Characterization of the novel, HLA-DRB1*07:147Q, identified by next-generation sequencing in a kidney donor.

HLA-DRB1*07:147Q differs from HLA-DRB1*07:01:01:01 by one nucleotide substitution leading to a premature stop codon in exon 4.

Kinetics of Torque Teno virus in heart transplant patients.

End-stage heart failure often requires heart transplantation as a life-prolonging treatment. Immunosuppressive therapy is necessary to avoid rejection, but is associated with serious adverse effects. New approaches are needed to monitor immune function in heart transplant patients. We here report the kinetics of Torque Teno Virus (TTV) after transplantation in a large cohort of heart transplant patients and examine its possible role in predicting rejection. We included 106 patients from Aarhus University Hospital and Oslo University Hospital. Patients were followed for 3 years with clinical assessments, biopsies, TTV measurements, and flowcytometric phenotyping. We observed TTV levels reaching a maximum 3 months after transplantation for all 106 patients, after which levels gradually declined. 38 patients (38 %) had biopsy-proven rejection within the first year. We did not find evidence of an association between TTV and serum trough levels, events of rejection, nor flow cytometric immunophenotype. We report data on a large cohort of heart transplant patients and contribute to the understanding of how TTV behaves in transplant patients. Despite not finding an association with rejection, our results provide important insights into the kinetics of TTV levels after transplantation, which may be useful in future studies of immune function in heart transplant patients.

Eosinophilic infiltration as the initial trace of acute mixed cellular and antibody mediated rejection in a heart transplant patient with concomitant immense epitope-associated HLA-antibody production: a case report.

Acute mixed cellular and antibody-mediated rejection (MR) has an estimated prevalence of 7.8%. However, knowledge of MR immune pathogenesis in cardiac graft rejection remains sparse. We report a case of acute MR in a heart transplant patient with a mutation in the MYH7 gene encoding the protein ß-myosin heavy chain, resulting in familial hypertrophic cardiomyopathy. The patient presented with substantial eosinophilic infiltration and extensive production of Human Leukocyte Antigen (HLA)-antibodies associated with shared epitopes. Eosinophilic infiltration in the endo- and myocardium was diagnosed in routine post-transplant biopsies stained with hematoxylin-eosin on day 6 after transplantation. On day 27, the patient presented with dyspnea, weight gain, increased pro-brain natriuretic peptide, and was hospitalized due to suspected acute rejection. Endomyocardial biopsies showed eosinophils in endo- and myocardium with additional lymphocytes and hyperplastic endothelium. Immunohistochemistry, including CD31/CD68 double stain confirmed endothelium-associated macrophages in capillaries and severe C4d positivity in the capillaries and endocardial endothelium. Lymphocytes were identified as primarily CD45+/CD3+ T cells with a concomitant few CD45+/CD20+ B cells. HLA-antibody analysis demonstrated a significant increase in 13 HLA-antibodies present in pre-transplant-serum, of which anti-B7 was donor-specific, and 23 strong de-novo HLA-class I antibodies of which anti-B62 was donor-specific. 72% of HLA-antibodies, including the two donor-specific antibodies, shared the same HLA antigen epitope; 43P+69A or 163L+167W. This is a case reporting both HLA-antibody and pathohistological data indicating the need for better understanding of interactions between cellular and antibody-mediated immune response mechanisms in graft rejection, and the significance of pre-transplant donor-specific antibodies during immunological pre-transplant risk assessment.

Two novel HLA class I alleles, HLA-C*04:489 and -C*07:01:115, were identified by next-generation sequencing.

The novel HLA-C*04:489 and -C*07:01:115 alleles were detected during routine HLA typing by next-generation sequencing.

Identification of the novel HLA class I allele, HLA-A*31:215, identified by Next Generation Sequencing.

The novel HLA allele HLA-A*31:215 differs from HLA-A*31:01:02:01 by one nucleotide substitution c.G832A in exon 4.

Scandiatransplant acceptable mismatch program-10 years with an effective strategy for transplanting highly sensitized patients.

