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Objective: Diagnosing lung injury is a challenge in lung transplantation. It has been unclear if a single biopsy specimen is truly representative of the entire organ. Our objective was to investigate lung inflammatory biomarkers using human lung tissue biopsies and ex vivo lung perfusion perfusate., Methods: Eight human donor lungs declined for transplantation were air inflated, flash frozen, and partitioned from apex to base. Biopsies were then sampled throughout the lung. Perfusate was sampled from 4 lung lobes in 8 additional donor lungs subjected to ex vivo lung perfusion. The levels of interleukin-6, interleukin-8, interleukin-10, and interleukin-1β were measured using quantitative reverse transcription polymerase chain reaction from lung biopsies and enzyme-linked immunosorbent assay from ex vivo lung perfusion perfusate., Results: The median intra-biopsy equal-variance P value was .50 for messenger RNA biomarkers in tissue biopsies. The median intra-biopsy coefficient of variance was 18%. In donors with no apparent focal injuries, the biopsies in each donor showed no difference in various lung slices, with a coefficient of variance of 20%. The exception was biopsies from the lingula and injured focal areas that demonstrated larger differences. Cytokines in ex vivo lung perfusion perfusate showed minimal variation among different lobes (coefficient of variance = 4.9%)., Conclusions: Cytokine gene expression in lung biopsies was consistent, and the biopsy analysis reflects the whole lung, except when specimens were collected from the lingula or an area of focal injury. Ex vivo lung perfusion perfusate also provides a representative measurement of lung inflammation from the draining lobe. These results will reassure clinicians that a lung biopsy or an ex vivo lung perfusion perfusate sample can be used to inform donor lung selection., (Copyright © 2023. Published by Elsevier Inc.)

Background: Aspiration is a relative contraindication to accepting donor lungs for transplant and is currently assessed by visual inspection of the airways via bronchoscopy. However, this method is limited as it does not assess for microaspiration. Bile acids measured in large airway bronchial wash (LABW) samples have been shown to be a marker of aspiration in lung transplant recipients. Herein, we investigate the utility of measuring total bile acids (TBA) in donor LABW to predict performance of donor lungs and recipient outcomes., Methods: TBA was measured in 605 consecutive lung donors at the Toronto Lung Transplant Program. TBA levels were compared in donor lungs deemed unsuitable for transplant, requiring further assessment on ex vivo lung perfusion (EVLP), and those suitable for direct transplantation using Mann-Whitney-U tests. Relationships between LABW TBA concentrations and recipient outcomes were evaluated using multivariable Cox-PH models and log-rank analysis., Results: Donor TBA was highest in lungs deemed unsuitable for transplant and correlated with clinical assessment of aspiration. LABW TBA concentration correlated with calcium, decreased pH, and increased pro-inflammatory mediators in EVLP perfusate. TBA cut-off of 1245 nM was able to differentiate donor lungs directly declined from those suitable for direct transplantation with a 91% specificity (AUROC: 73%). High donor TBA status was associated with the increased rate of primary graft dysfunction, longer time to extubation, and shorter time to chronic lung allograft dysfunction., Conclusions: In a large retrospective cohort, we observed that donor LABW TBA was associated with suitability of donor lungs for transplant, performance of the organ on EVLP, and adverse recipient outcomes., (Copyright © 2023 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Procuring a good quality transbronchial-biopsy sample is essential for diagnosing acute cellular rejection after lung transplantation (LT). Insufficient transbronchial-biopsy samples are graded "AX." We hypothesized that AX samples may be associated with a higher risk for chronic lung allograft dysfunction (CLAD) or death/retransplant, through a potential anatomic or physiologic underlying pulmonary process or because of undiagnosed acute cellular rejection episodes., Methods: We conducted a single-center, retrospective, cohort study drawn from all consecutive adult, first, bilateral LT between 1999 and 2015. We reviewed all biopsies obtained within the first year posttransplant and compared outcomes of patients with ≥1 AX to patients with no AX. Association of any AX or percent AX with time to CLAD or death/retransplant was assessed using Cox Proportional Hazards models., Results: The cohort consisted of 809 patients with a median of 6 (interquartile range 5-6) biopsies and 16.7% (interquartile range 0-25) AX samples within the first year posttransplant. Four hundred thirty-nine (54.3%) subjects had ≥1 AX sample obtained within the time period. Median time to CLAD or death/retransplant, from 1 year posttransplant, was 761 (320, 1587) and 1200 (662, 2308) days, respectively. In the multivariable analysis, there was no difference in risk for CLAD (hazard ratio = 1.05, 95% confidence interval, 0.87-1.28, P = 0.60), or death/retransplant (hazard ratio = 1.14, 95% confidence interval, 0.92-1.42, P = 0.24) between patients with ≥1 AX biopsy versus none. Among subjects with ≥1 AX, having >50% AX biopsies was not associated with outcome., Conclusions: This is the first study to demonstrate that AX biopsies are not associated with an increased risk of CLAD or death/retransplant after LT and may not require to repeat the biopsy., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation (LTx). Early detection or prediction of CLAD can lead to changes in patient management that, in turn, may improve prognosis. The purpose of this study was to investigate the utility of quantitative computed tomography (CT) lung density analysis in early prediction of CLAD., Methods: This retrospective cohort was drawn from all consecutive adult, first LTxs performed between 2006 and 2011. Post-transplant monitoring included scheduled surveillance bronchoscopies with concurrent pulmonary-functions tests and low-dose chest CT. Quantitative density metrics (QDM) derived from CT scans obtained at the time of 10%-19% decline in forced expiratory volume in 1 second (FEV1) were evaluated: 114 bilateral LTx recipients (66 with CLAD and 48 stable) and 23 single LTx recipients (11 with CLAD, 12 stable) were included in the analysis., Results: In both single and double LTx, at the time of 10%-19% drop in FEV1 from baseline, the QDM was higher in patients who developed CLAD within 3 years compared with those patients who remained stable for at least 3.5 years. The area under the receiver operating characteristic curve (AUC) was 0.89 for predicting CLAD in single LTx and 0.63 in bilateral LTx. A multipredictor AUC accounting for FEV1, QDM, presence of consolidation, and ground glass opacities increased the AUC to 0.74 in double LTx., Conclusions: QDM derived from a CT histogram at the time of early drop in FEV1 may allow prediction of CLAD in patients after single or double LTx.

