Tools for a personalized tacrolimus dose adjustment in the follow-up of renal transplant recipients. Metabolizing phenotype according to CYP3A genetic polymorphisms versus concentration-dose ratio.

BACKGROUND AND JUSTIFICATION: The strategy of the concentration-dose (C/D) approach and the different profiles of tacrolimus (Tac) according to the cytochrome P450 polymorphisms (CYPs) focus on the metabolism of Tac and are proposed as tools for the follow-up of transplant patients. The objective of this study is to analyse both strategies to confirm whether the stratification of patients according to the pharmacokinetic behaviour of C/D corresponds to the classification according to their CYP3A4/5 cluster metabolizer profile. MATERIALS AND METHODS: 425 kidney transplant patients who received Tac as immunosuppressive treatment have been included. The concentration/dose ratio (C/D) was used to divide patients in terciles and classify them according to their Tac metabolism rate (fast, intermediate, and slow). Based on CYP3A4 and A5 polymorphisms, patients were classified into 3 metabolizer groups: fast (CYP3A5*1 carriers and CYP34A*1/*1), intermediate (CYP3A5*3/3 and CYP3A4*1/*1) and slow (CYP3A5*3/*3 and CYP3A4*22 carriers). RESULTS: When comparing patients included in each metabolizer group according to C/D ratio, 47% (65/139) of the fast metabolizers, 85% (125/146) of the intermediate and only 12% (17/140) of the slow also fitted in the homonym genotype group. Statistically lower Tac concentrations were observed in the fast metabolizers group and higher Tac concentrations in the slow metabolizers when compared with the intermediate group both in C/D ratio and polymorphisms criteria. High metabolizers required approximately 60% more Tac doses than intermediates throughout follow-up, while poor metabolizers required approximately 20% fewer doses than intermediates. Fast metabolizers classified by both criteria presented a higher percentage of times with sub-therapeutic blood Tac concentration values. CONCLUSION: Determination of the metabolizer phenotype according to CYP polymorphisms or the C/D ratio allows patients to be distinguished according to their exposure to Tac. Probably the combination of both classification criteria would be a good tool for managing Tac dosage for transplant patients.

Herramientas para un ajuste de dosis de tacrolimus más personalizado en el seguimiento de los pacientes con transplante renal. Fenotipo metabolizador según polimorfismos genéticos del CYP3A vs. el cociente concentración-dosis / Tools for a personalized tacrolimus dose adjustment in the follow-up of renal transplant recipients: Metabolizing phenotype according to CYP3A genetic polymorphisms versus concentration–dose ratio

Antecedentes y justificación: La estrategia de la aproximación concentración-dosis (C/D) y los distintos perfiles del tacrolimus (Tac), según los polimorfismos del citocromo P450 (CYPs) se centran en el metabolismo de Tac y se plantean como herramientas para el seguimiento de los pacientes trasplantados. El objetivo de este estudio es comparar la exposición al Tac analizado según ambas estrategias. Materiales y métodos: Se han incluido 425 pacientes trasplantados renales. El cálculo del cociente concentración Tac/dosis (C/D) permitió dividir la población en terciles y clasificar los pacientes según su tasa de metabolismo del Tac en tres grupos (rápida, intermedia y lenta). Con base en los polimorfismos del CYP3A4 y A5, los pacientes se agruparon en metabolizadores rápidos (portadores del CYP3A5*1 y CYP34A *1/*1), intermedios (CYP3A5*3/3 y CYP3A4*1/*1) y lentos (CYP3A5 *3/*3 y portadores del CYP3A4*22). Resultados: Al comparar los pacientes de cada grupo metabolizador según los dos criterios, coincidieron 47% (65/139) de los metabolizadores rápidos, 85% (125/146) de los intermedios y solo 12% (17/140) de los lentos. Se observaron concentraciones de Tac estadísticamente menores en los metabolizadores rápidos y concentraciones mayores en los lentos, comparándolos con el grupo intermedio según el cociente C/D o según polimorfismos. Los metabolizadores rápidos requirieron alrededor de 60% más de dosis de Tac que los intermedios a lo largo del seguimiento, mientras que los lentos aproximadamente 20% menos de dosis que los intermedios. Los metabolizadores rápidos clasificados por ambos criterios presentan un porcentaje mayor de veces con valores de concentración de Tac en sangre infraterapéuticos... (AU)

