An LC-MS-based designated comparison method with similar performance to the Lp(a) reference measurement procedure to guide molar Lp(a) standardization.

BACKGROUND: The 2022 consensus statement of the European Atherosclerosis Society (EAS) on lipoprotein(a) (Lp(a)) recognizes the role of Lp(a) as a relevant genetically determined risk factor and recommends its measurement at least once in an individual's lifetime. It also strongly urges that Lp(a) test results are expressed as apolipoprotein (a) (apo(a)) amount of substance in molar units and no longer in confounded Lp(a) mass units (mg/dL or mg/L). Therefore, IVD manufacturers should transition to molar units. A prerequisite for this transition is the availability of an Lp(a) Reference Measurement Procedure (RMP) that allows unequivocal molecular detection and quantification of apo(a) in Lp(a). To that end an ISO 17511:2020 compliant LC-MS based and IFCC-endorsed RMP has been established that targets proteotypic peptides of apolipoprotein(a) (apo(a)) in Lp(a). The RMP is laborious and requires highly skilled operators. To guide IVD-manufacturers of immunoassay-based Lp(a) test kits in the transition from mass to molar units, a Designated Comparison Method (DCM) has been developed and evaluated. METHODS: To assess whether the DCM provides equivalent results compared to the RMP, the procedural designs were compared and the analytical performance of DCM and RMP were first evaluated in a head-to-head comparison. Subsequently, apo(a) was quantified in 153 human clinical serum samples. Both DCM and RMP were calibrated using external native calibrators that produce results traceable to SRM2B. Measurement uncertainty (MU) was checked against predefined allowable MU. RESULTS: The major difference in the design of the DCM for apo(a) is the use of only one enzymatic digestion step. The analytical performance of the DCM and RMP for apo(a) is highly similar. In a direct method comparison, equivalent results were obtained with a median regression slope 0.997 of and a median bias of - 0.2 nmol/L (- 0.2%); the intermediate imprecision of the test results was within total allowable error (TEa) (CVa of 10.2% at 90 nmol/L). CONCLUSIONS: The semi-automated, higher throughput, LC-MS-based method for Lp(a) meets the predefined analytical performance specifications and allowable MU and is hence applicable as a higher order Designated Comparison Method, which is ideally suited to guide IVD manufacturers in the transition from Lp(a) mass to molar units.

Quantitative protein mass-spectrometry requires a standardized pre-analytical phase.

OBJECTIVES: Quantitative protein mass-spectrometry (QPMS) in blood depends on tryptic digestion of proteins and subsequent measurement of representing peptides. Whether serum and plasma can be used interchangeably and whether in-vitro anticoagulants affect the recovery is unknown. In our laboratory serum samples are the preferred matrix for QPMS measurement of multiple apolipoproteins. In this study, we investigated the effect of different matrices on apolipoprotein quantification by mass spectrometry. METHODS: Blood samples were collected from 44 healthy donors in Beckton Dickinson blood tubes simultaneously for serum (with/without gel) and plasma (heparin, citrate or EDTA). Nine apolipoproteins were quantified according to standard operating procedure using value-assigned native serum calibrators for quantitation. Tryptic digestion kinetics were investigated in the different matrices by following formation of peptides for each apolipoprotein in time, up to 22 h. RESULTS: In citrate plasma recovery of apolipoproteins showed an overall reduction with a bias of -14.6%. For heparin plasma only -0.3% bias was found compared to serum, whereas for EDTA-plasma reduction was more pronounced (-5.3% bias) and variable with >14% reduction for peptides of apoA-I, A-II and C-III. Digestion kinetics revealed that especially slow forming peptides showed reduced formation in EDTA-plasma. CONCLUSIONS: Plasma anticoagulants affect QPMS test results. Heparin plasma showed comparable results to serum. Reduced concentrations in citrate plasma can be explained by dilution, whereas reduced recovery in EDTA-plasma is dependent on altered proteolytic digestion efficiency. The results highlight the importance of a standardized pre-analytical phase for accurate QPMS applications in clinical chemistry.

Development and Provisional Validation of a Multiplex LC-MRM-MS Test for Timely Kidney Injury Detection in Urine.

Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-ß1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05-30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.

