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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.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.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).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.

For patients with end-stage lung disease, lung transplantation is a lifesaving therapy. Currently however, the number of patients who require a transplant exceeds the number of donor lungs available. One of the contributing factors to this is the conservative mindset of physicians who are concerned about transplanting marginal lungs due to the potential risk of primary graft dysfunction. Ex vivo lung perfusion (EVLP) technology has allowed for the expansion of donor pool of organs by enabling assessment and reconditioning of these marginal grafts before transplant. Ongoing efforts to optimize the therapeutic potential of EVLP are underway. Researchers have adopted the use of different large and small animal models to generate translational preclinical data. This includes the use of rejected human lungs, pig lungs, and rat lungs. In this review, we summarize some of the key current literature studies relevant to each of the major EVLP model platforms and identify the advantages and disadvantages of each platform. The review aims to guide investigators in choosing an appropriate species model to suit their specific goals of study, and ultimately aid in translation of therapy to meet the growing needs of the patient population.

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.

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

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.

Donor lungs from the United States can be offered by US organ procurement organizations to Canada if no American centers accept them. The purpose of this study is to evaluate outcomes of patients undergoing transplant at a single center in Canada using declined lungs from the United States and to compare these outcomes to patients receiving lungs from Canadian donors.A single-center retrospective review of recipients receiving lung transplantation between January 2009 and October 2019 was performed. An Organ Procurement and Transplantation Network standard transplant analysis and research-limited dataset as of August 17, 2021, was provided by the United Network for Organ Sharing. De-identified patient-level data were extracted from the standard transplant analysis and research file to identify lung offers made by US organ procurement organizations, declined by US lung centers, and transplanted by the University Health Network within the study time frame. We divided the analysis into 2 groups: recipients receiving donor lungs from Canada and recipients receiving donor lungs from the United States. Donor and recipient characteristics between the 2 groups were compared. Primary end point was proportional survival over a 10-year period. Secondary end points included 30-day mortality, intensive care unit and hospital length of stay, severe primary graft dysfunction, and incidence of chronic lung allograft dysfunction.During the study period, 1424 lung transplants were performed at our center. Of these, 124 (8.7%) were performed using donors from the United States. The incidence of transplants using US donors increased from 5% (5 out of 102) in 2009 to 15% (30 out of 200) in 2018. US donors were younger (aged 41 vs 47 years; P = .004), less likely to be from donors after cardiac death (9.6% vs 20%; P = .008), had higher use of ex vivo lung perfusion (EVLP, 46% vs 27%; P = .0002), and higher incidence of positive nucleic acid test for hepatitis C (16% vs 0.7%; P = .0001). Although the incidence of EVLP utilization was higher in the US lungs versus Canada lungs, more than half of US lungs (54%) proceeded directly to transplantation. Similar short- and long-term outcomes were observed between the 2 groups, including overall survival (hazard ratio, 1.12; 95% CI, 0.85-1.47; P = .40) CONCLUSIONS: Lung transplantation using donor lungs declined by multiple centers in the United States resulted in similar short- and long-term outcomes compared with donor lungs offered in Canada.

Background: Effective treatment of respiratory infections continues to be a major challenge. In high doses (³160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent in preliminary studies. 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. Methods: 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

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.

Background Hemoglobin-based oxygen carriers (HBOCs) are potential alternatives to red blood cells in transfusions. Clinical trials using early versions of HBOCs noted adverse effects that appeared to result from removal of the vasodilator nitric oxide (NO). Previous reports suggest that size-enlarged HBOCs may avoid NO-rich regions along the vasculature and therefore not cause vasoconstriction and hypertension. Study design and methods Hemoglobin (Hb) bis-tetramers (bis-tetramers of hemoglobin that are prepared using CuAAC chemistry [BT-Hb] and bis-tetramers of hemoglobin that are specifically acetylated and prepared using CuAAC chemistry [BT-acHb]) can be reliably produced by a bio-orthogonal cyclo-addition approach. We considered that an HBOC derived from chemical coupling of two Hbs would be sufficiently large to avoid NO scavenging and related side effects. The ability of intravenously infused BT-Hb and BT-acHb to remain in the circulation without causing hypertension were determined in wild-type (WT) and diabetic (db/db) mouse models. Results In WT mice, the coupled oxygen-carrying proteins retained their function over several hours after administration. No significant changes in systolic blood pressure from baseline were observed after intravenous infusion of BT-Hb or BT-acHb in awake WT and db/db mice. In contrast, infusion of native Hb or cross-linked Hb tetramers in both animal models induced systemic hypertension. Conclusion The results of this study indicate that bis-tetrameric HBOCs derived from the bio-orthogonal cyclo-addition process are likely to overcome clinical issues that arise from NO scavenging by Hb derivatives.