Background and justification: The strategy of the concentration–dose (C/D) approach and the different profiles of tacrolimus (Tac) according to the cytochrome P450 polymorphisms (CYPs) focus on the metabolism of Tac and are proposed as tools for the follow-up of transplant patients. The objective of this study is to analyse both strategies to confirm whether the stratification of patients according to the pharmacokinetic behaviour of C/D corresponds to the classification according to their CYP3A4/5 cluster metabolizer profile. Materials and methods: Four hundred and twenty-five kidney transplant patients who received Tac as immunosuppressive treatment have been included. The concentration/dose ratio (C/D) was used to divided patients in terciles and classify them according to their Tac metabolism rate (fast, intermediate, and slow). Based on CYP3A4 and A5 polymorphisms, patients were classified into three metabolizer groups: fast (CYP3A5*1 and CYP34A*1/*1 carriers), intermediate (CYP3A5*3/3 and CYP3A4*1/*1) and slow (CYP3A5*3/*3 and CYP3A4*22 carriers). Results: When comparing patients included in each metabolizer group according to C/D ratio, 47% (65/139) of the fast metabolizers, 85% (125/146) of the intermediate and only 12% (17/140) of the slow also fitted in the homonym genotype group. Statistically lower Tac concentrations were observed in the fast metabolizers group and higher Tac concentrations in the slow metabolizers when compared with the intermediate group both in C/D ratio and polymorphisms criteria. High metabolizers required approximately 60% more Tac doses than intermediates throughout follow-up, while poor metabolizers required approximately 20% fewer doses than intermediates. Fast metabolizers classified by both criteria presented a higher percentage of times with sub-therapeutic blood Tac concentration values... (AU)

CYP3A5*3 and CYP3A4*22 Cluster Polymorphism Effects on LCP-Tac Tacrolimus Exposure: Population Pharmacokinetic Approach.

The aim of the study is to develop a population pharmacokinetic (PopPK) model and to investigate the influence of CYP3A5/CYP3A4 and ABCB1 single nucleotide polymorphisms (SNPs) on the Tacrolimus PK parameters after LCP-Tac formulation in stable adult renal transplant patients. The model was developed, using NONMEM v7.5, from full PK profiles from a clinical study (n = 30) and trough concentrations (C0) from patient follow-up (n = 68). The PK profile of the LCP-Tac formulation was best described by a two-compartment model with linear elimination, parameterized in elimination (CL/F) and distributional (CLD/F) clearances and central compartment (Vc/F) and peripheral compartment (Vp/F) distribution volumes. A time-lagged first-order absorption process was characterized using transit compartment models. According to the structural part of the base model, the LCP-Tac showed an absorption profile characterized by two transit compartments and a mean transit time of 3.02 h. Inter-individual variability was associated with CL/F, Vc/F, and Vp/F. Adding inter-occasion variability (IOV) on CL/F caused a statistically significant reduction in the model minimum objective function MOFV (p < 0.001). Genetic polymorphism of CYP3A5 and a cluster of CYP3A4/A5 SNPs statistically significantly influenced Tac CL/F. In conclusion, a PopPK model was successfully developed for LCP-Tac formulation in stable renal transplant patients. CYP3A4/A5 SNPs as a combined cluster including three different phenotypes (high, intermediate, and poor metabolizers) was the most powerful covariate to describe part of the inter-individual variability associated with apparent elimination clearance. Considering this covariate in the initial dose estimation and during the therapeutic drug monitoring (TDM) would probably optimize Tac exposure attainments.

Atiende y atento/a. Estrategias para mejorar la asistencia y el provecho de las clases presenciales / Attend and attentive. Strategies to improve attendance and use of on-site classes

Introducción: Recientemente se ha percibido una reducción significativa de la asistencia del alumnado a las clases de teoría del grado de Podología. El profesorado, consciente de la existente correlación positiva entre la asistencia y el rendimiento acadèmico, se ha propuesto adaptar las clases de teoría para hacerlas más atractivas para el alumnado. Métodos: Se plantearon cambios en dos niveles en las materias de Fisiología, Bioquímica y Biofísica en los cursos 2021-22 y 2022-23. Por un lado, se estructuraron las clases en bloques de 15-20 minutos, alternando la transmisión de información con actividades de participación activa. Por otro lado, se cambió el sistema de evaluación, introduciendo pequeñas actividades evaluativas en algunas clases teóricas. Resultados: Se alcanzó una asistencia superior al 75% en la mayoría de las clases. Las principales causas de absentismo descritas por los estudiantes fueron estudiar otras asignaturas y motivos de salud. El alumnado valoró tanto el formato de clase como el sistema de evaluación con una puntuación de notable. Se analizó si esta estrategia docente comportó mejoras en el rendimiento acadèmico mediante la comparación de las notas obtenidas con las notas de cursos anteriores, y no se observaron diferencias significativas. El profesorado manifestó que la estrategia docente, aunque satisfactoria, implicaba una mayor dedicación. Conclusión: En definitiva, la estrategia descrita para promover la asistencia a clase, aparte de dar más trabajo al profesorado, no ha significado una mejoría en el nivel de aprendizaje del alumnado, pero ha sido satisfactoria para el alumnado y el profesorado.(AU)

