CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

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.

Predictors of Urinary and Fecal Incontinence in Prefrail and Frail Older Adults: A Cross-Sectional Study of the FRAGSALUD Project.

BACKGROUND: Frailty is associated with urinary and fecal incontinence, which are common geriatric syndromes. This study aims to identify health factors associated with incontinence in prefrail or frail older adults living in the community. METHODS: This multicenter cross-sectional study included 225 older adults (75.0 ±â€…6.4 years) with prefrailty or frailty based on the 5-component Fried phenotype. Physical function was assessed using the Short Physical Performance Battery (SPPB). Physical activity, inactivity, and sleep were estimated using a wrist-worn accelerometer. Urinary or fecal incontinence was registered using the Barthel scale (urine and bowel items). Multivariable logistic regression analyses, with age as a covariate, were conducted to identify associations of incontinence. RESULTS: In our participants, 27% presented urinary or fecal incontinence with no sex differences (p = .266). Our results showed that age, daily medication count, and number of falls in the previous year independently predicted incontinence in frail and prefrail older adults (p < .05). Some Fried's criteria, including self-reported exhaustion, gait speed, and handgrip strength, were associated with the presence of incontinence (p < .05), but not Fried's classification. The SPPB total score and its isolated variables were significantly associated with the urinary and fecal incontinence (p < .05). However, none of the accelerometer outcomes showed significant associations with incontinence status. CONCLUSIONS: According to this study, age, number of medications, and falls (but not sex) are linked to urinary and fecal incontinence in frail or prefrail older adults living in the community, recommending the assessment of physical function using the SPPB rather than estimating daily physical activity, inactivity, or sleep.

Exploring the interplay of frailty, physical function, physical activity, nutritional status, and their association with quality of life and depressive symptoms in older adults with the frailty phenotype.

OBJECTIVE: This study aimed to explore the interplay between frailty, physical function, physical activity, nutritional status, and their impact on the quality of life and depressive status in older adults with frailty. METHODS: A cross-sectional study involving 235 pre-frail/frail older adults residing in Spanish communities was conducted. Frailty was assessed using Fried's criteria, physical function was evaluated using the Short Physical Performance Battery, and physical activity levels were measured via wrist-worn accelerometers. Nutritional status was determined using the Mini-Nutritional Assessment alongside anthropometric measurements. Quality of life was gauged using the EuroQoL 5-Dimension 5-Level, while depressive status was assessed using the Yesavage 15-item Geriatric Depression Scale. Multivariate linear regression and logistic regression analyses were employed to elucidate the associations of these factors with quality of life and depression. RESULTS: Our findings revealed significant correlations between various factors and quality of life. Notably, reported fatigue (ß = -0.276, p = 0.002), performance in the 4-m gait test (ß = -0.242, p = 0.001), the score on the short version of the Mini-Nutritional Assessment (ß = 0.312, p = 0.002), and engagement in light physical activity (ß = 0.180, p = 0.023) were all found to be associated with quality of life. In terms of depressive symptoms, the Mini-Nutritional Assessment score emerged as a protective factor (Odds ratio, OR: 0.812, p < 0.001), as did participation in moderate physical activity (OR: 0.988, p = 0.028). Conversely, fatigue (OR: 3.277, p = 0.003) and a slow gait speed (OR: 1.136, p = 0.045) were identified as risk factors for depressive symptoms. CONCLUSIONS: This study underscores the detrimental association of fatigue and slow gait speed on both quality of life and depressive status among older adults with frailty. In contrast, engaging in physical activity and addressing malnutrition risk emerge as critical protective factors for enhancing quality of life and ameliorating depressive symptoms in this population. CLINICAL TRIAL REGISTRATION: This is a study that uses cross-sectional data from a trial registered at ClinicalTrials.gov (Identifier: NCT05610605).

Associations Between Physical Activity and Inactivity Levels on Physical Function and Sleep Parameters of Older Adults With Frailty Phenotype.

