Heart rate response determines long term exercise capacity after heart transplantation

BACKGROUND: Exercise capacity after heart transplantation (HTx) remains limited despite normal left ventricular systolic function of the allograft. Various clinical and haemodynamic parameters are predictive of exercise capacity following HTx. However, the predictive significance of chronotropic competence has not been demonstrated unequivocally despite its immediate relevance for cardiac output. AIMS: This study assesses the predictive value of various clinical and haemodynamic parameters for exercise capacity in HTx recipients with complete chronotropic competence evolving within the first 6 postoperative months. METHODS: 51 patients were enrolled in this exercise study. Patients were included when at least >6 months after HTx and without negative chronotropic medication or factors limiting exercise capacity such as significant transplant vasculopathy or allograft rejection. Clinical parameters were obtained by chart review, haemodynamic parameters from current cardiac catheterisation, and exercise capacity was assessed by treadmill stress testing. A stepwise multiple regression model analysed the proportion of the variance explained by the predictive parameters. RESULTS: The mean age of these 51 HTx recipients was 55.4 +/- 13.2 yrs on inclusion, 42 pts were male and the mean time interval after cardiac transplantation was 5.1 +/- 2.8 yrs. Five independent predictors explained 47.5% of the variance observed for peak exercise capacity (adjusted R2 = 0.475). In detail, heart rate response explained 31.6%, male gender 5.2%, age 4.1%, pulmonary vascular resistance 3.7%, and body-mass index 2.9%. CONCLUSION: Heart rate response is one of the most important predictors of exercise capacity in HTx recipients with complete chronotropic competence and without relevant transplant vasculopathy or acute allograft rejection.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

Performance of gene-expression profiling test score variability to predict future clinical events in heart transplant recipients.

BACKGROUND A single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0-40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events. METHODS We defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group (death, re-transplantation or graft failure ≥315 days post-transplantation and within 3 years of the final GEP test) and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated. RESULTS The estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8). CONCLUSION In heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score. TRIAL REGISTRATION Clinicaltrials.gov identifier NCT00761787.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Restricted and conserved T-cell repertoires involved in allorecognition of class II major histocompatibility complex.

The nature of the alloreactive T-cell response is not yet clearly understood. These strong cellular responses are thought to be the basis of allograft rejection and graft-vs.-host disease. The question of the extent of responding T-cell repertoires has so far been addressed by cellular cloning, often combined with molecular T-cell receptor (TCR) analysis. Here we present a broad repertoire analysis of primed responder cells from mixed lymphocyte cultures in which two different DR1/3 responders were stimulated with DR3/4 cells. Repertoire analysis was performed by TCR spectratyping, a method by which T cells are analyzed on the basis of the complementarity-determining region 3 length of different variable region (V) families. Strikingly, both responders showed very similar repertoires when the TCR V beta was used as a lineage marker. This was not seen when TCR V alpha was analyzed. A different pattern of TCR V beta was observed if the stimulating alloantigen was changed. This finding indicates that alloreactive T cells form a specific repertoire for each alloantigen. Since conservation appears to be linked to TCR V beta, the question of different roles of alpha and beta chains in allorecognition is raised.

Sampling times for monitoring tacrolimus in stable adult liver transplant recipients

