Orthotopic Lung Transplantation Research Articles

Techniques for lung transplantation in the rat

The rat is an important model organism for lung transplantation research. Orthotopic rat lung transplantation is a complex procedure, which requires advanced microsurgical techniques. This technical paper describes in detail highly reproducible intraoperative microsurgical techniques and peri-operative management, including the donor procedure, recipient anesthetic management, the recipient operation and anticipated peri-operative complications.

Vein Suturing Results in Worse Lung Graft Outcomes Compared to the Cuff Method

Background: The rat orthotopic lung transplant model is not widely used yet because of the complexity of the procedure, in particular, venous anastomosis. Here, we performed a rat orthotopic lung transplantation using either the suture (ST) or cuff (CT) method for vein anastomosis. Objectives: To compare the vein ST and CT techniques in terms of operative time, success, recipient survival, and early histological outcomes was the objective of this study. Methods: A total of 24 left lung transplants in rats were performed. Twelve syngeneic (Lewis to Lewis) and 12 allogeneic (Brown-Norway to Lewis) lung transplants were performed using either the vein ST or the CT procedure. Arterial and bronchial anastomoses were performed with the CT technique. Graft histological damage was evaluated 3–7 days post-transplant in all rat lungs. Results: The surgical success rate was 75% in both the ST and CT groups. Failures related mainly to vein bleeding (n = 2 in the ST group) and thrombosis (n = 1 in the ST group; n = 2 in the CT group). Total ischemia time was longer in the ST group (122 ± 25 min in ST group vs. 83 ± 10 min in CT group, mean ± SD), due to prolonged warm ischemia time (60 ± 12 min in the ST group vs. 21 ± 5 min in the CT group, mean ± SD), reflecting the time required to complete the vein ST procedure. The prolonged warm ischemia time resulted in significantly higher vascular inflammation in syngeneic grafts (2.3 ± 1.2 ST group vs. 0 in the CT group, mean ± SD) and in increased severity of ischemia/reperfusion injury and acute graft rejection (3.6 ± 0.4 in the ST group vs. 2.6 ± 0.4 in the CT group, mean ± SD) in allogeneic lung transplants. Conclusions: The vein ST technique is a more time-consuming procedure than the CT method and the prolonged anastomosis time has a deleterious impact on transplant outcomes. These findings suggest that warm ischemia time – one of the modifiable transplant factors – should be considered a major risk factor in lung transplantation, particularly in the setting of donation after cardiac death.

A B cell-dependent pathway drives chronic lung allograft rejection after ischemia-reperfusion injury in mice.

Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplant (LT). Ischemia-reperfusion injury (IRI) promotes chronic rejection (CR) and CLAD, but the underlying mechanisms are not well understood. To examine mechanisms linking IRI to CR, a mouse orthotopic LT model using a minor alloantigen strain mismatch (C57BL/10 [B10, H-2b ] → C57BL/6 [B6, H-2b ]) and isograft controls (B6→B6) was used with antecedent minimal or prolonged graft storage. The latter resulted in IRI with subsequent airway and parenchymal fibrosis in prolonged storage allografts but not isografts. This pattern of CR after IRI was associated with the formation of B cell-rich tertiary lymphoid organs within the grafts and circulating autoantibodies. These processes were attenuated by B cell depletion, despite preservation of allograft T cell content. Our observations suggest that IRI may promote B cell recruitment that drives CR after LT. These observations have implications for the mechanisms leading to CLAD after LT.

