A bead-based multiplex assay covering all coronaviruses pathogenic for humans for sensitive and specific surveillance of SARS-CoV-2 humoral immunity.

Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Toxin exposure and HLA alleles determine serum antibody binding to toxic shock syndrome toxin 1 (TSST-1) of Staphylococcus aureus.

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1.

Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

Effectiveness of vaccines in preventing hospitalization due to COVID-19: A multicenter hospital-based case-control study, Germany, June 2021 to January 2022.

We included 852 patients in a prospectively recruiting multicenter matched case-control study in Germany to assess vaccine effectiveness (VE) in preventing COVID-19-associated hospitalization during the Delta-variant dominance. The two-dose VE was 89 % (95 % CI 84-93 %) overall, 79 % in patients with more than two comorbidities and 77 % in adults aged 60-75 years. A third dose increased the VE to more than 93 % in all patient-subgroups.

Left Ventricular Wall Reconstruction with Autologous Vascularized Tunica Muscularis of Stomach in a Porcine Pilot Model.

INTRODUCTION: Surgical replacement of dysfunctional cardiac muscle with regenerative tissue is an important option to combat heart failure. But, current available myocardial prostheses like a Dacron or a pericardium patch neither have a regenerative capacity nor do they actively contribute to the heart's pump function. This study aimed to show the feasibility of utilizing a vascularized stomach patch for transmural left ventricular wall reconstruction. METHODS: A left ventricular transmural myocardial defect was reconstructed by performing transdiaphragmatic autologous transplantation of a vascularized stomach segment in six Lewe minipigs. Three further animals received a conventional Dacron patch as a control treatment. The first 3 animals were followed up for 3 months until planned euthanasia, whereas the observation period for the remaining 3 animals was scheduled 6 months following surgery. Functional assessment of the grafts was carried out via cardiac magnetic resonance tomography and angiography. Physiological remodeling was evaluated histologically and immunohistochemically after heart explantation. RESULTS: Five out of six test animals and all control animals survived the complex surgery and completed the follow-up without clinical complications. One animal died intraoperatively due to excessive bleeding. No animal experienced rupture of the stomach graft. Functional integration of the heterotopically transplanted stomach into the surrounding myocardium was observed. Angiography showed development of connections between the gastric graft vasculature and the coronary system of the host cardiac tissue. CONCLUSIONS: The clinical results and the observed physiological integration of gastric grafts into the cardiac structure demonstrate the feasibility of vascularized stomach tissue as myocardial prosthesis. The physiological remodeling indicates a regenerative potential of the graft. Above all, the connection of the gastric vessels with the coronary system constitutes a rationale for the use of vascularized and, therefore, viable stomach tissue for versatile tissue engineering applications.

Biocompatibility of an apical ring plug for left ventricular assist device explantation: Results of a feasibility pre-clinical study.

BACKGROUND: Patients receiving left ventricle assist devices (LVADs) as bridge to recovery remain a minority with 1%-5% of LVADs explanted after improvement of myocardial function. Nevertheless, considering the growing population of patients supported with LVADs, an increasing demand of new explantation strategies is expected in the near future. A novel plug for LVAD explantation has been developed and its biocompatibility profile needs to be proved. This study tested the biocompatibility of this novel plug in an in vivo ovine model. METHODS: Six adult Blackhead Persian female sheep received plug implantation on the cardiac apex via minimally invasive approach and were clinically observed up to 90 days. Echocardiography was performed to detect thrombus formation or further plug-related complications. After the observation period, euthanasia was performed and samples including the plug and the surrounding tissues were obtained to be analyzed with correlative light and electron microscopy. Organ necrosis, ischemia and peripheral embolism were investigated. RESULTS: Three animals survived surgery and completed the follow-up time without experiencing clinical complications. Echocardiographic controls excluded the presence of an intracavitary thrombus in the left ventricle (LV). Autopsy confirmed no signs of local infection, LV thrombus or peripheral embolism. Light and electron microscopy revealed an intact epithelium covering a layer of connective tissue on the plug surface facing the heart lumen. CONCLUSIONS: This novel apical plug for LVAD explantation allows for endothelial and connective tissue growth on its ventricular side within 90 days from surgery. Further studies are required to fully demonstrate the biocompatibility of this apical plug and investigate the optimal anticoagulation regimen to be applied after implantation.

