In this prospective study we investigated a cohort after heart transplantation with a novel PCR-based approach with focus on treated rejection. Blood samples were collected coincidentally to biopsies, and both absolute levels of dd-cfDNA and donor fraction were reported using digital PCR. 52 patients (11 children and 41 adults) were enrolled (NCT03477383, clinicaltrials.gov), and 557 plasma samples were analyzed. 13 treated rejection episodes >14 days after transplantation were observed in 7 patients. Donor fraction showed a median of 0.08% in the cohort and was significantly elevated during rejection (median 0.19%, p < 0.0001), using a cut-off of 0.1%, the sensitivity/specificity were 92%/56% (AUC ROC-curve: 0.78). Absolute levels of dd-cfDNA showed a median of 8.8 copies/mL and were significantly elevated during rejection (median 23, p = 0.0001). Using a cut-off of 7.5 copies/mL, the sensitivity/specificity were 92%/43% for donor fraction (AUC ROC-curve: 0.75). The results support the feasibility of this approach in analyzing dd-cfDNA after heart transplantation. The obtained values are well aligned with results from other trials. The possibility to quantify absolute levels adds important value to the differentiation between ongoing graft damage and quiescent situations.

Purpose Cell-free DNA (cfDNA) has been used as a marker of rejection after heart transplantation (HTx). The commonly reported outcome is donor fraction (DF). However, cfDNA from donor (dd-cfDNA) and recipient (rd-cfDNA) can be quantified separately. We aimed to investigate the influence of donor and recipient-derived factors as well as intraoperative measures on levels of cfDNA. Methods Blood samples were collected in parallel to the first endomyocardial biopsy in 45 patients (8 children, 37 adults) after HTx. Levels of rd- and dd-cfDNA and DF were determined using digital PCR. Regression analyses were performed with respect to the influence of donor- (age, body surface area (BSA), gender) and recipient-derived factors (age, BSA, gender, kidney function pre-HTx, ventricular assist device (VAD) pre-HTx, previous heart operation) as well as perioperative measures (operation time, ischemic graft time, early rejection, presence of acute kidney injury). Results The first biopsy was taken on a mean of 11 days after HTx (median 10, range 5-14, IQR 9-12). Mean ischemic time was 180 min (median 180, range 62-394, IQR 118-215), mean operation time was 376 min (median 333, range 174-745, IQR 239-510). Mean rd-cf-DNA levels were 190,000 copies/μl (median 140,000, range 33,000-1,400,000, IQR 83,000-200,000), mean dd-cfDNA-levels 700 copies/μl (median 400, range 150-7000, IQR 270-620). Mean DF was 0.48 (median 0.33, range 0.09-5.3, IQR 0.2-0.5). In a multiple regression model, ischemic graft time was the only factor with a statistically significant impact on levels of dd-cfDNA, but not on DF. The biggest influence on rd-cfDNA-levels was the use of a VAD pre-HTx, but this was not statistically significant. Strong correlations were seen between age and BSA of donor and recipient. Conclusion The separate quantification of both rd- and dd-cfDNA, in contrast to solely reporting DF, allows for the distinction of factors mainly influencing the recipient (such as the pre-HTx use of VAD) and the donor (such as ischemic graft time). In this proof-of-concept study, most of the investigated measures did not reach statistical significance. The correlation of ischemic graft time and dd-cfDNA (but not DF), however, shows the possibility of precisely detecting graft injury

Allomap test was defined as ≥32 in patients ≤6 months and ≥34 in patients > 6 months post-HT.Positive Allosure test was defined as ≥0.12%.Positive biopsy included ISHLT Grades ≥1B.Results: 320 heart transplant recipients enrolled to this study.Mean age was 57.5 §14.0, 231 were males (72.0%).There was a total of 523 Heart-Care blood tests drawn and 148 biopsies were performed (Figure).Of 82 patients with elevated Allomap and Allosure, only 7 (8.5%) of them had a confirmed rejection episode.Among 71 patients with elevated Allosure with normal Allomap, only 2 (2.8%) patients had confirmed rejection.2 patients (4.3%) with negative Allomap and Allosure had rejection.During the study period 55 patients (17.2%) were admitted to the hospital for a total of 67 admissions.17 patients were admitted for rejection (5.3%).None of the patients died during the study period.Conclusion: Transitioning from invasive endomyocardial biopsy monitoring to a non-invasive HeartCare protocol in HT recipients seems feasible based on the high negative value of the combined Allomap and Allosure tests.However, further study is needed to follow the long-term outcomes and presence of rejection in patients post-transplant with this protocol.