The Time Has Come for Quantitative Protein Mass Spectrometry Tests That Target Unmet Clinical Needs.

Protein mass spectrometry (MS) is an enabling technology that is ideally suited for precision diagnostics. In contrast to immunoassays with indirect readouts, MS quantifications are multiplexed and include identification of proteoforms in a direct manner. Although widely used for routine measurements of drugs and metabolites, the number of clinical MS-based protein applications is limited. In this paper, we share our experience and aim to take away the concerns that have kept laboratory medicine from implementing quantitative protein MS. To ensure added value of new medical tests and guarantee accurate test results, five key elements of test evaluation have been established by a working group within the European Federation for Clinical Chemistry and Laboratory Medicine. Moreover, it is emphasized to identify clinical gaps in the contemporary clinical pathways before test development is started. We demonstrate that quantitative protein MS tests that provide an additional layer of clinical information have robust performance and meet long-term desirable analytical performance specifications as exemplified by our own experience. Yet, the adoption of quantitative protein MS tests into medical laboratories is seriously hampered due to its complexity, lack of robotization and high initial investment costs. Successful and widespread implementation in medical laboratories requires uptake and automation of this next generation protein technology by the In-Vitro Diagnostics industry. Also, training curricula of lab workers and lab specialists should include education on enabling technologies for transitioning to precision medicine by quantitative protein MS tests.

Mechanism of action of 4-substituted phenols to induce vitiligo and antimelanoma immunity.

Monobenzone is a 4-substituted phenol that can induce vitiligo and antimelanoma immunity. We investigated the influence of the chemical structure on the biological activity of a series of structurally related 4-substituted phenols. All phenols inhibited cellular melanin synthesis, and eight of ten phenols inhibited tyrosinase activity, using the MBTH assay. These phenols also induced glutathione (GSH) depletion, indicative of quinone formation and protein thiol binding, which can increase the immunogenicity of melanosomal proteins. Specific T-cell activation was found upon stimulation with phenol-exposed pigmented cells, which also reacted with unexposed cells. In contrast, 4-tertbutylphenol induced immune activation was not restricted to pigment cells, analogous to contact sensitization. We conclude that 4-substituted phenols can induce specific T-cell responses against melanocytes and melanoma cells, also acting at distant, unexposed body sites, and may confer a risk of chemical vitiligo. Conversely, these phenols may be applicable to induce specific antimelanoma immunity.

Automated Multiplex LC-MS/MS Assay for Quantifying Serum Apolipoproteins A-I, B, C-I, C-II, C-III, and E with Qualitative Apolipoprotein E Phenotyping.

BACKGROUND: Direct and calculated measures of lipoprotein fractions for cardiovascular risk assessment suffer from analytical inaccuracy in certain dyslipidemic and pathological states, most commonly hypertriglyceridemia. LC-MS/MS has proven suitable for multiplexed quantification and phenotyping of apolipoproteins. We developed and provisionally validated an automated assay for quantification of apolipoprotein (apo) A-I, B, C-I, C-II, C-III, and E and simultaneous qualitative assessment of apoE phenotypes. METHODS: We used 5 value-assigned human serum pools for external calibration. Serum proteins were denatured, reduced, and alkylated according to standard mass spectrometry-based proteomics procedures. After trypsin digestion, peptides were analyzed by LC-MS/MS. For each peptide, we measured 2 transitions. We compared LC-MS/MS results to those obtained by an immunoturbidimetric assay or ELISA. RESULTS: Intraassay CVs were 2.3%-5.5%, and total CVs were 2.5%-5.9%. The LC-MS/MS assay correlated (R = 0.975-0.995) with immunoturbidimetric assays with Conformité Européenne marking for apoA-I, apoB, apoC-II, apoC-III, and apoE in normotriglyceridemic (n = 54) and hypertriglyceridemic (n = 46) sera. Results were interchangeable for apoA-I ≤3.0 g/L (Deming slope 1.014) and for apoB-100 ≤1.8 g/L (Deming slope 1.016) and were traceable to higher-order standards. CONCLUSIONS: The multiplex format provides an opportunity for new diagnostic and pathophysiologic insights into types of dyslipidemia and allows a more personalized approach for diagnosis and treatment of lipid abnormalities.

Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

Quantifying protein measurands by peptide measurements: where do errors arise?

Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (∼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from ∼9% to ∼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally demonstrated specimen-specific biases associated with modified peptide sequences or alterations in protease activity. In conclusion, the presented overview of bias and imprecision components contributes to a better understanding of the sources of errors in MS-based protein quantification and provides valuable recommendations to assess and control analytical quality in concordance with the requirements for clinical use.

Metrological traceability in mass spectrometry-based targeted protein quantitation: a proof-of-principle study for serum apolipoproteins A-I and B100.

In this study, we have followed up on previous liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometry (MS) approaches for measurement of apolipoprotein (apo) A-I and apo B100 in serum aiming for implementation of a multiplexed assay in a clinical chemistry laboratory with full metrological traceability. Signature peptides were selected and detected by dynamic MRM, and stable isotope labeled (SIL)-peptides were used as internal standards. Five apo A-I and four apo B100 peptides were measured in serum digests with linearity (R(2)>0.992) in the physiologically relevant concentration ranges. Linearity with regard to protein concentration was ascertained at five concentration levels (R(2)>0.926 and R(2)>0.965, for the apo A-I and apo B100 peptides, respectively). Three native value-assigned sera were used as external calibrators for further method verification. Imprecision values on sample preparation and LC-MS/MS acquisition were below the established minimal specifications for apo A-I and apo B100 (5.0% and 5.3%, respectively). Correlation of LC-MS/MS results with immunoturbidimetric assay results, for normo- and hypertriglyceridemic samples, showed R(2)>0.944 for apo A-I and R(2)>0.964 for apo B100. This LC-MS/MS method has potential for clinical application in normo- and dyslipidemic patients. BIOLOGICAL SIGNIFICANCE: Measurement of apo A-I and apo B100 may offer an alternative to high and low density lipoprotein cholesterol (HDL-c and LDL-c) methods for cardiovascular disease risk assessment in dyslipidemic patients [1]. An LC-MS/MS method for apo A-I and apo B100 has the advantage of antibody independent and specific detection of protein signature peptides. The introduction of an LC-MS/MS method for apo A-I and apo B100 can serve as an example for many existing and newly developed (multiplex) biomarker methods in quantitative clinical chemistry proteomics (qCCP). Such LC-MS/MS methods should meet basic clinical chemistry principles with regard to test evaluation [2]. Criteria for imprecision should be pre-defined, e.g., based on biological variation. The use of commutable and traceable serum-based calibrators will improve inter-laboratory reproducibility of LC-MS/MS methods and may contribute to a more rapid transition of biomarker discovery to clinical utility with benefit for the patient treatment and improvement of general health care.

Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators.

Implementation of quantitative clinical chemistry proteomics (qCCP) requires targeted proteomics approaches, usually involving bottom-up multiple reaction monitoring-mass spectrometry (MRM-MS) with stable-isotope labeled standard (SIS) peptides, to move toward more accurate measurements. Two aspects of qCCP that deserve special attention are (1) proper calibration and (2) the assurance of consistent digestion. Here, we describe the evaluation of tryptic digestion efficiency by monitoring various signature peptides, missed cleavages, and modifications during proteolysis of apolipoprotein A-I and B in normo- and hypertriglyceridemic specimens. Absolute quantification of apolipoprotein A-I and B was performed by LC-MRM-MS with SIS peptide internal standards at two time points (4 and 20 h), using three native protein calibrators. Comparison with an immunoturbidimetric assay revealed recoveries of 99.4 ± 6.5% for apolipoprotein A-I and 102.6 ± 7.2% for apolipoprotein B after 4 h of trypsin digestion. Protein recoveries after 20 h trypsin incubation equaled 95.9 ± 6.9% and 106.0 ± 10.0% for apolipoproteins A-I and B, respectively. In conclusion, the use of metrologically traceable, native protein calibrators looks promising for accurate quantification of apolipoprotein A-I and B. Selection of rapidly formed peptides, that is, with no or minor missed cleavages, and the use of short trypsin incubation times for these efficiently cleaved peptides are likely to further reduce the variability introduced by trypsin digestion and to improve the traceability of test results to reach the desirable analytical performance for clinical chemistry application.

