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Background Normothermic ex vivo lung perfusion (EVLP) allows for functional assessment of donor lungs; thus has increased the use of marginal lungs for transplantation. To extend EVLP for advanced organ reconditioning and regenerative interventions, cellular metabolic changes need to be understood. We sought to comprehensively characterize the dynamic metabolic changes of the lungs during EVLP, and to identify strategies to improve EVLP. Methods Human donor lungs (n = 50) were assessed under a 4-hour Toronto EVLP protocol. EVLP perfusate was sampled at first (EVLP-1h) and fourth hour (EVLP-4h) of perfusion and were submitted for mass spectrometry-based untargeted metabolic profiling. Differentially expressed metabolites between the 2 timepoints were identified and analyzed from the samples of lungs transplanted post-EVLP (n = 42) to determine the underlying molecular mechanisms. Results Of the total 312 detected metabolites, 84 were up-regulated and 103 were down-regulated at EVLP-4h relative to 1h (FDR adjusted p ≥ |1.1|). At EVLP-4h, markedly decreased energy substrates were observed, accompanied by the increase in fatty acid β-oxidation. Concurrently, accumulation of amino acids and nucleic acids was evident, indicative of increased protein and nucleotide catabolism . The uniform decrease in free lysophospholipids and polyunsaturated fatty acids at EVLP-4h suggests cell membrane remodeling. Conclusions Untargeted metabolomics revealed signs of energy substrate consumption and metabolic by-product accumulation under current EVLP protocols. Strategies to supplement nutrients and to maintain homeostasis will be vital in improving the current clinical practice and prolonging organ perfusion for therapeutic application to further enhance donor lung utilization.

Ischemia-reperfusion (I/R)-induced lung injury undermines lung transplantation (LTx) outcomes by predisposing lung grafts to primary graft dysfunction (PGD). Necrosis is a feature of I/R lung injury. However, regulated necrosis (RN) with specific signaling pathways has not been explored in an LTx setting. In this study, we investigated the role of RN in I/R-induced lung injury. To study I/R-induced cell death, we simulated an LTx procedure using our cell culture model with human lung epithelial (BEAS-2B) cells. After 18 h of cold ischemic time (CIT) followed by reperfusion, caspase-independent cell death, mitochondrial reactive oxygen species production, and mitochondrial membrane permeability were significantly increased. N-acetyl-Leu-Leu-norleucinal (ALLN) (calpain inhibitor) or necrostatin-1 (Nec-1) [receptor interacting serine/threonine kinase 1 (RIPK1) inhibitor] reduced these changes. ALLN altered RIPK1/RIPK3 expression and mixed lineage kinase domain-like (MLKL) phosphorylation, whereas Nec-1 did not change calpain/calpastatin expression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was demonstrated to be downstream of calpain and regulate RIPK3 expression and MLKL phosphorylation during I/R. This calpain-STAT3-RIPK axis induces endoplasmic reticulum stress and mitochondrial calcium dysregulation. LTx patients’ samples demonstrate that RIPK1, MLKL, and STAT3 mRNA expression increased from CIT to reperfusion. Moreover, the expressions of the key proteins are higher in PGD samples than in non-PGD samples. Cell death associated with prolonged lung preservation is mediated by the calpain-STAT3-RIPK axis. Inhibition of RIPK and/or calpain pathways could be an effective therapy in LTx.

Objectives Ex vivo lung perfusion provides an innovative method to assess and repair donor lungs. The current Toronto ex vivo lung perfusion protocol can reliably and reproducibly preserve lungs for 12 hours. A longer ex vivo lung perfusion preservation time could enable the application of more advanced repair therapies and the rescue of more donor lungs for lung transplant. Our objective was to achieve stable 24-hour normothermic ex vivo lung perfusion. Methods We systematically examined 3 modifications of ex vivo lung perfusion perfusate administration in a large animal 24-hour ex vivo lung perfusion model. Pig lungs were assigned to 4 groups (n = 5 per group): (1) control; (2) continuous replacement of ex vivo lung perfusion perfusate; (3) modified feed, which used a modified solution to maintain perfusate osmolality by adjusting glucose and sodium levels; and (4) total parenteral nutrition, in which we added parenteral nutrition to the perfusate. Results Only 1 lung in the control group completed 24-hour ex vivo lung perfusion. However, 24-hour perfusion was achieved in 4 lungs in the continuous replacement group, 3 lungs in the modified feed group, and 4 lungs in the total parenteral nutrition group. The total parenteral nutrition group achieved significantly longer stable perfusion time compared with control (P = .03). Lung function was significantly improved and inflammatory cytokine production was reduced in the continuous replacement and total parenteral nutrition groups compared with control. Conclusions Modifications of ex vivo lung perfusion perfusate toward achieving a stable homeostatic state can extend perfusion time for up to 24 hours. Although these modifications allow for prolonged ex vivo lung perfusion, further research will be required to develop stable lung support beyond 24 hours.

