer an excellent road map to navigate the complex care of KTRs. [ABSTRACT FROM AUTHOR]

β Cell transcription factors such as forkhead box protein O1 (FoxO1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), pancreatic and duodenal homeobox 1, and neuronal differentiation 1, are dysfunctional in type 2 diabetes mellitus (T2DM). Posttransplant diabetes mellitus resembles T2DM and reflects interaction between pretransplant insulin resistance and immunosuppressants, mainly calcineurin inhibitors (CNIs). We evaluated the effect of tacrolimus (TAC), cyclosporine A (CsA), and metabolic stressors (glucose plus palmitate) on insulinoma β cells in vitro and in pancreata of obese and lean Zucker rats. Cells were cultured for 5 days with 100 μM palmitate and 22 mM glucose; CsA (250 ng/mL) or TAC (15 ng/mL) were added in the last 48 h. Glucose plus palmitate increased nuclear FoxO1 and decreased nuclear MafA. TAC in addition to glucose plus palmitate magnified these changes in nuclear factors, whereas CsA did not. In addition to glucose plus palmitate, both drugs reduced insulin content, and TAC also affected insulin secretion. TAC withdrawal or conversion to CsA restored these changes. Similar results were observed in pancreata of obese animals on CNIs. TAC and CsA, in addition to glucose plus palmitate, induced comparable inhibition of calcineurin and nuclear factor of activated T cells (NFAT); therefore, TAC potentiates glucolipotoxicity in β cells, possibly by sharing common pathways of β cell dysfunction. TAC-induced β cell dysfunction is potentially reversible. Inhibition of the calcineurin-NFAT pathway may contribute to the diabetogenic effect of CNIs but does not explain the stronger effect of TAC compared with CsA., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Liver transplantation (LT) has originally been designed to treat hepatobiliary malignancies. The initial results of LT for hepatocellular cancer (HCC) were, however, dismal this mainly due to the poor patient selection procedure. Better surgical and perioperative care and, especially, the refinement of selection criteria led to a major improvement of results, making HCC nowadays (again!) one of the leading indications for LT. This evolution is clearly shown by the innumerable reports aiming to further extend inclusion criteria for LT in HCC patients. Nonetheless, the vast majority of papers only deals with morphologic (tumour diameter and number) and (only recently) biologic (tumour markers and response to locoregional treatment) parameters to do so. Curiously enough, the role of both the immune competent state of the recipient as well as the impact of both immunosuppression (IS) type and load has been very poorly addressed in this context, even if it has been shown for a long time, based on both basic and clinical research, that they all play a key role in the outcome of any oncologic treatment and in the development of de novo as well as recurrent tumours. This chapter aims to give, after a short introductive note about the currently used inclusion criteria of HCC patients for LT and about the role of IS in carcinogenesis, a comprehensive overview of the actual literature related to the impact of different immunosuppressive drugs and schemes on outcome of LT in HCC recipients. Unfortunately, up to now solid conclusions cannot be drawn due to the lack of high-level evidence studies caused by the heterogeneity of the studied patient cohorts and the lack of prospectively designed and randomized studies. Based on long-term personal experience with immunosuppressive handling in LT some proposals for further clinical research and practice are put forward. The strategy of curtailing and minimising IS should be explored in the growing field of transplant oncology taking thereby into account the immunological privilege of the liver allograft. These strategies will become more and more compelling when further extending the indications in which adjuvant chemotherapy will probably become an inherent part of the therapeutic scheme of HCC liver recipients., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