normothermic temperatures, however, may aggravate graft injury, especially in endothelial cells.We hypothesized that replacing CSS with hypothermic, oxygenated perfusion (HOPE) provides cardioprotection by preserving the vasculature through the production of nitric oxide.Methods: Following anaesthesia, diaphragm transection and circulatory arrest in male Wistar rats to simulate DCD conditions, hearts underwent 21 min of warm, in-situ ischemia.Hearts were then subjected to either 30 min of CSS, HOPE, or HOPE with the presence of L-NAME (nitric oxide synthase inhibitor).Afterwards, hearts were reperfused ex-situ for 60 min under oxygenated, normothermic conditions.Results: Compared to CSS, HOPE hearts demonstrated higher cardiac function (determined by cardiac output, left ventricular work, as well as contraction and relaxation rates) after 60 min of reperfusion.Furthermore, preliminary results indicate a higher coronary vascular resistance at the end of the hypothermic perfusion period in hearts with L-NAME compared to HOPE alone.Early reperfusion coronary flow, an indicator of vascular function, tended to be higher in hearts subjected to HOPE compared to hearts treated with L-NAME or to the current clinical scenario (CSS).Conclusion: Preservation of vascular and contractile function with HOPE appears superior to the current clinical protocol (CSS).The increase in coronary flow during early reperfusion in HOPE hearts was abolished with the addition of L-NAME, indicating that the beneficial vascular effects of HOPE could be mediated by the production of nitric oxide.Consequently, we believe that HOPE holds great potential for preservation of cardiac grafts obtained with DCD.

Purpose Donor-derived cell-free DNA (dd-cfDNA) as a highly sensitive marker of rejection after heart transplantation (HTx) has gained emerging interest. Recent studies are based on sequencing techniques and use fractional abundance (dd-cfDNA as a fraction of total cfDNA) as their outcome. Here we present patient examples of the BIODRAFT-study (NCT03477383) based on PCR-techniques. Methods Blood samples are taken prospectively in parallel with endomyocardial biopsies (EMB) during the first year after HTx. dd-cfDNA is analyzed using targeted preamplification of 35 single nucleotide polymorphisms followed by digital droplet-PCR. Outcome is fractional abundance as well as total number of DNA copies, both from the donor and the recipient. Results 71 patients (57 adults, 14 children) are so far included and more than 500 blood samples analyzed. In otherwise stable patients, both fractional abundance and total DNA copies seem to follow biopsy patterns with respect to rejection. However, we could also identify different scenarios in which the distribution of cfDNA seems more complicated than hitherto described: Patients with primary graft failure, clinically silent CMV-infection or mild right heart failure show elevated levels of dd-cfDNA, thus complicating the interpretation of results. Some patients present with markedly elevated levels of their own cfDNA without obvious clinical reasons. Conclusion Besides the well-known concept of fractional abundance, our approach gives results that allow following absolute DNA copy numbers of both donor and recipient. This widens the horizon of cfDNA as a marker of graft injury after HTx, and gives an outlook to clinical scenarios with possible false positive (mimicking rejection) and, even worse, false negative results: high levels of recipient-cfDNA could mask ongoing rejection if only fractional abundance can be reported. The biology of cell-free DNA is complex and seems not yet fully understood. Donor-derived cell-free DNA (dd-cfDNA) as a highly sensitive marker of rejection after heart transplantation (HTx) has gained emerging interest. Recent studies are based on sequencing techniques and use fractional abundance (dd-cfDNA as a fraction of total cfDNA) as their outcome. Here we present patient examples of the BIODRAFT-study (NCT03477383) based on PCR-techniques. Blood samples are taken prospectively in parallel with endomyocardial biopsies (EMB) during the first year after HTx. dd-cfDNA is analyzed using targeted preamplification of 35 single nucleotide polymorphisms followed by digital droplet-PCR. Outcome is fractional abundance as well as total number of DNA copies, both from the donor and the recipient. 71 patients (57 adults, 14 children) are so far included and more than 500 blood samples analyzed. In otherwise stable patients, both fractional abundance and total DNA copies seem to follow biopsy patterns with respect to rejection. However, we could also identify different scenarios in which the distribution of cfDNA seems more complicated than hitherto described: Patients with primary graft failure, clinically silent CMV-infection or mild right heart failure show elevated levels of dd-cfDNA, thus complicating the interpretation of results. Some patients present with markedly elevated levels of their own cfDNA without obvious clinical reasons. Besides the well-known concept of fractional abundance, our approach gives results that allow following absolute DNA copy numbers of both donor and recipient. This widens the horizon of cfDNA as a marker of graft injury after HTx, and gives an outlook to clinical scenarios with possible false positive (mimicking rejection) and, even worse, false negative results: high levels of recipient-cfDNA could mask ongoing rejection if only fractional abundance can be reported. The biology of cell-free DNA is complex and seems not yet fully understood.

