Effect of everolimus-based drug regimens on CMV-specific T-cell functionality after renal transplantation: 12-month ATHENA subcohort-study results.

Post-transplant cytomegalovirus (CMV) infections and increased viral replication are associated with CMV-specific T-cell anergy. In the ATHENA-study, de-novo everolimus (EVR) with reduced-exposure tacrolimus (TAC) or cyclosporine (CyA) showed significant benefit in preventing CMV infections in renal transplant recipients as compared to standard TAC + mycophenolic acid (MPA). However, immunomodulatory mechanisms for this effect remain largely unknown. Ninety patients from the ATHENA-study completing the 12-month visit on-treatment (EVR + TAC n = 28; EVR + CyA n = 19; MPA + TAC n = 43) were included in a posthoc analysis. Total lymphocyte subpopulations were quantified. CMV-specific CD4 T cells were determined after stimulation with CMV-antigen, and cytokine-profiles and various T-cell anergy markers were analyzed using flow cytometry. While 25.6% of MPA + TAC-treated patients had CMV-infections, no such events were reported in EVR-treated patients. Absolute numbers of lymphocyte subpopulations were comparable between arms, whereas the percentage of regulatory T cells was significantly higher with EVR + CyA versus MPA + TAC (p = 0.019). Despite similar percentages of CMV-specific T cells, their median expression of CTLA-4 and PD-1 was lower with EVR + TAC (p < 0.05 for both) or EVR + CyA (p = 0.045 for CTLA-4) compared with MPA + TAC. Moreover, mean percentages of multifunctional CMV-specific T cells were higher with EVR + TAC (27.2%) and EVR + CyA (29.4%) than with MPA + TAC (19.0%). In conclusion, EVR-treated patients retained CMV-specific T-cell functionality, which may contribute to enhanced protection against CMV infections.

CYP3A5*3 and CYP2C8*3 variants influence exposure and clinical outcomes of tacrolimus-based therapy.

Aim: The influence of variants in pharmacokinetics-related genes on long-term exposure to tacrolimus (TAC)-based therapy and clinical outcomes was investigated. Patients & methods: Brazilian kidney recipients were treated with TAC combined with everolimus (n = 178) or mycophenolate sodium (n = 97). The variants in CYP2C8, CYP2J2, CYP3A4, CYP3A5, POR, ABCB1, ABCC2, ABCG2, SLCO1B1 and SLCO2B1 were analyzed. Main results:CYP3A5*3/*3 genotype influenced increase in TAC concentration from week 1 to month 6 post-transplantation (p < 0.05). The living donor and CYP2C8*3 variant were associated with reduced risk for delayed graft function (OR = 0.07; 95% CI = 0.03-0.18 and OR = 0.45; 95% CI = 0.20-0.99, respectively, p < 0.05). Conclusion: The CYP3A5*3 variant is associated with increased early exposure to TAC. Living donor and CYP2C8*3 variant seem to be protective factors for delayed graft function in kidney recipients.

A Nucleic Acid Nanostructure Built through On-Electrode Ligation for Electrochemical Detection of a Broad Range of Analytes.

For an assay to be most effective in point-of-care clinical analysis, it needs to be economical, simple, generalizable, and free from tedious workflows. While electrochemistry-based DNA sensors reduce instrumental costs and eliminate complicated procedures, there remains a need to address probe costs and generalizability, as numerous probes with multiple conjugations are needed to quantify a wide range of biomarkers. In this work, we have opened a route to circumvent complicated multiconjugation schemes using enzyme-catalyzed probe construction directly on the surface of the electrode. With this, we have created a versatile DNA nanostructure probe and validated its effectiveness by quantification of proteins (streptavidin, anti-digoxigenin, anti-tacrolimus) and small molecules (biotin, digoxigenin, tacrolimus) using the same platform. Tacrolimus, a widely prescribed immunosuppressant drug for organ transplant patients, was directly quantified with electrochemistry for the first time, with the assay range matching the therapeutic index range. Finally, the stability and sensitivity of the probe was confirmed in a background of minimally diluted human serum.

Tacrolimus Variability: A Cause of Donor-Specific Anti-HLA Antibody Formation in Children.