A Comprehensive View on the Human Antibody Repertoire Against Staphylococcus aureus Antigens in the General Population.

Our goal was to provide a comprehensive overview of the antibody response to Staphylococcus aureus antigens in the general population as a basis for defining disease-specific profiles and diagnostic signatures. We tested the specific IgG and IgA responses to 79 staphylococcal antigens in 996 individuals from the population-based Study of Health in Pomerania. Using a dilution-based multiplex suspension array, we extended the dynamic range of specific antibody detection to seven orders of magnitude, allowing the precise quantification of high and low abundant antibody specificities in the same sample. The observed IgG and IgA antibody responses were highly heterogeneous with differences between individuals as well as between bacterial antigens that spanned several orders of magnitude. Some antigens elicited significantly more IgG than IgA and vice versa. We confirmed a strong influence of colonization on the antibody response and quantified the influence of sex, smoking, age, body mass index, and serum glucose on anti-staphylococcal IgG and IgA. However, all host parameters tested explain only a small part of the extensive variability in individual response to the different antigens of S. aureus.

The Arabidopsis Plastidial Glucose-6-Phosphate Transporter GPT1 is Dually Targeted to Peroxisomes via the Endoplasmic Reticulum.

Studies on Glucose-6-phosphate (G6P)/phosphate translocator isoforms GPT1 and GPT2 reported the viability of Arabidopsis (Arabidopsis thaliana) gpt2 mutants, whereas heterozygous gpt1 mutants exhibited a variety of defects during fertilization/seed set, indicating that GPT1 is essential for this process. Among other functions, GPT1 was shown to be important for pollen and embryo-sac development. Because our previous work on the irreversible part of the oxidative pentose phosphate pathway (OPPP) revealed comparable effects, we investigated whether GPT1 may dually localize to plastids and peroxisomes. In reporter fusions, GPT2 localized to plastids, but GPT1 also localized to the endoplasmic reticulum (ER) and around peroxisomes. GPT1 contacted two oxidoreductases and also peroxins that mediate import of peroxisomal membrane proteins from the ER, hinting at dual localization. Reconstitution in yeast (Saccharomyces cerevisiae) proteoliposomes revealed that GPT1 preferentially exchanges G6P for ribulose-5-phosphate (Ru5P). Complementation analyses of heterozygous +/gpt1 plants demonstrated that GPT2 is unable to compensate for GPT1 in plastids, whereas GPT1 without the transit peptide (enforcing ER/peroxisomal localization) increased gpt1 transmission significantly. Because OPPP activity in peroxisomes is essential for fertilization, and immunoblot analyses hinted at the presence of unprocessed GPT1-specific bands, our findings suggest that GPT1 is indispensable in both plastids and peroxisomes. Together with its G6P-Ru5P exchange preference, GPT1 appears to play a role distinct from that of GPT2 due to dual targeting.

Technical report: xMAPr - High-dynamic-range (HDR) quantification of antigen-specific antibody binding.

Here, we demonstrate a high-dynamic-range quantification of antibody binding to single antigens in a multiplexed suspension bead array format. Using a dilution-based approach and the newly developed data analysis tool the quantitative dynamic range is increased by three orders of magnitude in the selected samples. The strong increase in dynamic range results in a more robust data basis for downstream analyses.

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation.

Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic.

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 105 S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.

* Six-Year-Old Sheep as a Clinically Relevant Large Animal Model for Aortic Valve Replacement Using Tissue-Engineered Grafts Based on Decellularized Allogenic Matrix.

Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in preclinical and clinical studies. However, in animal models valves have either been tested in juvenile models or in the clinically less relevant pulmonary valve position. In this study, we tested the grafts in the aortic valve (AV) position of 6-year-old sheep, as geriatric patients in need of an AV substitute due to calcification are the largest patient group benefiting from TE grafts. Decellularized AV (DAV; n = 4) and DAV additionally re-endothelialized with autologous cells (n = 3) were implanted in the AV position of 6-year-old female sheep. Function was investigated at implantation and explantation 12 months later. Regeneration capacity was analyzed by the repopulation degree of the graft with recipient's cells, by the generation of a new endothelial layer and by intracellular staining against pro-collagen type I. DAV and re-endothelialized AV demonstrated excellent function with only two valves developing mild insufficiencies (1°). Of the repopulating cells only few cells were identified as inflammation cells, while the majority was found to be interstitial cells producing procollagen type I. Endothelial coverage was found, but seemed to be reduced. The regenerative capacity of decellularized matrix is not only a feature exhibited when implanted in juvenile individuals but also is evident when implanted in the high-pressure AV position of older sheep, revealing the potential of TE grafts in age-advanced patients.