Ex vivo lung perfusion (EVLP) enables assessment of marginal donor lungs for transplantation, with similar clinical outcomes to conventional lung transplantation. We investigated whether cell death-related proteins in the EVLP perfusate could predict primary graft dysfunction (PGD) after transplantation.M30 (indicating epithelial apoptosis), M65 (indicating total epithelial cell death), and high mobility group box 1 (HMGB-1, related to cell death and inflammation) protein levels in EVLP perfusate were measured by enzyme-linked immunosorbent assay and correlated with clinical outcomes.From 100 sequential EVLP patients, 79 lungs were transplanted. Patients who were bridged with extracorporeal life support (ECLS, n = 6) or who received lobar/single lung (n = 25) were excluded. PGD grade 3 (partial pressure of arterial oxygen/fraction of inspired oxygen200 or need for ECLS) developed in 11 at any time within 72 hours after transplantation (PGD Group). PGD grade 3 did not develop in 34 patients (Control Group). M30 was significantly higher in the PGD Group than in the Control Group at 1 hour (PGD: 73.3 ± 24.9, control: 53.9 ± 15.9 U/liter; p0.01) and at 4 hours (PGD: 137.0 ± 146.6, Control: 72.4 ± 40.0 U/liter; p = 0.046) of EVLP. The increase of HMGB-1 from 1 to 4 hours of EVLP was significantly greater in the PGD Group (PGD: 37.0 ± 25.4, Control: 7.2 ± 16.8 ng/ml; p0.001). Higher levels of or a greater increase in M30 and a greater increase in HMGB-1 were associated with higher mortality in Cox regression.Levels of M30 and HMGB-1 in the EVLP perfusate correlate with PGD after lung transplantation and might therefore be useful biomarkers to improve donor lung assessment during EVLP.

BACKGROUND Gastroesophageal reflux disease (GERD) is a risk factor for chronic lung allograft dysfunction. Bile acids—putative markers of gastric microaspiration—and inflammatory proteins in the bronchoalveolar lavage (BAL) have been associated with chronic lung allograft dysfunction, but their relationship with GERD remains unclear. Although GERD is thought to drive chronic microaspiration, the selection of patients for anti-reflux surgery lacks precision. This multicenter study aimed to test the association of BAL bile acids with GERD, lung inflammation, allograft function, and anti-reflux surgery. METHODS We analyzed BAL obtained during the first post-transplant year from a retrospective cohort of patients with and without GERD, as well as BAL obtained before and after Nissen fundoplication anti-reflux surgery from a separate cohort. Levels of taurocholic acid (TCA), glycocholic acid, and cholic acid were measured using mass spectrometry. Protein markers of inflammation and injury were measured using multiplex assay and enzyme-linked immunosorbent assay. RESULTS At 3 months after transplantation, TCA, IL-1β, IL-12p70, and CCL5 were higher in the BAL of patients with GERD than in that of no-GERD controls. Elevated TCA and glycocholic acid were associated with concurrent acute lung allograft dysfunction and inflammatory proteins. The BAL obtained after anti-reflux surgery contained reduced TCA and inflammatory proteins compared with that obtained before anti-reflux surgery. CONCLUSIONS Targeted monitoring of TCA and selected inflammatory proteins may be useful in lung transplant recipients with suspected reflux and microaspiration to support diagnosis and guide therapy. Patients with elevated biomarker levels may benefit most from anti-reflux surgery to reduce microaspiration and allograft inflammation.