In a 24-month, multicenter, open-label, randomized trial, 715 de novo kidney transplant recipients were randomized at 10-14 weeks to convert to everolimus (n = 359) or remain on standard calcineurin inhibitor (CNI) therapy (n = 356; 231 tacrolimus; 125 cyclosporine), all with mycophenolic acid and steroids. The primary endpoint, change in estimated glomerular filtration rate (eGFR) from randomization to month 12, was similar for everolimus versus CNI: mean (standard error) 0.3(1.5) mL/min/1.73 2 versus -1.5(1.5) mL/min/1.73 2 (p = 0.116). Biopsy-proven acute rejection (BPAR) at month 12 was more frequent under everolimus versus CNI overall (9.7% vs. 4.8%, p = 0.014) and versus tacrolimus-treated patients (2.6%, p < 0.001) but similar to cyclosporine-treated patients (8.8%, p = 0.755). Reporting on de novo donor-specific antibodies (DSA) was limited but suggested more frequent anti-HLA Class I DSA under everolimus. Change in left ventricular mass index was similar. Discontinuation due to adverse events was more frequent with everolimus (23.6%) versus CNI (8.4%). In conclusion, conversion to everolimus at 10-14 weeks posttransplant was associated with renal function similar to that with standard therapy overall. Rates of BPAR were low in all groups, but lower with tacrolimus than everolimus., (© 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

SIMCER was a 6-mo, multicenter, open-label trial. Selected de novo liver transplant recipients were randomized (week 4) to everolimus with low-exposure tacrolimus discontinued by month 4 (n = 93) or to tacrolimus-based therapy (n = 95), both with basiliximab induction and enteric-coated mycophenolate sodium with or without steroids. The primary end point, change in estimated GFR (eGFR; MDRD formula) from randomization to week 24 after transplant, was superior with everolimus (mean eGFR change +1.1 vs. -13.3 mL/min per 1.73 m 2 for everolimus vs. tacrolimus, respectively; difference 14.3 [95% confidence interval 7.3-21.3]; p < 0.001). Mean eGFR at week 24 was 95.8 versus 76.0 mL/min per 1.73 m 2 for everolimus versus tacrolimus (p < 0.001). Treatment failure (treated biopsy-proven acute rejection [BPAR; rejection activity index score >3], graft loss, or death) from randomization to week 24 was similar (everolimus 10.0%, tacrolimus 4.3%; p = 0.134). BPAR was more frequent between randomization and month 6 with everolimus (10.0% vs. 2.2%; p = 0.026); the rate of treated BPAR was 8.9% versus 2.2% (p = 0.055). Sixteen everolimus-treated patients (17.8%) and three tacrolimus-treated patients (3.2%) discontinued the study drug because of adverse events. In conclusion, early introduction of everolimus at an adequate exposure level with gradual calcineurin inhibitor (CNI) withdrawal after liver transplantation, supported by induction therapy and mycophenolic acid, is associated with a significant renal benefit versus CNI-based immunosuppression but more frequent BPAR., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: In a phase 2 study, kidney transplant recipients of low immunologic risk who switched from a calcineurin inhibitor (CNI) to belatacept had improved kidney function at 12 months postconversion versus those continuing CNI therapy, with a low rate of acute rejection and no transplant loss., Study Design: 36-month follow-up of the intention-to-treat population., Setting & Participants: CNI-treated adult kidney transplant recipients with stable transplant function (estimated glomerular filtration rate [eGFR], 35-75mL/min/1.73m 2 )., Interventions: At 6 to 36 months posttransplantation, patients were randomly assigned to switch to belatacept-based immunosuppression (n=84) or continue CNI-based therapy (n=89)., Outcomes: Safety was the primary outcome. eGFR, acute rejection, transplant loss, and death were also assessed., Measurements: Treatment exposure-adjusted incidence rates for safety, repeated-measures modeling for eGFR, Kaplan-Meier analyses for efficacy., Results: Serious adverse events occurred in 33 (39%) belatacept-treated patients and 36 (40%) patients in the CNI group. Treatment exposure-adjusted incidence rates for serious infections (belatacept vs CNI, 10.21 vs 9.31 per 100 person-years) and malignancies (3.01 vs 3.41 per 100 person-years) were similar. More patients in the belatacept versus CNI group had any-grade viral infections (14.60 vs 11.00 per 100 person-years). No posttransplantation lymphoproliferative disorder was reported. Belatacept-treated patients had a significantly greater estimated gain in mean eGFR (1.90 vs 0.07mL/min/1.73m 2 per year; P for time-by-treatment interaction effect = 0.01). The probability of acute rejection was not significantly different for belatacept (8.38% vs 3.60%; HR, 2.50 [95% CI, 0.65-9.65; P=0.2). HR for the comparison of belatacept to the CNI group for time to death or transplant loss was 1.00 (95% CI, 0.14-7.07; P=0.9)., Limitations: Exploratory post hoc analysis with a small sample