Negative impact of recipient SPRED2 deficiency on transplanted lung in a mouse model

Ischemia-reperfusion injury (IRI) after lung transplantation mainly contributes to the development of primary graft dysfunction. The Sprouty-related EVH1-domain-containing (SPRED) protein family inhibits the mitogen activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway. Our study was aimed at examining the role of SPRED2 in IRI in mice that received orthotopic lung transplantation. Syngeneic mouse lung transplantation was performed in wild-type C57BL/6 J (WT) mice and Spred2 knockout (Spred2−/−) mice on the C57BL/6 J background from the WT donor. Four hours after reperfusion, blood gas analysis was performed, and lung grafts were sacrificed and analyzed. By using arterial oxygen tension measurements and histological evaluation using Lung Injury Score, we revealed more severe IRI in the grafts transplanted to Spred2−/− recipients, which manifested as exacerbated airway epithelial cell damage, interstitial edema with hemorrhage and neutrophil infiltration. Intragraft ERK1/2 activation and expression levels of proinflammatory cytokines and chemokines in Spred2−/− recipients were higher than those in WT recipients. SPRED2 plays an important role in protecting the lungs from IRI in lung transplantation recipients. We suggest that focused treatments suppressing the activity of the MAPK/ERK pathway in transplantation recipients could be the potential therapeutic option for the prevention of lung IRI.

Alpha-1 Antitrypsin Attenuates Acute Lung Allograft Injury in a Rat Lung Transplant Model.

Background.Ischemia-reperfusion injury (IRI) after lung transplantation triggers a cascade of inflammatory changes that can contribute to acute allograft injury. This influences both the short- and long-term survival of the lung allograft. Alpha-1 antitrypsin (AAT) is a protease inhibitor with known anti-inflammatory and immune-regulatory properties that mitigate tissue damage. This study explores the protective effects of AAT in the setting of IRI utilizing a rat lung transplant model.Methods.Orthotopic left single lung transplantation was performed from Lewis to Sprague-Dawley rats; recipients did not receive systemic immunosuppression. Before transplantation, the donor lungs were primed with either albumin (control) or AAT. Starting the day of transplantation, recipient rats also received either albumin (control) or AAT with subsequent doses administered over the next 7 days. On the eighth postoperative day, lung allografts were recovered and analyzed.Results.Degree of inflammatory infiltrate, as quantified by the allograft weight (g)/body weight (kg) ratio, was significantly reduced in the AAT-treated group compared with controls (3.5 vs 7.7, respectively, P < 0.05). Treatment with AAT also significantly decreased allograft necrosis in treated animals, as measured by a semiquantitative score that ranged from 0 to 4 (1.25 vs 4, P < 0.05). In addition, lymphocytes isolated from recipients treatment group showed significant proliferative inhibition via a mixed lymphocyte response assay in response to donor antigens.Conclusions.AAT attenuates acute allograft injury and necrosis in a rat model of lung transplantation, suggesting that AAT may play a role in reducing IRI-induced inflammation.

Detection of lung transplant rejection in a rat model using hyperpolarized [1-13 C] pyruvate-based metabolic imaging.

The current standard for noninvasive imaging of acute rejection consists of X-ray/CT, which derive their contrast from changes in ventilation, inflammation and edema, as well as remodeling during rejection. We propose the use of hyperpolarized [1-13 C] pyruvate MRI-which provides real-time metabolic assessment of tissue-as an early biomarker for tissue rejection. In this preliminary study, we used μCT-derived parameters and HP 13 C MR-derived biomarkers to predict rejection in an orthotopic left lung transplant model in both allogeneic and syngeneic rats. On day 3, the normalized lung density-a parameter that accounts for both lung volume (mL) and density (HU)-was -0.335 (CI: -0.598, -0.073) and-0.473 (CI: -0.726, -0.220) for the allograft and isograft, respectively (not significant, 0.40). The lactate-to-pyruvate ratios-derived from the HP 13 C MRI-for the allograft and isograft were 0.200 (CI: 0.161, 0.240) and 0.114 (CI: 0.074, 0.153), respectively (significant, 0.020). Both techniques showed tissue rejection on day 7. A separate sub-study revealed CD8+ cells as the primary source of the lactate-to-pyruvate signal. Our study suggests that hyperpolarized (HP) [1-13 C] pyruvate MRI is a promising early biomarker for tissue rejection that provides metabolic assessment in real time based on changes in cellularity and metabolism of lung tissue and the infiltrating inflammatory cells, and may be able to predict tissue rejection earlier than X-ray/CT.