Purpose This study explores feasibility and safety of using enzymes (FpGalNAc deacetylase & FpGalNase) to remove ABO-A antigens from donor lungs during ex vivo lung perfusion (EVLP). The enzymes have been reported to efficiently convert ABO-A blood to O (Withers, Nat Microbiol, 2019). Methods Human lungs (ABO-A1, n=5), declined for transplant, were treated with the enzymes during EVLP. Tissues were sampled before and after. The distribution and expression level of ABO antigens were analyzed by immunohistochemistry and flow cytometry. Lung function was monitored for side-effects. Immediate post-transplant immune response was simulated in an ex vivo model of hyperacute lung rejection, with introduction of ABO-O plasma (carrying ABO antibodies) as the surrogate for the recipient circulation (Fig.1H, n=1). The physiology and histology of lungs were analyzed for anti-A-induced immune responses. Results The histo-blood type A (BTA) antigens in the A1 lungs are observed primarily on endothelial and epithelial cells. BTA were cleared remarkably well from treated lungs (Fig.1A). The enzymes convert vascular BTA to the blood group H (BTH) antigens found in ABO-O tissue (Fig.1B). Over 97% of endothelial BTA antigens were removed within the typical 4h clinical EVLP timeframe using very low dose (1 µg/mL) enzymes (Fig.1C). No acute side-effects were seen (Fig.1D-G). The rejection model showed that the 3-hour treatment of human lung with low dose enzymes prevented the damage triggered by type O plasma (presumably due to anti-A antibodies) as observed in the physiology and histology of the control lung (Fig.1I-J). Conclusion Ex vivo enzymatic treatment can efficiently remove ABO-A antigen in donor lungs without acute side-effects. Preliminary results show that the treatment can prevent ABO mismatch-induced early damages. The treatment can potentially allow expansion of ABO-incompatible lung transplantation, leading to significant improvements in logistics and fairness of organ allocation.

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 2 remained

Purpose We explored the feasibility and safety of using recently developed enzymes (FpGalNAc deacetylase and FpGalNase) to remove histo-blood type antigens from donor lungs during ex vivo lung perfusion (EVLP). The enzymes have been reported to efficiently convert Type A whole blood to O (Withers, Nature Microbiology, 2019). Methods Human type A red blood cells (RBCs, n=5/group) and arteries (aorta and pulmonary arteries, n=3/group) were treated with enzyme-containing EVLP solution and lung preservation solution. Human donor lungs (type A) declined for transplantation were treated with the enzymes (1µg/mL) during normothermic EVLP for 1 and 3 h. Lung physiological parameters were monitored throughout treatment. Cell and tissue samples were taken before and after treatment. The distribution and expression levels of blood type antigens were analyzed by flow cytometry and immunohistochemistry. Results The enzymes achieved over 99% antigen removal from RBC within 1h at a dose of 4 µg/mL (Fig1A) in EVLP solution and lung preservation solution. The histo-blood type antigens in human arteries were observed primarily on endothelial cells - blood type A (BTA) antigens co-localized with endothelial marker CD31 (Fig1B, Pre-treatment). In vitro static treatment of arteries demonstrated 92±1% of BTA antigen removal with 1 µg/mL enzyme (Fig1B). Blood type antigen in human lungs were observed on both endothelial and epithelial cells. Within 1h of perfusion, human lungs treated with enzyme had remarkable clearance of intravascular BTA antigens (Fig1C). Similar results were observed after 3h treatment. No acute physiological side effects were observed from the enzymatic treatment. Conclusion Ex vivo enzymatic treatment can efficiently remove blood type antigen in donor lungs without acute side effects. This treatment has potential to be developed as an adjuvant therapy in ABO-incompatible organ transplantation, minimizing the need for recipient antibody removal procedures or augmented immunosuppression.