size., Conclusions: Switching patients from a CNI to belatacept may represent a safe approach to immunosuppression and is being further explored in an ongoing phase 3b trial., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age-specific effects of the calcineurin inhibitor tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T cells. Old recipient mice exhibited prolonged skin graft survival compared with young animals after TAC administration. More important, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce proinflammatory interferon-γ cytokine production and promote interleukin-10 production in old CD4 + T cells. In addition, TAC administration decreased interleukin-2 secretion in old CD4 + T cells more effectively while inhibiting the proliferation of CD4 + T cells in old mice. Both TAC-treated murine and human CD4 + T cells demonstrated an age-specific suppression of intracellular calcineurin levels and Ca 2+ influx, two critical pathways in T cell activation. Of note, depletion of CD8 + T cells did not alter allograft survival outcome in old TAC-treated mice, suggesting that TAC age-specific effects were mainly CD4 + T cell mediated. Collectively, our study demonstrates age-specific immunosuppressive capacities of TAC that are CD4 + T cell mediated. The suppression of calcineurin levels and Ca 2+ influx in both old murine and human T cells emphasizes the clinical relevance of age-specific effects when using TAC., (© 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

In renal transplantation, use of calcineurin inhibitors (CNIs) is associated with nephrotoxicity and immunosuppression with malignancies and infections. This trial aimed to minimize CNI exposure and total immunosuppression while maintaining efficacy. We performed a randomized controlled, open-label multicenter trial with early cyclosporine A (CsA) elimination. Patients started with basiliximab, prednisolone (P), mycophenolate sodium (MPS), and CsA. At 6 months, immunosuppression was tapered to P/CsA, P/MPS, or P/everolimus (EVL). Primary outcomes were renal fibrosis and inflammation. Secondary outcomes were estimated glomerular filtration rate (eGFR) and incidence of rejection at 24 months. The P/MPS arm was prematurely halted. The trial continued with P/CsA (N = 89) and P/EVL (N = 96). Interstitial fibrosis and inflammation were significantly decreased and the eGFR was significantly higher in the P/EVL arm. Cumulative rejection rates were 13% (P/EVL) and 19% (P/CsA), (p = 0.08). A post hoc analysis of HLA and donor-specific antibodies at 1 year after transplantation revealed no differences. An individualized immunosuppressive strategy of early CNI elimination to dual therapy with everolimus was associated with decreased allograft fibrosis, preserved allograft function, and good efficacy, but also with more serious adverse events and discontinuation. This can be a valuable alternative regimen in patients suffering from CNI toxicity., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Despite their clinical efficacy, concerns about calcineurin inhibitor (CNI) toxicity make alternative regimens that reduce CNI exposure attractive for renal transplant recipients. In this systematic review and meta-analysis, we assessed four CNI immunosuppression strategies (minimization, conversion, withdrawal, and avoidance) designed to reduce CNI exposure and assessed the impact of each on patient and allograft survival, acute rejection and renal function. We evaluated 92 comparisons from 88 randomized controlled trials and found moderate- to high-strength evidence suggesting that minimization strategies result in better clinical outcomes compared with standard-dose regimens; moderate-strength evidence indicating that conversion to a mammalian target of rapamycin inhibitor or belatacept was associated with improved renal function but increased rejection risk; and moderate- to high-strength evidence suggesting planned CNI withdrawal could result in improved renal function despite an association with increased rejection risk. The evidence base for avoidance studies was insufficient to draw meaningful conclusions. The applicability of the review is limited by the large number of studies examining cyclosporine-based strategies and low-risk populations. Additional research is needed with tacrolimus-based regimens and higher risk populations. Moreover, research is necessary to clarify the effect of induction and adjunctive agents in alternative immunosuppression strategies and should include more comprehensive and consistent reporting of patient-centered outcomes., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