Immediate graft performance after lung transplantation is associated with short- and long-term clinical outcomes. However, the biologic mechanism that determines outcomes is not fully understood.To investigate the impact of cell death signals at 24 and 48 hours after lung transplantation on short- and long-term clinical outcomes.Plasma samples were collected pretransplantation and at 24 and 48 hours after transplant from 60 bilateral lung transplant recipients. Ten patients had primary graft dysfunction (PGD) grade 3 (PaO2/FiO2 ratio200 or on extracorporeal membrane oxygenation support) at 72 hours after transplant (PGD group). The remaining 50 patients were defined as the control group. Levels of plasma M30 (signifying epithelial apoptosis), M65 (signifying epithelial apoptosis plus necrosis), and high-mobility group box 1 protein (HMGB-1; signifying necrosis of all cell types) were measured by ELISA and correlated with clinical outcomes. Survival analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Prediction accuracy of markers was assessed by calculated area under the curve of receiver operating characteristic graph.The PGD group had significantly higher M30 and M65 levels at 24 and 48 hours after transplant compared with the control group. There was no significant difference in HMGB-1. Area under the curve for 1-year survival was 0.86, 0.93, and 0.51 for M30, M65, and HMGB-1 at 48 hours, respectively. Survival analysis showed that higher M30 and M65 levels at 24 and 48 hours were significantly associated with worse survival. M65 at 48 hours remained significant even after adjustment for PGD. HMGB-1 was not significantly associated with survival.Recipient plasma concentration of epithelial cell death markers (M30, M65) after lung transplantation is negatively correlated with early graft performance and long-term survival.

IntroductionTransplantation of lungs from donation after circulatory death (DCD) in addition to donation after brain death (DBD) became routine worldwide to address the global organ shortage. The development ofex vivolung perfusion (EVLP) for donor lung assessment and repair contributed to the increased use of DCD lungs. We hypothesise that a better understanding of the differences between lungs from DBD and DCD donors, and between EVLP and directly transplanted (non-EVLP) lungs, will lead to the discovery of the injury-specific targets for donor lung repair and reconditioning.MethodsTissue biopsies from human DBD (n=177) and DCD (n=65) donor lungs, assessed with or without EVLP, were collected at the end of cold ischaemic time. All samples were processed with microarray assays. Gene expression, network and pathway analyses were performed using R, Ingenuity Pathway Analysis and STRING. Results were validated with protein assays, multiple logistic regression and 10-fold cross-validation.ResultsOur analyses showed that lungs from DBD donors have upregulation of inflammatory cytokines and pathways. In contrast, DCD lungs display a transcriptome signature of pathways associated with cell death, apoptosis and necrosis. Network centrality revealed specific drug targets to rehabilitate DBD lungs. Moreover, in DBD lungs, tumour necrosis factor receptor-1/2 signalling pathways and macrophage migration inhibitory factor-associated pathways were activated in the EVLP group.A panel of genes that differentiate the EVLP from the non-EVLP group in DBD lungs was identified.ConclusionThe examination of gene expression profiling indicates that DBD and DCD lungs have distinguishable biological transcriptome signatures.

IntroductionThe ex vivo lung perfusion (EVLP) technique has been developed to assess the function of marginal donor lungs and has significantly increased donor lung utilisation. EVLP has also been explored as a platform for donor lung repair through injury-specific treatments such as antibiotics or fibrinolytics. We hypothesised that actively expressed pathways shared between transplantation and EVLP may reveal common mechanisms of injury and potential therapeutic targets for lung repair prior to transplantation.Materials and methodsRetrospective transcriptomics analyses were performed with peripheral tissue biopsies from “donation after brain death” lungs, with 46 pre-/post-transplant pairs and 49 pre-/post-EVLP pairs. Pathway analysis was used to identify and compare the responses of donor lungs to transplantation and to EVLP.Results22 pathways were enriched predominantly in transplantation, including upregulation of lymphocyte activation and cell death and downregulation of metabolism. Eight pathways were enriched predominantly in EVLP, including downregulation of leukocyte functions and upregulation of vascular processes. 27 pathways were commonly enriched, including activation of innate inflammation, cell death, heat stress and downregulation of metabolism and protein synthesis. Of the inflammatory clusters, Toll-like receptor/innate immune signal transduction adaptor signalling had the greatest number of nodes and was central to inflammation. These mechanisms have been previously speculated as major mechanisms of acute lung injury in animal models.ConclusionEVLP and transplantation share common molecular features of injury including innate inflammation and cell death. Blocking these pathways during EVLP may allow for lung repair prior to transplantation.