This study investigated the relationship between physical activity, inactivity, physical function, and sleep in older adults with a frailty phenotype. A total of 184 pre-frail/frail older adults were included. Physical activity, inactive behavior, and sleep parameters were assessed using a wrist-worn accelerometer. Participants were categorized into four groups based on their levels of inactivity and physical activity. The results showed that individuals with lower levels of inactivity had better lower body mean velocity and sleep regularity than those with higher levels of inactivity. Physically active older adults exhibited faster gait speed and performed better in lower body strength tests than physically inactive participants. Further analysis revealed that specific combinations of inactivity and physical activity were associated with varying levels of physical function. The findings highlight the importance of physical activity and the negative impact of inactivity on physical function and sleep in older adults with a frailty phenotype.

Effects of an educational intervention on frailty status, physical function, physical activity, sleep patterns, and nutritional status of older adults with frailty or pre-frailty: the FRAGSALUD study.

Introduction: The prevalence of frailty is increasing worldwide, emphasizing the importance of prioritizing healthy ageing. To address this, cost-effective and minimally supervised interventions are being sought. This study aimed to assess the impact of an educational program on frailty status, physical function, physical activity, sleep patterns, and nutritional status in community-dwelling older adults with at least 1 Fried's frailty criteria. Methods: A 6-month multicentre randomized controlled trial was conducted from March 2022 to February 2023 in 14 health centres located in Cadiz and Malaga, Spain. The educational intervention consisted of 4 group sessions and 6 follow-up phone calls spread over 6 months. The program focused on educating participants about frailty and its impact on health, providing guidelines for physical activity, healthy dietary habits, cognitive training, psychological well-being and social activities. A total of 163 participants, divided into control (n = 80) and educational groups (n = 83) were assessed before and after the intervention. Results: The results showed a significant group-time interaction in the physical function evaluated with a large effect on Short Physical Performance Battery score (η2p = 0.179, -0.1 [-1.2-1.0] points for control group vs. 1.0 [0.0-3.0] points for educational group, p < 0.001), and an effect on the 4-meter gait test ((η2p = 0.122, 0.5 [0.1-0.0] s for control group vs. -0.4 [-0.5- -0.3] s for educational group, p < 0.001), and the 5-repetition sit-to-stand test (η2p = 0.136, 1.0 [0.0-1.2] s for control group vs. -4.3 [-7.0- -2.3] for educational group, p < 0.001). Additionally, the use of accelerometers to assess physical activity, inactivity, and sleep patterns revealed a significant small effect in the number of awakenings at night ((η2p = 0.040, 1.1 [-0.5-3.4] awakenings for control group vs. 0.0 [-2.2-0.0] awakenings for educational group, p = 0.009). The findings also highlighted a significant medium effect regarding malnutrition risk, which was assessed using the Mini-Nutritional Assessment score (η2p = 0.088, -0.7 [-2.3-1.5] points for control group vs. 1.5 [-0.5-3.0] points for educational group, p < 0.001). Discussion: Thus, the 6-month educational program effectively improved physical function, sleep patterns, and nutritional status compared to usual healthcare attendance in community-dwelling older adults with frailty or pre-frailty. These findings underscore the potential of minimally supervised interventions in promoting a healthy lifestyle in this vulnerable population.

Health factors associated with cognitive frailty in older adults living in the community.

Introduction: This study aims to investigate the health factors associated with cognitive frailty in frail and pre-frail older adults living in the community. Methods: A total of 233 older adults meeting Fried's criteria for pre-frailty or frailty were included. Cognitive status was evaluated using the Short Portable Mental Status Questionnaire. Health factors encompassed nutritional status (evaluated using the Mini Nutritional Assessment tool, body mass index, and waist, arm, and leg circumferences), physical function (assessed with the Short Physical Performance Battery), quality of life (measured with the total index of the EuroQoL 5-Dimension 5-Level questionnaire - EQoL-Index -, and the Visual-Analogue Scale - QoL-VAS - for today's health state), as well as sleep, physical activity, and inactivity estimated through wrist-worn accelerometers. Multivariable logistic regression analyses were conducted to identify potential predictors of cognitive frailty, considering age as a confounding factor. Results: Cognitive frail participants exhibited advanced age, heightened self-reported exhaustion, diminished overall physical performance, reduced leg perimeter, decreased engagement in moderate-to-vigorous physical activity, and higher levels of inactivity (all p<0.05). However, after adjusting for age, only QoL-VAS emerged as a cognitive frailty risk factor (Odds ratio: 1.024), while the EQoL-Index, calf perimeter, and levels of moderate-to-vigorous physical activity were identified as protective factors (Odds ratios: 0.025, 0.929, and 0.973, respectively). Discussion: This study highlights the complex relationship between non-modifiable factors such as age, and modifiable factors including quality of life, nutritional status, and physical activity in the development of cognitive frailty among older adults with a frailty phenotype living in the community.