BACKGROUND AND OBJECTIVES: The most important determinant of long-term graft survival in renal transplantation is adequate immunosuppression. Inadequate immunosuppression may lead to graft loss due to the presence of anti-HLA antibody. The aim of this study was to investigate the effect of variability in tacrolimus blood concentration on anti-HLA antibody development in pediatric recipients of living-donor renal transplants. METHODS: Pediatric recipients of living-donor renal transplants were retrospectively evaluated. Patients with a minimum of two years of follow-up who were administered tacrolimus were included in the study. Patients who had pretransplant anti-HLA antibody were excluded. Variability in tacrolimus blood concentration was assessed using the coefficient of variation ("tacrolimus CV") method. Tacrolimus CV was calculated separately for the first 6 months post-transplant, between 6 and 12 months post-transplant, and from the end of the first year post-transplant to the last follow-up. We constructed receiver operating characteristic (ROC) curves of the tacrolimus CV for each group to find the best cutoff value. RESULTS: A total of 67 patients (including 48 males; 72%) with a mean age of 15.16 ± 4.43 years were included in the study. Anti-HLA antibody positivity was detected in 12 patients (18%). More than three HLA mismatches and the presence of acute cellular rejection correlated with the development of anti-HLA antibody (p = 0.056, 0.009). Tacrolimus CVs for the three periods were 0.37 ± 0.11, 0.31 ± 0.18, and 0.35 ± 0.12, respectively. The cutoff value of tacrolimus CV for anti-HLA antibody development was calculated as 0.32 with a sensitivity of 90.91% and specificity of 50.94% [AUC (area under the curve) 0.713, p = 0.023]. During the second 6-month period and after a  year post-transplant, the percentage of patients with tacrolimus CV > 0.32 was significantly higher in the anti-HLA antibody positive group than in the antibody negative group (67% vs 31%, p = 0.027; 83% vs 47%, p = 0.033). The eGFR (estimated glomerular filtration rate) was similar for the anti-HLA antibody negative and positive groups (78.72 ± 2.86 vs 77.45 ± 8.08, p > 0.05). CONCLUSION: High tacrolimus concentration variability appears to be associated with anti-HLA antibody formation in pediatric recipients of living-donor renal transplants.

Differences in Peripheral Blood Lymphocytes between Brand-Name and Generic Tacrolimus Used in Stable Liver Transplant Recipients.

OBJECTIVES: In this study, peripheral blood lymphocytes were compared between a brand-name and a generic tacrolimus group in stable liver transplant recipients. SUBJECTS AND METHODS: Sixteen patients who underwent ABO-compatible living donor liver transplants between 2012 and 2013 and had stable graft function were included in this study. Ten patients received brand-name tacrolimus and 6 patients received generic tacrolimus. CD3, CD4, CD8, γδ, CD4+FoxP3+, and CD3-CD56+ T cells were analyzed in peripheral blood obtained preoperatively and 4, 8, 12, and 24 weeks after liver transplantation. Categorical variables were compared using a χ2 test or Fisher exact test, and continuous variables were compared using the Mann-Whitney U test. RESULTS: Regarding the baseline and perioperative characteristics, there were no statistically significant differences between the 2 groups. Immunosuppression also was not different. Subtype analysis of T-cell populations carried out in parallel showed similar levels of CD3, CD4, CD8, and γδT cells with brand-name tacrolimus and generic tacrolimus in stable liver transplant recipients. However, the levels of CD4+Foxp3+ and CD3-CD56+ T cells were higher in the brand-name tacrolimus group than in the generic tacrolimus group 8 weeks after transplantation (p < 0.05). CONCLUSIONS: The level of CD4+Foxp3+ T cells was higher in the brand-name tacrolimus group than in the generic tacrolimus group after transplantation. This finding showed that brand-name tacrolimus could have more potential immunosuppressive activity than generic tacrolimus regarding the contribution of CD4+Foxp3+ T cells to graft tolerance in liver transplant recipients.

Two cases of colon LST in transplanted liver patients.

Long-life immunosuppressive therapy increases the risk of de novo tumors in liver transplant recipients by decreasing the immune surveillance against malignant cells and oncogenic viruses. However, no cases of colon precancerous lesions have been reported in these subjects. Patient n. 1, a 73 yrs old male treated with calcineurin and purine synthesis inhibitors, showed at a per-protocol colono-scopy a 3 cm laterally spreading tumor (LST). Patient n. 2, a 73 yrs old male treated with calcineurin inhibitors, showed at a screening colonoscopy an LST occupying one third of the lumen circumference. Both subjects were asymptomatic, had been transplanted 14 years before, and in both cases, lesions showed severe dysplasia. LSTs represent 17.2% of advanced colorectal neoplasia (CRC) and risk factors are multifactorial. Immunosuppression may play a role which is however not completely understood. Based on this report, surveillance colonoscopy in liver transplanted patients should be considered.