Background Ex vivo lung perfusion (EVLP) is being increasingly applied as a method to evaluate and treat donor lungs for transplantation. However, with the previous limited worldwide experience, no studies have been able to evaluate the impact of indication for EVLP on organ utilization rates and recipient outcomes after lung transplantation (LTx). We examined these outcomes in a large-cohort, single-center series of clinical EVLP cases. Methods All EVLP procedures performed at our institution between October 2008 and December 2017 were examined. The EVLPs were divided into 4 groups based on the indication for the procedure: group 1, high-risk brain death donors (HR-BDD); group 2, standard-risk donation after cardiac death (S-DCD); group 3, high-risk donation after cardiac death (HR-DCD); and group 4, logistics (LOGISTICS, the need for prolongation of preservation time or organ retrieval by a different transplantation team). Results During the study period, a total of 1106 lung transplants were performed in our institution. In this period, 372 EVLPs were performed, 255 (69%) of which were accepted for transplantation, resulting in 262 transplants. Utilization rates were 70% (140 of 198) for group 1, 82% (40 of 49) for group 2, 63% (69 of 109) for group 3, and 81% (13 of 16) for group 4 (P = .42, Fisher's exact test). Recipient age (P = .27) and medical diagnosis (P = .31) were not different across the 4 groups. Kaplan–Meier survival by EVLP indication group demonstrated no differences. Thirty-day mortality was 2.1% in group 1, 5% in group 2, 2.9% in group 3, and 0% in group 4 (P = .87, Fisher's exact test). The median days of mechanical ventilation, intensive care unit stay, and hospital stay were 2, 4, and 21 in group 1; 2, 3, and 21 in group 2; 3, 5, and 28 in group 3; and 2, 4, and 17 in group 4 (P = .29, .17, and .09, respectively, Kruskal–Wallis rank-sum test). Conclusions Clinical implementation of EVLP has allowed our program to expand the annual lung transplantation activity by 70% in this time period. It has improved confidence in the utilization of DCD lungs and BDD lungs, with an average 70% utilization of post-EVLP treated donor lungs with excellent outcomes, while addressing significant challenges in donor lung assessment and the logistics of “real-life” clinical lung transplantation.

Purpose Gastroesophageal reflux disease (GERD) is prevalent after lung transplantation (LTx) and has been associated with allograft injury and chronic lung allograft dysfunction (CLAD) presumably through chronic microaspiration of bile acids and other gastric contents with subsequent innate immune activation. Anti-reflux surgery is widely used at some centers to treat GERD in LTx; however, its role in inflammation or aspiration is unknown. Our objective was to examine the effects of early anti-GERD surgery in LTx recipients on markers of microaspiration and inflammation in the bronchoalveolar lavage (BAL). Methods We identified 18 LTx recipients who underwent Nissen fundoplication within 6 months post LTx and who had BAL samples obtained within 3 months pre- and post-Nissen. BAL levels of IL-6, IL-8, S100A8, S100A12, CCL2, IL-1α, IL-1β, CCL5, sRAGE, and IL-12p70 were measured by multiplex assay. BAL levels of 3 most prevalent bile acids−cholic acid (CA), taurocholic acid (TCA), and glycocholic acid (GCA)−were measured by mass spectrometry. Values pre- and post-Nissen were compared using the paired Wilcoxon signed-rank test . Generalized Estimating Equation models were used to adjust for concurrent rejection, infection, and their treatment. Results IL-8, S100A8, IL-1α, IL-1β, CCL5, IL-12p70, and TCA were significantly reduced post-Nissen compared to pre-Nissen after adjustment (Figure 1). Other cytokines and bile acids were not significantly different between pre- and post-Nissen. However, most bile acid levels were very low; some patients had elevated bile acid levels pre-Nissen, which did not remain elevated post-Nissen. Conclusion Early anti-GERD surgery in LTx recipients is associated with a significant decrease in lung inflammation and may also decrease markers of microaspiration. Further studies are warranted to determine whether anti-reflux surgery can prevent allograft injury and CLAD in patients at risk.