Background: The application of mesenchymal stromal cell (MSC)-based therapy during ex vivo lung perfusion (EVLP) could repair injured donor lungs before transplantation. The aim of this study was to determine the efficacy of MSC therapy performed during EVLP on ischemia-reperfusion injury using a pig lung transplant model., Methods: Following 24 hours of cold storage, pig lungs were randomly assigned to 2 groups (n = 6 each), the control group without MSC vs the MSC group, where 5 × 10 6 cells/kg MSCs were delivered through the pulmonary artery during EVLP. After 12 hours of EVLP, followed by a 1-hour second cold preservation period, the left lung was transplanted and reperfused for 4 hours., Results: EVLP perfusate hepatocyte growth factor (HGF) level at 12 hours was significantly elevated in the MSC group compared with the control and was associated with a significant decrease in cell death markers, cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, in the MSC group. The MSC group showed significantly lower interleukin (IL)-18 and interferon gamma levels and a significantly higher IL-4 level in lung tissue at 12 hours of EVLP than the control group. After transplantation, the MSC group showed a significant increase in lung tissue HGF level compared with the control group, associated with a significantly reduced lung tissue wet-to-dry weight ratio. Lung tissue tumor necrosis factor-α level and pathological acute lung injury score were significantly lower in the MSC group than the control group., Conclusions: The administration of MSCs ameliorated ischemic injury in donor lungs during EVLP and attenuated the subsequent ischemia-reperfusion injury after transplantation., (Copyright © 2019. Published by Elsevier Inc.)