Donor-derived cell-free DNA (dd-cfDNA) has gained emerging interest as a highly specific marker of rejection after heart transplantation (HTx). However, the method is not established, in part due to technical issues: Sequencing techniques come with high cost and time delay, while PCR-based methods struggle with a low yield of cfDNA-harvest. Hitherto there has been a focus on either relative (dd-cfDNA versus all cfDNA, including recipient-derived cfDNA [rd-cfDNA] + dd-cfDNA) or absolute measurements of dd-cfDNA alone. We propose a novel approach involving a targeted pre-amplification step before digital droplet PCR using probe-based SNP-assays is performed, allowing for the measurement of both relative and absolute values of dd-cfDNA at the same time. This approach is currently being evaluated in an ongoing, prospective single-center study of HTx recipients.

The hippocampal dentate gyrus (DG) is an area of active proliferation and neurogenesis within the adult brain. The molecular events controlling adult cell genesis in the hippocampus essentially remain unknown. It has been reported previously that adult male and female rats from the strains Sprague Dawley (SD) and spontaneously hypertensive (SHR) have a marked difference in proliferation rates of cells in the hippocampal DG. To exploit this natural variability and identify potential regulators of cell genesis in the hippocampus, hippocampal gene expression from male SHR as well as male and female SD rats was analyzed using a cDNA array strategy. Hippocampal expression of the gene-encoding glucose-dependent insulinotropic polypeptide (GIP) varied strongly in parallel with cell-proliferation rates in the adult rat DG. Moreover, robust GIP immunoreactivity could be detected in the DG. The GIP receptor is expressed by cultured adult hippocampal progenitors and throughout the granule cell layer of the DG, including progenitor cells. Thus, these cells have the ability to respond to GIP. Indeed, exogenously delivered GIP induced proliferation of adult-derived hippocampal progenitorsin vivoas well asin vitro, and adult GIP receptor knock-out mice exhibit a significantly lower number of newborn cells in the hippocampal DG compared with wild-type mice. This investigation demonstrates the presence of GIP in the brain for the first time and provides evidence for a regulatory function for GIP in progenitor cell proliferation.

Background: Cardiac allograft vasculopathy (CAV) limits survival after cardiac transplantation. Tumor necrosis factor-alpha (TNF-alpha) may be a key factor in the development of CAV. Two bi-allelic polymorphisms associated with high TNF-alpha production have been identified in the TNF gene locus, TNFA1/2, at position -308 and TNFB1/2 at +252. We hypothesized that recipient TNFA2 and TNFB2 homozygosity is associated with the development of CAV after heart transplantation.Methods: TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase mini-sequencing in 70 cardiac transplant recipients. Recipients homozygous for TNFA2 or TNFB2 (Group A, n = 29) were compared with recipients heterozygous or homozygous for TNFA1 and TNFB1 (Group B, n = 41). Coronary arteriography was performed annually or when indicated. Cumulative freedom from CAV and survival was calculated according to the Kaplan-Meier test.Results: Mean follow-up was 3.8 +/- 0.3 years. In Group A, I I of 29 recipients (38%) developed CAV compared with 9 of 41 (22%) in Group B (p = 0.12). Cumulative freedom from CAV at 3 years was 42% in Group A and 80% in Group B (p = 0.043). In Group A, 11 of 29 recipients (38%) died during follow-up compared with 4 of 41 (10%) in Group B (p = 0.006). Cumulative survival at 3 years was 72% in Group A and 93% in Group B (p = 0.003).Conclusions: The results suggest that TNFA2 and TNFB2 allele homozygosity is associated with cardiac allograft vasculopathy and mortality in heart transplant recipients. Copyright (c) 2005 by the International Society for Heart and Lung Transplantation.