Purpose Gastroesophageal reflux disease (GERD) is thought to expose the lungs to refluxed gastric contents leading to injury and chronic lung allograft dysfunction (CLAD) in lung transplant recipients (LTRs). Prior small studies showed correlations between lung bile acids and CLAD/survival. The goal of this study was to determine whether bile acid levels in the bronchoalveolar lavage (BAL) correlate with GERD, concurrent lung allograft inflammation, or development of CLAD. Methods The cohort consisted of 25 GERD-positive and 50 GERD-negative adult sequential LTRs who underwent 24h pH/impedance reflux testing and BAL collection within 6 months post-transplant. Bile acids - taurocholic acid (TCA), glycocholic acid (GCA), and cholic acid (CA) - were measured using LC-MS/MS mass spectrometry; 10 cytokines were quantified using a multiplex assay. Levels were compared between groups using generalized linear models. Spearman correlations between biomarkers were assessed. The relationship between biomarkers and time to CLAD was examined using Cox proportional hazards models. Results TCA was significantly higher in BALs of GERD-positive patients compared to GERD-negative (0.4 vs. 0.1, p=0.04), with no significant differences in GCA, CA, or cytokines. Importantly, there were correlations (adjusted for 10 multiple comparisons) between TCA and GCA and inflammatory markers IL-6, CXCL8, CCL2, IL-1β, CCL5, IL-1α, and IL-12p70 (Fig 1A). Additionally, TCA (HR 1.37, p=0.04), IL-6 (HR 1.59, p=0.012), S100A8 (HR 1.39, p=0.033), and CCL2 (HR 1.41, p=0.03) at the time of the BAL were significantly associated with time to CLAD, independent of GERD status (Fig 1B shows additional Kaplan-Meier analysis). Conclusion BAL TCA correlates with GERD, concurrent innate immune inflammation, as well as risk of CLAD. We propose that BAL TCA levels, in conjunction with inflammatory marker measurements, may be useful in identifying LTRs with chronic aspiration of enteric contents that have a higher risk of subsequent adverse outcomes.

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.

Purpose We have applied cutting-edge mass cytometry (MC) technology to serial bronchoalveolar lavage (BAL) cells obtained from lung transplant recipients (LTRs) and have used it to compare the cellular composition of the BAL compartment in patients who remain stable or go on to develop lung allograft dysfunction (LAD), defined here as a drop, from any cause, in the forced expiratory volume in 1 second of 20% from the best post-transplant baseline value. Methods We have collected and cryopreserved BAL cells from 50 consecutive LTRs at their 3-, 6- and 9-month post-transplant surveillance bronchoscopies. Cryopreserved samples were then recovered and labeled with a panel of 37 heavy metal-tagged antibodies against surface markers and intracellular cytokines before MC. We applied t-distributed stochastic neighbor embedding (tSNE; Fig1 A) which assists with better visualization of multi-dimensinal analysis to identify both common and rare cell populations in the BAL. We further applied an algorithm called cluster identification, characterization and regression (CITRUS; Fig1 B) to allow unsupervised identification of differentially represented BAL cellular subsets between stable and LAD patients. Results Longitudinal analysis of the first 25 patients demonstrated different cell subsets and cytokine-producing cells, with substantial intra- and inter-patient variation. Our data identified an elevation in the frequency of CD4+ CD57+ PD-1+ cells separates LAD (18.41±4.89%) from stable patients (4.43±2.11%; Fig1 C). Emergence of CD57+ PD-1+ events among CD4+ T cells preceded LAD by up to several weeks (Fig1 D), suggesting that these cells may contribute to LAD development rather than simply correlating with it. Conclusion Our data suggest that emergence of CD4+ CD57+ PD-1+ T cells may predict and/or contribute to allograft dysfunction. Molecular and functional studies on this cell population are underway to obtain a better understanding of their role in lung transplant immunobiology.

Neutrophil extracellular traps (NETs) are detectable in donor ex vivo lung perfusate, and higher levels of NETs in perfusate are associated with worse recipient outcomes after transplanthttp://ow.ly/r4nM30nvZsK

Purpose The aim of this study was to evaluate the feasibility of messenger RNA (mRNA) and microRNA (miRNA) expression analysis by microarray using samples obtained by endobronchial ultrasonography-guided transbronchial needle aspiration (EBUS-TBNA). Description We isolated total RNA from 24 archived clinical EBUS-TBNA samples. The purified RNA that met the quality threshold was used for two different kinds of microarray analyses: whole transcript–based (WT) array for mRNA expression and miRNA expression array. Evaluation The RNA from 17 of the 24 samples (71%; 12 adenocarcinoma, 3 squamous cell carcinoma, 2 poorly differentiated carcinoma) met the quality threshold. After performing a one-way analysis of variance, we found genes with significant differences in histologic subtype ( p Conclusions Samples of EBUS-TBNA can be used for comprehensive WT and miRNA expression analysis using microarray technology.