Introduction: Recently, a significant reduction in student attendance to theory classes of the Podiatry degree has been perceived. The teaching staff, aware of the existing positive correlation between attendance and academic performance, has proposed to adapt the theory classes to make them more attractive for the students. Methods: Changes were proposed at two levels in the subjects of Physiology, Biochemistry and Biophysics in the academic years 2021-22 and 2022-23. On one hand, classes were structured in blocks of 15-20 minutes, alternating the transmission of information with active participation activities. On the other hand, the continuous assessment system was changed by introducing small evaluative activities in some theoretical classes. Results: Attendance over 75% was achieved in most of the classes. The main causes of absenteeism reported by students were studying other subjects and health reasons. The students rated both the class format and the evaluation system with a grade of B. An analysis was made of whether this teaching strategy led to improvements in academic performance by comparing the grades obtained with those of previous years, and no significant differences were observed. The faculty stated that the teaching strategy, although satisfactory, entailed a greater teaching load for a result that did not improve academic performance. Conclusions: In short, this strategy described to promote class attendance, apart from giving more work to the teaching staff, has not meant an improvement in the level of student learning. But it has been satisfactory for students and teachers.(AU)

A prospective controlled, randomized clinical trial of kidney transplant recipients developed personalized tacrolimus dosing using model-based Bayesian Prediction.

For three decades, tacrolimus (Tac) dose adjustment in clinical practice has been calculated empirically according to the manufacturer's labeling based on a patient's body weight. Here, we developed and validated a Population pharmacokinetic (PPK) model including pharmacogenetics (cluster CYP3A4/CYP3A5), age, and hematocrit. Our study aimed to assess the clinical applicability of this PPK model in the achievement of Tac Co (therapeutic trough Tac concentration) compared to the manufacturer's labelling dosage. A prospective two-arm, randomized, clinical trial was conducted to determine Tac starting and subsequent dose adjustments in 90 kidney transplant recipients. Patients were randomized to a control group with Tac adjustment according to the manufacturer's labeling or the PPK group adjusted to reach target Co (6-10 ng/ml) after the first steady state (primary endpoint) using a Bayesian prediction model (NONMEM). A significantly higher percentage of patients from the PPK group (54.8%) compared with the control group (20.8%) achieved the therapeutic target fulfilling 30% of the established superiority margin defined. Patients receiving PPK showed significantly less intra-patient variability compared to the control group, reached the Tac Co target sooner (5 days vs 10 days), and required significantly fewer Tac dose modifications compared to the control group within 90 days following kidney transplant. No statistically significant differences occurred in clinical outcomes. Thus, PPK-based Tac dosing offers significant superiority for starting Tac prescription over classical labeling-based dosing according to the body weight, which may optimize Tac-based therapy in the first days following transplantation.

The Effect of Intracellular Tacrolimus Exposure on Calcineurin Inhibition in Immediate- and Extended-Release Tacrolimus Formulations.

Despite intensive monitoring of whole blood tacrolimus concentrations, acute rejection after kidney transplantation occurs during tacrolimus therapy. Intracellular tacrolimus concentrations could better reflect exposure at the site of action and its pharmacodynamics (PD). Intracellular pharmacokinetic (PK) profile following different tacrolimus formulations (immediate-release (TAC-IR) and extended-release (TAC-LCP)) remains unclear. Therefore, the aim was to study intracellular tacrolimus PK of TAC-IR and TAC-LCP and its correlation with whole blood (WhB) PK and PD. A post-hoc analysis of a prospective, open-label, crossover investigator-driven clinical trial (NCT02961608) was performed. Intracellular and WhB tacrolimus 24 h time-concentration curves were measured in 23 stable kidney transplant recipients. PD analysis was evaluated measuring calcineurin activity (CNA) and simultaneous intracellular PK/PD modelling analysis was conducted. Higher dose-adjusted pre-dose intracellular concentrations (C0 and C24) and total exposure (AUC0-24) values were found for TAC-LCP than TAC-IR. Lower intracellular peak concentration (Cmax) was found after TAC-LCP. Correlations between C0, C24 and AUC0-24 were observed within both formulations. Intracellular kinetics seems to be limited by WhB disposition, in turn, limited by tacrolimus release/absorption processes from both formulations. The faster intracellular elimination after TAC-IR was translated into a more rapid recovery of CNA. An Emax model relating % inhibition and intracellular concentrations, including both formulations, showed an IC50, a concentration to achieve 50% CNA inhibition, of 43.9 pg/million cells.