The aim of this study was to determine the most informative sampling time(s) providing a precise prediction of tacrolimus area under the concentration-time curve (AUC). Fifty-four concentration-time profiles of tacrolimus from 31 adult liver transplant recipients were analyzed. Each profile contained 5 tacrolimus whole-blood concentrations (predose and 1, 2, 4, and 6 or 8 hours postdose), measured using liquid chromatography-tandem mass spectrometry. The concentration at 6 hours was interpolated for each profile, and 54 values of AUC(0-6) were calculated using the trapezoidal rule. The best sampling times were then determined using limited sampling strategies and sensitivity analysis. Linear mixed-effects modeling was performed to estimate regression coefficients of equations incorporating each concentration-time point (C0, C1, C2, C4, interpolated C5, and interpolated C6) as a predictor of AUC(0-6). Predictive performance was evaluated by assessment of the mean error (ME) and root mean square error (RMSE). Limited sampling strategy (LSS) equations with C2, C4, and C5 provided similar results for prediction of AUC(0-6) (R-2 = 0.869, 0.844, and 0.832, respectively). These 3 time points were superior to C0 in the prediction of AUC. The ME was similar for all time points; the RMSE was smallest for C2, C4, and C5. The highest sensitivity index was determined to be 4.9 hours postdose at steady state, suggesting that this time point provides the most information about the AUC(0-12). The results from limited sampling strategies and sensitivity analysis supported the use of a single blood sample at 5 hours postdose as a predictor of both AUC(0-6) and AUC(0-12). A jackknife procedure was used to evaluate the predictive performance of the model, and this demonstrated that collecting a sample at 5 hours after dosing could be considered as the optimal sampling time for predicting AUC(0-6).

Caractérisation de différents mécanismes immunologiques conduisant au dommage glomérulaire chez le greffé rénal avec rejet humoral; les effets des anticorps anti-HLA sur l’expression endothéliale et les niveaux sériques de thrombomoduline chez les patients transplantés

La greffe rénale est le meilleur traitement de l’insuffisance rénale terminale. Par contre, la perte prématurée du greffon est un problème majeur chez les greffés qui est due majoritairement au rejet. La classification de Banff reconnait 2 catégories de rejets : une réaction médiée par les anticorps (ABMR) et/ou une réaction cellulaire (TCMR). L’ABMR est caractérisé par le développement de novo d’anticorps contre le donneur (DSA) en circulation et des dommages histologiques, comme la glomérulopathie du transplant. De novo, les DSA anti-HLA-II sont plus fréquemment associés à la glomérulopathie du transplant que les anti-HLA-I et sont associés à un moins bon pronostic clinique. Toutefois, le mécanisme par lequel les anti-HLA-II sont plus dommageables demeure mal connu. Mon hypothèse est que les anticorps anti-HLA sont suffisants pour perturber l’hémostase de l’endothélium glomérulaire. Plus particulièrement, nous croyons que les anticorps anti-HLA-II, diminuent l’expression de la thrombomoduline (TBM), ce qui pourrait mener aux lésions endothéliales glomérulaires associées à la glomérulopathie du transplant. Pour évaluer cette hypothèse, j’ai utilisé un modèle in vitro d’endothélium glomérulaire humain et du sérum de patients transplantés rénaux. Nous avons observé que l’expression membranaire de la TBM augmentait de manière dosedépendante en présence d‘anti-HLA-I, mais pas anti-HLA-II. Toutefois, lors de la mesure intracellulaire nous avons observé une accumulation cytosolique en réponse à une stimulation par les anti-HLA-II. De plus, nous avons observé une association significative entre la présence de DSA circulants anti-HLA-II dans les patients transplantés rénaux et un faible taux de TBM sérique. Ces résultats indiquent que la liaison des anticorps anti-HLA-I et II produit des effets différents sur l’expression endothéliale de la TBM. Les anticorps anti-HLA-II pourraient être associés à un état prothrombotique qui pourrait expliquer l’occurrence plus élevée de lésions microangiopathiques dans l’allogreffe et la moins bonne condition observée chez les patients ayant ces anticorps.

