Orthotopic Left Lung Transplantation Research Articles

A Novel Rat Lung Transplantation Model Using Veno-Arterial Extracorporeal Membrane Oxygenation Support

ObjectivesExtracorporeal membrane oxygenation (ECMO) has become an important life support technique during lung transplantation. We aimed to develop a rat model for lung transplantation using veno-arterial (VA)-ECMO support. MethodsAdult male Sprague-Dawley (SD) rats weighing 400–450 g were used in this study. ECMO circuits were created by obtaining venous access from the femoral vein with subsequent extracorporeal oxygen exchange, which was then returned to the circulatory system through the left carotid artery (VA-ECMO). Simultaneously, the donor lungs were retrieved and immersed in cold, low-potassium dextran lung preservation solution. Orthotopic left lung transplantation supported by VA-ECMO was performed. Thereafter, a respiratory failure rat model was constructed using ventilation with a hypoxic and hypercapnic gas mixture, consisting of 6% O2, 8% CO2, and 86% N2, before lung transplantation. Similarly, left lung transplantation supported by VA-ECMO was performed in rats with respiratory failure. Arterial blood gas levels were measured at designated time points throughout the experiment. ResultsWe found that VA-ECMO provided sufficient oxygenation and carbon dioxide removal to allow for smooth left lung transplantation in healthy rats and those with respiratory failure. ConclusionWe established a rat model for lung transplantation using VA-ECMO. Left lung transplantation using VA-ECMO support is also feasible and safe in rat models of respiratory failure. These models provide efficient and economical models for translational medicine for lung transplantation using ECMO. Moreover, it will be invaluable to evaluate the physiological and pathophysiological roles of ECMO during lung transplantation.

A modified orthotopic left lung transplantation model in rats

To enhance the operability of the rat orthotopic left lung transplantation model, we implemented several improvements and meticulously detailed the procedure. One hundred and thirty-one healthy male Sprague Dawley rats, weighing between 250 and 300 g, were utilized, with 64 serving as donors, 64 as recipients, and 3 as sham controls. We employed a modified three-cuff technique for the orthotopic left lung transplantation. Notably, our modified perfusion method could prevent donor lung edema, while waist-shaped cuffs minimized suture slippage during anastomosis. Additionally, positioning the recipient rat in a slightly left-elevated supine position during anastomosis reduced tension on the lung hilum, thus mitigating the risk of vascular laceration. The introduction of a unique two-person anastomosis technique significantly reduced operation time and substantially improved success rates. Furthermore, maximizing inflation of donor lungs both during preservation and surgery minimized the occurrence of postoperative atelectasis. Various other procedural refinements contributed to the enhanced operability of our model. Sixty-four rat orthotopic left lung transplantations were performed with only one surgical failure observed. The acquisition time for donor lungs averaged (19 ± 4) minutes, while (11 ± 1) minutes were allocated for donor lung hilum anatomy and cuff installation. Recipient thoracotomy and left lung hilar anatomy before anastomosis required (24 ± 8) minutes, with anastomosis itself taking (31 ± 6) minutes. Remarkably, the survival rate at the 4-h postoperative mark stood at 96.7 %. Even six months post-operation, transplanted left rat lungs continued to exhibit proper inflation and contraction rhythms, displaying signs of chronic pathological changes. In summary, our modified rat model of orthotopic left lung transplantation demonstrates robust operability, significantly reducing surgical duration, improving operation success rates, and enhancing postoperative survival rates. Furthermore, its long-term survival capacity enables the simulation of acute and chronic disease processes following lung transplantation.

The nature of chronic rejection after lung transplantation: a murine orthotopic lung transplant study.

