Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

A machine-learning approach to human ex vivo lung perfusion predicts transplantation outcomes and promotes organ utilization.

Ex vivo lung perfusion (EVLP) is a data-intensive platform used for the assessment of isolated lungs outside the body for transplantation; however, the integration of artificial intelligence to rapidly interpret the large constellation of clinical data generated during ex vivo assessment remains an unmet need. We developed a machine-learning model, termed InsighTx, to predict post-transplant outcomes using n = 725 EVLP cases. InsighTx model AUROC (area under the receiver operating characteristic curve) was 79 ± 3%, 75 ± 4%, and 85 ± 3% in training and independent test datasets, respectively. Excellent performance was observed in predicting unsuitable lungs for transplantation (AUROC: 90 ± 4%) and transplants with good outcomes (AUROC: 80 ± 4%). In a retrospective and blinded implementation study by EVLP specialists at our institution, InsighTx increased the likelihood of transplanting suitable donor lungs [odds ratio=13; 95% CI:4-45] and decreased the likelihood of transplanting unsuitable donor lungs [odds ratio=0.4; 95%CI:0.16-0.98]. Herein, we provide strong rationale for the adoption of machine-learning algorithms to optimize EVLP assessments and show that InsighTx could potentially lead to a safe increase in transplantation rates.

Extension of Cold Static Donor Lung Preservation at 10°C.

Cold Static Donor Lung Preservation at 10°CDonor lungs for transplantation are currently stored on ice and transplanted as rapidly as possible. In an advance that may ease transplant logistics, Ali et al. report that prolonged storage at 10°C may lead to equivalent outcomes.

Evaluation of 10°C as the optimal storage temperature for aspiration-injured donor lungs in a large animal transplant model.

BACKGROUND: Our recent work has challenged 4°C as an optimal lung preservation temperature by showing storage at 10°C to allow for the extension of preservation periods. Despite these findings, the impact of 10°C storage has not been evaluated in the setting of injured donor lungs. METHODS: Aspiration injury was created through bronchoscopic delivery of gastric juice (pH: 1.8). Injured donor lungs (n = 5/group) were then procured and blindly randomized to storage at 4°C (on ice) or at 10°C (in a thermoelectric cooler) for 12 hours. A third group included immediate transplantation. A left lung transplant was performed thereafter followed by 4 hours of graft evaluation. RESULTS: After transplantation, lungs stored at 10°C showed significantly better oxygenation when compared to 4°C group (343 ± 43 mm Hg vs 128 ± 76 mm Hg, p = 0.03). Active metabolism occurred during the 12 hours storage period at 10°C, producing cytoprotective metabolites within the graft. When compared to lungs undergoing immediate transplant, lungs preserved at 10°C tended to have lower peak airway pressures (p = 0.15) and higher dynamic lung compliances (p = 0.09). Circulating cell-free mitochondrial DNA within the recipient plasma was significantly lower for lungs stored at 10°C in comparison to those underwent immediate transplant (p = 0.048), alongside a tendency of lower levels of tissue apoptotic cell death (p = 0.075). CONCLUSIONS: We demonstrate 10°C as a potentially superior storage temperature for injured donor lungs in a pig model when compared to the current clinical standard (4°C) and immediate transplantation. Continuing protective metabolism at 10°C for donor lungs may result in better transplant outcomes.

Metabolomic fingerprinting of porcine lung tissue during pre-clinical prolonged ex vivo lung perfusion using in vivo SPME coupled with LC-HRMS.

Normothermic ex vivo lung perfusion (NEVLP) has emerged as a modernized organ preservation technique that allows for detailed assessment of donor lung function prior to transplantation. The main goal of this study was to identify potential biomarkers of lung function and/or injury during a prolonged (19 h) NEVLP procedure using in vivo solid-phase microextraction (SPME) technology followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The use of minimally invasive in vivo SPME fibers for repeated sampling of biological tissue permits the monitoring and evaluation of biochemical changes and alterations in the metabolomic profile of the lung. These in vivo SPME fibers were directly introduced into the lung and were also used to extract metabolites (on-site SPME) from fresh perfusate samples collected alongside lung samplings. A subsequent goal of the study was to assess the feasibility of SPME as an in vivo method in metabolomics studies, in comparison to the traditional in-lab metabolomics workflow. Several upregulated biochemical pathways involved in pro- and anti-inflammatory responses, as well as lipid metabolism, were observed during extended lung perfusion, especially between the 11th and 12th hours of the procedure, in both lung and perfusate samples. However, several unstable and/or short-lived metabolites, such as neuroprostanes, have been extracted from lung tissue in vivo using SPME fibers. On-site monitoring of the metabolomic profiles of both lung tissues through in vivo SPME and perfusate samples on site throughout the prolonged NEVLP procedure can be effectively performed using in vivo SPME technology.

