Scandiatransplant acceptable mismatch program (STAMP) a bridge to transplanting highly immunized patients.

BACKGROUND: Highly immunized patients are a challenge for organ transplantation programs. One way of increasing the likelihood of transplantation in this group of patients is to expand the possible donations by defining acceptable HLA mismatches. In the Scandiatransplant Acceptable Mismatch Program (STAMP), a de-centralized approach has been implemented in 2009. AIMS: The program has been improved during the years from utilizing HLA-A, -B, -DR matching only to include typing of all deceased donors for HLA-A, -B, -C, -DRB1 and -DQB1. The calculation of a transplantability score (TS) has been introduced in order to take both HLA and AB0 into consideration resulting in a more realistic picture of the transplantability chance. MATERIALS AND METHODS: Patients were selected for eligibility and results of immunisation status were prepared in each of the 9 tissue typing laboratories, while access to the program is finally governed by a common steering group of immunologists and clinicians. RESULTS: In the period from March 2009 until February 2015, 96 patients were transplanted within this program. The mean recipient age was 49 years and 57% were females, 30% of the patients were first transplants and of these 93% were females. The majority of the patients had 2-5 HLA-A, -B. -DR mismatches. The allograft survival at 60 months was 79.1%. Applying the TS to the cohort confirmed that patients with a low TS score had longer waiting times. CONCLUSION: The program has matured during the years and now proves to be a valid approach for transplanting highly immunized patients.

Effect of remote ischemic conditioning on dendritic cell number in blood after renal transplantation--flow cytometry in a porcine model.

Delayed graft function after transplantation increases the risk of rejection. Remote ischemic conditioning (rIC) consists of repetitive, brief, non-damaging periods of ischemia in a limb. For reasons not fully understood, rIC protects the target organ against subsequent ischemia-reperfusion injury. Because ischemic endothelium attracts dendritic cells (DCs), we hypothesised that rIC protects the organ by "trapping" circulating DCs in the limb exposed to rIC. With fewer DCs thus available to infiltrate the graft, a strong T-cell mediated immune response toward the graft is less likely. To test this hypothesis, we measured the number of circulating DCs in a porcine model of renal transplantation with and without rIC. Brain death was induced in eight 65-kg donor pigs. After 22 h of cold ischemia, the kidneys were transplanted into sixteen 15-kg recipient pigs. The recipients were randomised to either non-rIC or rIC before reperfusion of the graft and observed 10 h after reperfusion. The number of DCs was determined by flow cytometry. DCs were identified on the basis of forward- and side-scatter characteristics of CD14-negative mononuclear cells with expression of CD172a. Dendritic cells were subclassified as either plasmacytoid (pDCs) (CD172a(dim), CD4(+), CD14(-)) or conventional (cDCs) (CD172a(high), CD4(-), CD14(-)). Remote ischemic conditioning did not affect the number of circulating cDCs or pDCs within the 10h after transplantation studied. Regardless of rIC, the number of pDCs decreased after graft reperfusion and then returned to baseline levels. In contrast, the number of circulating cDCs increased after reperfusion and later returned to baseline levels.

Gene expression analysis of calcineurin isoforms in T-lymphocytes--a method applied on kidney-transplant recipients.

BACKGROUND: Tacrolimus exerts its immunosuppressive effect through inhibition of the intracellular enzyme calcineurin phosphatase (CaN). In this study, we set-up a validated real-time PCR method to measure the gene expression of the two major isoforms of the catalytic subunit of CaN in T-lymphocytes. METHODS: 20 stable kidney-transplant recipients, 10 early kidney-transplant recipients and 10 healthy non-medicated subjects had blood drawn and T-lymphocytes were isolated using E-rosette gradient centrifugation method. The cell counts were analyzed by DNA quantification using Hoeschst 33285. Gene expressions were analyzed using real-time PCR for CaN Aalpha, CaN Abeta and the reference genes CD3E and PPIB. RESULTS: The real-time PCR method was found to be with high efficiencies and low intra- and inter-assay variabilities. No statistically significant differences were found in the gene expression levels of the two reference genes among the three groups. The two major isoforms of CaN A were expressed in equal amounts in the T-lymphocytes. CONCLUSION: We found no significant difference in the reference genes between the three groups, but looking at the data there was a trend towards an up-regulation of CD3E. PPIB appears to be the more stable of the two reference genes tested in our study.

Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model.

BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. CONCLUSIONS: CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Calcineurin activity in tacrolimus-treated renal transplant patients early after and 5 years after transplantation.

The pharmacodynamic (PD) action of tacrolimus (FK) within the T-cell is inhibition of calcineurin phosphatase (CaN). Determination of CaN activity provides us with an important PD marker. Eleven renal transplant patients treated with FK were investigated on day 14 following transplantation and 5 years later. Blood samples drawn before as well as 1, 2, 3, and 4 hours after oral intake of FK were analyzed for CaN activity and blood FK concentrations. Twenty healthy subjects had one blood sample drawn for CaN activity, which was measured as the release of (32)P from a phosphorylated peptide. Radioactivity of (32)P was quantitated by liquid scintillation counting with the results converted to units of CaN utilizing a calibration curve. On day 14, we observed significant inhibition of CaN activity at T:1, 2, and 3 compared with the predose level (P = .002; P = .015; P = .015). Furthermore, all measured CaN activities were significantly different from those observed in healthy nonmedicated subjects. In contrast, at 5 years posttransplant only the CaN activity at T:2 was significantly inhibited compared with the predose level (P = .02). Additionally, all CaN activities at this time were not significantly different from CaN activities in the healthy subjects. We were not able to demonstrate individual CaN activity profiles in the patients. The lack of CaN inhibition at 5 years after transplantation despite relevant drug concentrations, probably reflected the lower drug dose used long after transplantation. This result raises the question of whether CaN inhibition is necessary to hold graft function and whether FK possess CaN-independent mechanisms of action.

24-h monitoring of calcineurin phosphatase activity in healthy subjects.

The calcineurin inhibitors cyclosporine and tacrolimus are the cornerstone immunosuppressants used in solid organ transplantation. Studies investigating calcineurin (CaN) activity in renal transplanted patients have been published, but basic properties of the enzyme activity in healthy subjects remain to be described. The aim of this study was to investigate whether CaN displays circadian variation or sex difference is present in healthy subjects. Twenty subjects had blood samples drawn every 4 h for a 24-h period. CaN activity was determined in whole blood as the release of 32P from a phosphorylated peptide. Activity of the 32P was quantitated by liquid scintillation and results converted to units CaN utilizing a calibration curve. We found no circadian variation in CaN activity and no difference between the two sexes. The clinical importance of these findings is that blood samples for calcineurin activity can be drawn without taking the exact time of day into consideration, but only considering the time of drug intake.

Comparison of the temporal profile of calcineurin inhibition by cyclosporine and tacrolimus in renal transplant patients.

Calcineurin phosphatase (CaN) activity has been the focus of several recent studies on renal transplant patients as the calcineurin inhibitors tacrolimus (FK) and cyclosporine (CsA) are still the cornerstone in the immunosuppressive treatment. The aim of this study was to compare the temporal inhibition profiles of CaN using CsA or FK in two groups of renal transplant patients. Nineteen tacrolimus-treated and 10 cyclosporine-treated renal transplant patients had blood samples drawn before and at 1, 2, 3, 4, and 6 hours after ingestion of drug. CaN activity was measured as the release of 32P from a previously phosphorylated peptide and radioactivity quantitated by liquid scintillation counting. Results were expressed as units CaN. Blood concentrations of tacrolimus were determined with an IMx method and of CsA with an EMIT assay. FK-treated patients showed maximal inhibition of CaN activity at 1 to 3 hours, returning to the predose level 4 hours after drug intake. CsA-treated patients showed a gradual decrease in CaN activity with a nadir after 3 hours, failing to return to predose levels during the observation period. Both groups showed a significant rise in drug blood concentrations. To conclude, we have demonstrated that two widely used immunosuppressants, CsA and FK, show different CaN inhibitory profiles in renal transplant patients.

Calcineurin phosphatase activity and immunosuppression. A review on the role of calcineurin phosphatase activity and the immunosuppressive effect of cyclosporin A and tacrolimus.

The mode of immunosuppressive action of tacrolimus (FK506) and cyclosporin A has been elucidated. Both drugs bind to proteins in the cytoplasm to form complexes, which in turn inhibit the phosphatase activity of calcineurin, an important limiting step in the activation of T cells. The association between drug uptake (pharmacokinetics) and enzyme inhibition (pharmacodynamics) is under current investigation. Great variations in the correlation between blood drug levels and enzyme inhibition could indicate that monitoring calcineurin phosphatase activity for treatment might be superior to monitoring blood drug levels.