Top 10 Things Primary Care Physicians Should Know About Maintenance Immunosuppression for Transplant Recipients.

The success of organ transplantation allows many transplant recipients to return to life similar to nontransplant patients. Their need for regular health care, including preventive medicine, has switched the majority of responsibilities for their health care from transplant specialists to primary care physicians. To take care of transplant recipients, it is critical for primary care physicians to be familiar with immunosuppressive medications, their side effects, and common complications in transplant recipients. Ten subjects are reviewed here in order to assist primary care physicians in providing optimal care for transplant recipients.

Tacrolimus for the prevention and treatment of rejection of solid organ transplants.

Since its introduction to the antirejection armamentarium in 1994, tacrolimus has become the workhorse of transplant professionals for avoidance of solid organ transplant rejection. Not only does tacrolimus have potent immunosuppressive qualities that prevent rejection, but dosing is straight forward and it is generally well tolerated. However, in the long term, conditions such as calcineurin inhibitor nephrotoxicity can become a problem. A discussion of the compound, the pharmacokinetics, history, and current approved uses for tacrolimus is described. Indeed, tacrolimus is the most important drug for preventing transplant rejection. However, the increased appreciation for significant side effects, particularly in the long term, has led to building interest in new agents with different mechanisms of action and different metabolism.

Label-free quantification of Tacrolimus in biological samples by atomic force microscopy.

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose-response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.

Advantageous effects of immunosuppression with tacrolimus in comparison with cyclosporine A regarding renal function in patients after heart transplantation.

BACKGROUND: Nephrotoxicity is a serious adverse effect of calcineurin inhibitor therapy in patients after heart transplantation (HTX). AIM: In this retrospective registry study, renal function within the first 2 years after HTX in patients receiving de novo calcineurin inhibitor treatment, that is, cyclosporine A (CSA) or tacrolimus (TAC), was analyzed. In a consecutive subgroup analysis, renal function in patients receiving conventional tacrolimus (CTAC) was compared with that of patients receiving extended-release tacrolimus (ETAC). METHODS: Data from 150 HTX patients at Heidelberg Heart Transplantation Center were retrospectively analyzed. All patients were continuously receiving the primarily applied calcineurin inhibitor during the first 2 years after HTX and received follow-up care according to center practice. RESULTS: Within the first 2 years after HTX, serum creatinine increased significantly in patients receiving CSA (P<0.0001), whereas in patients receiving TAC, change of serum creatinine was not statistically significant (P=not statistically significant [ns]). McNemar's test detected a significant accumulation of patients with deterioration of renal function in the first half year after HTX among patients receiving CSA (P=0.0004). In patients receiving TAC, no significant accumulation of patients with deterioration of renal function during the first 2 years after HTX was detectable (all P=ns). Direct comparison of patients receiving CTAC versus those receiving ETAC detected no significant differences regarding renal function between patients primarily receiving CTAC or ETAC treatment during study period (all P=ns). CONCLUSION: CSA is associated with a more pronounced deterioration of renal function, especially in the first 6 months after HTX, in comparison with patients receiving TAC as baseline immunosuppressive therapy.

Immunosuppression with tacrolimus improved reproductive outcome of women with repeated implantation failure and elevated peripheral blood TH1/TH2 cell ratios.

PROBLEM: We evaluated the clinical efficacy of immunosuppressive treatment with tacrolimus for repeated implantation failure (RIF) patients who have elevated in T helper (Th1)/Th2 cytokine producing cell ratios. METHOD OF STUDY: This was a prospective cohort study of treatment for RIF patients (n = 42) with elevated peripheral blood Th1 (CD4(+) /IFN-γ(+) )/Th2 (CD4(+) /IL-4(+) ) cell ratios at the Sugiyama clinic between November 2011 and October 2013. Twenty-five patients were treated with tacrolimus (treatment group) and 17 received no treatment (control group). Treatment group received tacrolimus 2 days before embryo transfer and continued until the day of the pregnancy test, for a total of 16 days. The daily dose of tacrolimus (1-3 mg) was determined based on the degree of the Th1/Th2 cell ratio. RESULTS: The clinical pregnancy rate of the treatment group was 64.0%, which was significantly higher than that of the control group (0%) (P < 0.0001). In the treatment group, the miscarriage rate was 6.3%, the live birthrate was 60.0% (P < 0.0001). There was no significant side-effect from tacrolimus in treatment group. No one developed obstetrical complications during pregnancy. CONCLUSION: An immunosuppressive treatment using tacrolimus improved pregnancy outcome of repeated implantation failure patients with elevated Th1/Th2 ratios.

