T. Martinu

Ex Vivo Perfusion Treatment of Infection in Human Donor Lungs

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.

Successful Lung Transplantation from Hepatitis C Positive Donor to Seronegative Recipient

Lung transplantation using RNA+ hepatitis C (HCV+) donors to seronegative recipients is not currently performed due to the very high risk of transmission. Previous reports have shown poor survival when this practice was applied. The emergence of new direct‐acting antiviral drugs (DAA) suggests a high chance of sustained virologic response in immunocompetent patients. We report here successful transplantation of lungs from HCV+ donor to HCV− recipient. The recipient was an HCV− patient with chronic lung allograft dysfunction. Viral transmission occurred early posttransplant but excellent clinical outcomes were observed including elimination of HCV after 12 weeks of treatment using DAAs.

Halofuginone treatment reduces interleukin-17A and ameliorates features of chronic lung allograft dysfunction in a mouse orthotopic lung transplant model.

BACKGROUND Increasing evidence suggests that interleukin (IL)-17A plays an important role in chronic lung allograft dysfunction (CLAD), characterized by airway and lung parenchymal fibrosis, after lung transplantation. Halofuginone is a plant derivative that has been shown to inhibit Th17 differentiation. The purpose of this study was to examine the effect of halofuginone on CLAD development using a minor alloantigen‒mismatched mouse orthotopic lung transplant model. METHODS C57BL/6 recipient mice received an orthotopic left lung transplant from C57BL/10 donors, mismatched for minor antigens. Lung transplant recipients received daily intraperitoneal injections of 2.5 μg halofuginone or vehicle alone. Lung grafts were assessed on Days 7, 14, and 28 post-transplant. RESULTS Compared with control mice, on Day 28 post-transplant, lung grafts of mice treated with halofuginone showed a significant reduction in the percentage of obliterated airways (6.8 ± 4.7% vs 52.5 ± 13.8%, p < 0.01), as well as significantly reduced parenchymal fibrosis (5.5 ± 2.3% vs 35.9 ± 10.9%, p < 0.05). Immunofluorescent staining for IL-17A demonstrated a decreased number and frequency of IL-17A‒positive cells in halofuginone-treated lung grafts on Day 28, as compared with controls. Halofuginone treatment also decreased IL-17A and IL-22 transcripts at Day 14, transforming growth factor-β1 and matrix metalloproteinase-2 transcripts at Days 14 and 28. CONCLUSION The beneficial effect of halofuginone on development of airway and lung parenchymal fibrosis in the mouse lung transplant model highlights the important role of IL-17A in CLAD and merits further pre-clinical and clinical studies.

Lung Lavage and Surfactant Replacement During Ex Vivo Lung Perfusion for Treatment of Gastric Acid Aspiration–Induced Donor Lung Injury

BACKGROUND Ex vivo lung perfusion (EVLP) provides opportunities to treat injured donor lungs before transplantation. We investigated whether lung lavage, to eliminate inflammatory inhibitory components, followed by exogenous surfactant replacement, could aid lung recovery and improve post-transplant lung function after gastric aspiration injury. METHODS Gastric acid aspiration was induced in donor pigs, which were ventilated for 6 hours to develop lung injury. After retrieval and 10 hours of cold preservation, EVLP was performed for 6 hours. The lungs were randomly divided into 4 groups (n = 5, each): (1) no treatment (control), (2) lung lavage, (3) surfactant administration, and (4) lung lavage, followed by surfactant administration. After another 2-hour period of cold preservation, the left lung was transplanted and reperfused for 4 hours. RESULTS Physiologic lung function significantly improved after surfactant administration during EVLP. The EVLP perfusate from the lavage + surfactant group showed significantly lower levels of interleukin (IL)-1β, IL-6, IL-8, and secretory phospholipase A2. Total phosphatidylcholine was increased, and minimum surface tension was recovered to normal levels (≤5 mN/m) in the bronchioalveolar fluid after surfactant administration. Lysophosphatidylcholine in bronchioalveolar fluid was significantly lower in the lavage + surfactant group than in the surfactant group. Post-transplant lung function was significantly better in the lavage + surfactant group compared with all other groups. CONCLUSIONS Lung lavage, followed by surfactant replacement during EVLP, reduced inflammatory mediators and prevented hydrolysis of phosphatidylcholine, which contributed to the superior post-transplant function in donor lungs with aspiration injury.

A novel combined ex vivo and in vivo lentiviral interleukin-10 gene delivery strategy at the time of transplantation decreases chronic lung allograft rejection in mice