Processing and CD3+/CD19+ depletion of cadaveric vertebral bone marrow for primary immunodeficiency patients undergoing sequential bilateral orthotopic lung transplant (BOLT) and bone marrow transplant (BMT)

Background & Aim Patients with primary immune-deficiency (PID) may develop pulmonary complications and at that stage most are ineligible for either lung transplant (LTX) or bone marrow transplant (BMT) alone due to futility. We are conducting a first in human prospective trial, ClinicalTrials.gov: NCT01852370 where PID patients in lung failure -past the opportunity for curative BMT alone- may receive a 2 of 6 HLA antigen-matched cadaveric BOLT and minimum 8 weeks alter cryopreserved BMT from the same deceased UNOS donor. We hypothesized: 1) hematopoietic stem cells prepared from organ donor vertebral bodies (VB) would yield greater CD34+ cell dose compared to iliac crest aspirates-in part due to limited harvest time to avoid delay in the activity of organ recovery teams; 2) cryopreserved CD3/CD19 depleted bone marrow could generate durable hematopoiesis and donor derived immunity. Here we present the first 5 consecutive cases vertebral bone marrow (VBM) processing experience at Children's Hospital of Pittsburgh and subsequent CD3+/CD19+ cell depletion on the CliniMACS® device. With a threshold viability of 70% prior to cryopreservation the CD3/CD19-depleted product must contain 1 × 10E+6/kg CD34+ cells and 1 × 10E+5/kg CD3+ T cells to achieve durable engraftment and reduce the risk of GVHD respectively.® Methods, Results & Conclusion For details of pre/post processing, see Table. Since early 2017 we introduced chest/abdominal cavity wash with 1:1 saline-betadine mix and scrubbing T11-L4VB logs with chlora-prep that has eliminated bacterial contamination for the last 3 products. The first 2 were infused without adverse events following IRB/FDA approved antibiotic prophylaxis. Initially, processing of the VBs was arduous, using a scalpel and wire brush to clean the logs, then dissecting into small pieces with a mallet and osteotome. Since early 2017 a Stryker RemB saw has been used. A physical task that had taken 2 technologists 3 hours to complete has become almost effortless and one tech is freed up. The target pre-cryo CD34+ dose and viabilities have met target at all times, in fact CD34+ recovery has increased >70%, see Table. The entire process (dissection, crushing, filtering, CD3/CD19 depletion and cryopreservation) can be completed in 12 hours with 3 technologists. In summary, we have developed a complex sequence of procedures that even exceeded our target of 2E+06 CD34/kg pre-cryo dose (mean: 4.49E+06) leading to prompt and multilineage engraftment in 3 of the first 4 analyzable patients.

An obligatory role for club cells in preventing obliterative bronchiolitis in lung transplants.

Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell-mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions and poorly regenerate club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex-vivo analysis reveals a specific role for alloantigen-primed effector CD8+ T cells in preventing club cell proliferation and maintenance. Taken together, we demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a new model to identify the underlying mechanisms of OB.

Inhibition of mitochondrial autophagy protects donor lungs for lung transplantation against ischaemia-reperfusion injury in rats via the mTOR pathway.

Impaired mitochondrial function is a key factor attributing to lung ischaemia‐reperfusion (IR) injury, which contributes to major post‐transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3‐MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia‐reoxygenation (H/R) were monitored after 3‐MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3‐MA inhibited cell apoptosis of donor lung to alleviate I/R‐induced lung injury as well as inhibited H/R‐induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R‐ and H/R‐mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R‐induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury.