Purpose Post-transplant CMV reactivation from latently-infected lung allografts is extremely prevalent and results in inferior long-term outcomes. CMV latently infected cells ubiquitously express a viral surface protein, US28. We aimed to evaluate the therapeutic effects of a novel immunotoxin (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV transmission and reactivation post-transplantation. Methods Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected pre and post EVLP and subjected to an in vitro assay designed to evaluate viral reactivation capacity (Fig 1A). Based on US28’s homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied evaluating cell death markers of CD34+ and CD14+ using flow cytometry, and the ratio of cells post:pre perfusion were calculated. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. Results Delivery of F49A-FTP during EVLP showed no acute toxic events demonstrated by no significant differences in lung physiology and levels of IL-8, IL1-b and IFN-γ between the groups. Furthermore, while control lung tissue demonstrated a median increase from baseline of 32% (Range -16% to +112.5%) in viral reactivation, treated lungs demonstrated a significant median reduction in CMV reactivation of 76% (Range -15% to -99.9%) (Fig 1B, p=0.0087). No off-target effects were noted upon F49A-FTP delivery, demonstrated by similar amounts of cell death between the groups (Fig 1C). Conclusion We demonstrate that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation and could potentially be used to treat donor organs prior to transplant. Ongoing in vivo reactivation experiments using the EVLP treated samples in a humanized mouse will further validate these findings.

The vasoactivity of circulating cross-linked hemoglobin is consistent with the acellular protein penetrating the endothelial lining of blood vessels where hemoglobin can bind nitric oxide, the signal for relaxation of the muscles that surround blood vessels. In an important contrast, derivatives of bis-tetramers that are produced from hemoglobin by chemical coupling do not cause vasoconstriction in animal models. Presumably, they are unable to enter the endothelia where hemoglobin tetramers bind to nitric oxide. In addition, hemoglobin bis-tetramers can produce nitric oxide in circulation through their intrinsic nitrite reductase activity. Examination of this activity for hemoglobin-derived bis-tetramers that are acetylated at lysyl amino groups in their α subunits reveals enhanced activity (k = 2.21 M(-1) s(-1)) compared to that of nonacetylated bis-tetramers (k = 0.70 M(-1) s(-1)). Plots of nitrite reductase activities as a function of the corresponding oxygen affinities of certain allosteric-state-stabilized derivatives reveal a significant correlation, providing a basis for interpretation of the correlated functions.

Purpose Microbial infection in donor lungs decreases organ utilization in transplantation. Previous studies have demonstrated high-dose gaseous Nitric Oxide (gNO) to be effective against bacteria, however, this concept has not been studied in the context of lung transplantation. The present study explores the antimicrobial effects and the safety of continuously inhaled high-dose gNO. Methods Effects of gNO on strains of P. aeruginosa, S. aureus, E. coli and Burkholderia cepacia (purchased from American Type Culture Collection) were performed in vitro. Agar plates were inoculated and transferred into either control (medical air) or treatment chamber (medical air + 200 ppm gNO) for 6 h. Safety of continuously inhaled high-dose gNO were studied in a porcine normothermic ex vivo lung perfusion (EVLP) model. Donor lungs with minimal cold ischemia (2h) were randomized (n=4 each) into control (normal ventilation setting) and treatment (normal ventilation setting + high-dose of gNO) group. Physiologic and biologic measures were monitored over 12 h of EVLP. Results gNO significantly reduced all strains of bacteria when compared to control (n=3 per group, Fig.1A): P. aeruginosa (5.8 ±1.27 vs 9.2 ±0.40 Log10CFU/mL, p= 0.01), S. aureus (5.6 ±0.65 vs 9.1 ±0.66 Log10CFU/mL, p= 0.002), E. coli (5.3 ±1.20 vs 9.4 ±0.47 Log10CFU/mL, p= 0.005) and Burkholderia cepacia (7.0 ±0.51 vs 8.9 ±0.57 Log10CFU/mL, p= 0.01). No significant adverse effects in lung function during EVLP were observed in lungs receiving 12h of continuous high-dose of gNO (Fig. 1B). Inflammatory cytokines at 1, 6 and 12h were similar between control and treatment (Fig. 1C). NO2 levels were Conclusion Continuous high dose gNO at 200 ppm is effective towards reducing common respiratory pathogens in vitro. Inhaled high-dose gNO appears to be safe during 12h EVLP. We are further exploring this treatment using infected human lungs. If successful high dose gNO could be part of EVLP clinical protocols.