Superior rejection profile during the first 24 months after heart transplantation under tacrolimus as baseline immunosuppressive regimen.

BACKGROUND: The use of tacrolimus (TAC) in patients after heart transplantation (HTX) has increased over the last few years. AIM: In this retrospective study, we evaluated the effects of a TAC (conventional and extended-release TAC)-based immunosuppressive therapy regarding rejection profile in comparison to a cyclosporine A (CSA)-based regimen in patients after HTX. METHODS: The data of 233 patients who underwent HTX at the Heidelberg Heart Transplantation Center from May 1998 until November 2010 were retrospectively analyzed. Primary immunosuppressive therapy was changed from a CSA (n=114) to a TAC (n=119)-based regimen in February 2006 according to center routine. Follow-up period was 2 years post-HTX. Primary endpoint was time to first biopsy-proven rejection requiring therapy. In all patients, routine follow-up at the Heidelberg Heart Transplantation Center was mandatory. RESULTS: Multivariate risk factor analysis regarding time to first rejection episode showed no statistically significant differences regarding recipient age, donor age, recipient sex, donor sex, sex mismatch, ischemic time, and diagnosis leading to HTX between the two groups (all P= not statistically significant). Time to first biopsy-proven rejection was significantly longer in the TAC group (intention-to-treat analysis, n=233, log-rank test P<0.0001; per-protocol analysis, n=150, log-rank test P=0.0003). In patients who underwent a change of primary immunosuppression (n=49), a significantly longer time to first biopsy-proven rejection was also found in the primary TAC subgroup (log-rank test P=0.0297). Further subgroup analysis in the TAC subgroups showed no statistically significant differences in time to biopsy-proven rejection under extended-release TAC compared to conventional TAC (intention-to-treat analysis, log-rank test P=0.1736). CONCLUSION: Our study demonstrated that a TAC-based primary immunosuppressive therapy is superior to a CSA-based immunosuppressive regimen in patients after HTX regarding time to first biopsy-proven rejection.

Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay.

The golden standard for functional evaluation of immunodeficiencies is the incorporation of [(3)H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [(3)H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA+SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.

Sandwich assay for tacrolimus using 2 antitacrolimus antibodies.

BACKGROUND: Although detection of natural haptens by antihapten antibodies in sandwich assay format has the theoretical advantages of high analytical specificity and sensitivity, this type of assay has not been reported because of the seemingly insurmountable task of avoiding steric hindrance between the 2 bindings. This is especially true for ring-structured hydrophobic haptens. The macrolide drug tacrolimus (FK506, Prograf®, 804 Da) is such a hapten. Here we show the detection of tacrolimus using 2 antitacrolimus monoclonal antibodies in a sandwich assay. METHODS: Both antibodies were developed by use of an intact tacrolimus molecule covalently linked to a carrier protein but via 2 different positions separated by 10 carbon atoms. Epitope analysis based on drug analog binding was used to show no overlap between the binding sites of the 2 antibodies, indicating the 10-carbon separation resulted in 2 distinct epitopes. The distinct epitopes suggested that the drug might be approachable by the antibodies from 2 separate directions, which predicted simultaneous binding as in sandwich formation. RESULTS: This prediction was confirmed in sandwich ELISA and affinity column-mediated immunoassay formats. The assay demonstrated good imprecision and significantly lower metabolite cross-reactivity than competitive assay counterparts. Comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using 55 whole-blood samples from transplant patients with tacrolimus concentrations ranging from 0.9 to 29.5 ng/mL showed a linear regression: sandwich = 0.99 × LC-MS/MS + 0.10 ng/mL, r = 0.991, Sy|x = 1.08 ng/mL. CONCLUSIONS: This work demonstrates that a highly specific sandwich assay using 2 antihapten antibodies is feasible for the measurement of a hapten drug.

Post-transplant food allergy in children is associated with liver and not with renal transplantation: a monocentric comparative study.

