Drawing networks of rejection - a systems biological approach to the identification of candidate genes in heart transplantation.

Technological development led to an increased interest in systems biological approaches to characterize disease mechanisms and candidate genes relevant to specific diseases. We suggested that the human peripheral blood mononuclear cells (PBMC) network can be delineated by cellular reconstruction to guide identification of candidate genes. Based on 285 microarrays (7370 genes) from 98 heart transplant patients enrolled in the Cardiac Allograft Rejection Gene Expression Observational study, we used an information-theoretic, reverse-engineering algorithm called ARACNe (algorithm for the reconstruction of accurate cellular networks) and chromatin immunoprecipitation assay to reconstruct and validate a putative gene PBMC interaction network. We focused our analysis on transcription factor (TF) genes and developed a priority score to incorporate aspects of network dynamics and information from published literature to supervise gene discovery. ARACNe generated a cellular network and predicted interactions for each TF during rejection and quiescence. Genes ranked highest by priority score included those related to apoptosis, humoural and cellular immune response such as GA binding protein transcription factor (GABP), nuclear factor of κ light polypeptide gene enhancer in B-cells (NFκB), Fas (TNFRSF6)-associated via death domain (FADD) and c-AMP response element binding protein. We used the TF CREB to validate our network. ARACNe predicted 29 putative first-neighbour genes of CREB. Eleven of these (37%) were previously reported. Out of the 18 unknown predicted interactions, 14 primers were identified and 11 could be immunoprecipitated (78.6%). Overall, 75% (n= 22) inferred CREB targets were validated, a significantly higher fraction than randomly expected (P < 0.001, Fisher's exact test). Our results confirm the accuracy of ARACNe to reconstruct the PBMC transcriptional network and show the utility of systems biological approaches to identify possible molecular targets and biomarkers.

Relationship between a validated molecular cardiac transplant rejection classifier and routine organ function parameters.

BACKGROUND: As acute cellular cardiac allograft rejection is a systemic process affecting the entire organism, we hypothesized that scores of a peripheral blood mononuclear cell gene expression profiling (GEP) test developed and validated to rule out International Society of Heart and Lung Transplantation (ISHLT) grade > or = 3A/2R acute cellular cardiac allograft rejection also reflects biologically plausible changes of the routinely assessed clinical parameters. METHODS: We retrospectively analyzed 76 patients who underwent GEP testing, at the time of their routine clinical follow-up in our Institution between February 1, 2006 and January 31, 2007. Data were analyzed with t-test, nonparametric tests, bivariate Spearman's correlation, and multivariate linear regression modeling. RESULTS: More activated GEP-score correlated with longer corrected QT (QTc)-interval (r = 0.377, p = 0.001, n = 63), longer QRS duration (r = 0.231, p = 0.03, n = 66), higher heart rate (r = 0.221, p = 0.037, n = 66), higher serum creatinine (r = 0.26, p = 0.01, n = 75), higher gamma-glutamyl transferase (GGT) GGT (r = 0.266, p = 0.037, n = 46), lower pulmonary artery oxygen saturation (r = -0.313, p = 0.003, n = 76), lower platelet count (r = -0.372, p = 0.001, n = 74), lower monocyte count (r = -0.208, p = 0.040, n = 72), and lower high-density lipoprotein (HDL) HDL level (r = -0.242, p = 0.041, n = 53). Multivariate analysis showed a significant amount of variance in the GEP score independently explained by the variability of QTc-interval (beta = 1.998, p = 0.001) and platelet count (beta = -1.540, p = 0.017). Post hoc analysis of the 11 individual GEP-classifier genes showed WDRA40 (p = 0.02) and ras homolog gene family, member U (RHOU) RHOU (p = 0.01) independently related to mixed venous O(2)Sat%. CONCLUSION: A GEP test developed and validated to detect the absence of cardiac rejection correlates with electrocardiographic and hemodynamic cardiac parameters as well as renal, hepatic, bone marrow, and lipid metabolism parameters suggesting a complex relationship between rejection, leukocytes, and organ function within the continuum between alloimmunological quiescence and rejection.