Risk and Protective Factors for Frailty in Pre-Frail and Frail Older Adults.

This study aims to evaluate the differences in body composition, physical function, and physical activity between pre-frail/frail older adults and to detect risk and protective factors against frailty and physical frailty. Fried's criteria for frailty and physical frailty using the short-performance physical battery (SPPB) were measured in 179 older participants (75.3 ± 6.4 years old). Body weight, height, and waist, arm, and leg circumferences were obtained as body composition variables. Daily accelerometer outcomes (physical activity and inactivity) were obtained. Pre-frail participants showed overall better physical function and spent more time in physical activity and less time in long inactivity periods than frail participants (p < 0.05). Risk frailty factors were higher waist perimeter (Odds Ratio [OR]: 1.032, 95%CI: 1.003-1.062), low leg performance (OR: 1.025, 95%CI: 1.008-1.043), and inactivity periods longer than 30 min (OR:1.002, 95%CI: 1.000-1.005). Protective factors were standing balance (OR:0.908, 95%CI: 0.831-0.992) and SPPB score (OR: 0.908, 95%CI: 0.831-0.992) for frailty, handgrip strength (OR: 0.902, 95%CI: 0.844-0.964) for physical frailty, and light (OR: 0.986, 95%CI: 0.976-0.996) and moderate-to-vigorous (OR: 0.983, 95%CI: 0.972-0.996) physical activity for both. Our findings suggest that handgrip strength, balance, and physical activity are protective frailty factors and can be monitored in pre-frail older adults. Moreover, poor lower body performance and long inactivity periods are frailty risk factors, which highlights their importance in frailty assessment.

A biomarker assay to risk-stratify patients with symptoms of respiratory tract infection.

BACKGROUND: Patients who present to an emergency department (ED) with respiratory symptoms are often conservatively triaged in favour of hospitalisation. We sought to determine if an inflammatory biomarker panel that identifies the host response better predicts hospitalisation in order to improve the precision of clinical decision making in the ED. METHODS: From April 2020 to March 2021, plasma samples of 641 patients with symptoms of respiratory illness were collected from EDs in an international multicentre study: Canada (n=310), Italy (n=131) and Brazil (n=200). Patients were followed prospectively for 28 days. Subgroup analysis was conducted on confirmed coronavirus disease 2019 (COVID-19) patients (n=245). An inflammatory profile was determined using a rapid, 50-min, biomarker panel (RALI-Dx (Rapid Acute Lung Injury Diagnostic)), which measures interleukin (IL)-6, IL-8, IL-10, soluble tumour necrosis factor receptor 1 (sTNFR1) and soluble triggering receptor expressed on myeloid cells 1 (sTREM1). RESULTS: RALI-Dx biomarkers were significantly elevated in patients who required hospitalisation across all three sites. A machine learning algorithm that was applied to predict hospitalisation using RALI-Dx biomarkers had a mean±sd area under the receiver operating characteristic curve of 76±6% (Canada), 84±4% (Italy) and 86±3% (Brazil). Model performance was 82±3% for COVID-19 patients and 87±7% for patients with a confirmed pneumonia diagnosis. CONCLUSIONS: The rapid diagnostic biomarker panel accurately identified the need for inpatient care in patients presenting with respiratory symptoms, including COVID-19. The RALI-Dx test is broadly and easily applicable across many jurisdictions, and represents an important diagnostic adjunct to advance ED decision-making protocols.

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).

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.

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.

Deceased-donor lobar lung transplant: A successful strategy for small-sized recipients.