Chronic rejection is a major complication post-transplantation. Within lung transplantation, chronic rejection was considered as airway centred. Chronic Lung Allograft Dysfunction (CLAD), defined to cover all late chronic complications, makes it more difficult to understand chronic rejection from an immunological perspective. This study investigated the true nature, timing and location of chronic rejection as a whole, within mouse lung transplantation. 40 mice underwent an orthotopic left lung transplantation, were sacrificed at day 70 and evaluated by histology and in vivo µCT. For timing and location of rejection, extra grafts were sacrificed at day 7, 35, 56 and investigated by ex vivo µCT or single cell RNA (scRNA) profiling. Chronic rejection originated as innate inflammation around small arteries evolving toward adaptive organization with subsequent end-arterial fibrosis and obliterans. Subsequently, venous and pleural infiltration appeared, followed by airway related bronchiolar folding and rarely bronchiolitis obliterans was observed. Ex vivo µCT and scRNA profiling validated the time, location and sequence of events with endothelial destruction and activation as primary onset. Against the current belief, chronic rejection in lung transplantation may start as an arterial response, followed by responses in venules, pleura, and, only in the late stage, bronchioles, as may be seen in some but not all patients with CLAD.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75mg/h) monotherapy or co-solvent only (50% hydroxypropyl-β-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Development of an experimental technique for orthotopic left lung transplantation in a rabbit model

Objective: to develop, master and evaluate the efficiency of an isolated lung transplantation (LT) technique on a rabbit animal model using Perfadex Plus® solution for cold static storage. Materials and methods. Scottish Giant rabbits (n = 20) were used in this study and divided into two groups: donors and recipients. Donor lungs were preserved with Perfadex Plus® solution and stored for 6 hours at 4 °C. Recipient animals underwent unilateral orthotopic left LT. The postoperative follow-up period was 24 hours. Laboratory and instrumental control with assessment of blood gas composition, lactate level, ventilation parameters, and central hemodynamic parameters, was performed during the follow-up. Chest X-ray in direct projection was performed twice, and at the end of follow-up, material was taken for histologic examination. Results. We obtained a high oxygenation index in the post-transplant period (>350 at p < 0.023), as well as physiological indicators of lactate (3 ± 0.3 mmol/L at p < 0.002) and peak inspiratory pressure (15 ± 1 cmH2O, p < 0.001). Radiological examination showed no radiological signs of severe primary graft dysfunction in all cases (mean RALE score 1), which was confirmed by histological studies. Conclusion. Left LT in rabbits is possible, the LT technique on a biological rabbit model using Perfadex Plus® solution is valid and efficient with the achievement of satisfactory gas exchange, ventilation and metabolism parameters.

Protective Effect of Calpain Inhibition During Cold Ischemia on Ischemia-reperfusion Injury After Lung Transplantation.

Necroptosis, one of the types of regulated necrosis, causes ischemia-reperfusion (IR) lung injury. N-acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain inhibitor, is known to attenuate necroptosis and apoptosis, and the purpose of this study was to evaluate the protective effect of ALLN during cold ischemia against IR injury in a rat lung transplant model. Male Lewis rats (250-350 g) were divided into 3 groups: sham group (n = 4), nontransplantation; control group (n = 8), transplantation with IR lung injury; and ALLN group (n = 8), transplantation with IR lung injury/ALLN. Rats in the sham group underwent a simple thoracotomy, and the remaining 2 groups of rats underwent an orthotopic left lung transplant. Cold ischemic time was 15 h. After 2 h of reperfusion, physiological function, inflammatory cytokine expression, pathway activation, and the degrees of necroptosis and apoptosis were evaluated. Lung gas exchange (PaO 2 /FiO 2 ) was significantly better, and pulmonary edema was significantly improved in the ALLN group compared with the control group ( P = 0.0009, P = 0.0014). Plasma expression of interleukin-1β was significantly lower in the ALLN group than in the control group ( P = 0.0313). The proportion of necroptotic and apoptotic cells was significantly lower in the ALLN group than in the control group ( P = 0.0009), whereas the proportion of apoptotic cells remained unchanged ( P = 0.372); therefore, the calpain inhibitor was thought to suppress necroptosis. The administration of ALLN during cold ischemia appears to improve IR lung injury in a lung transplant animal model via the inhibition of necroptosis.

