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Long-term outcomes after lung transplantation remain inferior to those of other solid organ groups. The significance of eosinophils detected on transbronchial biopsies (TBBx) after lung transplantation and their relationship to long-term outcomes remain unknown. A retrospective single-center cohort study was performed of patients transplanted between January 01, 2001, and July 31, 2018, who had at least 1 TBBx with evaluable parenchymal tissue. Multivariable Cox proportional hazard models were used to assess the associations between eosinophil detection and: all-cause mortality and Chronic Lung Allograft Dysfunction (CLAD). 8887 TBBx reports from 1440 patients were reviewed for the mention of eosinophils in the pathology report. 112 (7.8%) patients were identified with eosinophils on at least one TBBx. The median (95% CI) survival time for all patients was 8.28 (7.32-9.31) years. Multivariable analysis, adjusted for clinical variables known to affect post-transplant outcomes, showed that the detection of eosinophils was independently associated with an increased risk of death (HR 1.51, 95% CI 1.24-1.85, p < 0.01) and CLAD (HR 1.35, 95% CI 1.07-1.70, P = 0.01). Eosinophils detected in TBBx are associated with an increased risk of CLAD and death. There may be benefit in specifically reporting the presence of eosinophils in TBBx reports and incorporating their presence in clinical decision-making.

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.

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.

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.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.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.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 -β [P0.001, AUC (area under the curve) = 0.86] at 1 hour. The best markers to differentiate PGD3 cases from controls were interleukin-8 (P0.001, AUC = 0.93) and growth-regulated oncogene-α (P = 0.001, AUC = 0.89) at 4 hours of EVLP.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.

We and others have demonstrated that acellular normothermic ex vivo lung perfusion of high-risk donor lungs can result in posttransplant outcomes equivalent to that of contemporaneous lung transplantation using standard donor lungs. However, the mechanism of this effect remains unclear. Given the restoration of cellular metabolic activity during normothermic perfusion, one possibility is that of lung healing via natural innate recovery mechanisms. We explored this by examining the gene expression changes occurring in human lungs during ex vivo lung perfusion. Human lungs clinically rejected for transplantation were perfused for 12 hours of EVLP with biopsies taken at the start, at 1 hour, at 3 hours, and then every 3 hours thereafter to 12 hours. Temporal changes were identified in 2585 genes using the Short Time-series Expression Miner and used for pathway analysis. Despite increases in endothelial markers of inflammation, circulating leukocyte cell-specific gene expression fell over 12 hours of ex vivo lung perfusion (EVLP), suggesting an interrupted inflammation response secondary to washout of circulating leukocytes. Analysis of these gene changes suggests lung recovery follows specific stages: cellular death, cellular preservation, cellular reorganization, and cellular invasion. EVLP may improve posttransplant lung function by washout of leukocytes and facilitating innate mechanisms of repair.

Objective: To identify potential biomarkers during ex vivo lung perfusion (EVLP) using metabolomics approach. Summary background data: EVLP increases the number of usable donor lungs for lung transplantation (LTx) by physiologic assessment of explanted marginal lungs. The underlying paradigm of EVLP is the normothermic perfusion of cadaveric lungs previously flushed and stored in hypothermic preservation fluid, which allows the resumption of active cellular metabolism and respiratory function. Metabolomics of EVLP perfusate may identify metabolic profiles of donor lungs associated with early LTx outcomes. Methods: EVLP perfusate taken at 1and 4 hperfusion were collected from 50 clinical EVLP cases, and submitted to untargeted metabolic profiling with mass spectrometry. The findings were correlated with early LTx outcomes. Results: Following EVLP, 7 cases were declined for LTx. In the remaining transplanted cases, 9 cases developed primary graft dysfunction (PGD) 3. For the metabolic profile at EVLP-1h, a logistic regression model based on palmitoyl-sphingomyelin, 5-aminovalerate, and decanoylcarnitine yielded a receiver operating characteristic (ROC) curve with an area under the curve (AUC) of 0.987 in differentiating PGD 3 from Non-PGD 3 outcomes. For the metabolic profile at EVLP-4h, a logistic regression model based on N2-methylguanosine, 5-aminovalerate, oleamide, and decanoylcarnitine yielded a ROC curve with AUC 0.985 in differentiating PGD 3 from non-PGD 3 outcomes. Conclusions: Metabolomics of EVLP perfusate revealed a small panel of metabolites highly correlated with early LTx outcomes, and may be potential biomarkers that can improve selection of marginal lungs on EVLP. Further validation studies are needed to confirm these findings.

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) enables assessment of marginal donor lungs for transplantation. We aimed to discover biomarkers in EVLP perfusate that could predict development of primary graft dysfunction (PGD). From September 2008 to August 2013, 100 clinical EVLPs were performed. Eleven patients developed PGD grade 3 within 72 h after transplant. The non-PGD group consisted of 34 patients without PGD grade 3. Nonbilateral lung transplants or transplant after extracorporeal life support were excluded from analyses. Soluble intercellular adhesion molecule 1 (sICAM-1), soluble VCAM-1 (sVCAM-1), and soluble E selectin (sE-selectin) levels, as markers of endothelial activation, were measured in the perfusate of EVLP by enzyme-linked immunosorbent assay and were correlated with clinical outcome. Levels of sICAM-1 at 1 h and sVCAM-1 at 1 and 4 h were significantly higher in the PGD group compared with the non-PGD group. The sE selectin levels were not statistically different between the study groups. Higher levels of sVCAM-1 at 1 and 4 h were statistically significantly associated with PGD either alone or after adjustment for other PGD risk factors. These adhesion molecules may help identify donor lungs at higher risk of PGD during EVLP.

Microchip sensors enable rapid, molecular-level profiling of donated lungs for transplant assessment., Biomarker profiling is being rapidly incorporated in many areas of modern medical practice to improve the precision of clinical decision-making. This potential improvement, however, has not been transferred to the practice of organ assessment and transplantation because previously developed gene-profiling techniques require an extended period of time to perform, making them unsuitable in the time-sensitive organ assessment process. We sought to develop a novel class of chip-based sensors that would enable rapid analysis of tissue levels of preimplantation mRNA markers that correlate with the development of primary graft dysfunction (PGD) in recipients after transplant. Using fractal circuit sensors (FraCS), three-dimensional metal structures with large surface areas, we were able to rapidly (

Although multiple gene and protein expression have been extensively profiled in human pulmonary arterial hypertension (PAH), the mechanism for the development and progression of pulmonary hypertension remains elusive. Analysis of the global metabolomic heterogeneity within the pulmonary vascular system leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways in the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to increased ATP synthesis for the vascular remodeling process in severe pulmonary hypertension. These identified metabolites may serve as potential biomarkers for the diagnosis of PAH. By profiling metabolomic alterations of the PAH lung, we reveal new pathogenic mechanisms of PAH, opening an avenue of exploration for therapeutics that target metabolic pathway alterations in the progression of PAH.

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.