In March 2009, the Scandiatransplant acceptable mismatch program (STAMP) was introduced as a strategy toward improving kidney allocation to highly sensitized patients. Patients with a transplantability score ≤ 2% are potential candidates for the program. Samples are analyzed and acceptable antigens (HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, DPA1) are defined by the local tissue typing laboratory and finally evaluated by a steering committee. In the matching algorithm, patients have the highest priority when the donor's antigens are all among the recipient's own or acceptable HLA antigens. In the period from 2009 to 2020, we have transplanted 278 highly sensitized kidney patients through the program. The graft survival of the STAMP patients was compared with 9002 deceased donor kidney-transplanted patients, transplanted in the same time period. The 10-year graft survival was 73.4% (95% CI: 60.3-90.0) for STAMP and 82.9% (95% CI: 81.6-84.3) for the reference group. (p = .2). In conclusion, the 10-year allograft survival demonstrates that the STAMP allocation algorithm is immunological safe. The program is continuously monitored and evaluated, and the introduction of matching for all HLA loci is a huge improvement to the program and demonstrate technical adaptability as well as clinical flexibility in a de-centralized organization.

The novel HLA class I allele, HLA-B*14:110, identified by next-generation sequencing.

The novel HLA-allele B*14:110, differs from B*14:02:01:01, by one nucleotide substitution, c.T247A in exon 2.

[The Nordic kidneyexchangeprogramme].

This review describes the ScandiaTransplant Kidney Exchange Programme and the background of renal exchange programmes gaining popularity worldwide, possibilities and limitations of the programmes, the ethical aspects and perspectives. The first kidney exchanges between Danish and Swedish countries were performed in 2019, and until now 23 exchanges and transplantations have been performed. All surgical procedures have been performed simultaneously and/or coordinated at different hospitals in Scandinavia, and the kidney grafts were transported between the participating units.

Detecting mismatched donor HLA types from allograft biopsies - An easily applicable tool for improved individualized risk assessment.

Short-term allograft survival has improved among solid organ transplant (SOT) patients. An increasing number of SOT patients are prepared for re-transplantation because of chronic allograft failure. Lack of HLA typing or incomplete HLA typing of previous donors complicates pretransplant risk assessment, as repeated HLA mismatches may be missed. In addition, a complete HLA type of the donor is essential in the diagnosis of antibody-mediated rejection. We aimed to determine donor HLA types from allograft biopsies from kidney, heart and liver grafts. Graft biopsies were obtained from 13 kidney, heart and liver transplanted patients. HLA typing was performed using q-PCR. Alleles of both donor and recipient origin were detected, and donor HLA type was concluded by deducting known HLA types of the recipient. For all 13 patients, we were able to determine mismatched donor HLA alleles from graft material. These results are promising, because they enable better individualized risk assessment.

Donor-specific anti-HLA antibodies by solid phase immunoassays: advantages and technical concerns.

The detection of donor-specific antibodies (DSAs) is a cornerstone in the immunological risk assessment prior to organ transplantation. The detection methods have developed rapidly during the last decade, and the evidence for clinical interpretation of results obtained by solid phase immunoassays (SPI) is slowly accumulating. Nevertheless, technical limitations and theoretical concerns still mean that "expert opinions" govern clinical decision-making when results of bead-based arrays are applied in immunological risk assessment prior to transplantation. This article underlines the prognostic value of SPI in the immunized recipient of an organ transplant while cautioning uncritical clinical interpretation of mean fluorescence intensity (MFI) as a quantitative parameter in organ transplantation based on documented as well as theoretical shortcomings of the method. The role of SPI-based detection of anti-HLA antibodies in clinical transplantation diagnostics is summarized and put into perspective of the Sensitization in Transplantation: Assessment of Risk (STAR) working group report 2017.

Survival, graft function, and incidence of allograft vasculopathy in heart transplant patients receiving adverse risk profile donor hearts.