Normothermic <i>ex vivo</i> lung perfusion using a developed solution followed by orthotopic left lung transplantation (experimental study)

The continued unavailability of adequate organs for transplantation to meet the existing demand has resulted in a major challenge in transplantology. This is especially felt in lung transplantation (LTx). LTx is the only effective method of treatment for patients with end-stage lung diseases. Normothermic ex vivo lung perfusion (EVLP) has been proposed to increase the number of donor organs suitable for transplant – EVLP has proven itself in a number of clinical trials. The ability to restore suboptimal donor lungs, previously considered unsuitable for transplantation, can improve organ functionality, and thus increase the number of lung transplants. However, widespread implementation of ex vivo perfusion is associated with high financial costs for consumables and perfusate.Objective: to test the developed solution on an ex vivo lung perfusion model, followed by orthotopic LT under experimental conditions.Materials and methods. The experiment included lung explantation stages, static hypothermic storage, EVLP and orthotopic left LTx. Perfusion was performed in a closed perfusion system. We used our own made human albumin-based perfusion solution as perfusate. Perfusion lasted for 2 hours, and evaluation was carried out every 30 minutes. In all cases, static hypothermic storage after perfusion lasted for 4 hours. The orthotopic single-lung transplantation procedure was performed using assisted circulation, supplemented by membrane oxygenation. Postoperative follow-up was 2 hours, after which the experimental animal was euthanized.Results. Respiratory index before lung explantation was 310 ± 40 mmHg. The PaO2/FiO2 ratio had positive growth dynamics throughout the entire EVLP procedure. Oxygenation index was 437 ± 25 mm Hg after 120 minutes of perfusion. Throughout the entire EVLP procedure, there was a steady decrease in pulmonary vascular resistance (PVR). Initial PVR was 300 ± 100 dyn×s/cm5; throughout the EVLP, PVR tended to fall, reaching 38,5 ± 12 dyn×s/cm5 at the end of perfusion.Conclusion. A safe and effective EVLP using our perfusate is possible. The developed orthotopic left lung transplantation protocol under circulatory support conditions, supplemented by membrane oxygenation, showed it is efficient and reliable.

LncRNA H19 aggravates primary graft dysfunction after lung transplantation via KLF5-mediated activation of CCL28

The present study aims to elucidate the possible involvement of H19 in primary graft dysfunction (PGD) following lung transplantation (LT) and the underlying mechanism. The transcriptome data were obtained through high-throughput sequencing analysis, and the differential long noncoding RNAs and messenger RNAs were screened for coexpression analysis. The interaction among H19, KLF5 and CCL28 was analyzed. A hypoxia-induced human pulmonary microvascular endothelial cell injury model was established, in which H19 was knocked down to elucidate its effect on the lung function, inflammatory response, and cell apoptosis. An orthotopic left LT model was constructed for in vivo mechanistic validation. High-throughput transcriptome sequencing analysis revealed the involvement of the H19/KLF5/CCL28 signaling axis in PGD. Silencing of H19 reduced inflammatory response and thus improved PGD. CCL28 secreted by human pulmonary microvascular endothelial cells after LT recruited neutrophils and macrophages. Mechanistic investigations indicated that H19 augmented the expression of CCL28 by binding to the transcription factor KLF5. Abundant expression of CCL28 reversed the alleviating effect of H19 silencing on PGD. In conclusion, the results point out that H19 exerts a promoting effect on PGD through increasing KLF5 expression and the subsequent CCL28 expression. Our study provides a novel insight into the mechanism of action of H19.

Research Highlights.

Research Highlights.

337.11: Adenovirus-Mediated Adiponectin Gene Transfer in a Rat Model of Orthotopic Left Lung Transplantation

337.11: Adenovirus-Mediated Adiponectin Gene Transfer in a Rat Model of Orthotopic Left Lung Transplantation

Activation of Humoral Immunity during the Pathogenesis of Experimental Chronic Lung Allograft Dysfunction.