Tacrolimus CYP3A Single-Nucleotide Polymorphisms and Preformed T- and B-Cell Alloimmune Memory Improve Current Pretransplant Rejection-Risk Stratification in Kidney Transplantation.

Achieving fast immunosuppression blood exposure after kidney transplantation is key to abrogating both preformed and de novo anti-donor humoral and cellular alloresponses. However, while tacrolimus (TAC) is the cornerstone immunosuppressant inhibiting adaptive alloimmunity, its blood exposure is directly impacted by different single-nucleotide polymorphisms (SNPs) in CYP3A TAC-metabolizing enzymes. Here, we investigated how functional TAC-CYP3A genetic variants (CYP3A4*22/CYP3A5*3) influence the main baseline clinical and immunological risk factors of biopsy-proven acute rejection (BPAR) by means of preformed donor-specific antibodies (DSAs) and donor-specific alloreactive T cells (DSTs) in a large European cohort of 447 kidney transplants receiving TAC-based immunosuppression. A total of 70 (15.7%) patients developed BPAR. Preformed DSAs and DSTs were observed in 12 (2.7%) and 227 (50.8%) patients, respectively. According to the different CYP3A4*22 and CYP3A5*3 functional allele variants, we found 4 differential new clusters impacting fasting TAC exposure after transplantation; 7 (1.6%) were classified as high metabolizers 1 (HM1), 71 (15.9%) as HM2, 324 (72.5%) as intermediate (IM), and 45 (10.1%) as poor metabolizers (PM1). HM1/2 showed significantly lower TAC trough levels and higher dose requirements than IM and PM (p < 0.001) and more frequently showed TAC underexposure (<5 ng/ml). Multivariate Cox regression analyses revealed that CYP3A HM1 and IM pharmacogenetic phenotypes (hazard ratio (HR) 12.566, 95% CI 1.99-79.36, p = 0.007, and HR 4.532, 95% CI 1.10-18.60, p = 0.036, respectively), preformed DSTs (HR 3.482, 95% CI 1.99-6.08, p < 0.001), DSAs (HR 4.421, 95% CI 1.63-11.98, p = 0.003), and delayed graft function (DGF) (HR 2.023, 95% CI 1.22-3.36, p = 0.006) independently predicted BPAR. Notably, a significant interaction between T-cell depletion and TAC underexposure was observed, showing a reduction of the BPAR risk (HR 0.264, 95% CI 0.08-0.92, p = 0.037). Such variables except for DSAs displayed a higher predictive risk for the development of T cell-mediated rejection (TCMR). Refinement of pretransplant monitoring by incorporating TAC CYP3A SNPs with preformed DSAs as well as DSTs may improve current rejection-risk stratification and help induction treatment decision-making.

The Role of Inflammasomes in Glomerulonephritis.

The inflammasome is an immune multiprotein complex that activates pro-caspase 1 in response to inflammation-inducing stimuli and it leads to IL-1ß and IL-18 proinflammatory cytokine production. NLRP1 and NLRP3 inflammasomes are the best characterized and they have been related to several autoimmune diseases. It is well known that the kidney expresses inflammasome genes, which can influence the development of some glomerulonephritis, such as lupus nephritis, ANCA glomerulonephritis, IgA nephropathy and anti-GBM nephropathy. Polymorphisms of these genes have also been described to play a role in autoimmune and kidney diseases. In this review, we describe the main characteristics, activation mechanisms, regulation and functions of the different inflammasomes. Moreover, we discuss the latest findings about the role of the inflammasome in several glomerulonephritis from three different points of view: in vitro, animal and human studies.

TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A.

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

Influence of the Circadian Timing System on Tacrolimus Pharmacokinetics and Pharmacodynamics After Kidney Transplantation.

