Gene expression profiling to study racial differences after heart transplantation.

BackgroundThe basis for increased mortality after heart transplantation in African Americans and other non-Caucasian racial groups is poorly defined. We hypothesized that increased risk of adverse events is driven by biologic factors. To test this hypothesis in the Invasive Monitoring Attenuation through Gene Expression (IMAGE) study, we determined whether the event rate of the primary outcome of acute rejection, graft dysfunction, death, or retransplantation varied by race as a function of calcineurin inhibitor (CNI) levels and gene expression profile (GEP) scores.MethodsWe determined the event rate of the primary outcome, comparing racial groups, stratified by time after transplant. Logistic regression was used to compute the relative risk across racial groups, and linear modeling was used to measure the dependence of CNI levels and GEP score on race.ResultsIn 580 patients monitored for a median of 19 months, the incidence of the primary end point was 18.3% in African Americans, 22.2% in other non-Caucasians, and 8.5% in Caucasians (p < 0.001). There were small but significant correlations of race and tacrolimus trough levels to the GEP score. Tacrolimus levels were similar among the races. Of patients receiving tacrolimus, other non-Caucasians had higher GEP scores than the other racial groups. African American recipients demonstrated a unique decrease in expression of the FLT3 gene in response to higher tacrolimus levels.ConclusionsAfrican Americans and other non-Caucasian heart transplant recipients were 2.5-times to 3-times more likely than Caucasians to experience outcome events in the Invasive Monitoring Attenuation through Gene Expression study. The increased risk of adverse outcomes may be partly due to the biology of the alloimmune response, which is less effectively inhibited at similar tacrolimus levels in minority racial groups

Gene Expression Signatures of Peripheral Blood Mononuclear Cells during the Early Post-Transplant Period in Patients Developing Cardiac Allograft Vasculopathy

Background. Cardiac allograft vasculopathy (CAV) is a major cause of graft loss and death after heart transplantation. Currently, no diagnostic methods are available during the early post-transplant period to accurately identify patients at risk of CAV. We hypothesized that PBMC gene expression profiles (GEP) can identify patients at risk of CAV. Methods. We retrospectively analyzed a limited set of whole-genome PBMC microarrays from 10 post-transplant patients who did (n = 3) or did not (n = 7) develop advanced grade CAV during their long-term follow-up. We used significance analysis of microarrays to identify differentially expressed genes and High-Throughput GoMiner to assess gene ontology (GO) categories. We corroborated our findings by retrospective analysis of PBMC real-time PCR data from 33 patients. Results. Over 300 genes were differentially expressed (FDR < 5%), and 18 GO-categories including “macrophage activation”, “Interleukin-6 pathway”, “NF-KappaB cascade”, and “response to virus” were enriched by these genes (FDR < 5%). Out of 8 transcripts available for RT-PCR analysis, we confirmed 6 transcripts (75.0%) including FPRL1, S100A9, CXCL10, PRO1073, and MMP9 (P < .05). Conclusion. Our pilot data suggest that GEP of PBMC may become a valuable tool in the evaluation of patients at risk of CAV. Larger prospectively designed studies are needed to corroborate our hypothesis

Unexplained Graft Dysfunction after Heart Transplantation—Role of Novel Molecular Expression Test Score and QTc-Interval: A Case Report

In the current era of immunosuppressive medications there is increased observed incidence of graft dysfunction in the absence of known histological criteria of rejection after heart transplantation. A noninvasive molecular expression diagnostic test was developed and validated to rule out histological acute cellular rejection. In this paper we present for the first time, longitudinal pattern of changes in this novel diagnostic test score along with QTc-interval in a patient who was admitted with unexplained graft dysfunction. Patient presented with graft failure with negative findings on all known criteria of rejection including acute cellular rejection, antibody mediated rejection and cardiac allograft vasculopathy. The molecular expression test score showed gradual increase and QTc-interval showed gradual prolongation with the gradual decline in graft function. This paper exemplifies that in patients presenting with unexplained graft dysfunction, GEP test score and QTc-interval correlate with the changes in the graft function

Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse Brain

The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies

Many heart transplant biopsies currently diagnosed as no rejection have mild molecular antibody-mediated rejection-related changes

[Abstract] Background: The Molecular Microscope (MMDx) system classifies heart transplant endomyocardial biopsies as No-rejection (NR), Early-injury, T cell-mediated (TCMR), antibody-mediated (ABMR), mixed, and possible rejection (possible TCMR, possible ABMR). Rejection-like gene expression patterns in NR biopsies have not been described. We extended the MMDx methodology, using a larger data set, to define a new "Minor" category characterized by low-level inflammation in non-rejecting biopsies. Methods: Using MMDx criteria from a previous study, molecular rejection was assessed in 1,320 biopsies (645 patients) using microarray expression of rejection-associated transcripts (RATs). Of these biopsies, 819 were NR. A new archetypal analysis model in the 1,320 data set split the NRs into NR-Normal (N = 462) and NR-Minor (N = 359). Results: Compared to NR-Normal, NR-Minor were more often histologic TCMR1R, with a higher prevalence of donor-specific antibody (DSA). DSA positivity increased in a gradient: NR-Normal 24%; NR-Minor 34%; possible ABMR 42%; ABMR 66%. The top 20 transcripts distinguishing NR-Minor from NR-Normal were all ABMR-related and/or IFNG-inducible, and also exhibited a gradient of increasing expression from NR-Normal through ABMR. In random forest analysis, TCMR and Early-injury were associated with reduced LVEF and increased graft loss, but NR-Minor and ABMR scores were not. Surprisingly, hearts with MMDx ABMR showed comparatively little graft loss. Conclusions: Many heart transplants currently diagnosed as NR by histologic or molecular assessment have minor increases in ABMR-related and IFNG-inducible transcripts, associated with DSA positivity and mild histologic inflammation. These results suggest that low-level ABMR-related molecular stress may be operating in many more hearts than previously estimated