Alloreactive and autoreactive antibodies have been associated with the development of chronic lung allograft dysfunction (CLAD), but their pathogenic role is disputed. Orthotopic left lung transplantation was performed in the Fischer-344 to Lewis rat strain combination followed by the application of ciclosporine for 10 days. Four weeks after transplantation, lipopolysaccharide (LPS) was instilled into the trachea. Lungs were harvested before (postoperative day 28) and after LPS application (postoperative days 29, 33, 40, and 90) for histopathological, immunohistochemical, and Western blot analyses. Recipient serum was collected to investigate circulating antibodies. Lung allografts were more strongly infiltrated by B cells and deposits of immunoglobulin G and M were more prominent in allografts compared to right native lungs or isografts and increased in response to LPS instillation. LPS induced the secretion of autoreactive antibodies into the circulation of allograft and isograft recipients, while alloreactive antibodies were only rarely detected. Infiltration of B cells and accumulation of immunoglobulin, which is observed in allografts treated with LPS but not isografts or native lungs, might contribute to the pathogenesis of experimental CLAD. However, the LPS-induced appearance of circulating autoreactive antibodies does not seem to be related to CLAD, because it is observed in both, isograft and allograft recipients.

Antibodies against complement component C5 prevent antibody-mediated rejection after lung transplantation in murine orthotopic models with skin-graft-induced pre-sensitization

ObjectiveAntibody-mediated rejection (AMR) could induce acute or chronic graft failure during organ transplantation. Several reports have shown that anti-C5 antibodies are effective against AMR after kidney transplantation. However, few reports have assessed the efficacy of anti-C5 antibodies against AMR after lung transplantation. Therefore, this study aimed to evaluate the efficacy of this novel therapy against AMR after lung transplantation.MethodsBALB/c and C57BL/6 mice were used as donors and recipients. One group was pre-sensitized (PS) by skin transplantation 14 days before lung transplantation. The other group was non-sensitized (NS). Orthotopic left-lung transplantation was performed in both groups. Animals were killed at 2 or 7 days after lung transplantation and evaluated for histopathology, C4d immunostaining, and serum donor-specific antibodies (DSAs) (n = 5 per group). Isograft (IS) models with C57BL/6 mice were used as controls. To evaluate the efficacy of C5 inhibition, other animals, which received similar treatments to those in the PS group, were treated with anti-C5 antibodies, cyclosporine/methylprednisolone, anti-C5 antibodies/cyclosporine/methylprednisolone, or isotype-matched irrelevant control monoclonal antibodies (n = 5 per group).ResultsTwo days after lung transplantation, the NS group exhibited mild, localized graft-rejection features (rejection score: 0.45 ± 0.08, p = 0.107). The PS group exhibited AMR features with a significantly higher rejection score (2.29 ± 0.42, p = 0.001), C4d vascular-endothelium deposition, and substantial presence of serum DSA. On day 7 after lung transplantation, both groups showed extensive graft alveolar wall destruction, and high acute-rejection scores. Mice receiving anti-C5 antibodies or anti-C5/antibodies/cyclosporine/methylprednisolone demonstrated significantly lower acute-rejection scores (0.63 ± 0.23, p = 0.002; 0.59 ± 0.22, p = 0.001, respectively) than those receiving isotype control antibodies.ConclusionsMurine orthotopic allograft lung transplant models met the clinical diagnosis and pathogenesis classification criteria of AMR. In these models, anti-C5 antibodies suppressed AMR. Therefore, anti-C5 therapy may be effective against AMR after lung transplantation.

Mapping the Diversity of the Innate Immune System over Time After Left Lung Transplantation in Mice