Development of Novel Murine Antibody Mediated Rejection Model after Orthotopic Lung Transplant

Purpose Antibody mediated rejection (AMR) has been playing an important role in organ transplantation inducing acute or chronic graft failure, which directly influence the survival of graft. There is little knowledge about AMR in lung transplant compared with other organ. Animal disease model is warranted to elucidate mechanism and development of novel drug for AMR, hence our purpose of this study is to establish murine AMR model of lung transplant. Methods As allograft model, BALB/c (Major histocompatibility complex; MHC H2b) and C57BL/6 (MHC H2b) were used for donor and recipient in order to detect and enhance AMR. Study groups were divided into two groups, including pre-sensitized by skin transplant (PS) group (n=10) and non-sensitized (NS) group (n=10). Skin transplant was performed 14days before lung transplant only in PS group, and orthotopic left lung transplant were performed in both groups. They were sacrificed at 2day or 7day post lung transplant, and evaluated for histopathology including acute rejection score, C4d immunostaining, and donor specific antibody (DSA) in serum. Isograft model (IS) using C57/BL6 murine was used for control. Results NS group showed only localized rejection features on their graft and rejection score (0.36±0.13, p=0.13; Fig a, b) at day 2 after lung transplant, on the other hand, PS group showed clinical features of AMR, including high rejection score (2.19±0.27, p=0.003), C4d deposition on vascular endothelium, and DSA in serum at day 2 after lung transplant. On day 7 after lung transplant, the both groups showed extensive destruction of alveolar wall in graft, high acute rejection score (NS 3.25±0.18, p Conclusion The murine orthotopic allograft lung transplant model satisfied criteria of AMR. The allograft model pre-sensitized by skin transplant showed enhanced AMR at early period post lung transplant; therefore this model could be useful to examine pathogenesis of AMR and development of new therapy for AMR.

Prolonged Cold-Ischemia Increases Reactive Oxygen Species and Activates Autophagy by Enhancing Glycolysis in the Graft through the mTOR Signaling Pathway in Orthotopic Lung Transplantation of Rats

Purpose Ischemia-reperfusion (I/R) injury continues to plague the field of graft transplantation and is a major contributor to early graft dysfunction or failure after organ transplantation. The aim of the present study is to investigate the mechanisms by which prolonged CI influences the autophagy in the graft lung, in which the mTOR signaling pathway was involved. Methods Orthotopic lung transplantation models were induced in rats, whose lungs were preserved under CI conditions for varying durations. Autophagy activities, energy metabolism, and reactive oxygen species (ROS) level were determined at each time point. The perfusion treatments of inhibitor (3-Methyladenine [3-MA]) and activator (rapamycin) of autophagy and inhibitor of oxidative phosphorylation (oligomycin [OM]) were performed to verify the relationships among mTOR signaling pathway and autophagy in the cold I/R injury. Results Autophagy was induced by CI preservation, which was peaked at 12 h after CI. Additionally, a coexistence of reduced oxidative phosphorylation and elevated ROS level was observed in rat lung tissues. Increased activities of key glycolytic enzymes, glucose consumption and lactic acid production followed with prolonged CI, which became stable after a 9 h period of CI. Of importance, the perfusion treatment of 3-MA or OM decreased ROS level and Beclin-1, PINK1, Parkin, and AMPK expression, but increased mTOR expression; while the perfusion treatment of rapamycin induced a reverse tendency. Conclusion These findings demonstrate that activated autophagy during prolonged CI preservation leads to poor oxidative phosphorylation, robust glycolysis, and ROS level rise in the graft lung via the mTOR signaling pathway.