AIM: To clarify if use of adverse cardiovascular risk profile (ARP) grafts is associated with impaired long-term outcomes after heart transplantation (HTx). METHODS: Survival was obtained from Scandia Transplant and a local database. ARP DONOR INCLUSION CRITERIA: ≥55 years, diabetes mellitus, arterial hypertension, hypoxemia-induced death, impaired left ventricular (LV) ejection fraction. ARP donors were compared to donors not meeting the eligibility criteria. Sub-analyses were made for donor age. RESULTS: In total, 302 HTxs were performed in 296 patients from 31 December 1992 to 11 August 2016. Median survival was 16.5 years (95% CI, 14.3-22.9), there was no difference between profiles (HR 0.63 (95% CI, 0.33-1.19), P = 0.15). LV systolic function was significantly better in ARP donors (P < 0.05). Freedom from cardiac allograft vasculopathy (CAV) was comparable between profiles, HR 0.9 (95% CI 0.5-1.5). Donor age predisposes to CAV (high to low age: HR 2.8 (95% CI 1.7-4.5), P < 0.0001). Median survival was comparable in patients receiving allograft ≥55 and <55 years (HR 0.77 (95% CI 0.4-1.4), P = 0.38). CONCLUSION: Long-term survival and graft function were excellent in patients receiving ARP grafts. Older grafts were associated with CAV but did not influence survival. Thus, the strategy of expanding availability using ARP grafts seems safe.

Donor-specific antibodies are associated with micro- and macrovascular coronary disease, restrictive myocardial damage, and poor outcome in heart-transplanted patients.

AIMS: We examined the relationship between donor-specific HLA antibody (DSA) presence and graft function, hemodynamics, cardiac allograft vasculopathy (CAV), and major adverse cardiac events (MACE) in stable long-term heart-transplanted (HTx) patients. METHODS: Sera from 79 patients (median 7.5 years after HTx) were analyzed for DSA presence. Graft function was evaluated by echocardiography and right heart catheterization. CAV burden was determined by coronary angiography, optical coherence tomography (OCT), and coronary flow velocity reserve (CFVR). Patients were prospectively followed after DSA assessment. MACE included significant CAV progression, heart failure, treated rejection, and cardiovascular death. RESULTS: Sixty patients had no DSA, and 19 patients were sensitized. The vasculopathy burden by angiography, OCT, and CFVR was more pronounced in DSA-positive patients than in DSA-negative patients. DSA-positive patients had higher pulmonary capillary wedge pressure (16 [8; 21] vs 9 mm Hg [7; 11], P<.05) and right atrial pressure (8 [6; 9] vs 4 mm Hg [2; 6], P<.01) and lower global longitudinal strain (-13% [-10; -15] vs -16% [-14; -17], P<.01) than DSA-negative patients. DSA presence was a strong MACE predictor (HR 4.7 (95% CI 2.0-11.4), P<.001). CONCLUSIONS: DSA-positive patients had higher vasculopathy burden, higher filling pressures, and lower longitudinal myocardial deformation than DSA-negative patients. The DSA presence was a strong MACE predictor.

Protein a Immunoadsorption May Hamper the Decision to Transplant Due to Interference With CDC Crossmatch Results.

Transplanting immunized patients requires immunological monitoring in the pretransplant phase to follow reduction of donor specific HLA antibodies (DSA) after Staphylococcus aureus protein A (SPA) immunoadsorption (IA) or therapeutic plasma exchange followed by IVIG and Rituximab administration. Pretreatment aims to significantly reduce DSA strength. The Tissue Typing Lab at Aarhus University Hospital performs immunological monitoring of approximately 150 kidney transplantation patients per year from two transplant centers. From 2012 to 2013, we experienced seven patients desensitized using SPA IA, initially presenting negative cytotoxic complement dependent (CDC) T-cell crossmatches but positive B and T cell flowcytometric crossmatch, who despite significant DSA reduction developed weakly positive CDC T-cell crossmatch shortly prior to transplantation. We hypothesised that leached SPA during IA could be the cause, as the complication was not observed in patients who received plasma exchanges. We found that the positive CDC was not donor specific and SPA column material incubated with control serum reproduced a positive CDC T-cell crossmatch. Finally, we detected leached SPA in one of the patient samples using a highly sensitive time-resolved fluorescent assay. In conclusion, the results emphasize the importance of carefully considering CDC crossmatch results subsequent to IA, before a planned transplantation is either postponed or cancelled. J. Clin. Apheresis 32:163-169, 2017. © 2016 Wiley Periodicals, Inc.