Successful 3-day lung preservation using a cyclic normothermic ex vivo lung perfusion strategy.

BACKGROUND: Cold static preservation (CSP) at higher temperatures (10°C) has been recently shown as an optimal strategy up to 24-36h of preservation. Here, we hypothesized that alternating 10°C static storage with cycles of normothermic ex vivo lung perfusion (EVLP) would provide conditions for cellular "recharge", allowing for multi-day lung preservation. METHODS: Donor lungs from male Yorkshire pigs were preserved using 10°C CSP with two cycles of 4h EVLP. After a total of 3 days of preservation, a left lung transplant was performed followed by 4h of graft evaluation. As controls, 2 lungs were preserved solely with continuous 10°C preservation for 3 days and transplanted. FINDINGS: For animals receiving lungs preserved using a cyclic EVLP protocol, lung function and histological structures were stable and the recipient systemic partial pressure of oxygen/fraction of inspired oxygen (P/F Ratio) after excluding the contralateral lung was 422 ± 61 mmHg. In contrast, lungs preserved solely in continuous cold static storage at 10°C for 72h developed massive lung failure, resulting in recipient death. Metabolomic analysis revealed that EVLP plays a critical role in the re-vitalization of key central carbon energy metabolites (Glucose, Succinate, N-Acetyl Aspartate) and reducing the expression of the inflammasome activation marker CASP1. INTERPRETATION: In conclusion, we demonstrate for the first time the feasibility of 3-day lung preservation leading to excellent early post-transplant outcomes. The thoughtful combination of cold storage (10°C) and intermittent EVLP can open new opportunities in organ transplantation. FUNDING: This work was supported by the UHN Foundation (Grant#1013612).

High Doses of Inhaled Nitric Oxide as an Innovative Antimicrobial Strategy for Lung Infections.

Since the designation of nitric oxide as "Molecule of the Year" in 1992, the scientific and clinical discoveries concerning this biomolecule have been greatly expanding. Currently, therapies enhancing the release of endogenous nitric oxide or the direct delivery of the exogenous compound are recognized as valuable pharmacological treatments in several disorders. In particular, the administration of inhaled nitric oxide is routinely used to treat patients with pulmonary hypertension or refractory hypoxemia. More recently, inhaled nitric oxide has been studied as a promising antimicrobial treatment strategy against a range of pathogens, including resistant bacterial and fungal infections of the respiratory system. Pre-clinical and clinical findings have demonstrated that, at doses greater than 160 ppm, nitric oxide has antimicrobial properties and can be used to kill a broad range of infectious microorganisms. This review focused on the mechanism of action and current evidence from in vitro studies, animal models and human clinical trials of inhaled high-dose nitric oxide as an innovative antimicrobial therapy for lung infections.

Centralized Organ Recovery and Reconditioning Centers.

An increased focus on improving efficiency and decreasing costs has resulted in alternative models of donor management and organ recovery. The specialized donor care facility model provides highly efficient and cost-effective donor care at a free-standing facility, resulting in improved organ yield, shorter ischemic times, decreased travel, and fewer nighttime operations. Ex vivo lung perfusion (EVLP) improves utilization of extended criteria donor lungs, and centralized EVLP facilities have the potential to increase transplant volumes for smaller transplant programs in specified geographic regions. These alternative models are increasingly being used in the United States to improve waitlist mortality and combat the ongoing donor organ shortage.

Near-infrared fluorescence imaging during ex vivo lung perfusion: Noninvasive real-time evaluation of regional lung perfusion and edema.

OBJECTIVE: Ex vivo lung perfusion (EVLP) is an excellent platform to evaluate donor lung function before transplantation, but novel methods are needed to accurately confirm transplant quality. Near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) has been used in various clinical perioperative applications to evaluate tissue perfusion. We used NIRF imaging during pig and human EVLP to evaluate donor lung perfusion and edema. METHODS: Pig lungs with various degrees of lung injury (n = 10) and human lungs rejected from clinical transplantation (n = 3) were imaged during EVLP using intravascular ICG and a SPY Elite (Stryker) NIRF imaging unit. Optimal ICG and imaging conditions, and perfusion and edema quantification methods, were established. Pig lung transplants with extended graft preservation (n = 5) and control native lungs (n = 6) were also imaged. RESULTS: A single ICG dose resulted in sustained donor lung NIRF throughout the EVLP. Even and homogenous ICG signal was demonstrated in areas of normal lung. Low NIRF was present in regions with poor tissue perfusion, and rapid, intense ICG accumulation occurred in damaged and edematous areas. Segmental perfusion defects were common in the peripheral and elevated regions of the lungs, and serial imaging showed gradual perfusion recovery during EVLP. Impaired microvascular reperfusion, indicated by a decreased NIRF ingress rate, was detected in transplanted pig lungs early after reperfusion. CONCLUSIONS: NIRF imaging enables noninvasive real-time evaluation of lung perfusion and edema during EVLP. Prospective clinical studies are needed to determine the role of NIRF imaging in donor lung assessment and selection, and prediction of posttransplant outcomes.