Background: Chronic pulmonary graft-versus-host disease (cpGVHD) after hematopoietic cell transplant (HCT) manifests as progressive airway and parenchymal lung fibrosis. On the basis of our prior data, mice that undergo allogeneic HCT with Tbet-knockout donors (AlloTbet) have increased lung Th17 cells and IL-17A and develop fibrosis resembling human cpGVHD. The role of IL-17A in posttransplant pulmonary fibrosis remains incompletely understood. We hypothesized that IL-17A is necessary for development of murine cpGVHD in this model., Methods: AlloTbet mice received weekly intraperitoneal anti-IL-17A or IgG (200 μg/mouse) starting 2 weeks post-HCT and were sacrificed after week 5. Histologic airway and parenchymal fibrosis were semiquantitatively graded in a blinded fashion. Lung cells and proteins were measured by flow cytometry, ELISA, and multicytokine assays., Results: Anti-IL-17A modestly decreased airway and parenchymal lung fibrosis, along with a striking reduction in pulmonary neutrophilia, IL-6, MIP-1α, MIP-1β, CXCL1, and CXCL5 in AlloTbet mice. Additionally, anti-IL-17A decreased CCL2, inflammatory monocytes and macrophages, and Th17 cells., Conclusions: In the setting of murine AlloHCT with Tbet donors, IL-17A blockade decreases fibrotic features of cpGVHD. This may be mediated by the observed reduction in neutrophils or specific lung monocyte and macrophage populations or alternatively via a direct effect on fibroblasts. Collectively, our results further suggest that anti-IL-17A strategies could prove useful in preventing alloimmune-driven fibrotic lung diseases.

Background: It is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data., Results: Using three mock communities across a range of densities ranging from 8E+ 02 to 8E+ 09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+ 05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+ 04 and 8E+ 06 16S copies/ml. For the lowest densities, below 8E+ 03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples., Conclusion: Relative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+ 04 16S copies/ml.

Background: Bronchoalveolar lavage (BAL) has proven to be very useful to monitor the lung allograft after transplantation. In addition to allowing detection of infections, multiple BAL analytes have been proposed as potential biomarkers of lung allograft rejection or dysfunction. However, BAL collection is not well standardized and differences in BAL collection represent an important source of variation. We hypothesized that there are systematic differences between sequential BALs that are relevant to BAL analysis., Methods: As part of 126 consecutive bronchoscopies in lung transplant recipients, two sequential BALs (BAL1 and BAL2) were performed in one location during each bronchoscopy by instilling and suctioning 50 ml of normal saline twice into separate containers. Cell concentration, viability and differentials, Surfactant Protein-D (SP-D), Club Cell Secretory Protein (CCSP), and levels of CXCL10, IL-10, CCL2, CCL5, VEGF-C, RAGE, CXCL9, CXCL1, IL-17A, IL-21, PDGF, and GCSF were compared between BAL1 and BAL2., Results: Total cell concentration did not differ between BAL1 and BAL2; however, compared to BAL2, BAL1 had more dead cells, epithelial cells, neutrophils, and higher concentrations of airway epithelium-derived CCSP and inflammatory markers. BAL2 had a higher concentration of SP-D compared to BAL1., Conclusion: In this study performed in lung transplant recipients, we show that sequential BALs represent different lung compartments and have distinct compositions. BAL1 represents the airway compartment with more epithelial cells, neutrophils, and epithelium-derived CCSP. Conversely, BAL2 samples preferentially the distal bronchoalveolar space with greater cell viability and higher SP-D. Our findings illustrate how the method of BAL collection can influence analyte concentrations and further emphasize the need for a standardized approach in translational research involving BAL samples.

Background: Ischemia-reperfusion injury related to lung transplantation is a major contributor to early postoperative morbidity and mortality. We hypothesized that donation after cardiac death donor lungs experience warm ischemic conditions that activate different injurious mechanisms compared with donor lungs that undergo prolonged cold ischemic conditions., Methods: Rat donor lungs were preserved under different cold ischemic times (CIT) (12 hours or 18 hours), or under warm ischemia time (WIT) (3 hours) after cardiac death, followed by single left lung transplantation. Lung function was analyzed during the 2-hour reperfusion period. Microscopic injury, cell death, energy status, and inflammatory responses were assessed., Results: Pulmonary oxygenation function was significantly worse in both 18hCIT and WIT groups, accompanied by higher peak airway pressure, acute lung injury scores, and expression of cell death markers compared with the 12hCIT control group. In lung tissue, reperfusion induced increased expression levels of interleukin (IL)-1α, IL-1β, IL-6, and chemokines CCL2, CCL3, CXCL1, and CXCL2 in CIT lungs. Notably, these changes were much lower in the WIT group. Additionally, plasma levels of IL-6, IL-18, CCL2, and vascular endothelial growth factor were significantly higher, and adenosine triphosphate levels were significantly reduced in warm versus cold ischemic lungs., Conclusions: Compared with 12hCIT, posttransplant pathophysiology deteriorated similarly in both 18hCIT and WIT groups. However, tissue adenosine triphosphate levels and inflammatory profiling differed between warm versus cold ischemic donor lungs. These differences should be carefully considered when developing specific therapeutic strategies to reduce ischemia-reperfusion injury in lung transplantation.