Characterization of human and Staphylococcus aureus proteins in respiratory mucosa by in vivo- and immunoproteomics.

Staphylococcus aureus is a Gram-positive opportunistic bacterium which can be found as a commensal in the nares of about 50% of the human population. Besides asymptomatic carriage, S. aureus has also been found to colonize nasal polyps, a subform of chronic rhinosinusitis, in 60 to 100% of cases, and even reside intracellularly in nasal polyp tissue. The aim of this study was to shed light on the behavior of S. aureus in the human airways by analyzing S. aureus-specific proteins in nasal polyp tissue from patients with chronic rhinosinusitis and to characterize the immunogenic potential of the identified (mainly secreted) proteins. As a result, in total >600 S. aureus proteins were identified by high resolution mass spectrometry or multiple reaction monitoring. Of those roughly 180 are typically localized in the membrane, surface exposed or secreted. For 115 S. aureus proteins, partially also detected in vivo by mass spectrometry, IgA- and IgG-specific antibody signals were profiled. Strong antibody signals were predominantly found for surface expose or secreted proteins. SIGNIFICANCE: In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue. We discovered bacterial proteins involved in invasion of tissue, virulence, bacterial signal transduction or acquisition of nutrients. Some of the detected superantigens and Spls are known to provoke secretion of a broad spectrum of cytokines. Therefore, our manuscript contains new information about the invasion of S. aureus in nasal polyp tissue and its protein-specific immunogenicity.

Defects in Peroxisomal 6-Phosphogluconate Dehydrogenase Isoform PGD2 Prevent Gametophytic Interaction in Arabidopsis thaliana.

We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision.

Decellularized aortic allografts versus pulmonary autografts for aortic valve replacement in the growing sheep model: haemodynamic and morphological results at 20 months after implantation.

OBJECTIVES: Pulmonary autografts (PAs) represent the substitute of choice for aortic valve (AV) replacement, especially in children and young adults. Similarly, decellularized aortic valve allografts (DAVAs) have shown excellent mid-term function when implanted in the systemic circulation. The aim of this study was to compare the performance of DAVAs with that of pulmonary autografts after a Ross procedure in the growing sheep model. METHODS: AV root replacement was performed in female lambs (25 ± 3.4 kg) using either DAVAs (n = 5) or pulmonary autografts (n = 5) as in the Ross procedure. Sheep undergoing the Ross procedure received a decellularized pulmonary allograft in place of pulmonary valve. Haemodynamics was investigated by echocardiography and magnetic resonance imaging. The roots were explanted at 20 months and examined by histology to determine the degree of repopulation and quality of the extracellular matrix, and by immunohistochemistry to characterize the repopulating cells. RESULTS: The mean valve diameter increased from 16 to 21 and from 16 to 25 mm in DAVAs and PAs, respectively. At explantation, one PA and one DAVA exhibited moderate insufficiency. Significant differences in transvalvular gradient were only found in PAs between implantation and prior to explantation. The cusps of all implants were soft, pliable and showed no major signs of degeneration. In the decellularized allografts, cell repopulation occurred at the wall and cusp level with a well-maintained, three-layered cusp structure. Ventricular cusp surface of decellularized allografts was more strongly repopulated than the arterial surface. Cusps were covered with cells positive for endothelial markers and were also repopulated by interstitial cells. CONCLUSIONS: DAVAs and PAs provide adequate haemodynamics after AV replacement in the growing sheep. While decellularized grafts are repopulated by endothelial and interstitial cells, autografts maintain in general their native cell distribution. Maintenance of valvular competence during enlargement of the valve ring is, in our opinion, representative of the capacity for physiological growth in both graft types.

Successful matrix guided tissue regeneration of decellularized pulmonary heart valve allografts in elderly sheep.

