Increased Arginase Expression and Decreased Nitric Oxide in Pig Donor Lungs after Normothermic Ex Vivo Lung Perfusion.

An established pig lung transplantation model was used to study the effects of cold ischemia time, normothermic acellular ex vivo lung perfusion (EVLP) and reperfusion after lung transplantation on l-arginine/NO metabolism in lung tissue. Lung tissue homogenates were analyzed for NO metabolite (NOx) concentrations by chemiluminescent NO-analyzer technique, and l-arginine, l-ornithine, l-citrulline and asymmetric dimethylarginine (ADMA) quantified using liquid chromatography-mass spectrometry (LC-MS/MS). The expression of arginase and nitric oxide synthase (NOS) isoforms in lung was measured by real-time polymerase chain reaction. EVLP preservation resulted in a significant decrease in concentrations of NOx and l-citrulline, both products of NOS, at the end of EVLP and after reperfusion following transplantation, compared to control, respectively. The ratio of l-ornithine over l-citrulline, a marker of the balance between l-arginine metabolizing enzymes, was increased in the EVLP group prior to reperfusion. The expression of both arginase isoforms was increased from baseline 1 h post reperfusion in EVLP but not in the no-EVLP group. These data suggest that EVLP results in a shift of the l-arginine balance towards arginase, leading to NO deficiency in the lung. The arginase/NOS balance may, therefore, represent a therapeutic target to improve lung quality during EVLP and, subsequently, transplant outcomes.

Hemodynamic performance and clinical outcome of pericardial Perimount Magna and Porcine Hancock-II valves in aortic position.

BACKGROUND: We investigated hospital and midterm outcome of patients operated for an aortic valve replacement (AVR) with a pericardial Perimount or a Porcine Hancock-II valve. METHODS: We analyzed 353 patients with Perimount Magna (n = 189) or Hancock-II valves (n = 164). Echocardiographic data, hospital outcome, and follow-up were collected and compared. The role of the type of valve on perioperative and midterm outcome was investigated. RESULTS: Mean age was 75.3 ± 6.8 and 74.3 ± 7.1 years (P = .17) for Perimount and Hancock-II group, respectively. Fifty-four Perimount (28.6%) and 24 patients with Hancock-II (14.6%) required urgent procedures (P = .002), including six type-A dissections and five endocarditis. EuroSCORE-II was 3.1 ± 2.7% (Perimount) and 2.7 ± 2.2% (Hancock-II). Combined procedures were performed in 115 Perimount (60.8%) and 71 patients with Hancock (43.3%); redo procedures counted for 1% and 2.4%, respectively (P = .42). Mean valve size was 23.2 ± 1.8 mm for pericardial and 23.6 ± 1.9 mm for porcine valves (P = .08). Hospital mortality (6.3% vs 2.4%; P = .05), kidney failure (11.6% vs 9.8%; P = .73), and new pacemaker implantation rates (6.3% vs 3.0%; P = .21) were higher in the Perimount group reflecting the fact that more urgent, combined, and critical procedures were implanted with a Perimount Magna. Overall, 51 patients died over 60 months (34 Perimount, 17 Hancock), corresponding to a mortality of 5.3 per 100-persons year (95% confidence interval [CI]: 3.8-7.4) and 3.0 (95% CI: 1.8-4.8), respectively. Survival at 5 years was 76% (95% CI: 68-82) and 83% (95% CI: 74-89) in the Perimount and Hancock groups (log-rank test; P = .099). CONCLUSIONS: We confirm a good clinical outcome of patients with AVR with modern pericardial or a porcine bioprosthesis. Despite better hemodynamic, the Perimount does not improve the midterm clinical outcome compared with the porcine valve.

Safety and Efficacy of Ex Vivo Donor Lung Adenoviral IL-10 Gene Therapy in a Large Animal Lung Transplant Survival Model.