Despite significant progress in the field of transplant immunology, acute cellular rejection (ACR) remains a very frequent complication after lung transplantation (LTx), with almost 30% of LTx recipients experiencing at least one episode of treated ACR during the first year of follow-up. Most episodes respond to the first-line immunosuppressive treatment and are rarely a direct cause of death. However, the association of ACR with later adverse outcomes, such as chronic lung allograft dysfunction, bronchial stricture, and infectious complications associated with the intensification of immunosuppression, negatively impacts long-term survival. The burden imposed on patients and health-care resources is even higher in cases of refractory or recurrent ACR, which accelerates lung function decline. Although important laboratory and clinical research conducted over the last two decades has improved our understanding of the mechanisms underlying ACR, there are still many uncertainties about the risk factors for ACR, the optimal monitoring strategies, and the prediction of long-term outcomes. These knowledge gaps contribute to the large variability in clinical practice among LTx centers, which renders multicenter studies of ACR challenging. In this review, we summarize current evidence on the epidemiology, pathogenesis, and risk factors of ACR. We describe diagnostic and therapeutic approaches that are currently used in the clinical practice and also review promising diagnostic tools that are under investigation. Associations between ACR and other adverse outcomes of LTx are examined. Finally, within each topic of discussion, we highlight the main areas of controversy and opportunities for future research., Competing Interests: The authors have no conflicts of interest to disclose., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Several mouse lung transplantation (Tx) models have been proposed for the study of chronic airway fibrosis (CAF), the most prevalent complication seen in human lung transplant recipients, termed chronic lung allograft dysfunction. Alternatively, it has been called for to establish an experimental animal model for restrictive allograft syndrome, another phenotype of chronic lung allograft dysfunction. However, these mouse transplant models exhibit significant heterogeneity in consistency and reproducibility. We therefore aimed at reevaluating current available models., Methods: Four different Tx combinations were used that manifest CAF: 2 minor antigen-mismatched Tx combinations (MINOR, donor: C57BL/10, recipient: C57BL/6J); or MINOR-N using recipient C57BL/6N, major histocompatibility antigen-mismatched immunosuppressed Tx (MAJOR, donor: BALB/c, recipient: C57BL/6J), and syngeneic Tx (donor and recipient: C57BL/6J) as control. The recipients were harvested and analyzed at week 8. Oxygenation, histology, reverse transcription polymerase chain reaction, and magnetic resonance imaging were performed to analyze outcome of those models., Results: The most prominent manifestation of CAF, thickest subepithelial fibrotic changes, worst oxygenation, and the most severe acute rejection were detected in the MAJOR group compared with all other (P < 0.05). Gene expressions of TNF-α and TGF-β1 were higher, and IL-10 was lower in the MAJOR group. Immunohistochemistry found pleuroparenchymal fibrotic change in both the MAJOR and MINOR-J groups., Conclusions: We propose the major mismatch model under mild immunosuppression as the most suitable model for studying posttransplant CAF, and both the major and minor mismatch models for the restrictive phenotype.