Ex vivo enzymatic treatment converts blood type A donor lungs into universal blood type lungs.

Donor organ allocation is dependent on ABO matching, restricting the opportunity for some patients to receive a life-saving transplant. The enzymes FpGalNAc deacetylase and FpGalactosaminidase, used in combination, have been described to effectively convert group A (ABO-A) red blood cells (RBCs) to group O (ABO-O). Here, we study the safety and preclinical efficacy of using these enzymes to remove A antigen (A-Ag) from human donor lungs using ex vivo lung perfusion (EVLP). First, the ability of these enzymes to remove A-Ag in organ perfusate solutions was examined on five human ABO-A1 RBC samples and three human aortae after static incubation. The enzymes removed greater than 99 and 90% A-Ag from RBCs and aortae, respectively, at concentrations as low as 1 µg/ml. Eight ABO-A1 human lungs were then treated by EVLP. Baseline analyses of A-Ag in lungs revealed expression predominantly in the endothelial and epithelial cells. EVLP of lungs with enzyme-containing perfusate removed over 97% of endothelial A-Ag within 4 hours. No treatment-related acute lung toxicity was observed. An ABO-incompatible transplant was then simulated with an ex vivo model of antibody-mediated rejection using ABO-O plasma as the surrogate for the recipient circulation using three donor lungs. The treatment of donor lungs minimized antibody binding, complement deposition, and antibody-mediated injury as compared with control lungs. These results show that depletion of donor lung A-Ag can be achieved with EVLP treatment. This strategy has the potential to expand ABO-incompatible lung transplantation and lead to improvements in fairness of organ allocation.

Safety of continuous 12-hour delivery of antimicrobial doses of inhaled nitric oxide during ex vivo lung perfusion.

INTRODUCTION: High-dose nitric oxide (NO) has been shown effective against a variety of micro-organisms in vitro, including common bacteria found in donor organs. However, clinical obstacles related to its implementation in vivo are the formation of methemoglobin and the accumulation of toxic nitrogen compounds. Ex vivo lung perfusion (EVLP) is a platform that allows for organ maintenance with an acellular perfusion solution, thus overcoming these limitations. The present study explores the safety of continuous high-dose inhaled (iNO) during EVLP for an extended period of 12 hours. METHODS: Lungs procured from Yorkshire pigs were randomized into control (standard ventilation) and treatment (standard ventilation + 200 ppm iNO) groups, then perfused with an acellular solution for 12 hours (n = 4/group). Lung physiology and biological markers were evaluated. RESULTS: After 12 hours of either standard EVLP or EVLP + 200 ppm iNO, we did not notice any significant physiologic difference between the groups: pulmonary oxygenation (P = .586), peak airway pressures (P = .998), and dynamic (P = .997) and static (P = .908) lung compliances. In addition, no significant differences were seen among proinflammatory cytokines measured in perfusate and lung tissue. Importantly, most common toxic compounds were kept at safe levels throughout the treatment course. CONCLUSIONS: High-dose inhaled NO delivered continuously over 12 hours appears to be safe without inducing any significant pulmonary inflammation or deterioration in lung function. These findings support further efficacy studies to explore the use of iNO for the treatment of infections in donor lungs during EVLP.

Ex vivo treatment of cytomegalovirus in human donor lungs using a novel chemokine-based immunotoxin.

BACKGROUND: Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfusion (EVLP). METHODS: HCMV seropositive human lungs were placed on EVLP alone or EVLP + 1mg/L of F49A-FTP for 6 hours (n = 6, each). CD14+ monocytes isolated from biopsies pre and post EVLP underwent HCMV reactivation assay designed to evaluate viral reactivation capacity. Off-target effects of F49A-FTP were studied evaluating cell death markers of CD34+ and CD14+ cells using flow cytometry. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. RESULTS: We demonstrate that lungs treated ex-vivo with F49A-FTP had a significant reduction in HCMV reactivation compared to controls, suggesting successful targeting of latent virus (76% median reduction in F49A-FTP vs 15% increase in controls, p = 0.0087). Furthermore, there was comparable cell death rates of the targeted cells between both groups, suggesting no off-target effects. Ex-vivo lung function was stable over 6 hours and no differences in key inflammatory cytokines were observed demonstrating safety of this novel treatment. CONCLUSIONS: Ex-vivo F49A-FTP treatment of human lungs targets and kills latent HCMV, markedly attenuating HCMV reactivation. This approach demonstrates the first experiments targeting latent HCMV in a donor organ with promising results towards clinical translation.