Impacto de los Niveles de Tacrolimus en el Rechazo al Injerto y la Tasa de Filtración Glomerular en Pacientes con Trasplante Hepático realizado en la FCI-IC entre 2009 y 2014

Introducción: El tacrolimus es el medicamento de elección para evitar el rechazo al injerto hepático. Su dosis se ajusta a partir de los niveles séricos que se toman periódicamente para asegurar rango terapéutico. Además, niveles elevados se asocian con disfunción renal postrasplante. Sin embargo, no hay consenso frente a los niveles adecuados para pacientes con trasplante hepático. Objetivo: Determinar la relación entre los niveles de tacrolimus y la presencia de rechazo agudo al injerto hepático en pacientes con trasplante hepático realizado en la Fundación Cardioinfantil – Instituto de Cardiología (FCI-IC). Determinar la relación entre los niveles de tacrolimus y la TFG en pacientes con trasplante hepático realizado en la FCI-IC. Métodos: Estudio observacional tipo cohorte histórica en pacientes adultos con trasplante hepático realizado en la FCI-IC entre 2009-2014. Resultados: No se encontró una asociación estadísticamente significativa entre los niveles de tacrolimus y la presencia de rechazo agudo, en sus diferentes definiciones (OR=1,02, p=0,14 y OR=1,01, p=0,29) incluso al ajustar por otras covariables (OR=1,03, p=0,10 y OR=1,02, p=0,25). No fue posible corroborar el diagnóstico con biopsia porque no todos la tenían. Si bien la relación entre los niveles de tacrolimus y la TFG fue estadísticamente significativa (p≤0,001), tiene bajo impacto clínico, pues la TFG disminuyó menos de un punto por cada incremento en 1 ng/ml en los niveles de tacrolimus. Conclusiones: Se necesitan más estudios para establecer la relación entre la exposición a tacrolimus y estos desenlaces para definir si es seguro disminuir su dosis con el fin de reducir los eventos adversos.

Tratamiento del rechazo mediado por anticuerpos en pacientes trasplantados renales revisión sistemática

Antecedentes: El trasplante renal es la mejor alternativa terapéutica para la enfermedad renal crónica terminal. Los medicamentos inmunosupresores previenen el rechazo. El rechazo mediado por anticuerpos es frecuente y disminuye la función y duración del injerto. Objetivo: Evaluar sistemáticamente la evidencia disponible relacionada con la eficacia y seguridad del tratamiento para el rechazo mediado por anticuerpos en pacientes trasplantados renales. Metodologia: Revisión sistemática en bases de datos MEDLINE, EMBASE, Scopus y Biblioteca virtual de la salud. Literatura gris google scholar, google academico, www.clinicaltrialsregister.eu, and https://clinicaltrials.gov/. Búsqueda manual referencias artículos pre-seleccionados así como de revisiones previamente publicadas. Se siguieron las recomendacioes guia PRISMA para la identificacion de artículos potenciales, tamizaje y selección teniendo en cuenta los criterios de inclusion. Extracción datos de acuerdo a las variables, revisión calidad de los artículos elegidos utilizando evaluación riesgo de segos de Cochrane. Resultados: Se seleccionaron 9 ensayos clínicos publicados entre 1980 y 2016, incluyeron 222 pacientes (113 brazo de intervención y 109 en el control), seguimiento promedio 16 meses. Intervenciones evaluadas plasmaféresis, inmunoadsorción y rituximab. Hubo una amplia heterogeneidad en la definición de criterios de inclusión, criterios diagnósticos de rechazo y medidas de evaluación de eficacia de las intervenciones. Tres estudios encontraron diferencias estadísticamente significativas entre los grupos de tratamiento. Conclusiones: La evidencia sobre la eficacia de los tratamientos del rechazo mediado por anticuerpos en injertos renales es de baja calidad. Son necesarios ensayos clínicos controlados para poder definir el tratamiento óptimo de estos pacientes.

Time-Dependent Specificity of Immunopathologic (C4d-CD68) and Histologic Criteria of Antibody-Mediated Rejection for Donor-Specific Antibodies and Allograft Dysfunction in Heart Transplantation.