Ex vivo normothermic lung perfusion (EVLP) is a novel platform and method developed to facilitate functional assessment and implementation of advanced therapies for donor lungs prior to transplantation. This study aimed to determine the safety and immunological and functional benefits of ex vivo adenoviral human interleukin-10 (AdhIL-10) gene delivery to prevent the development of primary graft dysfunction in a large animal survival model. Pig donor lungs were retrieved, preserved for 6 h at 4°C, and then randomly assigned to four groups: (1) AdhIL-10 gene therapy: 12 h EVLP + AdhIL-10 intra-bronchial delivery; (2) EVLP-control: 12 h EVLP; (3) Vector-control: 12 h EVLP + adenoviral vector intra-bronchial delivery; and (4) prolonged hypothermic preservation: additional 12 h of cold ischemia. The left lung was then transplanted and evaluated. The recipients were recovered and kept alive until day 7 post-transplant under standard triple immunosuppression. Plasma levels of hIL-10 were detected in the treatment group throughout the 7 days. Analysis of peripheral blood obtained after transplant showed no signs of hematological, renal, or hepatic toxicity in the AdhIL-10 group. The immediate post-transplant lung function was significantly better in the EVLP-control and AdhIL-10 groups. Gas exchange at day 7 was superior in allografts from the AdhIL-10 group, and the histologic inflammation score was significantly lower. Lymphocytes from AdhIL-10 group harvested from mediastinal lymph nodes at day 7 post-transplantation and co-cultured with donor lymphocytes showed significantly less interferon gamma production in an Enzyme-Linked ImmunoSpot assay when compared with non-treatment groups. It has been demonstrated in this preclinical large animal survival study that ex vivo treatment with AdhIL-10 is safe and improves post-transplant lung function over EVLP alone. Improved function and an immunological advantage in both the innate and adaptive immune responses have been demonstrated.

The role of the endothelin-1 pathway as a biomarker for donor lung assessment in clinical ex vivo lung perfusion.

BACKGROUND: Normothermic ex vivo lung perfusion (EVLP) is a preservation technique that allows reassessment of donor lungs before transplantation. We hypothesized that the endothelin-1 (ET-1) axis would be associated with donor lung performance during EVLP and recipient outcomes after transplantation. METHODS: ET-1, Big ET-1, endothelin-converting enzyme (ECE), and nitric oxide (NO) metabolites were quantified in the perfusates of donor lungs enrolled in a clinical EVLP trial. Lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcomes defined as absence of primary graft dysfunction (PGD) Grade 3 (PGD3) ; (II) PGD3: bilateral lung transplantation with PGD3 any time within 72 hours; and (III) Declined: lungs rejected after EVLP. RESULTS: There were 25 lungs in Group I, 7 in Group II, and 16 in Group III. At 1 and 4 hours of EVLP, the perfusates of Declined lungs had significantly higher levels of ET-1 (3.1 ± 2.1 vs. 1.8±2.3 pg/ml, p = 0.01; 2.7 ± 2.2 vs. 1.3 ± 1.1 pg/ml, p = 0.007) and Big ET-1 (15.8 ± 14.2 vs. 7.0 ± 6.5 pg/ml, p = 0.001; 31.7 ± 17.4 vs. 19.4 ± 9.5 pg/ml, p = 0.007) compared with Controls. Nitric oxide metabolite concentrations were significantly higher in Declined and PGD3 lungs than in Controls. For cases of donation after cardiac death, PGD3 and Declined lungs had higher ET-1 and Big ET-1 levels at 4 hours of perfusion compared with Controls. At this time point, Big ET-1 had excellent accuracy to distinguish PGD3 (96%) and Declined (92%) from Control lungs. CONCLUSIONS: In donation after cardiac death lungs, perfusate ET-1 and Big ET-1 are potential predictors of lung function during EVLP and after lung transplantation. They were also associated with non-use of lungs after EVLP and thus could represent useful biomarkers to improve the accuracy of donor lungs selection.

Protein expression profiling predicts graft performance in clinical ex vivo lung perfusion.