Purpose Following lung transplantation, the surgical intervention with ischemic periods and the foreign allograft are the first to trigger a response from the innate immune system, which represents the primary nonspecific defense against “non-self”. In this study, we investigated the diversity of the emerging innate immune cells and the timing of their appearance in a murine orthotopic left lung transplant model. Methods Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6). All mice received daily immunosuppression of 10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone. Serial sacrifice was performed at day 1, 7 and 35 post-transplantation (n=6 at each time point for each group). Left transplanted lungs were harvested, single cell suspension was made and absolute numbers of innate immune cells were quantified by flow cytometry. Results Following transplantation, isografts and allografts showed an increase in neutrophil (p=0.017) and tissue resident NK cell (p=0.002) numbers at day 1, and in eosinophil (p=0.002) numbers at day 7 post-lung transplant. Granulocytes demonstrated similar patterns in isografts and allografts. Allografts demonstrated an increase in antigen presenting cells i.e. patrolling monocytes (Ly6C-) (p=0.002), interstitial macrophages (p<0.0001) and dendritic cells (p=0.005) at day 7 compared to isografts. Classical NK cells were increased in allografts at day 7 versus isografts (p=0.03). All cell numbers returned to baseline isograft values at day 35 post-transplant, except for dendritic cells (p=0.0045). Conclusion Diverse innate immune cell types show different patterns over time post-lung transplantation depending on transplant induced ischemia-reperfusion injury and/or alloimmune response against the foreign graft. These differences may be very important to dissect the various pathophysiological processes triggered by lung transplantation. Following lung transplantation, the surgical intervention with ischemic periods and the foreign allograft are the first to trigger a response from the innate immune system, which represents the primary nonspecific defense against “non-self”. In this study, we investigated the diversity of the emerging innate immune cells and the timing of their appearance in a murine orthotopic left lung transplant model. Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6). All mice received daily immunosuppression of 10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone. Serial sacrifice was performed at day 1, 7 and 35 post-transplantation (n=6 at each time point for each group). Left transplanted lungs were harvested, single cell suspension was made and absolute numbers of innate immune cells were quantified by flow cytometry. Following transplantation, isografts and allografts showed an increase in neutrophil (p=0.017) and tissue resident NK cell (p=0.002) numbers at day 1, and in eosinophil (p=0.002) numbers at day 7 post-lung transplant. Granulocytes demonstrated similar patterns in isografts and allografts. Allografts demonstrated an increase in antigen presenting cells i.e. patrolling monocytes (Ly6C-) (p=0.002), interstitial macrophages (p<0.0001) and dendritic cells (p=0.005) at day 7 compared to isografts. Classical NK cells were increased in allografts at day 7 versus isografts (p=0.03). All cell numbers returned to baseline isograft values at day 35 post-transplant, except for dendritic cells (p=0.0045). Diverse innate immune cell types show different patterns over time post-lung transplantation depending on transplant induced ischemia-reperfusion injury and/or alloimmune response against the foreign graft. These differences may be very important to dissect the various pathophysiological processes triggered by lung transplantation.

Prolonged Cold Ischemia and Indirect Alloimmunity Are Fundamental for the Development of Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Lung transplantation has the worst long-term survival in solid organ transplantation due to chronic lung allograft dysfunction (CLAD). Lymphatic vessels play central roles in alloimmunity by bridging innate and adaptive immune response. The purpose of this project is to study the role of lymphatic endothelium in CLAD development and to analyse the impact of prolonged donor cold ischemia on CLAD development in the experimental lung transplantation in mouse. <h3>Methods</h3> Single mismatch orthotopic left lung transplantation (left lungs from HLA-A2 knock-in mice on a C57BL/6J background or C57BL/6J as donor mice were orthotopically transplanted to C57BL/6J recipient mice) was used as a CLAD model. Donor lungs were induced to 4 hours cold ischemia at 4°C. CLAD development was analysed by Masson's trichrome staining. Lymphatic vessels were stained by anti-podoplanin and anti-LYVE-1 antibodies. Pro-inflammatory macrophages were stained by anti-iNOS antibody, anti-inflammatory macrophages were stained by anti-CD206 antibody. VEGFC source in macrophages were identified by dual immunofluorescence. <h3>Results</h3> Short cold allograft ischemia (1h, 4°C) resulted in the development of lymphocytic bronchiolitis (LB) 8 weeks after the surgery. Prolonged cold allograft ischemia resulted in the development of lymphocytic bronchiolitis (LB) already one week after the surgery and led to the late stage CLAD 8 weeks after reperfusion. All histological features of bronchiolitis obliterate syndrome and restrictive allograft syndrome (RAS) were observed in 8-weeks allogeneic lungs and were similar to human findings. The lungs from syngeneic recipients showed no pathological changes. In the allogeneic recipients, the lymphatic density increased during the CLAD development. VEGFC expression was observed in iNOS and CD206 positive macrophages. <h3>Conclusion</h3> Prolonged cold lung ischemia seems to facilitate the development of experimental late stage CLAD. Indirect alloimmunity and deteriorated ischemia-reperfusion injury are fundamental for the late stage CLAD development. Experimental allogeneic lung transplantation with prolonged cold ischemia leads to a lymphatic phenotype similar to human RAS. Proinflammatory and anti-inflammatory macrophages are likely to drive lymphangiogenesis by increased VEGFC expression.