In vivo repopulation of decellularized allografts with recipient cells leads to a positive remodeling of the graft matrix in juvenile sheep. In light of the increasing number of heart valve replacements among older patients (>65 years), this study focused on the potential for matrix-guided tissue regeneration in elderly sheep. Pulmonary valve replacement was performed in seven-year old sheep using decellularized (DV), decellularized and CCN1-coated (RV), or decellularized and in vitro reendothelialized pulmonary allografts (REV) (n=6, each group). CCN1 coating was applied to support re-endothelialization. In vitro re-endothelialization was conducted with endothelial-like cells derived from peripheral blood. Echocardiograms of all grafts showed adequate graft function after implantation and at explantation 3 or 6 months later. All explants were macroscopically free of thrombi at explantation, and revealed repopulation of the allografts on the adventitial side of valvular walls and proximal in the cusps. Engrafted cells expressed vimentin, sm α-actin, and myosin heavy chain 2, while luminal cell lining was positive for vWF and eNOS. Cellular repopulation of valvular matrix demonstrates the capacity for matrix-guided regeneration even in elderly sheep but is not improved by in vitro endothelialization, confirming the suitability of decellularized matrix for heart valve replacement in older individuals.

In vivo degradation of magnesium alloy LA63 scaffolds for temporary stabilization of biological myocardial grafts in a swine model.

Synthetic or biological patch materials used for surgical myocardial reconstruction are often fragile. Therefore, a transient support by degradable magnesium scaffolds can reduce the risk of dilation or rupture of the patch until physiological remodeling has led to a sufficient mechanical durability. However, there is evidence that magnesium implants can influence the growth and physiological behavior of the host's cells and tissue. Hence, we epicardially implanted scaffolds of the magnesium fluoride-coated magnesium alloy LA63 in a swine model to assess biocompatibility and degradation kinetics. Chemical analysis of the pigs' organs revealed no toxic accumulation of magnesium ions in the skeletal muscle, myocardium, liver, kidney, and bone of the pigs 1, 3, and 6 months postimplantation. The implants were surrounded by a fibrous granulation tissue, but no signs of necrosis were histologically evaluable. A sufficiently slow degradation rate of the magnesium alloy scaffold can be demonstrated via micro-computed tomography investigation. We conclude that stabilizing scaffolds of the magnesium fluoride-coated magnesium alloy LA63 can be used for epicardial application because no significant adverse effects to myocardial tissue were noted. Thus, degradable stabilizing scaffolds of this magnesium alloy with a slow degradation rate can extend the indication of innovative biological and synthetic patch materials.

Orthotopic replacement of aortic heart valves with tissue-engineered grafts.

AIMS: Heart valve tissue engineering aims to create a graft with improved durability compared to routinely used valve substitutes. This study presents the function and morphological changes of a tissue-engineered aortic valve (TEV) compared to the cryopreserved valve (CPV), aortic valve (AV) allografts in an orthotopic position in sheep. METHODS AND RESULTS: Ovine AV conduits (n=5) were decellularized with detergents. Autologous endothelial cells (ECs) were seeded onto the valve surface and cultured under physiological conditions using a high pulsatile flow. Grafts were implanted as a root with reimplantation of coronary ostia in sheep. Crystalloid cardioplegia and isogenic blood transfusions from previous sacrificed sheep were used. Only antiplatelet aggregation therapy was used postoperatively. CPVs (n=4) served as controls. The grafts were investigated for function (echocardiography, magnetic resonance investigation), morpho/histological appearance, graft rejection, and calcification at 3 months. Decellularization led to cell-free scaffolds with preserved extracellular matrices, including the basement membrane. TEVs were covered with ECs expressing typical endothelial markers. Neither dilatation, stenosis, reductions of cusp mobility nor a significant transvalvular gradient, were observed in the TEV group. Explanted valves exhibited normal morphology without signs of inflammation. An endothelial monolayer covered cusps and the valve sinus. In the CPV group, sporadic, macroscopic, calcified degeneration with mild AV insufficiency was noted. Histology revealed signs of rejection and incipient calcification of the tissue. CONCLUSION: Tissue-engineered AV based on decellularized valve allografts satisfy short-term requirements of the systemic circulation in sheep. Although results of long-term experiments are pending, the lack of degenerative traits thus far, makes these grafts a promising alternative for future aortic heart valve surgery.