Acute antibody-mediated rejection after ABO-incompatible kidney transplantation treated successfully with antigen-specific immunoadsorption.

ABO-incompatible kidney transplantation is possible after pre-treatment with rituximab, intravenous immunoglobulin and basiliximab combined with tacrolimus, mycophenolate mofetil and prednisolone. We report on the first patient treated with this protocol who developed acute antibody-mediated rejection (Banff grade II with IgG deposits) caused by ABO antibodies (anti-B). Anti-rejection treatment with anti-B-specific immunoadsorption, intravenous immunoglobulin and methylprednisolone efficiently cleared deposited IgG from the kidney allograft and re-established normal kidney function. We suggest that ABO-incompatible kidney transplantation complicated by acute antibody-mediated rejection, caused by ABO antibodies, may successfully be treated with this regime.

The calcineurin activity profiles of cyclosporin and tacrolimus are different in stable renal transplant patients.

Cyclosporin and tacrolimus remain the cornerstone immunosuppressive drugs in organ transplantation. Dosing and monitoring these drugs is based on pharmacokinetic protocols, but measuring a pharmacodynamic parameter, calcineurin phosphatase (CaN) activity, could be a valuable supplement in determining optimal doses. Forty stable renal transplant patients were investigated three times in a 6-month period. Blood samples were drawn at 0, 1, 2, 3 and 4 h after oral intake of tacrolimus (FK) or cyclosporin at days 1 and 180. At day 90, one blood sample at trough level (FK) or C2 level (cyclosporin A, CsA) was drawn. 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 with CaN. We demonstrated that calcineurin activity profiles at days 1 and 180 were the same for both drugs. Furthermore, we found that patients treated with tacrolimus or cyclosporin displayed different calcineurin activity profiles. We found that cyclosporin displayed greater calcineurin inhibition than tacrolimus. We have demonstrated that the two drugs exert significantly different effects on calcineurin activity in renal transplant patients with stable, well-functioning grafts and that tacrolimus-treated patients can maintain good, stable graft function with minimal CaN inhibition.

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays.

Cyclosporine exhibits a wide spectrum of metabolites that vary considerably in the extent to which they interfere with the various parent drug monitoring immunoassays. There is no consensus regarding the clinical significance of metabolites. Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites in this perspective? The aim of the present study was to determine the concentration of cyclosporine (by means of three different assay methods) and the four most significant metabolites (AM1, AM4N, AM9, and AM1C) in relation to calcineurin phosphatase inhibition. Twelve randomly selected cyclosporine-treated renal transplant patients were included in the study. Blood samples were drawn before, 1, 2, 3, 4, 6, 8, and 12 hr after oral intake of cyclosporine. Parent drug and metabolites were determined by liquid chromatography/tandem mass spectrometry (LC/MSMS). Additionally, cyclosporine concentration was determined by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition correlates strongly with parent cyclosporine metabolites concentrations determined by all three assay methods. Determination methods that took metabolites into consideration exhibit stronger correlations with calcineurin phosphatase inhibition (sum of cyclosporin plus metabolites r=-0.93, LC/MSMS; pFPIA r=-0.94, P

Role of metabolites and calcineurin inhibition on C2 monitoring in renal transplant patients.