Objective Our objective was to develop a rapid‐onset and durable gene‐delivery strategy that is applicable at the time of transplant to determine its effects on both acute rejection and chronic lung allograft fibrosis using a mouse orthotopic lung transplant model. Methods C57BL/6 mice received an orthotopic left lung transplant from syngeneic donors or C57BL/10 donors. By using syngeneic lung transplantation, we established a novel gene transfer protocol with lentivirus luciferase intrabronchial administration to the donor lung ex vivo before transplantation. This strategy was applied in allogeneic lung transplantation with lentivirus engineering expression of human interleukin‐10 or lentivirus luciferase (control). Results Bioluminescent imaging revealed that the highest early transgene expression was achieved when lentivirus luciferase was administered both directly into the donor lung graft ex vivo before implantation and subsequently to the recipient in vivo daily on post‐transplant days 1 to 4, compared with post‐transplant in vivo administration only (days 0 to 4). Our previous work with adenoviral interleukin‐10 gene therapy indicates that early interleukin‐10 expression in the allograft is desirable. Therefore, we selected the combined protocol for human interleukin‐10 encoding lentiviral vector therapy. In the allogeneic transplant setting, ex vivo and in vivo human interleukin‐10 encoding lentiviral vector therapy reduced acute rejection grade (2.0 vs 3.0, P < .05) at day 28. The percentage of fibrotic obliterated airways was reduced in the human interleukin‐10 encoding lentiviral vector–treated group (10.9% ± 7.7% vs 40.9% ± 9.3%, P < .05). Conclusions Delivery of lentiviral interleukin‐10 gene therapy, using a novel combined ex vivo and in vivo delivery strategy, significantly improves acute and chronic rejection in the mouse lung transplant model.

Upregulation of alveolar neutrophil enzymes and long pentraxin-3 in human chronic lung allograft dysfunction subtypes

From the Latner Thoracic Surgery Research Laboratories, Department of Surgery, Toronto General Hospital Research Institute, University Health Network, and Institute of Medical Science, Faculty of Medicine, and Division of Respirology, University of Toronto, Toronto, Ontario, Canada; the Department of Thoracic Surgery, Kansai Medical University, Hirakata, Osaka, Japan; and Department of Thoracic Surgery, University of Tokyo Graduate School of Medicine, Hongo, Tokyo, Japan. Disclosures: Authors have nothing to disclose with regard to commercial support. Supported by the Canadian Institutes of Health Research (MOP-190953), Canadian Cystic Fibrosis Foundation (grant 2387), and Ontario Ministry of Research and Innovation (grant GL2-01-019). T.S. was supported by Research Fellowships of the Japan Society for Promotion of Science for Young Scientists. Received for publication Dec 26, 2017; accepted for publication Feb 17, 2018; available ahead of print March 21, 2018. Address for reprints: Shaf Keshavjee, MD, MSc, Toronto General Hospital, 200 Elizabeth St, 9N946, Toronto, Ontario M5G 2C4, Canada (E-mail: shaf.keshavjee@uhn.ca). J Thorac Cardiovasc Surg 2018;155:2774-6 0022-5223/

Donor Bronchial Wash Bile Acid and Suitability of Donor Lungs for Transplantation

36.00 Copyright 2018 by The American Association for Thoracic Surgery https://doi.org/10.1016/j.jtcvs.2018.02.039 Upregulated neutrophil elastase and long pentraxin-3 in restrictive allograft syndrome.

Effects of Warm Versus Cold Ischemic Donor Lung Preservation on the Underlying Mechanisms of Injuries during Ischemia and Reperfusion

Figure 1 Donor bronchial wash (BW) bile acid level was significantly elevated in the declined lungs, as compared with the transplanted lungs (p 1⁄4 0.01). The bar represents the median value. Aspiration of gastric content could result in a chemical pneumonitis, which likely varies with pH, contents, and amount of fluid aspirated. The degree of gastric aspiration– induced lung graft dysfunction during ex-vivo assessment and after transplant is difficult to estimate at the time of donor organ retrieval, considering that the severity of gastric aspiration–induced lung injury ranges from mild, sub-clinical chemical pneumonitis with favorable post-transplant outcome, to progressive pulmonary injury with severe primary graft dysfunction (PGD) after transplantation. A major problem in this regard is the absence of a prognostic biomarker to identify severely injured donor lungs due to gastric aspiration, which would not be suitable for transplantation. Ex-vivo lung perfusion (EVLP) has emerged as an innovative platform to evaluate lung function and provide a platform for targeted precise treatment for injured donor lungs outside the body before transplantation. In this study, we sought to examine the severity of gastric aspiration–induced lung injury in donors using a bile acid assay in donor bronchial wash (BW) and evaluate the association between bile acid levels and the quality of donor lungs assessed during EVLP. This study was conducted with the approval from the research ethics board University of Toronto Health Network. Donor bronchial wash (BW) samples were collected from January 2013 to May 2015. We defined the technique of BW collection as follows: 20 ml of normal saline instilled through a flexible bronchoscope and aspirated back from the mainstem bronchus at a targeted side that should be chosen from the affected areas, based on radiologic and bronchoscopic examination, at the time of organ retrieval in the donor operating room. A bile acid assay using a spectrophotometric enzymatic cycling assay kit (BQ Kits, Inc., San Diego, CA) was performed using the Cobas diagnostic platform (Roche, Rotkreuz, Switzerland), which takes 7 minutes to perform. Clinical suspicion of gastric aspiration was based on a history of witnessed gastric aspiration or a combination of

Acute Cellular Rejection: Is It Still Relevant?

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)-1&agr;, IL-1&bgr;, 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.

Pentraxin 3 deficiency enhances features of chronic rejection in a mouse orthotopic lung transplantation model

&NA; 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.