Antifibrinolytics attenuate inflammatory gene expression after cardiac surgery.

OBJECTIVES: Anti-inflammatory effects of tranexamic acid and aprotinin, used to abate perioperative blood loss, are reported and might be of substantial clinical relevance. The study of messenger ribonucleic acid synthesis provides a valuable asset in evaluating the inflammatory pathways involved. METHODS: Whole-blood messenger ribonucleic acid expression of 114 inflammatory genes was compared pre- and postoperatively in 35 patients randomized to receive either placebo, tranexamic acid, or aprotinin. These results were further confirmed by reverse transcription-polymerase chain reaction. RESULTS: Of the 23 genes exhibiting independently altered postoperative gene expression levels, 8 were restricted to the aprotinin group only (growth differentiation factor 3, interleukin 19, interleukin 1 family member 7, transforming growth factor α, tumor necrosis factor superfamily 10, tumor necrosis factor superfamily 12, tumor necrosis factor superfamily 13B, vascular endothelial growth factor α), whereas both aprotinin and tranexamic acid altered gene expression of 3 genes as compared with placebo (FMS-related tyrosine kinase 3 ligand, growth differentiation factor 5, interferon-α8). In general, less upregulation of pro-inflammatory, and more upregulation of anti-inflammatory, genes was observed for patients treated with antifibrinolytics. Gene expression affected by aprotinin coded mostly for proteins that function through serine proteases. CONCLUSIONS: This study demonstrates that the use of tranexamic acid and aprotinin results in altered inflammatory pathways on the genomic expression level. We further demonstrate that the use of aprotinin leads to significant attenuation of the immune response, with several inhibitory effects restricted to the use of aprotinin only. The results aid in a better understanding of the targets of these drugs, and add to the discussion on which antifibrinolytic can best be used in the cardiac surgical patient.

Effects of arsenite and UVA-1 radiation on calcineurin signaling.

Calcineurin is a Ca(2+)-dependent serine/threonine phosphatase and the target of the immunosuppressive drugs cyclosporin and tacrolimus, which are used in transplant recipients to prevent rejection. Unfortunately, the therapeutic use of this drugs is complicated by a high incidence of skin malignancy, which has set off a number of studies into the role of calcineurin signaling in skin, particularly with respect to cell cycle control and DNA repair. Both UVA1 radiation and arsenic species are known to promote skin cancer development via production of reactive oxygen species. In light of the well-documented sensitivity of calcineurin to oxidative stress, we examined and compared the effects of UVA1 and arsenite on calcineurin signaling. In this paper, we show that physiologically relevant doses of UVA1 radiation and low micromolar concentrations of arsenite strongly inhibit calcineurin phosphatase activity in Jurkat and skin cells and decrease NFAT nuclear translocation in Jurkat cells. The effects on calcineurin signaling could be partly prevented by inhibition of NADPH oxidase in Jurkat cells or increased dismutation of superoxide in Jurkat and skin cells. In addition, both UVA1 and arsenite decreased NF-κB activity, although at lower concentrations, arsenite enhanced NF-κB activity. These data indicate that UVA1 and arsenite affect a signal transduction route of growingly acknowledged importance in skin and that calcineurin may serve as a potential link between ROS exposure and impaired tumor suppression.

Coat color dilution in mice because of inactivation of the melanoma antigen MART-1.

Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocyte-specific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and/or maintenance.

Molecular diagnostics of calcineurin-related pathologies.

BACKGROUND: The Ca(2+)-dependent protein phosphatase enzyme calcineurin (Cn) (protein phosphatase 3) is best known for its role as director of the adaptive immune response. One of its principal substrates is the nuclear factor of activated T cells (NFAT), which translocates to the nucleus after dephosphorylation to mediate gene transcription. Drugs targeting Cn (the Cn inhibitors tacrolimus and cyclosporin A) have revolutionized posttransplantation therapy in allograft recipients by considerably reducing rejection rates. CONTENT: Owing primarily to intensive study of the side effects of the Cn inhibitors, the unique importance of Cn and Cn/NFAT signaling in the normal physiological processes of many other cell and tissue types is becoming more evident. During the last decade, it has become clear that an extensive and diverse array of clinical conditions can be traced back, at least in part, to a disturbed Cn-signaling axis. Hence, both diagnostics and therapeutic monitoring could benefit from a technique that conveniently reads out Cn/NFAT operative status. SUMMARY: This review outlines the current knowledge on the pathologic conditions that have calcineurin as a common denominator and reports on the progress that has been made toward successfully applying Cn and Cn/NFAT activity markers in molecular diagnostics.