Purpose Donor to recipient CMV mismatch leads to high incidence of CMV infection post lung transplant leading to inferior long-term patient outcomes. CMV latently infected cells ubiquitously express a surface marker, US28. We aimed to evaluate the therapeutic effects of a novel fusion toxin protein (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV reactivation post transplantation. Methods Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected to evaluate viral reactivation using an in vitro assay that induces latent CMV to reactivate to an infectious phase. Since US28 has 38% homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied using flow cytometry for cell death assessment of CD34+ and CD14+ cells. Lung function on EVLP was evaluated as a safety endpoint. Results F49A-FTP was delivered through the PA on EVLP with no acute toxic events based on physiological parameters (Figure 1A). We performed an in vitro assay using tissue samples obtained after EVLP to stimulate CMV to reactivate in order to assess efficacy of the drug. After primary culture from lung tissue samples, relative reactivation events (assessed with IFF staining for IE protein of lytic CMV, Figure 1B) occurred in 100% of controls vs. only 0.04±0.02% of treated lungs. For off-target analysis, we performed flow cytometry and calculated the ratio of post:pre perfusion CD34+ and CD14+ cells (Figure 1C). There were no changes in number of cells for both groups: CD34+ ratio 0.8±0.5 vs. 0.6±0.5 (control and F49A-FTP, respectively, p=0.4974); CD14+ ratio 1.54±0.74 vs. 1.66±1.13 (control and F49A-FTP, respectively, p=0.8660). Conclusion Our study demonstrates that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation. Ongoing ex vivo and in vivo reactivation experiments will confirm these findings.

Lanthanide ions are readily bound to ion-exchange resins. The lanthanide-containing resins serve as immobilized catalysts for the biomimetic monoacylation of diols in water using acyl phosphate monoesters as acylation agents. This method provides an efficient route for recovering the catalyst in the process of modifying RNA derivatives and carbohydrates.

Cross-linked human hemoglobins have been evaluated for clinical use as circulating oxygen carriers. However, their induction of vasoactivity was sufficiently problematic to lead to the cessation of clinical trials. The source of vasoactivity is likely to be endothelial extravasation causing the scavenging of endogenous nitric oxide. It was recently shown that species that consist of two coupled hemoglobin tetramers do not evoke vasoactivity in a sensitive murine model. Presumably these materials are too large to extravasate. In order to make this class of material more readily available, there is a need for improved methods that can form a cross-linked bis-tetramer without producing smaller species at the same time. A potentially efficient route to cross-linking and coupling two Hb tetramers is through phase-directed copper-catalyzed azide alkyne cycloaddition (PDCuAAC). However, introduction of the necessary azide-containing cross-link gives mixtures of tetrameric and bis-tetrameric proteins, as the PDCuAAC process appears to be limited to only those proteins where the cross-link containing the azide is exclusively within the β-subunits. In order to block formation of the azide cross-link within the α-subunits, subunit-specific introduction of the azide is necessary. This is achieved by blocking reaction at the reactive amino groups of the β-subunits in the site that binds the allosteric activator 2,3-diphosphoglycerate (DPG) with inositol hexaphosphate (IHP), permitting α-selective acetylation with acetyl 3,5-dibromosalicylate. After removal of IHP, reaction with an anionic cross-linker containing an azide group occurs within the β-subunits. The resulting α-acetylated β-β'-cross-linked hemoglobin azide (acHb-N3) undergoes efficient PDCuAAC with bis-alkynes to produce cross-linked bis-tetramers. Analysis of circular dichroism spectra of the modified species shows that there is little change in the structure of the globin chains as a result of the chemical modifications. The oxygenation properties are consistent with those needed for effective oxygenation in circulation, while the bis-tetrameric structure is sufficiently large to avoid extravasation and depletion of nitric oxide.