Introduction: Tacrolimus is the backbone immunosuppressant after solid organ transplantation. Tacrolimus has a narrow therapeutic window with large intra- and inter-patient pharmacokinetic variability leading to frequent over- and under-immunosuppression. While routine therapeutic drug monitoring (TDM) remains the standard of care, tacrolimus pharmacokinetic variability may be influenced by circadian rhythms. Our aim was to analyze tacrolimus pharmacokinetic/pharmacodynamic profiles on circadian rhythms comparing morning and night doses of a twice-daily tacrolimus formulation. Methods: This is a post-hoc analysis from a clinical trial to study the area under curve (AUC) and the area under effect (AUE) profiles of calcineurin inhibition after tacrolimus administration in twenty-five renal transplant patients. Over a period of 24 h, an intensive sampling (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 12.5, 13, 13.5, 14, 15, 20, and 24 h) was carried out. Whole blood and intracellular tacrolimus concentrations and calcineurin activity were measured by UHPLC-MS/MS. Results: Whole blood and intracellular AUC12-24 h and Cmax achieved after tacrolimus night dose was significantly lower than after morning dose administration (AUC0-12 h) (p < 0.001 for both compartments). AUE0-12 h and AUE12-24 h were not statistically different after morning and night doses. Total tacrolimus daily exposure (AUC0-24 h), in whole blood and intracellular compartments, was over-estimated when assessed by doubling the morning AUC0-12 h data. Conclusion: The lower whole blood and intracellular tacrolimus concentrations after night dose might be influenced by a distinct circadian clock. This significantly lower tacrolimus exposure after night dose was not translated into a significant reduction of the pharmacodynamic effect. Our study may provide conceptual bases for better understanding the TDM of twice-daily tacrolimus formulation.

Sustained Inhibition of Calcineurin Activity With a Melt-Dose Once-daily Tacrolimus Formulation in Renal Transplant Recipients.

Tacrolimus (Tac) is the cornerstone calcineurin inhibitor in transplantation. Extended-release Meltdose formulation (Tac-LCP) offers better bioavailability compared with immediate-release formulation (Tac-IR). We postulated that the less fluctuating pharmacokinetic (PK) profile of Tac-LCP might maintain a sustained inhibition of calcineurin activity (CNA) between dose intervals. Higher concentrations (peak plasma concentration (Cmax )) after Tac-IR may not result in a more potent CNA inhibition due to a capacity-limited effect. This study was aimed at evaluating the pharmacodynamic (PD)/PK profiles of Tac-IR compared with Tac-LCP. An open-label, prospective, nonrandomized, investigator-driven study was conducted. Twenty-five kidney transplant recipients receiving Tac-IR were switched to Tac-LCP. Before and 28 days after conversion, intensive CNA-PD and PK sampling were conducted using ultra-high-performance liquid chromatography-tandem accurate mass spectrometry. PD nonlinear mixed effects model was performed in Phoenix-WinNonlin. Statistically significant higher Cmax (P < 0.001) after Tac-IR did not result in lower CNA as compared with after Tac-LCP (P = 0.860). Tac-LCP showed a statistically more maintained CNA inhibition between dose intervals (area under the effect-time curve from 0 to 24 hours (AUE0-24h )) compared with Tac-IR, in which CNA returned to predose levels after 4 hours of drug intake (373.8 vs. 290.5 pmol RII·h/min·mg prot, Tac-LCP vs. Tac-IR; P = 0.039). No correlation was achieved between any PD and PK parameters in any formulations. Moreover, Tac concentration to elicit a 50% of the maximum response (half-maximal inhibitory concentration) was 9.24 ng/mL. The higher Cmax after Tac-IR does not result in an additional CNA inhibition compared with Tac-LCP attributable to a capacity-limited effect. Tac-LCP may represent an improvement of the PD of Tac due to the more sustained CNA inhibition during dose intervals.

Role of Gastrointestinal Hormones as a Predictive Factor for Long-Term Diabetes Remission: Randomized Trial Comparing Metabolic Gastric Bypass, Sleeve Gastrectomy, and Greater Curvature Plication.