Metabolomic fingerprinting of porcine lung tissue during pre-clinical prolonged ex vivo lung perfusion using in vivo SPME coupled with LC-HRMS / 药物分析学报

Normothermic ex vivo lung perfusion(NEVLP)has emerged as a modernized organ preservation tech-nique that allows for detailed assessment of donor lung function prior to transplantation.The main goal of this study was to identify potential biomarkers of lung function and/or injury during a prolonged(19 h)NEVLP procedure using in vivo solid-phase microextraction(SPME)technology followed by liquid chromatography-high resolution mass spectrometry(LC-HRMS).The use of minimally invasive in vivo SPME fibers for repeated sampling of biological tissue permits the monitoring and evaluation of biochemical changes and alterations in the metabolomic profile of the lung.These in vivo SPME fibers were directly introduced into the lung and were also used to extract metabolites(on-site SPME)from fresh perfusate samples collected alongside lung samplings.A subsequent goal of the study was to assess the feasibility of SPME as an in vivo method in metabolomics studies,in comparison to the traditional in-lab metabolomics workflow.Several upregulated biochemical pathways involved in pro-and anti-inflammatory responses,as well as lipid metabolism,were observed during extended lung perfusion,especially between the 11th and 12th hours of the procedure,in both lung and perfusate samples.However,several unstable and/or short-lived metabolites,such as neuroprostanes,have been extracted from lung tissue in vivo using SPME fibers.On-site monitoring of the metabolomic profiles of both lung tissues through in vivo SPME and perfusate samples on site throughout the prolonged NEVLP procedure can be effectively performed using in vivo SPME technology.

A novel pre-clinical strategy to deliver antimicrobial doses of inhaled nitric oxide.

Effective treatment of respiratory infections continues to be a major challenge. In high doses (≥160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent, including its efficacy in vitro for coronavirus family. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb <6%. Both groups were ventilated continuously for 6 hours, then the animals were weaned from sedation, mechanical ventilation and followed for 3 days. During treatment, and on the third post-operative day, physiologic assessments were performed to monitor lung function and other significative markers were assessed for potential pulmonary or systemic injury. No significant change in lung function, or inflammatory markers were observed during the study period. Both gas exchange function, lung tissue cytokine analysis and histology were similar between treated and control animals. During treatment, levels of metHb were maintained <6% by administration of MB, and NO2 remained <5 ppm. Additionally, considering extrapulmonary effects, no significant changes were observed in biochemistry markers. Our findings showed that high-dose iNO delivered continuously over 6 hours with adjuvant MB is clinically feasible and safe. These findings support the development of investigations of continuous high-dose iNO treatment of respiratory tract infections, including SARS-CoV-2.

Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.

Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.

Static lung storage at 10°C maintains mitochondrial health and preserves donor organ function.

Cold static preservation on ice (~4°C) remains the clinical standard of donor organ preservation. However, mitochondrial injury develops during prolonged storage, which limits the extent of time that organs can maintain viability. We explored the feasibility of prolonged donor lung storage at 10°C using a large animal model and investigated mechanisms related to mitochondrial protection. Functional assessments performed during ex vivo lung perfusion demonstrated that porcine lungs stored for 36 hours at 10°C had lower airway pressures, higher lung compliances, and better oxygenation capabilities, indicative of better pulmonary physiology, as compared to lungs stored conventionally at 4°C. Mitochondrial protective metabolites including itaconate, glutamine, and N-acetylglutamine were present in greater intensities in lungs stored at 10°C than at 4°C. Analysis of mitochondrial injury markers further confirmed that 10°C storage resulted in greater protection of mitochondrial health. We applied this strategy clinically to prolong preservation of human donor lungs beyond the currently accepted clinical preservation limit of about 6 to 8 hours. Five patients received donor lung transplants after a median preservation time of 10.4 hours (9.92 to 14.8 hours) for the first implanted lung and 12.1 hours (10.9 to 16.5 hours) for the second. All have survived the first 30 days after transplantation. There was no grade 3 primary graft dysfunction at 72 hours after transplantation, and median post-transplant mechanical ventilation time was 1.73 days (0.24 to 6.71 days). Preservation at 10°C could become the standard of care for prolonged pulmonary preservation, providing benefits to both patients and health care teams.