Endothelial Stabilization Reduces the Effects of Brain Death Induced Acute Lung Injury Post-Transplant

Purpose Donor Brain death (BD) provokes activation of inflammatory pathways resulting in acute lung injury (BD-ALI), characterized by the breakdown of endothelial and epithelial barrier function. These injuries are further propagated in the early transplant period by ischemia reperfusion injury (IRI) which together adversely affect post-transplant outcomes. We have developed and characterized a novel small molecule gap/tight junction stabilizer, aCT1, which we have shown to improve cellular communication and integrity. Herein we explored the use of aCT1 as a pretreatment therapeutic to protect donor lungs from the effects of BD-ALI. Methods A mouse model of BD and orthotopic left lung transplant (LTx) were utilized to examine the effects of aCT1 on barrier function stabilization in BD and subsequent impact on LTx outcomes. Following BD induction donors were nebulized with aCT1 or vehicle and ventilated for a further 3 hours. Lungs were then removed to be flushed with and stored in Perfadex solution at 4°C. After 18 hours of cold storage the right lung was processed for histological analysis and the left lung transplanted into CD45.1 B6 mice. Transplanted lungs were then harvested 6 hours post-Tx for analysis of graft injury via histopathology and protein quantification. For controls a standard living donor (LD) model was used for comparison. Results After 3 hrs of BD induction and 18 hours cold ischemia, lungs had BD-ALI features which included significant increases in immune cell infiltration, edema, and endothelial activation. Treatment with aCT1 had no impact on BD-ALI features. 6 hrs post-Tx lungs from BD donors had significantly increased IRI as compared to LD controls, as determined by increased immune cell infiltration and pathological assessment of injury. Treatment of BD donors with aCT1 significantly reduced this BD injury profile, with pathological injury not dissimilar to that seen in LD controls (BD+aCT1 3 vs. BD 8.25, BD+aCT1 3 vs. LD 3, respectively. p Conclusion These data support gap junction stabilization as a viable option for protecting donor lungs against BD-ALI exacerbated ischemia reperfusion injury.

Circulating Exosomes with Lung Self-Antigens, Kα1Tubulin and Collagen V, and Immune Responses to Lung Self-Antigens Precedes Histological Evidence of Chronic Rejection Following Orthotopic Murine Lung Transplantation

Purpose Lung transplantation (LTx) is a treatment option for end-stage pulmonary diseases. Despite improvements in surgical techniques and post-transplant (Tx) immunosuppression, LTx continues to have one of the poorest long-term outcomes compared to other solid organ Tx, with five year survival of about 50%. Methods Using an orthotopic single LTx model of B6D2F1/J to DBA/2J strain combination, we demonstrate that >80% of transplanted animals developed histological signs of chronic rejection by day 30 of the transplanted lung but not native. We collected blood at days 7, 14, 28, and 30 and serum was tested by ELISA for development of antibodies to lung self-antigens (SAgs), Collagen V (Col-V) and K-alpha 1 Tubulin (Kα1T). Results Significantly elevated levels of antibodies to Col-V and Kα1T were demonstrable on day 14 when there was no histological evidence for chronic rejection of the transplanted lungs. Circulating exosomes were isolated from sera collected on days 7 and 14 which demonstrated increased levels of lung SAgs by western blots in the exosomes isolated from day 14 sera. These results clearly demonstrate that not only antibodies to lung SAgs are present in sera from day 14, but also circulating exosomes with Col-V and Ka1T are easily demonstrable in sera collected on day-14. Semi-quantitation of western blot by densitometry further demonstrated significantly increased levels of exosomes with lung SAgs starting from day 14 until sacrifice on day 30. Splenocytes isolated following sacrifice demonstrated specific reactivity to lung SAgs with significantly high frequency of IFN-γ (Col-V 109±2.5, Kα1T 101±5.5) and TNF-α (Col-V 39±1.5, Kα1T 53±5.5) secreting cells by ELISPOT. Conclusion Taken together our results demonstrate that circulating exosomes with lung SAgs and de novo development of antibodies to lung SAgs occurs prior to histological evidence of chronic rejection in this murine model of chronic rejection following orthotopic single LTx suggesting that circulating exosomes with lung SAgs can contribute in the pathogenesis of chronic rejection following LTx. Further, our data suggests that circulating exosomes with lung SAgs could be a potential biomarker for patients at risk for developing chronic rejection following human LTx.