In order to identify patients at risk for developing post-transplant lymphoproliferative disease (PTLD), a sensitive nested RT-PCR method for detection of EBNA1 gene expression in peripheral blood cells was used. EBNA1 expression in peripheral blood samples from 60 organ recipients was analyzed and compared with 24 healthy controls in a retrospective study. Overall, EBNA1-positive samples were detected at least once in 43% of the transplant patients with post-transplant lymphoproliferative disease, in 18% of the other transplant patients and in none of the healthy controls. The odds ratio for EBNA1 expression in patients with post-transplant lymphoproliferative disease was 3.42 (95% CI=1.02-11.54) compared to other transplant recipients. Together with normal EBV Q promoter initiated EBNA1 transcripts, an alternatively spliced form was expressed in peripheral blood cells in the above-mentioned transplant patients. This transcript lacks the U leader exon in the 5'-untranslated region (UTR). We have previously identified and characterized a functional internal ribosome entry site, the EBNA IRES, in the untranslated U leader exon of EBNA1. Transfection experiments with EBNA1 coding plasmids followed by Western blot showed that the EBNA IRES promotes cap-independent translation and increases the EBNA1 protein level. The alternative EBNA1 transcript lacking this function is expressed in the majority of the investigated EBNA1-positive patient samples as well as in some EBV-positive B-cell lines. Alternative splicing in this form gives EBV potential to regulate the translation of EBNA1 by modifying the 5' UTR. These findings indicate a new mechanism for EBNA1 expression in vivo.

BACKGROUND:Tumor necrosis factor-alpha (TNF-alpha), a key factor in the inflammatory cascade, has been implicated in coronary artery disease. Two biallelic polymorphisms in the TNF gene locus (TNFA at position -308 and TNFB at +252) may influence TNF-alpha production. Individuals with the rare TNFA2 allele or TNFB2 homozygosity have augmented TNF-alpha production. We investigated the genotypes associated with increased TNF-alpha production in coronary artery bypass grafting (CABG) patients and if these genotypes influence the magnitude of the postoperative inflammatory response.METHODS:TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase minisequencing in 86 CABG patients. Plasma concentrations of TNF-alpha, IL-6 and C3a and C-reactive protein (CRP) were analyzed before and after surgery in 45 of the patients and compared with genetically high and low TNF-alpha producers.RESULTS:Thirty percent of the patients carried the TNFA2 allele and 45% were TNFB2 homozygous. The allelic frequencies were TNFA1/TNFA2=0.84/0.16 and TNFB1/TNFB2=0.32/0.68. Pre- and postoperative levels of TNF-alpha, IL-6, C3a and CRP did not differ significantly between genetically high and low TNF-alpha producers.CONCLUSIONS:The frequency of high TNF-alpha producing genotypes in a CABG population was comparable to that previously reported from normal populations. Furthermore, we found no evidence that the investigated TNF-alpha gene polymorphisms influence postoperative inflammatory response after uncomplicated coronary surgery.

The study was designed to investigate the influence of hydroxyurea (HU) treatment on PRV-1 expression. Eighteen newly diagnosed patients with essential thrombocythemia (ET) or polycythemia vera (PV) were included. HU significantly increased PRV-1 gene expression in the early stage of treatment.

Essential thrombocythaemia (ET) is a heterogeneous disorder with respect to plasma erythropoietin concentration at diagnosis and clonality of haematopoiesis. Polycythaemia rubra vera-1 (PRV-1) positivity, i.e. PRV-1 mRNA overexpression, is known to be present in the vast majority of patients with polycythaemia vera and also in some patients with ET. In the present study, PRV-1 expression was quantified by real-time polymerase chain reaction in 70 ET patients; 17 of them (24%) were found to be PRV-1 positive. Ten of the 17 PRV-1 positive ET patients had experienced thromboembolic complications compared with 14 of 53 PRV-1 negative patients, the difference between the two groups being statistically significant (P=0.02). In addition, the frequency of total vascular complications, thromboembolic events and major bleedings, was significantly higher in the group of PRV-1 positive as compared with PRV-1 negative ET patients (P=0.03). The time from diagnosis of ET to the requirement of platelet-lowering therapy was significantly shorter in PRV-1 positive compared with PRV-1 negative ET patients (P=0.014). It can be concluded that PRV-1 positive patients appear to suffer from a more aggressive disorder with increased risk for vascular complications and a greater need for platelet-lowering therapy, compared with PRV-1 negative ET patients.