PURPOSE: Long-term studies comparing the mechanisms of different bariatric techniques for T2DM remission are scarce. We aimed to compare type 2 diabetes (T2DM) remission after a gastric bypass with a 200-cm biliopancreatic limb (mRYGB), sleeve gastrectomy (SG), and greater curvature plication (GCP), and to assess if the initial secretion of gastrointestinal hormones may predict metabolic outcomes at 5 years. MATERIAL AND METHODS: Forty-five patients with mean BMI of 39.4(1.9)kg/m2 and T2DM with HbA1c of 7.7(1.9)% were randomized to mRYGB, SG, or GCP. Anthropometric and biochemical parameters, fasting concentrations of PYY, ghrelin, glucagon, and AUC of GLP-1 after SMT were determined prior to and at months 1 and 12 after surgery. At 5-year follow-up, anthropometrical and biochemical parameters were determined. RESULTS: Total weight loss percentage (TWL%) at year 1 and GLP-1 AUC at months 1 and 12 were higher in the mRYGB than in the SG and GCP. TWL% remained greater at 5 years in mRYGB group - 27.32 (7.8) vs. SG - 18.00 (10.6) and GCP - 14.83 (7.8), p = 0.001. At 5 years, complete T2DM remission was observed in 46.7% after mRYGB vs. 20.0% after SG and 6.6% after GCP, p < 0.001. In the multivariate analysis, shorter T2DM duration (OR 0.186), p = 0.008, and the GLP-1 AUC at 1 month (OR 7.229), p = 0.023, were prognostic factors for complete T2DM remission at 5-year follow-up. CONCLUSIONS: Long-term T2DM remission is mostly achieved with hypoabsortive techniques such as mRYGB. Increased secretion of GLP-1 after surgery and shorter disease duration were the main predictors of T2DM remission at 5 years.

Mycophenolic acid interferes the transcriptional regulation and protein trafficking of maturation surface markers in dendritic cells.

BACKGROUND: The ability of dendritic cells (DCs) to regulate adaptive immunity makes them interesting cells to be used as therapeutic targets modulating alloimmune responses. Mycophenolic acid (MPA) is an immunosuppressor commonly used in transplantation, and its effect on DCs has not been fully investigated. METHODS: Monocyte-derived DCs were obtained from healthy volunteers and cultured for 7 days. Cells were treated with MPA on day 2 and matured by lipopolysaccharide (LPS) stimulation. Functionality of mature DC (mDCs) was evaluated by allogeneic mixed lymphocytes reaction. Surface expression of maturation markers (CD40, CD83, CD86, and ICAM-1) was analyzed in both immature DCs (iDCs) and mDCs by flow cytometry. To assess transcriptional regulation and protein subcellular location, RT-PCR and confocal microscopy were used, respectively. RESULTS: MPA decreased surface expression of all maturation markers in mDCs and significantly abrogated DCs-induced allogeneic T-cell proliferation after MPA pre-treatment. In iDCs, the reduced surface protein expression after MPA paralleled with mRNA downregulation of their genes. In mDCs, the mRNA levels of ICAM-1, CD40 and CD83 were enhanced in MPA-treated mDCs with an increase in the expression of CD83 and ICAM-1 near the Golgi compared to non-treated mDCs. In contrast, mRNA levels of CD86 were diminished after MPA treatment. CONCLUSIONS: The reduced surface markers expression in mDCs exerted by MPA produced a decline in their capacity to activate immune responses. Moreover, the inhibition of guanosine-derived nucleotide biosynthesis by MPA treatment leads to DC maturation interference by two mechanisms depending on the marker, transcriptional downregulation or disrupted intracellular protein trafficking.

Long-Term Effects in Bone Mineral Density after Different Bariatric Procedures in Patients with Type 2 Diabetes: Outcomes of a Randomized Clinical Trial.

There is scant evidence of the long-term effects of bariatric surgery on bone mineral density (BMD). We compared BMD changes in patients with severe obesity and type 2 diabetes (T2D) 5 years after randomization to metabolic gastric bypass (mRYGB), sleeve gastrectomy (SG) and greater curvature plication (GCP). We studied the influence of first year gastrointestinal hormone changes on final bone outcomes. Forty-five patients, averaging 49.4 (7.8) years old and body mass index (BMI) 39.4 (1.9) kg/m2, were included. BMD at lumbar spine (LS) was lower after mRYGB compared to SG and GCP: 0.89 [0.82;0.94] vs. 1.04 [0.91;1.16] vs. 0.99 [0.89;1.12], p = 0.020. A higher percentage of LS osteopenia was present after mRYGB 78.6% vs. 33.3% vs. 50.0%, respectively. BMD reduction was greater in T2D remitters vs. non-remitters. Weight at fifth year predicted BMD changes at the femoral neck (FN) (adjusted R2: 0.3218; p = 0.002), and type of surgery (mRYGB) and menopause predicted BMD changes at LS (adjusted R2: 0.2507; p < 0.015). In conclusion, mRYGB produces higher deleterious effects on bone at LS compared to SG and GCP in the long-term. Women in menopause undergoing mRYGB are at highest risk of bone deterioration. Gastrointestinal hormone changes after surgery do not play a major role in BMD outcomes.