Triptolide Attenuates Graft Inflammation During Ex Vivo Lung Perfusion

Purpose Ex vivo lung perfusion (EVLP) has been proposed as a platform for lung graft assessment and preservation. However, its proinflammatory milieu may be associated with deterioration in graft quality which may contribute to posttransplant graft dysfunction. Triptolide (TL), a diterpenoid triepoxide, has recently been shown to have a therapeutic potential in various disease states due to its anti-inflammatory properties. Based on this, we sought to assess the impact of TL on graft preservation during EVLP as well as associated posttransplant outcomes. Methods Using rat heart-lung blocs, EVLP was performed with Steen solution containing 100 nM of TL at 37°C for 4 hours. Graft evaluation included physiologic, metabolic and functional parameters. Tissue inflammatory profile and endoplasmic reticulum (ER) stress markers were assessed. Following 4 hours of EVLP, orthotopic single lung transplantation was performed using 3-cuff technique. Results were compared with those of lung grafts undergoing EVLP without TL. Results Physiologic and functional parameters of lung grafts on EVLP with TL were better than those without treatment (Figure A). Glucose consumption was significantly decreased in lungs treated with TL. Levels of proinflammatory cytokine mRNA were lower in TL-treated lungs which is attributed to inhibition of nuclear factor κB (NF-κB) signaling. In addition, TL improved ER stress during EVLP as evidenced by diminished ER stress markers. Most importantly, posttransplant graft function was significantly better in the TL group compared to untreated group (Figure C). Conclusion Treatment of lung grafts with TL during EVLP may serve as a potential compound for enhanced graft preservation. These studies warrant further investigation into its potential protective effect and may have implications for improving posttransplant outcomes.

Elevetad Cysteine-Protease Activity in Bronchiolitis Obliterans Syndrome

Purpose Lung transplantation (LTx) is the final treatment option for patients with end-stage lung diseases, however, 50% of LTx patients do not survive the first 5 years due to chronic lung allograft dysfunction (CLAD). Bronchiolitis obliterans syndrome (BOS) is the most common CLAD phenotype, characterized by mononuclear cell infiltration, airway epithelial cell dysfunction, and peribronchial collagen deposition that lead to progressive bronchiolar occlusion and pulmonary function decline. The mechanisms underlying BOS development remain poorly described. Here, we investigated the role of proteases in tissue remodeling in BOS. Methods Bronchoalveolar lavage fluid (BALF) and lung tissue from LTx patients with and without BOS were analyzed by quantitative proteomics, Western blotting (WB), FRET based activity assay, ELISA, and immunostaining. Orthotopic lung transplantation of single mismatched lungs in mice was used as a model of CLAD. BALF and lung tissue were analyzed up to 2 months after LTx by WB and tissue staining. Results Quantitative proteomics of human BALF showed down-regulation of cystatin C (p = 0.0001) and cystatin S (p = 0.0188), members of the cysteine-protease inhibitor superfamily that inhibit cysteine protease activity. WB and protease activity assays further confirmed increased amount and activities of cysteine proteases in human BALF from BOS patients. Histological analysis of lungs from BOS patients demonstrated accumulation of these proteases in the peribronchiolar and perivascular compartment characterized by mononuclear infiltration and fibrosis. Histological analysis of the CLAD mouse model confirmed a significant increase of the expression of cysteine-proteases in the peribronchiolar area starting from seven days after LTx. Conclusion Here, we demonstrate that BOS is associated with an increase in the amount and activity of cysteine-proteases. Their accumulation in the peribronchiolar and perivascular area, which clearly shows signs of fibrosis, justifies further investigation into the role of these proteases in the pathogenesis, early detection, and outcome prediction of BOS.

Constrictive Pericarditis after Lung Transplant: Zebra or Horse?