This prospective, randomized, double-blind, placebo-controlled study evaluated the effects of ramipril on urinary protein excretion in renal transplant patients treated with sirolimus following conversion from a calcineurin inhibitor. Patients received ramipril or placebo for up to 6 weeks before conversion and 52 weeks thereafter. Doses were increased if patients developed proteinuria (urinary protein/creatinine ratio ≥0.5); losartan was given as rescue therapy for persistent proteinuria. The primary end point was time to losartan initiation. Of 295 patients randomized, 264 met the criteria for sirolimus conversion (ramipril, 138; placebo, 126). At 52 weeks, the cumulative rate of losartan initiation was significantly lower with ramipril (6.2%) versus placebo (23.2%) (p < 0.001). No significant differences were observed between ramipril and placebo for change in glomerular filtration rate from baseline (p = 0.148) or in the number of patients with biopsy-confirmed acute rejection (13 vs. 5, respectively; p = 0.073). One patient in the placebo group died due to cerebrovascular accident. Treatment-emergent adverse events were consistent with the known safety profile of sirolimus and were not potentiated by ramipril co-administration. Ramipril was effective in reducing the incidence of proteinuria for up to 1 year following conversion to sirolimus in maintenance renal transplant patients., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

There is no literature on the use of belatacept for sensitized patients or regrafts in kidney transplantation. We present our initial experience in high immunologic risk kidney transplant recipients who were converted from tacrolimus to belatacept for presumed acute calcineurin inhibitor (CNI) toxicity and/or interstitial fibrosis/tubular atrophy. Six (mean age = 40 years) patients were switched from tacrolimus to belatacept at a median of 4 months posttransplant. Renal function improved significantly from a peak mean estimated glomerular filtration rate (eGFR) of 23.8 ± 12.9 mL/min/1.73 m(2) prior to the switch to an eGFR of 42 ± 12.5 mL/min/1.73 m(2) (p = 0.03) at a mean follow-up of 16.5 months postconversion. No new rejection episodes were diagnosed despite a prior history of rejection in 2/6 (33%) patients. Surveillance biopsies performed in 5/6 patients did not show subclinical rejection. No development of donor-specific antibodies (DSA) was noted. In this preliminary investigation, we report improved kidney function without a concurrent increase in risk of rejection and DSA in six sensitized patients converted from tacrolimus to belatacept. Improvement in renal function was noted even in patients with chronic allograft fibrosis without evidence of acute CNI toxicity. Further studies with protocol biopsies are needed to ensure safety and wider applicability of this approach., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Vascularized composite allografts (VCAs) are technically feasible. Similar to other organ transplants, VCAs are hampered by the toxicity and incomplete efficacy associated with conventional immunosuppression. Complications attributable to calcineurin inhibitors remain prevalent in the clinical cases reported to date, and these loom particularly large given the nonlifesaving nature of VCAs. Additionally, acute rejection remains almost ubiquitous, albeit controllable with current agents. Costimulation blockade offers the potential to provide prophylaxis from rejection without the adverse consequences of calcineurin-based regimens. In this study, we used a nonhuman-primate model of VCA in conjunction with immunosuppressive regimens containing combinations of B7-specific costimulation blockade with and without adhesion blockade with LFA3-Ig to determine what adjunctive role these agents could play in VCA transplantation when combined with more conventional agents. Compared to tacrolimus, the addition of belatacept improved rejection free allograft survival. The combination with LFA3-Ig reduced CD2(hi) memory T cells, however did not provide additional protection against allograft rejection and hindered protective immunity. Histology paralleled clinical histopathology and Banff grading. These data provide the basis for the study of costimulation blockade in VCA in a relevant preclinical model., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Calcineurin inhibitor cyclosporine is widely used as an immunosuppressant