Background-Tissue engineering using in vitro-cultivated autologous vascular wall cells is a new approach to biological heart valve replacement. In the present study, we analyzed a new concept to process allogenic acellular matrix scaffolds of pulmonary heart valves after in vitro seeding with the use of autologous cells in a sheep model. Methods and Results-Allogenic heart valve conduits were acellularized by a 48-hour trypsin/EDTA incubation to extract endothelial cells and myofibroblasts. The acellularization procedure resulted in an almost complete removal of cells.After that procedure, a static reseeding of the upper surface of the valve was performed sequentially with autologous myofibroblasts for 6 days and endothelial cells for 2 days, resulting in a patchy cellular restitution on the valve surface.The in vivo function was tested in a sheep model of orthotopic pulmonary valve conduit transplantation. Three of 4 unseeded control valves and 5 of 6 tissue-engineered valves showed normal function up to 3 months. Unseeded allogenic acellular control valves showed partial degeneration (2 of 4 valves) and no interstitial valve tissue reconstitution. Tissue-engineered valves showed complete histological restitution of valve tissue and confluent endothelial surface coverage in all cases. Immunohistological analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (␣-actin), and matrix synthesis (procollagen I). There were histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, but these were not found in valve and pulmonary artery tissue. Conclusions-The in vitro tissue-engineering approach using acellular matrix conduits leads to the in vivo reconstitution of viable heart valve tissue.

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.Epstein-Barr virus (EBV)-induced posttransplant lymphoproliferative disorders (PTLD) are a rare but often fatal complication of immunosuppression after bone marrow and organ transplantation. Clinically, PTLD ranges from an infectious mononucleosis-like syndrome-which might respond to reduction of immunosuppression-to a primary extranodal presentation of the disease with a poor prognosis (17). Early diagnosis of PTLD is still a prerequisite for successful treatment, despite advances in therapy (9, 17). Recent studies have demonstrated a direct relationship between the extent of EBV load in peripheral blood mononuclear cells (PBMC) and the risk of developing PTLD (1,2,11,(20)(21)(22)24). Although PTLD patients usually exhibit uncommonly high levels of EBV DNA, new evidence that an increased viral load alone might not be related to PTLD development has emerged (18). Thus, an accepted level of EBV load predictive of PTLD development has not been established as yet.Conventional PCR-based quantification of viral DNA only allows semiquantitative results, and time-consuming hybridization and blotting steps are necessary after amplification (18,22,26,27). These and other disadvantages of conventional quantification have now been overcome by the real-time TaqMan PCR technology using the ABI Prism 7700 sequence detection system (SDS) (8). This technology has already been applied to the detection and quantification of EBV in plasma and cellular DNA (13,16,19,28). Quantification of cell-associated viruses, however, has remained imprecise, sinc...

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EB viral load measurements in plasma and PBMC of patients using RQ-PCR are superior to serology and are a powerful tool for monitoring transplanted patients.

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Hepatitis B infection after heart transplantation leads to chronic liver disease in the majority of the affected patients, causing cirrhosis in more than 55% within the first decade after transplantation. Liver failure is a common cause of death in the infected group of patients. Active HBV vaccination is mandatory for all organ transplant candidates, in particular before heart transplantation.

Progression of CAV is a complex process, modified by changes in the vascular geometry. Especially within the 1st year after HTX, luminal loss is influenced not only by an increase in plaque area but by a decrease in total vessel volume as well.