Peripheral blood mononuclear cell transcriptome profiles suggest T-cell immunosuppression after uncomplicated mechanical circulatory support device surgery.

Mechanical circulatory support device (MCSD) surgery in patients with advanced heart failure patients is often complicated by infections that are linked to altered cell-mediated immunity. Using a transcriptome-wide peripheral blood mononuclear cell (PBMC) gene expression profiling approach, we analyzed expression patterns directly before and after MCSD implantation in 11 patients who had an uncomplicated course after MCSD implantation (Day 0-24 hours before, Day 1-24 hours after, and Day 7-1 week after implantation). Data were analyzed using Significance Analysis of Microarrays (SAM) and High-Throughput GoMiner on post-implantation profiles (Day 1, Day 7) in comparison with baseline (Day 0). Day1 profiles included differential expression of 821 genes (SAM, FDR <0.1, fold change >1.5), enriching >60 Gene Ontology (GO) categories. Grouping by component genes revealed GO clusters, which we term "interleukin related" (primarily upregulated), "T-cell related" (primarily downregulated), and "apoptosis related" (both up- and down-regulated genes). Day 7 profiles included GO categories related to repair processes. In conclusion, transcriptome-wide expression profiling of PBMCs suggests a response pattern to MCSD implantation of inflammatory activation and simultaneous T-cell suppression.

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.

Managing drugs and devices in patients with permanent ventricular assist devices.

Patients will be considered for destination mechanical circulatory support device (MCSD) implantation when all other organ-saving treatment options have failed and they are not eligible for heart transplantation. Current medical evidence suggests that only for those patients who are inotrope-dependent and therefore likely have a 1-year survival probability without MCSD implantation of less than 50%, MCSD intervention will add to survival and quality-of-life benefit. Suitable candidates for MCSD are those patients who have a high risk of dying from heart failure but acceptable noncardiac risk. Evaluation of patients for MCSD requires a systematic and critical review of all organ systems and of the psychosocial situation. Specifically, right ventricular function and risk of right ventricular failure should be evaluated before planning destination MCSD implantation. Treatment will focus on prompt recovery from MCSD implantation, maintaining optimal treatment for heart failure, and preventing/treating MCSD complications, including infection, bleeding, coagulopathy, right heart failure, and device dysfunction. MCSD programs should be organized as an advanced heart failure center directed by specialized heart failure cardiologists, surgeons expert at implant and management of MCSD, specialized nurses, social workers, psychologists, financial experts, and physical therapists. MCSD practice is based on a patient-centered theory, with an appropriate understanding of the respective roles of the physician and the patient during their iterative encounters in which the patient is an autonomous person making responsible personal health decisions while the health care team is providing continued expert and empathic counseling about various options, based on systematic outcomes research (eg, by participation in the Interagency Registry for Mechanically Assisted Circulatory Support - MCSD database ).

Cardiac transplantation: any role left?

The authors analyze the question of whether heart transplantation still has a role in the current era of complex technologies. To achieve this objective, the authors first discuss the known benefits of different therapeutic modalities currently available for patients who have end-stage heart failure, including pharmacologic management, electrophysiologic therapies, high-risk surgical strategies, implantation of mechanical circulatory support device therapy, and heart transplantation. The authors then evaluate the current developments and future perspectives in the field that may influence the likelihood of heart transplantation to remain the therapeutic modality of choice for end-stage heart failure.

Destination therapy: does progress depend on left ventricular assist device development?

The role of therapy using mechanical circulatory support devices has evolved rapidly over the last two decades. New developments in the field achieved smaller adverse events, but, currently, only minor improvements in survival were observed in published observational data. The authors discuss the development of mechanical circulatory support devices as a "destination therapy" option for patients who have end-stage heart failure and are ineligible for heart transplantation as it relates to left ventricular assist device development.