in clinic. However, mounting evidence has shown that cyclosporine hinders tolerance induction by dampening Tregs. Therefore, it is of paramount importance to overcome this pitfall. Kaempferol was reported to inhibit DC function. Here, we found that kaempferol delayed islet allograft rejection. Combination of kaempferol and low-dose, but not high-dose, of cyclosporine induced allograft tolerance in majority of recipient mice. Although kaempferol plus either dose of cyclosporine largely abrogated proliferation of graft-infiltrating T cells and their CTL activity, both proliferation and CTL activity in mice treated with kaempferol plus low-dose, but not high-dose, cyclosporine reemerged rapidly upon treatment withdrawal. Kaempferol increased CD4+FoxP3+ Tregs both in transplanted mice and in vitro, likely by suppressing DC maturation and their IL-6 expression. Reduction in Tregs by low dose of cyclosporine was reversed by kaempferol. Kaempferol-induced Tregs exhibited both allospecific and non-allospecific suppression. Administering IL-6 abrogated allograft tolerance induced by kaempferol and cyclosporine via diminishing CD4+FoxP3+ Tregs. Thus, for the first time, we demonstrated that kaempferol promotes transplant tolerance in the presence of low dose of cyclosporine, which allows for sufficient Treg generation while minimizing side effects, resulting in much-needed synergy between kaempferol and cyclosporine., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Mammalian target of rapamycin (mTOR) inhibitors have been used in transplantation with the hope of minimizing calcineurin inhibitor (CNI)-induced nephrotoxicity. However, mTOR inhibitors are also associated with a range of side effects, including metabolic complications. We aimed to determine the risks of metabolic complications after the conversion from CNI to mTOR inhibitor postkidney transplant. A systematic search in PubMed up to September 2013 identified nine relevant trials (a total of 2323 patients). The primary end points were the relative risks (RRs) of new-onset diabetes after transplant (NODAT) and hypercholesterolemia. The overall RRs of NODAT and hypercholesterolemia associated with mTOR inhibitors were 1.32 (95% confidence interval [CI] 0.92-1.87) and 2.15 (95% CI 1.35-3.41), respectively, compared with CNI-based regimen. Subgroup analyses revealed no differences in the incidence of NODAT or hypercholesterolemia between sirolimus- versus everolimus-based regimen, or between early versus late conversion. Analyses of secondary outcomes revealed a higher risk of acute rejection, proteinuria and anemia, but no difference in the risk of opportunistic infections after mTOR inhibitor conversion. In conclusion, the conversion from CNI to mTOR inhibitor in low-to-moderate risk kidney transplant recipients was associated with nonsignificant trend toward increased risk of NODAT and significant increase in hypercholesterolemia, acute rejection, proteinuria and anemia., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

In a randomized, comparative study of cardiac transplant patients with mild-to-moderate renal insufficiency, conversion from calcineurin inhibitors (CNIs) to sirolimus improved renal function at 1 year versus continuing CNIs, with an attendant risk of biopsy-confirmed acute rejection (BCAR). Post hoc analyses were conducted to identify predictors of BCAR and GFR improvement associated with conversion. Patients with proteinuria >500 mg/day were excluded. Univariate and multivariate regression analyses tested 13 parameters for BCAR and six for GFR improvement. In 57 sirolimus-treated patients, mean daily mycophenolate mofetil (MMF) dose was lower in those with versus without BCAR (1000 vs. 1420 mg; p = 0.014). Receiver operating characteristic analysis identified MMF dose ≤1000 mg/day as the optimal cutoff to predict BCAR. Multivariate analysis confirmed low MMF dose (odds ratio: 9.94; p = 0.007) and non-white race (odds ratio: 15.3; p = 0.06) were independently associated with BCAR. GFR improvement was evaluated in intent-to-treat patients (n = 116). Significant interaction was detected between treatment effect and preexisting diabetes status (univariate p = 0.077; multivariate p = 0.022), indicating greater beneficial effect of sirolimus in those without preexisting diabetes. These findings suggest that sirolimus is more effective in improving GFR in patients without preexisting diabetes, and adequate MMF doses are needed for sirolimus conversion., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)