Rationale: Despite increasing evidence about the role of donor-specific human leukocyte antigen (HLA) antibodies in transplant outcomes, the incidence and impact of de novo donor-specific antibodies (dnDSA) after lung transplantation remains unclear., Objectives: To describe the incidence, characteristics, and impact of dnDSA after lung transplantation., Methods: We investigated a single-center cohort of 340 lung transplant recipients undergoing transplant during 2008 to 2011. All patients underwent HLA-antibody testing quarterly pretransplant and at regular intervals over the first 24 months after transplant. The patients received modified immunosuppression depending on their pretransplant sensitization status. Risk factors for dnDSA development, as well as the associations of dnDSA with patient survival and chronic lung allograft dysfunction (CLAD), were determined using multivariable analysis., Measurements and Main Results: The cumulative incidence of dnDSA was 47% at a median of 86 days (range, 44-185 d) after lung transplantation. Seventy-six percent of recipients with dnDSA had DQ-DSA. Male sex and the use of ex vivo lung perfusion were associated with an increased risk of dnDSA, whereas increased HLA-DQB1 matching was protective. DQ-dnDSA preceded or coincided with the diagnosis of CLAD in all cases. Developing dnDSA (vs. no dnDSA) was associated with a twofold increased risk of CLAD (hazard ratio, 2.04; 95% confidence interval, 1.13-3.69). This association appeared to be driven by the development of DQ-dnDSA., Conclusions: dnDSA are common after lung transplantation, with the majority being DQ DSA. DQ-dnDSA are associated with an increased risk of CLAD. Strategies to prevent or treat DQ-dnDSA may improve outcomes for lung transplant recipients.

Ex vivo lung perfusion (EVLP) is a platform to treat infected donor lungs with antibiotic therapy before lung transplantation. Human donor lungs that were rejected for transplantation because of clinical concern regarding infection were randomly assigned to two groups. In the antibiotic group (n = 8), lungs underwent EVLP for 12 h with high-dose antibiotics (ciprofloxacin 400 mg or azithromycin 500 mg, vancomycin 15 mg/kg, and meropenem 2 g). In the control group (n = 7), lungs underwent EVLP for 12 h without antibiotics. A quantitative decrease in bacterial counts in bronchoalveolar lavage (BAL) was found in all antibiotic-treated cases but in only two control cases. Perfusate endotoxin levels at 12 h were significantly lower in the antibiotic group compared with the control group. EVLP with broad-spectrum antibiotic therapy significantly improved pulmonary oxygenation and compliance and reduced pulmonary vascular resistance. Perfusate endotoxin levels at 12 h were strongly correlated with levels of perfusates tumor necrosis factor α, IL-1β and macrophage inflammatory proteins 1α and 1β at 12 h. In conclusion, EVLP treatment of infected donor lungs with broad-spectrum antibiotics significantly reduced BAL bacterial counts and endotoxin levels and improved donor lung function., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Respiratory viral infections cause significant morbidity and increase the risk for chronic pulmonary graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). Our overall hypothesis is that local innate immune activation potentiates adaptive alloimmunity. In this study, we hypothesized that a viral pathogen-associated molecular pattern (PAMP) alone can potentiate pulmonary GVHD after allogeneic HCT. We, therefore, examined the effect of pulmonary exposure to polyinosinic:polycytidylic acid (poly I:C), a viral mimetic that activates innate immunity, in an established murine HCT model. Poly I:C-induced a marked pulmonary T cell response in allogeneic HCT mice as compared to syngeneic HCT, with increased CD4+ cells in the lung fluid and tissue. This lymphocytic inflammation persisted at 2 weeks post poly I:C exposure in allogeneic mice and was associated with CD3+ cell infiltration into the bronchiolar epithelium and features of epithelial injury. In vitro, poly I:C enhanced allospecific proliferation in a mixed lymphocyte reaction. In vivo, poly I:C exposure was associated with an early increase in pulmonary monocyte recruitment and activation as well as a decrease in CD4+FOXP3+ regulatory T cells in allogeneic mice as compared to syngeneic. In contrast, intrapulmonary poly I:C did not alter the extent of systemic GVHD in either syngeneic or allogeneic mice. Collectively, our results suggest that local activation of pulmonary innate immunity by a viral molecular pattern represents a novel pathway that contributes to pulmonary GVHD after allogeneic HCT, through a mechanism that includes increased recruitment and maturation of intrapulmonary monocytes., (Copyright © 2010 Elsevier B.V. All rights reserved.)