BACKGROUND: In heart transplantation, antibody-mediated rejection (AMR) is diagnosed and graded on the basis of immunopathologic (C4d-CD68) and histopathologic criteria found on endomyocardial biopsies (EMB). Because some pathologic AMR (pAMR) grades may be associated with clinical AMR, and because humoral responses may be affected by the intensity of immunosuppression during the first posttransplantation year, we investigated the incidence and positive predictive values (PPV) of C4d-CD68 and pAMR grades for clinical AMR as a function of time. METHODS: All 564 EMB from 40 adult heart recipients were graded for pAMR during the first posttransplantation year. Clinical AMR was diagnosed by simultaneous occurrence of pAMR on EMB, donor specific antibodies and allograft dysfunction. RESULTS: One patient demonstrated clinical AMR at postoperative day 7 and one at 6 months (1-year incidence 5%). C4d-CD68 was found on 4,7% EMB with a "decrescendo" pattern over time (7% during the first 4 months vs. 1.2% during the last 8 months; P < 0.05). Histopathologic criteria of AMR occurred on 10.3% EMB with no particular time pattern. Only the infrequent (1.4%) pAMR2 grade (simultaneous histopathologic and immunopathologic markers) was predictive for clinical AMR, particularly after the initial postoperative period (first 4 months and last 8 months PPV = 33%-100%; P < 0.05). CONCLUSION: In the first posttransplantation year, AMR immunopathologic and histopathologic markers were relatively frequent, but only their simultaneous occurrence (pAMR2) was predictive of clinical AMR. Furthermore, posttransplantation time may modulate the occurrence of C4d-CD68 on EMB and thus the incidence of pAMR2 and its relevance to the diagnosis of clinical AMR.

Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode

Introduction: Tim-3 is a Th1 lymphocytes membrane protein with inhibitory function. Its ligand, galectin-9, was recently identified and it is expressed in some lymphocyte subpopulation. In addition, endothelial cells and fibroblasts can also express galectin-9 according to the local cytokine milieu. Both molecules can act as important regulatory tools in the immune system. Aim: Evaluate the expression of these immunoregulatory molecules inside kidney allografts during acute rejection episodes. Methods: By using a quantitative polymerase chain reaction assay, we measured the levels of messenger RNA (mRNA) for galectin-9 and Tim-3 in 21 samples obtained at allograft nephrectomy. Five samples received the histological diagnosis of acute non-vascular rejection (ANVR), twelve of acute vascular rejection (AVR), and five of loss of non-immune cause (LNIC; as control). As cytolytic response markers we measured mRNA levels of granzyme B, interferon-gamma and perforin. The statistic analysis was performed using one way analysis of variance (ANOVA) and Pearson correlation. Results: The mean levels of Tim-3 mRNA expression were 13.99 +/- 6.99 for LNIC, 48.13 +/- 54.47 for RACNV and 238.63 +/- 333.14 for RAV (p = 0.004). For galectin-9, the mean values were 0.57 +/- 0.49 for LNIC, 0.66 +/- 0.36 for RACNV and 2.34 +/- 1.62 for RAV (p = 0.006). Furthermore, there was a positive correlation between both molecules (r = 0.526, p = 0.016). Also. granzyme B, perforin and interferon-gamma mRNA expression were different among the three groups. Conclusion: Messenger RNA level expressions of all the studied molecules were higher inside allografts with more severe rejection. Moreover, there was a positive correlation between galectin-9 and Tim-3 mRNA levels. The simultaneous expression of galectin-9 and Tim-3 may indicate an immunoregulatory function, during the ongoing cytotoxic response. (C) 2008 Elsevier B.V. All rights reserved.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Multifocal Renal Allograft Biopsy: Impact on Therapeutic Decisions