Ex vivo lung perfusion (EVLP) allows for the evaluation and treatment of donor lungs before transplant. Different EVLP strategies have been described using either an open left atrium (LA) (pressure of 0 mm Hg) or closed LA (pressure of 5 mm Hg). We hypothesized that maintaining a physiologic positive LA pressure during EVLP is protective to the lung.Pig lungs were flushed with Perfadex, retrieved and stored at 4°C for 4 hours [short cold ischemic time (CIT), n = 10] or 18 hours (prolonged CIT, n = 8). Subsequently, lungs underwent normothermic EVLP for 12 hours using either an open or closed LA technique. A linear mixed effect model was used to compare functional parameters between the 2 groups.After short CIT, 12-hour EVLP could not be completed in 4 of 5 open atrium cases due to significant pulmonary edema. Lung injury was evident in this group after 7 hours of EVLP, demonstrating an increase in pulmonary vascular resistance (p0.001) and peak inspiratory pressure (p = 0.001), and a decrease in lung compliance (p0.001) and perfusate oxygenation (p = 0.04). In contrast, in the closed atrium group, all lungs completed 12 hours of EVLP with stable functional parameters. At the end of the experiment, the wet/dry ratio (p = 0.015) and lung edema score (p = 0.02) were significantly worse in the open LA group compared with the closed LA EVLP group. Similar findings were observed in the prolonged CIT group.The use of a closed atrial technique to create a controlled positive LA during EVLP leads to significantly less edema and superior lung physiology.

Purpose: Ex-Vivo Lung Perfusion (EVLP) is an emerging technology to reas- sess marginal donor lungs for transplantation. We have previously reported that cell death related perfusate bio-markers in EVLP are potentially predic- tive for primary graft dysfunction (PGD). Our objective was to investigate whether EVLP perfusate cell death markers could be used to predict post- transplant long term survival. Methods: Caspase-cleaved cytokeratin 18 (M30) and HMGB-1 levels in the perfusate of EVLP of marginal donor lungs were measured by quantitative enzyme linked immunosorbent assay, and were correlated with short and long term clinical outcome. Results: From 100 sequential clinical EVLP cases, 79 lungs were selected to be transplanted. Recipients that were bridged with pre-transplant extra- corporeal life support system were excluded from this analysis. 11 patients (PGD group) developed PGD 3 at any time within 72 h after transplant following bilateral lung transplant (BLTx). 34 patients (Control group) did not develop PGD grade 3 after BLTx. Levels of M30 were signifi- cantly higher in PGD group compared to Control group at 1 h (Control 53.9±15.9 vs. PGD 73.3± 24.9 U/L, p= 0.004) and 4 h (Control 72.4±40.0 vs. PGD 137.0±146.6 U/L; p= 0.04) of EVLP . The increase of HMGB-1 from 1 h to 4 h of EVLP (delta HMGB-1) in PGD group was signifi- cantly higher than Control (Control 7.2±16.8 vs. PGD 37.0±25.4 ng/ml; p= 0.0002). Uni variate Cox regression analysis revealed that higher M30 at 1 h (HR= 1 .03 (95%CI 1.004-1.058) per 1 U/L; p= 0 .025) and 4 h (HR= 1.009 (95%CI 1.002-1.016) per 1 U/L; p= 0.009), and higher delta HMGB-1 (HR= 1.033 (95%CI 1.006-1.061) per 1 ng/ml; p= 0.016) are sig- nificantly predictive for survival. After adjustment for PGD using bivariate cox regression analyses, M30 at 4h remained significant (p= 0.04) and the other markers were not statistically significant (M30 at 1 h; p= 0.13; delta HMGB-1; p= 0 .10). The significance of M30 at 4 h seen after adjustment for PGD may suggest that the marker provides additional predictive power for survival above that predicted by PGD status alone. Conclusion: We have demonstrated a correlation of cell death markers in the EVLP perfusate with PGD outcome and long term survival. Further research and validation is required to determine which of these biomarkers will be important in repertoire for real time predictive evaluation of the ex vivo perfused donor lung.