Changes in Bone Mineral Density in Patients with Type 2 Diabetes After Different Bariatric Surgery Procedures and the Role of Gastrointestinal Hormones.

BACKGROUND: To compare changes in bone mineral density (BMD) in patients with morbid obesity and type 2 diabetes (T2D) a year after being randomized to metabolic gastric bypass (mRYGB), sleeve gastrectomy (SG), and greater curvature plication (GCP). We also analyzed the association of gastrointestinal hormones with skeletal metabolism. METHODS: Forty-five patients with T2D (mean BMI 39.4 ± 1.9 kg/m2) were randomly assigned to mRYGB, SG, or GCP. Before and 12 months after surgery, anthropometric, body composition, biochemical parameters, fasting plasma glucagon, ghrelin, and PYY as well as GLP-1, GLP-2, and insulin after a standard meal were determined. RESULTS: After surgery, the decrease at femoral neck (FN) was similar but at lumbar spine (LS), it was greater in the mRYGB group compared with SG and GCP - 7.29 (4.6) vs. - 0.48 (3.9) vs. - 1.2 (2.7)%, p < 0.001. Osteocalcin and alkaline phosphatase increased more after mRYGB. Bone mineral content (BMC) at the LS after surgery correlated with fasting ghrelin (r = - 0.412, p = 0.01) and AUC for GLP-1 (r = - 0.402, p = 0.017). FN BMD at 12 months correlated with post-surgical fasting glucagon (r = 0.498, p = 0.04) and insulin AUC (r = 0.384, p = 0.030) and at LS with the AUC for GLP-1 in the same time period (r = - 0.335, p = 0.049). However, in the multiple regression analysis after adjusting for age, sex, and BMI, the type of surgery (mRYGB) remained the only factor associated with BMD reduction at LS and FN. CONCLUSIONS: mRYGB induces greater deleterious effects on the bone at LS compared with SG and GCP, and gastrointestinal hormones do not play a major role in bone changes.

Validation and evaluation of four sample preparation methods for the quantification of intracellular tacrolimus in peripheral blood mononuclear cells by UHPLC-MS/MS.

Rejection and toxicity occur despite monitoring of tacrolimus blood levels during clinical routine. The intracellular concentration in lymphocytes could be a better reflection of the tacrolimus exposure. Four extraction methods for tacrolimus in peripheral blood mononuclear cells were validated and evaluated with UHPLC-MS/MS. Methods based on protein precipitation (method 1), solid phase extraction (method 2), phospholipids and proteins removal (method 3) and liquid-liquid extraction (method 4) were evaluated on linearity, lower limit of quantification (LLOQ), imprecision and bias. Validation was completed for the methods within these requirements, adding matrix effect and recovery. Linearity was 0.126 (LLOQ)-15 µg/L, 0.504 (LLOQ)-15 µg/L and 0.298 (LLOQ)-15 µg/L with method 1, 2 and 3, respectively. With method 4 non-linearity and a LLOQ higher than 0.504 µg/L were observed. Inter-day imprecision and bias were ≤4.6%, ≤10.9%; ≤6.8%, ≤-11.2%; ≤9.4%, ≤10.3% and ≤44.6%, ≤23.1%, respectively, with methods 1, 2, 3 and 4. Validation was completed for method 1 and 3 adding matrix effect (7.6%; 15.0%) and recovery (8.9%; 10.8%), respectively. The most suitable UHPLC-MS/MS method for quantification of intracellular tacrolimus was protein precipitation due to the best performance characteristics and the least time-consuming rate and complexity.

Measurement of calcineurin activity in peripheral blood mononuclear cells by ultra-high performance liquid chromatography-tandem mass spectrometry. Renal transplant recipients application (pharmacodynamic monitoring).