UNLABELLED: Food allergy is increasingly reported after paediatric liver transplantation. The underlying physiopathological mechanism remains incompletely understood. Therefore, we aimed to determine the incidence, clinical presentation, possible risk factors, and prognosis of post-transplant food allergy in children currently followed after liver and renal transplantation. The study population consists of 49 liver and 21 renal transplant patients transplanted between the age of 22 months and 15 years. Data were collected retrospectively from medical records and via a doctor's questionnaire taken from the parents in a monocentric setting. Post-transplant food allergy has developed in 13 liver transplant patients and in none of the renal transplant recipients. Within the liver transplant group, median age at liver transplantation is significantly lower in the food-allergic (10 months) versus non-food-allergic group (3.3 years; p = 0.002). The use of tacrolimus as primary maintenance immunosuppression is associated with food allergy (p = 0.032) and mean donor age is significantly lower in the food-allergic group (p = 0.009). Compared to the renal transplant group, median age at transplantation is significantly lower in the liver patients (p < 0.001). No significant differences are found in primary immunosuppressive regimens between renal and liver transplant patients. CONCLUSION: Post-transplant food allergy is an important clinical problem in children after liver transplantation which does not affect renal transplant patients despite similar immunosuppressive regimens. Within the group of liver transplant recipients, tacrolimus use, young age at time of transplant and younger donor age were associated with the development of food allergy.

Effects of surface camouflaged islet transplantation on pathophysiological progression in a db/db type 2 diabetic mouse model.

To investigate the inhibition effects of pancreatic islet transplantation on the progression of obese type 2 diabetes, we analyzed the effects of surface camouflaged islet transplantation on delaying the disease progression in a db/db diabetic mouse model. Surface camouflaged islets using 6-arm-PEG-catechol were transplanted in db/db diabetic mice. The fat accumulation and toxicity in the liver, the expansion of islets in the pancreas, and the size change of abdominal adipocyte were analyzed. In addition, the blood glucose control, insulin levels and immunohistochemical staining of recovered tissues were analyzed after transplantation. Then co-administration of anti-CD154 monoclonal antibody and Tacrolimus (IT group) deterred the pathophysiological progression of obese type 2 diabetes. At day 3 of transplantation, the serum insulin concentration of IT group was increased compared to the db/db diabetic mice group. The immunohistochemical studies demonstrated that the mass of 6-arm-PEG-catechol grafted islet was preserved in the transplantation site for 14 days. Surface modification using 6-arm-PEG-catechol effectively inhibited the immune cell infiltration and activation of host immune cells when immunosuppressive drug was given to the db/db type 2 diabetes mice. Therefore, 6-arm-PEG-catechol grafted islets effectively restored the insulin secretion in islet recipients and prevented the disease progression in type 2 diabetes.

Calcineurin inhibitors in liver transplantation - still champions or threatened by serious competitors?

Current strategies for immunosuppression in liver transplant (LT) recipients include the design of protocols targeting a more individualized approach to reduce risk factors such as renal failure, cardiovascular complications and malignancies. Renal injury in LT recipients may be often multifactorial and is associated with increased risk of post-transplant morbidity and mortality. The quest for low toxicity immunosuppressive regimens has been challenging and resulted in CNI minimization protocols or CNI withdrawal and conversion to mycophenolate mofetil (MMF) and/or mammalian target of rapamycin inhibitor-based immunosuppressive regimens. Use of antibody induction to delay CNI administration may be an option in particular in immunocompromized, critically ill patients with high MELD scores. Protocols including MMF introduction and concomitant CNI minimization have the potential to recover renal function even in the medium and long term after LT. We review on hot topics in the prevention and management of acute and chronic renal injury in LT patients. For this purpose, we present and critically discuss results from immunosuppressive studies published in the current literature or presented at recent LT meetings.

Treg cell resistance to apoptosis in DNA vaccination for experimental autoimmune encephalomyelitis treatment.

BACKGROUND: Regulatory T (Treg) cells can be induced with DNA vaccinations and protect mice from the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Tacrolimus (FK506) has been shown to have functions on inducing immunosuppression and augmenting apoptosis of pathologic T cells in autoimmune disease. Here we examined the therapeutic effect of DNA vaccine in conjunction with FK506 on EAE. METHODOLOGY/PRINCIPAL FINDINGS: After EAE induction, C57BL/6 mice were treated with DNA vaccine in conjunction with FK506. Functional Treg cells were induced in treated EAE mice and suppressed Th1 and Th17 cell responses. Infiltrated CD4 T cells were reduced while Treg cells were induced in spinal cords of treated EAE mice. Remarkably, the activated CD4 T cells augmented apoptosis, but the induced Treg cells resisted apoptosis in treated EAE mice, resulting in alleviation of clinical EAE severity. CONCLUSIONS/SIGNIFICANCE: DNA vaccine in conjunction with FK506 treatment ameliorates EAE by enhancing apoptosis of CD4 T cells and resisting apoptosis of induced Treg cells. Our findings implicate the potential of tolerogenic DNA vaccines for treating MS.