Approximately 45% of newly diagnosed patients with essential thrombocythaemia (ET) demonstrate subnormal plasma erythropoietin (EPO) concentrations, which constitutes a risk factor for occlusive vascular events. In 58 ET patients, a possible association between polycythaemia rubra vera-1 (PRV-1) overexpression and subnormal plasma EPO was investigated, which was always measured prior to the institution of platelet lowering agents. At the time when PRV-1 expression was measured, 28 of 58 (48%) ET patients had received platelet lowering treatment. PRV-1 expression was measured by quantitative real-time reverse transcription-polymerase chain reaction assay of mRNA extracted from purified peripheral blood buffy coat. The cycle threshold (CT) value of PRV-1 was determined and was divided with the CT value for the housekeeping GAPDH gene transcript. A quotient <0.93 was defined as PRV-1 positive. Of the ET patients 12 of 58 (21%) were PRV-1 positive and 19 of 58 (33%) demonstrated subnormal plasma EPO. In the 58 ET patients there was a significant association between low plasma EPO and PRV-1 positive results (P = 0.001). The 30 ET patients who had not received any platelet lowering treatment showed a significant (P = 0.005) relation between PRV-1 positivity and subnormal plasma EPO. No such relationship was present in the 28 ET patients who had received prior treatment with the above drugs (P = 0.147).

UNLABELLED:Cardiopulmonary bypass induces a systemic inflammatory response characterized by alterations in cardiopulmonary function. Mediators for this morbidity are the cytokines tumor necrosis factor (TNF)-alpha and interleukins. A genomic polymorphism within the TNF locus is associated with increased TNF-alpha levels and high mortality in severe trauma and sepsis. We assessed the relationship of biallelic polymorphisms of the TNF locus in patients undergoing elective cardiac surgery to release of proinflammatory cytokines and cardiopulmonary morbidity. TNF genotypes, plasma concentrations of TNF-alpha, interleukin-6, and cardiopulmonary morbidity were studied in 95 unselected, consecutive patients undergoing routine cardiac surgery. TNF genotypes were determined by the solid-phase minisequencing method. Patients homozygous for the TNFB2 allele (n = 42) displayed larger peak concentrations of TNF-alpha (11.3 +/- 1.3 versus 7.8 +/- 0.7 pg/mL; P = 0.013) and interleukin-6 (153 +/- 27 versus 87 +/- 7 pg/mL; P = 0.010) when compared with patients homozygous or heterozygous for TNFB1 (n = 53). The TNFB2 homozygotes had a higher incidence of left ventricular dysfunction (31% versus 9%; P = 0.029; odds ratio 3.84 [95% confidence interval, 1.40-24.3]), postoperative pulmonary dysfunction (24% versus 6%; P = 0.016; odds ratio 5.21 [95% confidence interval, 1.49-18.3]), and a lower pulmonary oxygenation index (29 +/- 1.9 versus 36.1 +/- 1.8; P = 0.013). Patients homozygous for the TNFB2 allele may develop an enhanced systemic inflammatory response with an increased risk of cardiopulmonary morbidity after cardiac surgery. IMPLICATIONS:The associations between tumor necrosis factor (TNF) gene polymorphism, plasma cytokines, and cardiopulmonary function after elective cardiac surgery were evaluated. Patients homozygous for the TNFB2 allele displayed larger concentrations of TNF-alpha and interleukin-6 and had an increased risk of developing left ventricular and pulmonary dysfunction compared with TNFB1 homo- or heterozygotes.

In a previous report we demonstrated Epstein-Barr virus expression in cutaneous squamous cell carcinomas from heart transplant recipients. In a comparative study, skin lesions from renal allograft recipients were investigated for the presence of Epstein-Barr virus. Thirty cutaneous squamoproliferative lesions from 10 kidney transplant recipients were examined for Epstein-Barr virus-specific gene expression. The techniques used for detection were polymerase chain reaction, in situ hybridization and immunohistochemistry. Epstein-Barr virus DNA was not detected by polymerase chain reaction in the neoplasias, and only single Epstein-Barr virus-positive carcinoma cells were shown by in situ hybridization in three cases of infiltrative squamous cell carcinomas. Immunohistochemistry for Epstein-Barr virus latent membrane protein 1 showed a negative result in all samples. These findings differ from our earlier investigations of cutaneous squamous cell carcinomas from heart transplant recipients where Epstein-Barr virus expressions were common. This may indicate that the part Epstein-Barr virus plays in the development of post-transplant, cutaneous squamoproliferative disorders is related to type of organ transplantation and/or grade of immunosuppression.

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE ε2, ε3 and ε4) and has important functions in nerve development and repair. Inheritance of the ε4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The ε4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of ε3 was significantly increased (P=0.005), suggesting that ε4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the ε4 allele in natural selection.