BACKGROUND: Therapeutic drug monitoring of calcineurin inhibitor, tacrolimus (TAC), is routinely used in post-transplantation. Currently, measurement of calcineurin activity has been proposed as a promising clinical tool to evaluate efficacy and to optimize drug dosing. The main aim of our study was to develop a method to measure phosphatase calcineurin activity (CNA) in peripheral blood mononuclear cells (PBMCs) by ultra-high performance liquid chromatography-tandem mass spectrometry and to validate it following FDA and EMA guidelines. METHODS: This methodology is based on monitoring the Ca2+-dependent dephosphorylation of a phosphopeptide substrate. CNA was evaluated in 5 healthy volunteers and in 5 renal transplant patients receiving twice-daily formulation of TAC before drug intake. Moreover, we studied pharmacodynamic effect of TAC and blood concentrations of TAC in different drug dose intervals (0, 1, 3, 6 and 12 h). RESULTS: Our results showed linearity in the range 0.04-2.00 µM with a lower limit of quantification of 0.04 µM. Coefficients of variation and absolute relative biases were <4.3% and 10.3%, respectively. The mean recovery for peptide was 91.6 ±â€¯4.0%. Matrix effect study displayed ion suppression, and no carry-over and interferences were observed. There were no differences in CNA between healthy and TAC-treated patients. Furthermore, CNA showed maximum inhibition at 1 h after drug intake when TAC reached the highest blood concentration. CONCLUSIONS: This method improves the extraction phase of PBMCs and achieves faster determination compared to other techniques, bringing us closer to be applied in daily laboratory practice.

ANRIL as a genetic marker for cardiovascular events in renal transplant patients - an observational follow-up cohort study.

Cardiovascular disease is the leading cause of morbidity and mortality in kidney transplant recipients. Several single-nucleotide polymorphisms (SNPs) in the ANRIL gene pathway have been associated with cardiovascular events (CE). The main objective was to ascertain whether ANRIL (rs10757278) and CARD8 (rs2043211) SNPs could mediate susceptibility to CE. This was an observational follow-up cohort study of renal transplant recipients at Bellvitge University Hospital (Barcelona) from 2000 to 2014. A total of 505 recipients were followed up until achievement of a CE. Patients who did not achieve the endpoint were followed up until graft loss, lost to follow-up or death. Survival analysis was used to ascertain association between genetic markers, clinical data, and outcome. Fifty-three patients suffered a CE after renal transplantation. Results showed a significant association between ANRIL SNP and CE. Homozygous GG for the risk allele showed higher risk for CE than A carriers for the protective allele [HR = 2.93(1.69-5.11), P < 0.0001]. This effect was maintained when it was analyzed in combination with CARD8, suggesting that CARD8 SNP could play a role in the ANRIL mechanism. However, our study does not clarify the molecular mechanism for the CARD8 SNP regulation by ANRIL. ANRIL SNP may predispose to the development of CE after successful kidney transplantation.

PI3K-Akt signaling controls PFKFB3 expression during human T-lymphocyte activation.

Lymphocyte activation is associated with rapid increase of both the glycolytic activator fructose 2,6-bisphosphate (Fru-2,6-P2) and the enzyme responsible for its synthesis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFKFB3 gene, which encodes for the most abundant PFK-2 isoenzyme in proliferating tissues, has been found overexpressed during cell activation in several models, including immune cells. However, there is limited knowledge on the pathways underlying PFKFB3 regulation in human T-lymphocytes, and the role of this gene in human immune response. The aim of this work is to elucidate the molecular mechanisms of PFKFB3 induction during human T-lymphocyte activation by mitotic agents. The results obtained showed PFKFB3 induction during human T-lymphocyte activation by mitogens such as phytohemagglutinin (PHA). PFKFB3 increase occurred concomitantly with GLUT-1, HK-II, and PCNA upregulation, showing that mitotic agents induce a metabolic reprograming process that is required for T-cell proliferation. PI3K-Akt pathway inhibitors, Akti-1/2 and LY294002, reduced PFKFB3 gene induction by PHA, as well as Fru-2,6-P2 and lactate production. Moreover, both inhibitors blocked activation and proliferation in response to PHA, showing the importance of PI3K/Akt signaling pathway in the antigen response of T-lymphocytes. These results provide a link between metabolism and T-cell antigen receptor signaling in human lymphocyte biology that can help to better understand the importance of modulating both pathways to target complex diseases involving the activation of the immune system.

Liver Glucokinase(A456V) Induces Potent Hypoglycemia without Dyslipidemia through a Paradoxical Induction of the Catalytic Subunit of Glucose-6-Phosphatase.

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GK(A456V), described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GK(A456V) was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GK(A456V) overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GK(A456V)-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.