BACKGROUND: Many transplantation centres have switched to C2 monitoring of cyclosporin-treated renal transplant patients. The rationale is that the C2 correlates best with AUC0-4 (area under the concentration-time curve), which again correlates with rejection and nephrotoxicity. It has also been demonstrated that calcineurin phosphatase is inhibited maximally 1-2 h after intake of cyclosporin in patients receiving their first dose. Cyclosporin is metabolized to many compounds, which may influence the results of immunoassays. Some metabolites may have immunosuppressive activity. METHODS: Cyclosporin metabolites were added to whole blood from healthy volunteers and the calcineurin phosphatase activity (CaN) was determined. Twenty renal transplant patients at varying times after transplantation had blood samples drawn in the morning before and 1, 2, 3 and 4 h after intake of their usual dose of cyclosporin microemulsion. Whole blood samples were analysed by liquid chromatography/tandem mass spectrometry for cyclosporin blood concentration and for the cyclosporin metabolites AM1, AM9, AM1c and AM4n. All samples were analysed for CaN utilizing a 32P-labelled 19 amino-acid peptide. RESULTS: The concentrations of AM1c and AM4n were very low and cannot contribute to CaN inhibition. The ratio of AM1 and AM9 to cyclosporin was high before intake of the drug, but it was much lower during the following 4 h. The 2-h values of cyclosporin were the best predictors of AUC0-4. Calcineurin phosphatase was most inhibited in the 2-h samples and the 2-h value of CaN was the best predictor of CaN AUC0-4. The correlation with calcineurin inhibition seemed better for cyclosporin plus metabolites than for cyclosporin. CONCLUSIONS: Samples collected at 2 h are the best predictors of AUC0-4 for both cyclosporin and calcineurin inhibition. The impact of metabolites appears to be small; however, the temporal profile of calcineurin inhibition seemed to follow cyclosporin plus metabolites better than cyclosporin alone.

Validation of the calcineurin phosphatase assay.

BACKGROUND: The calcineurin inhibitors cyclosporine and tacrolimus are used as primary immunosuppressive drugs in transplant patients. Measuring calcineurin phosphatase (CaN) activity is a proposed pharmacodynamic approach to optimize dosing of these drugs. METHODS: Whole blood samples were obtained from 10 patients treated with calcineurin inhibitors and 20 healthy volunteers and frozen at -80 degrees C. CaN activity was measured by its ability to dephosphorylate a 19-amino acid peptide previously phosphorylated with [gamma-(32)P]ATP. Radioactivity was quantified by liquid scintillation, and results were converted from cpm to U of CaN. Validation of the assay included enzyme kinetics, linearity, precision (at low and normal CaN activities), analytical recovery, and limit of detection. RESULTS: The enzyme followed simple Michaelis-Menten-type kinetics: V(max) was estimated as 240 nmol (32)P x L(-1) x min(-1) and K(m) as 70 micromol/L. The assay was linear within the concentration range examined. Analytical recovery varied from 68% to 72%. The total analytical SD was 0.059 and 0.053 U of CaN for high and low CaN activity, respectively. The within-day SD for high and low activity was 0.032 and 0.039 U of CaN, respectively. The limit of detection was 0.04 U of CaN, which is far below the values measured in patients treated with CaN inhibitors. CONCLUSIONS: In addition to the pharmacokinetic monitoring applied today, the CaN assay can be used to monitor patients treated with calcineurin inhibitors, hopefully leading to prolonged graft survival.

Blood tacrolimus levels and calcineurin phosphatase activity early after renal transplantation.

The toxicity of tacrolimus (FK) despite therapeutic levels (trough) has led us to investigate its relationship with the inhibition of calcineurin (CaN) in recently transplanted renal patients. Twenty-one patients taking FK had blood drawn on day 3 and 14 at 0,1,2,3,4 and 6h. CaN activity was measured by its ability to cleave 32P from a previously radiolabeled phosphorylated 19-amino acid peptide. Radioactivity was quantitated and results were converted to units CaN. FK concentration was measured simultaneously. Maximal suppression of CaN occurred after 2h on both days. Unlike FK levels, CaN activity returned to predose levels by 6h. Comparing mean CaN activity at time 0 with each subsequent time showed statistical significance at hours 1, 2 and 3 on each day. Comparing mean FK concentrations, similarly, revealed statistical significance at all hours. Area under CaN activity curve (AUCCaN) vs. mean FK levels failed to show significance. However, comparing AUCCaN with mean CaN activity was significant throughout. CaN capacity at time 0 and 6h (day 14) resulted in the best estimate of CaN inhibition. Prior to steady-state (day 3), the best estimate occurred at 2h. No single FK concentration seemed to be a reliable indicator of CaN inhibition.