G6b-B cell surface inhibitory receptor expression is highly restricted to CD4+ T-cells and induced by interleukin-4-activated STAT6 pathway.

The G6b-B gene encodes a novel cell surface receptor of the immunoglobulin superfamily that activates inhibitory signaling pathways by triggering SHP-1/SHP-2 via immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic domain. We previously identified decreased G6b-B expression in peripheral blood mononuclear cells (PBMC) during acute cellular cardiac allograft rejection. We studied the expression of G6b-B in different human mononuclear cell populations and its regulation. Real-time polymerase chain reaction (PCR) revealed that G6b-B mRNA is higher in CD4+ T cells or monocytes, but is not different between CD25+ CD4+ T cells and CD25- CD4+ T cells. G6b-B mRNA was increased in CD4+ T cells in presence of interleukin-4 in dose- and time-dependent manners. To understand the regulatory mechanism, we analyzed a 1.9-kb 5'-flanking region of the G6b-B translation start site and found a putative cis-acting element for Signal Transducer and Activator of Transcription (STAT)-6. Luciferase-reporter-gene-assay and electrophoretic mobility shift assays identified the STAT6 site as necessary for the induction of G6b-B by IL-4. Our study demonstrates that G6b-B expression is highly restricted to peripheral CD4+ T cells and up-regulated by the IL-4-induced STAT6 pathway, strongly suggesting that G6b-B is involved in regulation of the immune response by CD4+ T cell-mediated and IL-4 induced regulatory mechanisms.

Cellular coating of the left ventricular assist device textured polyurethane membrane reduces adhesion of Staphylococcus aureus.

OBJECTIVE: Infections are among the most common and serious complications of ventricular assist device implantation. These infections generally occur within the first 2 months after surgery. The basis for this high incidence of infection is not well established, so a murine intravascular infection model was developed with aortic implantation of the textured polyurethane patch material currently used in HeartMate ventricular assist devices (Thoratec Corporation Pleasanton, Calif). METHODS: Polyurethane patch material was placed in the wall of the mouse descending aorta. Mice were then infected with Staphylococcus aureus 1 or 14 days after implantation. In vitro adhesion studies were conducted with polyurethane membranes coated with endothelial cells and membranes coated with fibrinogen. RESULTS: Mice were susceptible to infection in both dose- and time-dependent fashions. The patch material was significantly more susceptible to infection at day 1 than day 14. Immunohistologic and morphologic studies demonstrated that the CD31+ cells deposited on the membrane surface phenotypically appeared to be endothelial cells. In vitro adhesion studies of polyurethane membranes coated with endothelial cells showed them to be less susceptible to S. aureus binding than were membranes coated with fibrinogen. CONCLUSION: Textured polyurethane membranes are less susceptible to infection as cellular deposition occurs. The time frame within which these membranes become populated with cellular material is consistent with the time-dependent clinical incidence of infection. Cellular coating of polyurethane may provide a strategy for reducing the risk of infection.

The role of Staphylococcus aureus adhesins in the pathogenesis of ventricular assist device-related infections.

Ventricular assist devices (VADs) are an important form of therapy for end-stage congestive heart failure. However, infection of the VAD, which is often caused by Staphylococcus aureus, poses a major threat to survival. Using a novel in vitro binding assay with VAD membranes and a heterologous lactococcal system of expression, we identify 3 S. aureus proteins--clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as the main factors involved in adherence to VAD polyurethane membranes. Adherence is greatly diminished by long implantation times, reflecting a change in topological features of the VAD membrane, and is primarily mediated by the FnBPA domains in the staphylococcal proteins. We also compare the adherence of S. aureus mutant strains and show that other staphylococcal components appear to be involved in adherence to VAD membranes. Finally, we demonstrate that ClfA, FnBPA, and FnBPB mediate bacterial infection of implanted murine intra-aortic polyurethane patches.