Objective. There are no data to support the suggestion that samples removed from one segment of the transplanted kidney are representative of the whole graft. The aim of this study was to compare the histological differences between biopsies obtained from different portions of the renal allograft and their impact on treatment recommendations. Patients and Methods. Two hundred percutaneous biopsies were performed on kidney allografts and samples were collected from the upper and lower poles (100 kidneys). All samples were randomized and blindly reviewed. We obtained the discordance rates between the poles for the grading of acute rejection and for the diagnosis of nephrotoxicity due to immunosuppression. We also checked if the differences found were sufficient to call for different clinical recommendations. These values were compared with the intrapathologist variation rates. Results. In 70 kidneys adequate sampling was obtained from both poles. The diagnosis of acute rejection were made in 1.7. The discordance rate between the upper and lower poles was 82.3% (kappa = 0.34), higher than the intrapathologist variation (P =.002). Nephrotoxicity was found in 14 kidneys. The discordance rate between the upper and lower poles was 28.6% (kappa = 0.88), with no difference compared with the intrapathologist variation. In 14 of the 70 kidneys (25.7%), discordances between poles had impact on clinical recommendations, most of these cases due to different gradings of acute rejection (78%). This number was higher than the intrapathologist variation (P =.04). Conclusions. The histopathological changes in the kidney allograft are not always homogeneous. This heterogeneity may affect the therapeutic recommendations.

Analysis of Different Staining Techniques for C4d Detection in Renal Allograft Biopsies

Capillary C4d deposition has been recognized as a marker of antibody-mediated rejection (AMR). Although the detection of capillary C4d by means of immunofluorescence (IF) in cryostat sections is well established, frozen tissue is not always available, thus limiting the diagnosis of AMR. The aim of the present study was to analyze different techniques for C4d staining and the prevalence of C4d in renal allograft biopsies. Detection of C4d was carried out using IF or immunohistochemistry (IHC) on frozen and paraffin sections of renal allograft biopsies available from the same patients. Biopsies obtained from 20 patients were classified into 3 groups: no rejection, acute rejection, and chronic allograft nephropathy (CAN). The capillary C4d deposition prevalence in frozen-IF, considered the gold standard technique for C4d detection, was 45% (9/20 cases). Compared with frozen-IF, the frozen-IHC technique presented an 85% concordance rate (17/20 cases; r =.70; P <.001; sensitivity = 77.8%; specificity = 90.9%). The paraffin-IF technique showed similar results, with an 80% concordance rate (16/20 cases; r =.64; P <.005; sensitivity = 55.6%; specificity = 100%), whereas C4d detection occurred in only 65% of paraffin-IHC cases (13/20; r =.30; not significant; sensitivity = 66.7%; specificity = 63.6%). No capillary C4d deposition was detected in cases without evidence of rejection. However, 4/7 cases (57%) of acute rejection were C4d positive. In the CAN group, 5111 cases (45%) were C4d positive. In conclusion, these results demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.

Induction of Indoleamine 2,3-Dioxygenase by Gene Delivery in Allogeneic Islets Prolongs Allograft Survival

Indoleamine 2,3-dioxygenase (IDO), an enzyme that plays a critical role in fetomaternal tolerance, exerts immunoregulatory functions suppressing T-cell responses. The aims of this study were to promote IDO expression in rat islets using a nonviral gene transfer approach, and to analyze the effect of the in vivo induction of IDO in a model of allogeneic islet transplantation. The IDO cDNA was isolated from rat placenta, subcloned into a plasmid and transfected into rat islets using Lipofectamine. The efficiency of transfection was confirmed by qRT-PCR and functional analysis. The in vivo effect of IDO expression was analyzed in streptozotocin-induced diabetic Lewis rats transplanted with allogeneic islets under the renal capsule. Transplantation of IDO-allogeneic islets reversed diabetes and maintained metabolic control, in contrast to transplantation of allogeneic nontransfected islets, which failed shortly after transplantation in all animals. Graft survival of allograft islets transfected with IDO transplanted without any immunosuppression was superior to that observed in diabetic rats receiving nontransfected islets. These data demonstrated that IDO expression induced in islets by lipofection improved metabolic control of streptozotocin-diabetic rats and prolonged allograft survival.