Purpose Normothermic ex vivo lung perfusion (EVLP) is a new method to assess and improve donor lungs prior to committing to transplant (Tx). We have increased the time a lung can stably undergo EVLP from 4 to 12 h in the lab. To discover donor lung biomarkers that can predict the likelihood of poor outcome, we used a genome-wide RNA expression study focused on phenotypes related to EVLP performance. Methods and Materials We collected brain death donor lung biopsies at end of cold ischemic time which were unable to stably complete extended (12 h) EVLP (n=4), which stably completed 12 h EVLP and passed clinical criteria for Tx, despite not undergoing Tx (n=4), and which stably completed 4 h EVLP, had Tx and produced good recipient outcome (n=12). Samples were analyzed on Affymetrix U133plus2 microarrays. Results Initial analysis suggested that lungs able to complete 12 h EVLP are similar to lungs with good outcome Tx. In contrast, lungs failing to complete 12 h EVLP form a distinct group. Further analysis treated the former 2 groups as a single group (control) for comparison with the latter group (fail). Using a false discovery rate (FDR) Figure 1 shows a hierarchically clustered heatmap of these genes, demonstrating the distinctiveness of the fail lungs. Of these genes, 119 had relative fold > 2x, 278 had no overlapping intensity values (AUC=1), and 29 were both > 2x and AUC=1. These may be highly predicative biomarkers of lung quality. Conclusions We find that extended EVLP may be a good indicator of Tx outcome. Differential expression between extreme EVLP performance phenotypes indicates large transcript differences and has provided several potential mRNA biomarkers of Tx lung failure or success.

Purpose Normothermic Ex Vivo Lung Perfusion (EVLP) has increased our clinical lung transplantation (LTx) activity by 20% by salvaging marginal lungs, with satisfactory clinical outcomes. Rewarming these lungs on EVLP from 4°C to 37°C normalizes metabolic activity, and studying the dynamic metabolomic profile may help us understand lung injury and predict the ultimate function of the transplanted lung. Metabolic profiling of EVLP perfusate may elucidate the metabolic changes of donor lungs on EVLP. Methods and Materials Donor lungs from 28 BDD and 22 DCD donors were assessed on EVLP for 4 hours prior to LTx. For each lung, EVLP perfusate was sampled at 1 hr and 4 hr perfusion, and global metabolomic profiling was performed with Mass Spectrometry. We examined the results over time and with respect to BDD vs. DCD status. Results Global metabolomics detected 312 metabolites. Statistical analysis with Random Forest (RF) classification showed a dramatic difference in metabolite profiles between EVLP 1hr and 4hr with a 96% accuracy in predicting whether a sample was taken at the start or end of EVLP perfusion. Interestingly, the metabolic profiles grouped by donor status were sufficiently different to be able to predict with RF whether a sample belongs to BDD or DCD donor, with an accuracy of 80% at 1hr and 78% at 4 hr EVLP. Conclusions As we search for opportunities to personalize the diagnosis and treatment of the donor lung, it is evident that EVLP perfusate is easily accessible and is imminently suitable for metabolomics. The changes in metabolites in donor lungs as EVLP progresses confirms that cellular metabolism is active and modified over time in normothermic EVLP. The metabolomic profile demonstrates significant differences between BDD and DCD donor lungs during EVLP as well as dynamic changes over time. Further work is required to determine which of these metabolic changes are important to guide diagnosis, treatment and outcome prediction in EVLP donor lungs used for LTx.

Purpose: Bile acids (BA) in bronchoalveolar lavage (BAL) have been recognized as a specific aspiration biomarker which predict the development of lung allograft dysfunction. The aims of this study were to determine the correlation of BA levels in bronchial wash (BW) and BAL, and to investigate whether BA levels in BW could be a useful biomarker to determine the suitability of donor lungs for transplantation (LTx). Methods: BA were measured in post-transplant patients’ 74 BW and 74 BAL samples, concurrently collected from January to May, 2013. BW fluid was collected from proximal airway, aspirated by bronchoscope following the injection of 20 ml saline. BA were measured with an enzymatic cycling assay and modified standard curve which provided an accurate detection level between 0.05 and 10 μM. BA assay and quantitative microbiological culture were performed in 33 donors’ BW samples (9 declined and 24 used for LTx), collected from January to May, 2013. Results: Recipients’ BA in BW were strongly correlated with those in BAL (Spearman r = 0.71, P < 0.0001). BA were undetectable in 14 BW and 14 BAL with concurrent collection. Among the remaining samples, 42 (70%) BW showed higher BA levels than BAL. BA levels in BW were significantly higher than those in BAL (median BA levels in BW: 0.05 μM , and BAL: 0.03 μM; P = 0.0002). Donors’ BA levels were significantly higher in the declined cases, compared to the transplant cases (median BA levels: declined: 0.73 μM, and transplant: 0.125 μM; P = 0.0246). No significant difference was found in BA levels between bacterial cultured cases (n= 13) and not-cultured cases (n= 11). Infiltration of neutrophils within alveolar space or interstitial inflammation was observed histologically in the declined cases with high BA. Conclusion: A standardized technique of BA assay seems to be a sensitive method to detect an injurious gastro-esophageal reflux in both recipients and donors. BA levels in BW can be a useful biomarker to help determining suitability of donor lungs for transplantation.