Time to Try Something New: Establishing Rat Models of Lung Transplantation Under ECMO Support

<h3>Purpose</h3> Extracorporeal membrane oxygenation (ECMO) has emerged as an important preoperative, perioperative, and postoperative life-support requisite in lung transplantation. Therefore, we aimed to develop rat models of lung transplantation under veno-arterial (VA) and veno-venous (VV) ECMO support. <h3>Methods</h3> Adult male Sprague-Dawley rats weighing 400-450g (recipients, n=10; donors, n=10) were used. The ECMO circuits were initiated by venous drainage from the femoral vein with extracorporeal oxygen exchange and returned to the blood circulation through the right carotid artery (VA-ECMO) or external jugular vein (VV-ECMO). Before ECMO implantation, 5ml heparinized blood from the donor rat was drawn into tubes. Simultaneously, donor lungs were obtained after pulmonary arterial perfusion with low potassium dextran lung preservation solution. ECMO-supported orthotopic left lung transplantation was performed by using cuff technique. Arterial blood gases were measured at designated intervals throughout the experiments. <h3>Results</h3> We found that VA- and VV-ECMO provided sufficient oxygenation to support a smooth left single lung transplantation procedure. The intraoperative hemodynamic and blood gas parameters were maintained stable. <h3>Conclusion</h3> In this study, we first established rat models of lung transplantation under VA and VV ECMO support. This model provides an efficient, economical, and feasible platform for studying lung transplantation with ECMO. This model can be preclinically applied to unravel significant insights in support of further translational models in large animal and human lung transplantation.

Endothelial Cell Injury and Activation in a Murine Model of Left Lung Transplantation

<h3>Purpose</h3> Endothelial cells are the first non-self-barrier encountered by the recipient's immune system after allograft transplantation and thus are involved early during alloimmune response. Our goal was to study processes of endothelial cell injury/activation in a mouse model of lung transplantation. <h3>Methods</h3> Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6), both received daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone) and were serially sacrificed at day 1, 7 and 35 post-transplant (n=6/timepoint/group). Left transplanted lungs were made into single cell suspension and absolute cell numbers were quantified by flow cytometry for CD31+ endothelial cells, CD45+ leukocytes and MHCI/II, VCAM-1 and ICAM-1 for cell activation. One additional allograft and isograft were scanned with high resolution (HR) <i>ex vivo</i> microCT (µCT) to visualize airways and blood vessels at day 70 post transplantation. <h3>Results</h3> Endothelial cell numbers decreased from day 1 until day 7 (p=0.03) in allografts and recovered slightly at day 35. While in isografts, endothelial cells seemed to increase non-significantly over time. In contrast, leukocytes significantly increased at day 7 in allografts versus day 1 (p=0.036) and isografts at day 7 (p=0.003). Leukocytes remained stable in isografts over time (p=0.3). Mean fluorescence intensity (MFI) was increased for MHC I/II and VCAM-1 (p=0.003; p<0.0001; p=0.04) on endothelial cells, the MFI of MHC I/II and ICAM-1 on leukocytes was increased in allografts versus isografts (p=0.0002; p=0.006; p=0.002). HR µCT showed normal airway morphology while lumen of arteries and veins was narrowed in allograft compared to isograft. Arterioles and venules were occluded in allograft. <h3>Conclusion</h3> Destruction of endothelial cells may represent the very first onset of alloimmune response post lung transplantation and warrant further investigations towards diagnostic and therapeutic approaches.