SdrF, a Staphylococcus epidermidis Surface Protein, Contributes to the Initiation of Ventricular Assist Device Driveline–Related Infections

Staphylococcus epidermidis remains the predominant pathogen in prosthetic-device infections. Ventricular assist devices, a recently developed form of therapy for end-stage congestive heart failure, have had considerable success. However, infections, most often caused by Staphylococcus epidermidis, have limited their long-term use. The transcutaneous driveline entry site acts as a potential portal of entry for bacteria, allowing development of either localized or systemic infections. A novel in vitro binding assay using explanted drivelines obtained from patients undergoing transplantation and a heterologous lactococcal system of surface protein expression were used to identify S. epidermidis surface components involved in the pathogenesis of driveline infections. Of the four components tested, SdrF, SdrG, PIA, and GehD, SdrF was identified as the primary ligand. SdrF adherence was mediated via its B domain attaching to host collagen deposited on the surface of the driveline. Antibodies directed against SdrF reduced adherence of S. epidermidis to the drivelines. SdrF was also found to adhere with high affinity to Dacron, the hydrophobic polymeric outer surface of drivelines. Solid phase binding assays showed that SdrF was also able to adhere to other hydrophobic artificial materials such as polystyrene. A murine model of infection was developed and used to test the role of SdrF during in vivo driveline infection. SdrF alone was able to mediate bacterial adherence to implanted drivelines. Anti-SdrF antibodies reduced S. epidermidis colonization of implanted drivelines. SdrF appears to play a key role in the initiation of ventricular assist device driveline infections caused by S. epidermidis. This pluripotential adherence capacity provides a potential pathway to infection with SdrF-positive commensal staphylococci first adhering to the external Dacron-coated driveline at the transcutaneous entry site, then spreading along the collagen-coated internal portion of the driveline to establish a localized infection. This capacity may also have relevance for other prosthetic device–related infections

Molecular states associated with dysfunction and graft loss in heart transplants

[Abstract] Background: We explored the changes in gene expression correlating with dysfunction and graft failure in endomyocardial biopsies. Methods: Genome-wide microarrays (19,462 genes) were used to define mRNA changes correlating with dysfunction (left ventricular ejection fraction [LVEF] ≤ 55) and risk of graft loss within 3 years postbiopsy. LVEF data was available for 1,013 biopsies and survival data for 779 patients (74 losses). Molecular classifiers were built for predicting dysfunction (LVEF ≤ 55) and postbiopsy 3-year survival. Results: Dysfunction is correlated with dedifferentiation-decreased expression of normal heart transcripts, for example, solute carriers, along with increased expression of inflammation genes. Many genes with reduced expression in dysfunction were matrix genes such as fibulin 1 and decorin. Gene ontology (GO) categories suggested matrix remodeling and inflammation, not rejection. Genes associated with the risk of failure postbiopsy overlapped dysfunction genes but also included genes affecting microcirculation, for example, arginase 2, which reduces NO production, and endothelin 1. GO terms also reflected increased glycolysis and response to hypoxia, but decreased VEGF and angiogenesis pathways. T cell-mediated rejection was associated with reduced survival and antibody-mediated rejection with relatively good survival, but the main determinants of survival were features of parenchymal injury. Both dysfunction and graft loss were correlated with increased biopsy expression of BNP (gene NPPB). Survival probability classifiers divided hearts into risk quintiles, with actuarial 3-year postbiopsy survival >95% for the highest versus 50% for the lowest. Conclusions: Dysfunction in transplanted hearts reflects dedifferentiation, decreased matrix genes, injury, and inflammation. The risk of short-term loss includes these changes but is also associated with microcirculation abnormalities, glycolysis, and response to hypoxia

Redefining the molecular rejection states in 3230 heart transplant biopsies: relationships to parenchymal injury and graft survival

[Abstract] The first-generation Molecular Microscope (MMDx) system for heart transplant endomyocardial biopsies used expression of rejection-associated transcripts (RATs) to diagnose not only T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) but also acute injury. However, the ideal system should detect rejection without being influenced by injury, to permit analysis of the relationship between rejection and parenchymal injury. To achieve this, we developed a new rejection classification in an expanded cohort of 3230 biopsies: 1641 from INTERHEART (ClinicalTrials.gov NCT02670408), plus 1589 service biopsies added to improve the power of the machine learning algorithms. The new system used 6 rejection classifiers instead of RATs and generated 7 rejection archetypes: No rejection, 48%; Minor, 24%; TCMR1, 2.3%; TCMR2, 2.7%; TCMR/mixed, 2.7%; early-stage ABMR, 3.9%; and fully developed ABMR, 16%. Using rejection classifiers eliminated cross-reactions with acute injury, permitting separate assessment of rejection and injury. TCMR was associated with severe-recent injury and late atrophy-fibrosis and rarely had normal parenchyma. ABMR was better tolerated, seldom producing severe injury, but in later biopsies was often associated with atrophy-fibrosis, indicating long-term risk. Graft survival and left ventricular ejection fraction were reduced not only in hearts with TCMR but also in hearts with severe-recent injury and atrophy-fibrosis, even without rejection