OBJECTIVES: Lobar lung transplantation (LLTx) from deceased donors is a potential solution for donor-recipient size mismatch for small sized recipients. We reviewed our institutional experience to compare outcomes after LLTx to standard lung transplantation (LTx). METHODS: We retrospectively reviewed transplants in our institution from January 2000 to December 2017. LLTx early- and long-term outcomes were compared with LTx. Additional analysis of outcomes was performed after dividing the cohort into 2 eras (era 1, 2000-2012; era 2, 2013-2017). RESULTS: Among the entire cohort (1665), 75 were LLTx (4.5%). Compared with LTx, LLTx were more frequently bridged to transplant with extracorporeal life support or mechanical ventilation and were transplanted in a rapidly deteriorating status (respectively, 20% vs 4.4%, P = .001; 22.7% vs 7.9, P < .001; and 41.3% vs 26.5%, P = .013). LLTx had longer intensive care unit and hospital lengths of stay (respectively, median 17 vs 4 days, and 45 vs 23, both P < .001), and greater 30-day mortality (13.3% vs 4.3%, P = .001) and 90-day mortality (17.3% vs 7.2%, P = .003). In era 2, despite a significantly greater 30-day mortality (10.8% vs 2.8%, P = .026), there was no significant difference in 90-day mortality between LLTx and LTx (13.5% vs 5.1%, P = .070). Overall survival at 1, 3, and 5 years was not significantly different between LLTx and LTx (73.2% vs 84.4%, 56.9% vs 68.4% and 50.4% vs 55.8, P = .088). CONCLUSIONS: Although LLTx is a high-risk procedure, both mid- and long-term survival are comparable with LTx in all cohorts in the modern era. LLTx therefore represents a valuable surgical option for small-sized recipients.

Pig lung transplant survival model.

Although lung transplant is a life-saving therapy for some patients, primary graft dysfunction (PGD) is a leading cause of mortality and morbidity soon after a transplant. Ischemia reperfusion injury is known to be one of the most critical factors in PGD development. PGD is by definition an acute lung injury syndrome that occurs during the first 3 d following lung transplantation. To successfully translate laboratory discoveries to clinical practice, a reliable and practical large animal model is critical. This protocol describes a surgical technique for swine lung transplantation and postoperative management for a further 3 d post transplant. The protocol includes the background and rationale, required supplies, and a detailed description of the donor operation, transplant surgery, postoperative care, and sacrifice surgery. A pig lung transplant model is reliably produced in which the recipients survive for 3 d post transplant. This 3-d survival model can be used by lung transplant researchers to assess the development of PGD and to test therapeutic strategies targeting PGD. In total, the protocol requires 5 h for the surgeries, plus ~2 h in total for the postoperative care.

α1-Anti-trypsin improves function of porcine donor lungs during ex-vivo lung perfusion.

BACKGROUND: Ex-vivo lung perfusion (EVLP), a technique for donor lung assessment, also represents a platform for donor lung repair and immunomodulation. α1-Anti-trypsin (A1AT), a medication used to treat emphysema in A1AT-deficient patients, has anti-inflammatory properties and has been shown to attenuate ischemia-reperfusion injury in rat and pig lung transplants. The objective of this study was to determine whether administration of A1AT during EVLP can improve donor lung quality after prolonged hypothermic preservation. METHODS: Pig donor lungs were retrieved, preserved at 4°C for 24 hours, and then subjected to normothermic EVLP for 12 hours using the Toronto protocol. The treatment group (n = 6) received 3 mg/ml A1AT (Zemaira) in the EVLP perfusate, acellular Steen solution. The control group (n = 6) was perfused with Steen solution only. Physiologic functions and gas exchange were measured hourly. Pulmonary edema, lung injury, apoptosis and inflammatory mediators were evaluated in lung tissues and perfusate. RESULTS: A1AT treatment significantly reduced pulmonary arterial pressure, pulmonary vascular resistance and airway pressure changes from the baseline when compared with controls. A1AT treatment significantly improved both dynamic and static pulmonary compliance, and change in partial pressure of oxygen (ΔPO2) between the left atrium and the pulmonary artery. Furthermore, A1AT treatment also significantly reduced pulmonary edema (wet-to-dry ratio), pulmonary cell apoptosis and pro-inflammatory cytokine levels (interleukin-1α and -8) in the perfusate. CONCLUSION: Treatment of 24-hour-preserved pig donor lungs with A1AT during EVLP resulted in improved physiologic function, reduced pulmonary edema and inflammation and decreased cell death. Our findings suggest that treatment of donor lungs during EVLP with A1AT is a promising strategy to attenuate early lung injury and improve donor lung function before lung transplantation.