UVA1 radiation inhibits calcineurin through oxidative damage mediated by photosensitization.

The protein phosphatase calcineurin has been gradually revealing itself as the central controller of our immune response, although it is involved in a wide array of signaling pathways related to cellular development and cell cycle progression. As such, calcineurin is an attractive, yet delicate, therapeutic target for the prevention of allograft rejection and treatment of several inflammatory skin conditions. However, calcineurin activity is not only sensitive to immunosuppressants such as cyclosporin A and tacrolimus, but also subject to modulation by reactive oxygen species. We have recently shown, both in vivo and in vitro, that UVA1 radiation suppresses calcineurin activity. In this paper, we present evidence that this activity loss is due to singlet oxygen and superoxide generated by photosensitization and show that a closely related phosphatase, PP2A, is not affected. Furthermore, a survey of this damage reveals oxidation of several Met and Cys residues as well as an overall conformational change. These findings provide a mechanistic basis for the hypothesis that UVA1 and calcineurin inhibitors both affect the same signal transduction pathway in skin.

A spectrophotometric assay for routine measurement of mammalian target of rapamycin activity in cell lysates.

The mammalian target of rapamycin (mTOR) is an important mediator in the PI3K/AKT signaling pathway. mTOR is the target of immunosuppressive drugs, such as rapamycin and everolimus, that are used in transplant patients but also for the treatment of various cancers. We have developed a method for mTOR activity measurement in cell lysates that measures the phosphorylation of p70 S6 kinase by an enzyme linked immunosorbent assay (ELISA) protocol. Using an optimized lysis composition, activity could be measured in the peripheral blood mononuclear cells (PBMCs) isolated from blood. For the PBMCs, intra- and interassay variations of 7 and 10%, respectively, were found using one lot number of the kit. With different lot numbers, the interassay variation increased up to 21%. Activity remained constant for PBMC pool samples on storage for a period of more than 7 months. Activity could also be measured in CD3+ T-cells isolated from blood. In vitro experiments revealed maximum mTOR inhibition of 30% in PBMCs and 44% in T-cells. The in vitro inhibition in PBMCs could also be demonstrated by Western blotting. The mTOR activity measurements may be used to show in vivo inhibition in renal allograft patients during everolimus treatment and to study mTOR activity in various (tumor) cell types.

Everolimus and sirolimus antagonize tacrolimus based calcineurin inhibition via competition for FK-binding protein 12.

The calcineurin inhibitors cyclosporin A and tacrolimus and the inhibitors of the mTOR, sirolimus and everolimus bind immunophilins that are required for their immunosuppressive action. In contrast to cyclosporin A, tacrolimus and the mTOR inhibitors (MTIs) share common immunophilins, the FK506-binding proteins (FKBPs). We investigated the immunosuppressive interactions of MTIs on tacrolimus based immune suppression, since insights in immunological drug-drug interactions can be very relevant for optimization of immunosuppressive regimens in allograft transplantation medicine. Isolated peripheral blood mononuclear cells from healthy volunteers were incubated with combinations of MTIs and calcineurin inhibitors and when monitored for calcineurin activity and IL-2 excretion after mitogen stimulation, tacrolimus IC(50) concentrations shifted to higher concentrations in the presence of MTIs. This antagonism was absent for cyclosporin A, reproducible for 10 healthy volunteers (p<0.001) and stronger for sirolimus than for everolimus. When cell lysate was treated with and without MTI, tacrolimus and FKBP12, FKBP12 could increase calcineurin inhibition by tacrolimus and reverse the MTI antagonism for both MTIs. These results demonstrate that FKBP12 can be rate limiting for calcineurin inhibition at high tacrolimus concentrations and that the antagonism of sirolimus and everolimus on tacrolimus based immune suppression is mediated via saturation of FKBP12.