Right lung transplantation with a left-to-right inverted anastomosis in a rat model

ObjectiveRight lung transplantation in rats has been attempted occasionally, but the technical complexity makes it challenging to apply routinely. Additionally, basic research on inverted lobar lung transplantation is scarce because of the lack of a cost-effective experimental model. We first reported right lung transplantation in a rat model using left-to-right inverted anastomosis to imitate the principle of clinically inverted lung transplantation. MethodsRight lung transplantation was performed in 10 consecutive rats. By using a 3-cuff technique, the left lung of the donor rat was implanted into the right thoracic cavity of the recipient rat. The rat lung graft was rotated 180° along the vertical axis to achieve anatomic matching of right hilar structures. Another 10 consecutive rats had received orthotopic left lung transplantation as a control. ResultsAll lung transplantation procedures were technically successful without intraoperative failure. One rat (10%) died of full pulmonary atelectasis after right lung transplantation, whereas all rats survived after left lung transplantation. No significant difference was observed in heart-lung block retrieval (8.6 ± 0.8 vs 8.4 ± 0.9 minutes), cuff preparation (8.3 ± 0.9 vs 8.7 ± 0.9 minutes), or total procedure time (58.2 ± 2.6 vs 56.6 ± 2.1 minutes) between the right lung transplantation and standard left lung transplantation groups (P > .05), although the cold ischemia time (14.2 ± 0.9 vs 25.5 ± 1.7 minutes) and warm ischemia time (19.8 ± 1.5 vs 13.7 ± 1.8 minutes) were different (P < .001). ConclusionsRight lung transplantation with a left-to-right inverted anastomosis in a rat model is technically easy to master, expeditious, and reproducible. It can potentially imitate the principle of clinically inverted lung transplantation and become an alternative to standard left lung transplantation.

Resolution of Post-Lung Transplant Ischemia-Reperfusion Injury is Mediated via Resolvin D1-Alx/FPR2 and Maresin 1-LGR6 Receptor Signaling

Purpose Dysregulation of inflammation-resolution pathways can lead to post-lung transplant (LTx) ischemia-reperfusion injury (IRI) and allograft dysfunction. We investigated the role of ω-3-derived specialized pro-resolving lipid mediators, i.e. Resolvin D1 (RvD1) and Maresin-1 (MaR1), and the respective receptor signaling in the resolution of lung IRI. Methods RvD1 and MaR1 expressions were analyzed in bronchoalveolar lavage (BAL) from patients undergoing LTx on post-operative days 0, 1 and 7 using mass spectrometry and ELISA. C57BL/6 mice underwent sham surgery or lung IRI (1hr left lung ischemia followed by 6hr reperfusion) using an in vivo hilar ligation model. RvD1 and/or MaR1 (100ng/kg each; given intratracheally 1hr prior to IR) was administered to mice that were previously treated with/without siRNA for FPR2 (receptor for RvD1) or LGR6 (receptor for MaR1; 10μg given i.p. each). Orthotopic left lung transplants were performed between Balb/C brain dead donors and C57Bl/6 recipients to analyze FPR2 and LGR6 receptor expressions at 6hrs and 48hrs post-transplant. Macrophage (MH-S) and epithelial (MLE12) cells were exposed to hypoxia/reoxygenation (HR; 3hr/3hr or 24hrs) followed by cytokine analysis. Groups (n=3-10) were compared using ANOVA with post-hoc Tukey's test. Results RvD1 and MaR1 levels were significantly increased in BAL of LTx patients on day 7 compared to days 0 and 1. In the hilar ligation IR model, concomitant treatment with recombinant RvD1 and MaR1 significantly attenuated lung dysfunction after 6hrs compared to either treatment or IR alone, that was abolished by pre-treatment with siRNA for FPR2 and LGR6, respectively. Lung inflammation (IL-17, TNF-α, CXCL1, HMGB1), edema (wet/dry ratio) and injury (neutrophil infiltration) were significantly attenuated in mice treated with RvD1+MaR1 compared to either treatment or IR alone. FPR2 and LGR6 expressions were increased in lung tissue after 48hrs compared to 6hrs post-LTx. RvD1 attenuated HMGB1 and TNF-α secretion in HR-exposed MH-S cells that was abolished by siRNA for FPR2, and MaR1 treatment mitigated CXCL1 secretion by HR-exposed MLE12 cells that was abolished by siRNA for LGR6. Conclusion Our results suggest that resolution of lung IRI is mediated by RvD1-FPR2 and MaR1-LGR6 signaling pathways on macrophages and epithelial cells, respectively.