OBJECTIVES: To study the impact of ex vivo lung perfusion (EVLP) on cytokines, chemokines, and growth factors and their correlation with graft performance either during perfusion or after transplantation. BACKGROUND: EVLP is a modern technique that preserves lungs on normothermia in a metabolically active state. The identification of biomarkers during clinical EVLP can contribute to the safe expansion of the donor pool. METHODS: High-risk brain death donors and donors after cardiac death underwent 4 to 6 hours EVLP. Using a multiplex magnetic bead array assay, we evaluated analytes in perfusate samples collected at 1 hour and 4 hours of EVLP. Donor lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcome [absence of primary graft dysfunction- (PGD) grade 3]; (II) PGD3: bilateral transplantation with PGD grade 3 anytime within 72 hours; (III) Declined: lungs unsuitable for transplantation after EVLP. RESULTS: Of 50 cases included in this study, 27 were in Control group, 7 in PGD3, and 16 in Declined. From a total of 51 analytes, 34 were measurable in perfusates. The best marker to differentiate declined lungs from control lungs was stem cell growth factor -ß [P < 0.001, AUC (area under the curve) = 0.86] at 1 hour. The best markers to differentiate PGD3 cases from controls were interleukin-8 (P < 0.001, AUC = 0.93) and growth-regulated oncogene-α (P = 0.001, AUC = 0.89) at 4 hours of EVLP. CONCLUSIONS: Perfusate protein expression during EVLP can differentiate lungs with good outcome from lungs PGD3 after transplantation. These perfusate biomarkers can be potentially used for more precise donor lung selection improving the outcomes of transplantation.

Incidence and severity of primary graft dysfunction after lung transplantation using rejected grafts reconditioned with ex vivo lung perfusion.

OBJECTIVES: Ex vivo lung perfusion (EVLP) is a novel technique used to evaluate and recondition marginal or rejected grafts. Primary graft dysfunction (PGD) is a major early complication after lung transplantation (LTx). The use of marginal or initially rejected grafts may increase its incidence and severity. The aim of this study is to evaluate the incidence of PGD after LTx using rejected grafts reconditioned with EVLP. METHODS: PGD has been evaluated immediately after LTx (t0) and after 72 h (t72) in patients receiving standard (Group A) or reconditioned (Group B) grafts. EVLP was performed using a controlled acellular perfusion according to the Toronto technique. RESULTS: From July 2011 to February 2013, 36 LTxs have been performed: 28 patients (21 M/7 F, mean age 51.7 ± 14.7 years) in Group A and 8 (6 M/2 F, mean age 46.6 ± 9.8 years) in Group B (successful recondition rate of 73%, 8 of 11 cases). Incidence rate of PGD 3 at t0 and at t72 (Group A versus Group B) was 50 vs 37% (P = NS) and 25 vs 0% (P = NS), respectively. Post-transplant extracorporeal membrane oxygenation was required in 5 and 2 patients in Groups A and B, respectively (P = NS). CONCLUSIONS: The use of initially rejected grafts treated with EVLP does not increase the incidence and severity of PGD after LTx. Although comparison of PGD 3 incidence in the two groups did not reach a statistical difference, all EVLP patients suffering from severe PGD early after transplant recovered normal lung function at 72 h, suggesting a protective role of EVLP against PGD occurrence and severity.

Physiologic assessment of the ex vivo donor lung for transplantation.

BACKGROUND: The evaluation of donor lungs by normothermic ex vivo acellular perfusion has improved the safety of organ utilization. However, this strategy requires a critical re-evaluation of the parameters used to assess lungs during ex vivo perfusion compared with those traditionally used to evaluate the donor lung in vivo. Using a porcine model, we studied the physiology of acellular lung perfusion with the aim of improving the accuracy of clinical ex vivo evaluation. METHODS: Porcine lungs after 10 hours of brain death and 24 hours of cold ischemia and uninjured control lungs were perfused for 12 hours and then transplanted. PaO2, compliance, airway pressure and pulmonary vascular resistance were measured. Ventilation with 100% nitrogen and addition of red blood cells to the perfusate were used to clarify the physiologic disparities between in vivo blood perfusion and ex vivo acellular perfusion. RESULTS: During 12 hours of ex vivo perfusion, injured lungs developed edema with decreased compliance and increased airway pressure, but ex vivo PO2 remained stable. After transplantation, injured lungs demonstrated high vascular resistance and poor PaO2. A reduced effect of shunt on ex vivo lung perfusion PO2 was found to be attributable to the linearization of the relationship between oxygen content and PO2, which occurs with acellular perfusate. CONCLUSIONS: Ex vivo PO2 may not be the first indication of lung injury and, taken alone, may be misleading in assessing the ex vivo lung. Thus, evaluation of other physiologic parameters takes on greater importance.