Purpose Chronic lung allograft dysfunction (CLAD) remains a major cause of mortality and morbidity after transplantation. We have noted restrictive allograft syndrome (RAS) as a novel subtype of CLAD. We aimed to further characterize human CLAD subtypes by profiling damage-associated molecular pattern molecules in bronchoalveolar lavage fluid (BALF). Methods and Materials 17 consecutively identified specimens from patients with CLAD (5 RAS, 12 bronchiolitis obliterans syndrome (BOS) ) and 12 controls from patients with no CLAD were included. All the BALF specimens from CLAD cases were taken after their clinical onset. CLAD and its subtypes were defined based on pulmonary function test results. S100A8, S100A9, S100A12, S100P and high mobility group box-1 (HMGB-1) expression were measured in BALF by enzyme-linked immunosorbent assay. Results All of S100A8, S100A9, S100A12 and S100P were upregulated in RAS compared to controls (p figure 1 ] Conclusions In CLAD patients, we have noted a distinct difference in the expression of S100 family proteins in BALF. It appears that S100A9 is associated with RAS and not BOS. Hopefully, further characterization of molecular pathways involved in the development of CLAD subtypes will help to develop more accurate diagnostics and specifically directed therapies.

Purpose To study the impact of normothermic ex vivo lung perfusion (EVLP) on cytokines, chemokines and growth factors and their correlation with graft performance either during perfusion or after implantation. Methods and Materials High-risk brain death donors and donors after cardiac death underwent 4-6 hours EVLP with intent to transplant. Using a multiplex magnetic bead array assay we evaluated multiple analytes in perfusate samples. Donor lungs were divided into three grous: I. Control: bilateral transplantation with good early outcome (absence of PGD grade 3); II. PGD3: bilateral lung transplantation with PGD grade 3 anytime within 72 hours; III. Declined: lungs rejected following EVLP. Single-lung transplants and patients bridged to LTx with extracorporeal life support were excluded. Results Of 36 cases included in this study, 17 were in group I, 6 in group II and 13 in group III. Of a total of 51 analytes, 30 were measurable in perfusates. While markers from samples at 1 hour had higher capacity for discrimination between groups I and III, at the end of EVLP there were more analytes significantly higher in group II compared to I. The best markers to differentiate declined lungs from controls lungs were IL-1ra (p=0.005, AUC 0.80) and SCGF-b (p Figure ) Conclusions Perfusate protein expression can differentiate lungs with good outcome from lungs with a poor outcome (PGD 3) after transplantation as well as lungs that were declined during EVLP.

Purpose Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by vascular obstruction and right ventricular failure. Currently there is no systemic study to disclose abnormal metabolic pathways for PAH. We hypothesized that analysis of the global metabolome and related gene expression in PAH will disclose multiple metabolic changes in PAH disease. The goal of this study was to characterize biochemical and gene profiles observed in lung tissue samples collected from PAH patients after lung transplantation. Methods and Materials Global biochemical genetic profiles were determined and compared across the normal (n=8) and PAH (n=8) cohorts. The normal and PAH lung samples from lung Tx were analyzed on the GC/MS and LC/MS/MS platforms. Welch’s two-sample t-test was used to identify biochemical between experimental groups. mRNA from normal and native PAH lung were isolated and Affymetrix HGU133 Plus 2 arrays were run for each array of independently patient lung sample ( P Results The present dataset comprises a total of 376 compounds of identified biochemical. The PAH tissue showed 93 compounds exhibited altered metabolites (p ≤ 0.05) and were marked by changes in arginine and glucose metabolism, disruptions in mitochondrial oxidation and the TCA cycle, and alterations in bile acids, heme metabolism, oxidative stress, and inflammation compared to healthy subjects. There are significant expression changes of the genes encoding to the key enzymes of the entire metabolisms in PAH. Conclusions The altered PAH metabolisms are correlated with metabolic enzyme related gene expression that lead to the aberrant production or consumption of glucose, amino acids, nucleotides, and lipids. The abnormal regulation of widespread metabolic pathways in PAH may be an important part of PAH pathogenesis. Designing a new strategy for therapeutic targets on the multiple metabolic pathways may open new revenue of treatment of PAH in the future.