Development of a Regulatory T Cell-Permissive Immunosuppression Protocol in a Rat Model of Ex Vivo Lung Perfusion Followed by Lung Transplantation

<h3>Purpose</h3> CD4⁺CD25^highFoxp3⁺ regulatory T cells (Treg) can establish immunologic tolerance in animal models but are hampered by immunosuppressive drugs. IL-2-anti-IL-2 complexes (IL-2c) promote Treg expansion in vivo even with concomitant calcineurin inhibitor administration. Here, we combined recipient IL-2c therapy with pre-transplant Treg administration during ex vivo lung perfusion (EVLP), to test the hypothesis that this strategy would facilitate expansion and function of the Tregs within the graft. <h3>Methods</h3> We performed rat orthotopic left lung transplantation (OLT) experiments using Fischer 344 (F344, RT-1^lv1) donors and Wistar Kyoto (WKy, RT-1^l) recipients. Expanded WKy Tregs were administered to the F344 donor lungs during EVLP. Some animals received IL-2c (30 ug recombinant mouse IL-2 and 150 ug anti-mouse IL-2, given i.p. once on day 0) and/or Tacrolimus (Tac, 0.1mg/kg/day, 7 days). Treatment groups were: no treatment (n=7), Tac (n=5), IL-2c/Tac (n=4), Treg (n=6), and Treg/IL-2c/Tac (n=5). Necropsy was performed 7 days post-transplant. <h3>Results</h3> Graft CD25^highFoxp3⁺ content increased as a result of Treg therapy (p<0.001, Fig 1). ISHLT A and B grades were attenuated in the IL-2c/Tac and Treg/IL-2c/Tac groups (p<0.001, Fig 2-3). Graft Treg content and Treg-to-effector T cell ratio (Treg/Teff) at day 7 was increased by Treg/IL-2c/Tac (p<0.001, p=0.025, Fig 4). <h3>Conclusion</h3> Treg therapy increased intragraft Treg content, and IL-2c/Tac further increased intragraft Tregs 7 days after transplantation, with reductions observed in A and B rejection scores. The combination of graft-directed Treg cell therapy with a Treg-permissive immunosuppressive strategy may be a viable therapeutic approach for effective pre-transplant graft immunomodulation in lung transplantation.

79664 Complement Driven Auto-Reactive Antibodies in Lung Transplantation

ABSTRACT IMPACT: Our work unveils a novel mechanism of ischemia repurfusion injury driven by pre-existing autoimmunity following lung transplant and a potential therapeutic strategy for blocking complement-dependent injury thereby reducing risk of lung transplant rejection. OBJECTIVES/GOALS: Our goal was to determine if pre-existing autoimmune autoantibodies, such as those resulting from cigarette smoke (CS), contribute to graft rejection in lung transplantation (LTx) and if autoreactive-mediated graft injury is complement-dependent. METHODS/STUDY POPULATION: For in vivo experiments, we utilized our emphysema mouse model. Briefly, eight-week-old C57BL/6J mice are exposed to 3R4F reference cigarette smoke 5 hours per day, 5 days a week for 6 months. Upon completion, cigarette smoked (CS) mice and control (NS) mice received syngeneic orthotopic left-lung transplant from age-matched C57BL/6J donors. To determine if pre-existing autoreactivity mediated graft injury was complement-dependent we treated CS-LTx mice with a novel, bifunctional complement inhibitor. Autoantibody levels were measured by ELISA and lung injury was assessed by blinded histopathological analyses. Complement inhibition was verified by immunofluorescence. RESULTS/ANTICIPATED RESULTS: We found that CS-exposure leads to production of autoreactive antibodies towards extracellular matrix (ECM) components and contributes to graft injury. Interestingly, LTx into CS exposed mice further increased de-novo ECM autoantibody development. Lastly, treatment with our novel, bifunctional complement inhibitor blocked autoantibody spreading and significantly reduced graft rejection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These data demonstrate that smoking induces pre-LTx autoreactivity to ECM proteins that promotes graft injury following LTx. Furthermore, complement inhibition reduces autoantibody production and protects the graft from injury.