Purpose Constrictive pericarditis has become an increasingly recognized long-term post-operative complication of lung transplantation. Heightened clinical suspicion, improved diagnostic imaging and effective surgical treatment of the disease have led to progressive awareness of the pathology. We present our institutional experience with constrictive pericarditis in an effort to investigate the etiology and natural history of the disease. Methods From October 2005 to October 2018, 1,234 patients underwent orthotopic lung transplantation at Duke University Hospital. A prospectively maintained institutional database was queried to identify incident patients and determine baseline clinical data. At a median of 13.8 months (IQR 4.6-124 months), 10 patients (0.8%) developed constrictive pericarditis. Simple descriptive statistics were used to describe cohort characteristics and identify variables associated with constrictive pericarditis after lung transplantation. Results The indication for transplantation at index operation was idiopathic pulmonary fibrosis in 9 of 10 patients (1.2% of the 760 restrictive lung disease patients transplanted in the same time period). All ten patients presented with worsening dyspnea and pleural effusions. Right heart catheterization confirmed constrictive physiology in all cases. Nine patients underwent pericardiectomy with improvement in cardiovascular hemodynamics and resolution of symptoms with no 30-day perioperative mortality. Conclusion Diagnosis of constrictive pericarditis should be considered in patients with new onset heart failure symptoms and/or recurrent pleural effusions within 2 years of lung transplantation. Idiopathic pulmonary fibrosis may be associated with increased risk for constrictive pericarditis. Pericardiectomy is a safe and effective treatment for post-transplant constrictive pericarditis.

Fractional Flow Reserve Guided Coronary Revascularization in Lung Transplant Recipients

Purpose Coronary artery disease is a common incidental finding on coronary angiography performed as a work up for lung transplantation listing. Management of asymptomatic intermediate severity coronary artery disease (40-70% epicardial stenosis) and how it impacts outcomes post-transplant is largely empirical and based on local practice patterns and perceptions. Fractional flow reserve (FFR) guided percutaneous coronary intervention (PCI) is a proven strategy in patients with stable medically refractory angina pectoris. Here we report outcomes post lung transplantation in patients undergoing FFR guided revascularization for asymptomatic intermediate coronary artery disease. Methods We reviewed all lung transplant patients who received diagnostic coronary angiography in our institution since 1994. Patients with greater than 70% stenosis were excluded. Patients with intermediate severity coronary artery disease (40-70% angiographic stenosis) were managed with FFR guided coronary revascularization with either CABG at the time of lung transplantation or pre-lung transplantation PCI. FFR less than 0.80 was considered significant. Survival and major adverse cardiac outcomes (non-fatal myocardial infarction or stroke) were compared with those who underwent lung transplantation with no coronary revascularization. Results Among 370 consecutive lung transplant recipients at our institution, 25 (6.7%) underwent coronary revascularization (10 with simultaneous FFR guided CABG, 13 with simultaneous CABG and 2 with PCI prior to listing). 13 lung transplant recipients with non-FFR guided simultaneous CABG were excluded. Median survival was 1.51 years in those who didn't need coronary revascularization and 2.20 years in those who received FFR guided coronary revascularization (p=0.678). Major adverse cardiac event rates on follow up were also similar in the two groups (17.4% in no CABG vs 20% in FFR guided CABG, p= 0.849) at a median follow up of 31 months. Conclusion Concurrent coronary artery disease if managed with a FFR based coronary revascularization strategy CABG, is not associated with worse survival in orthotopic lung transplant recipients.

Nr5a2 promotes cancer stem cell properties and tumorigenesis in nonsmall cell lung cancer by regulating Nanog.

Lung cancer has the highest mortality rate due to late diagnosis and high incidence of metastasis. Cancer stem cells (CSCs) are a subgroup of cancer cells with self‐renewal capability similar to that of normal stem cells (NSCs). While CSCs may play an important role in cancer progression, mechanisms underlying CSC self‐renewal and the relationship between self‐renewal of the NSCs and CSCs remain elusive. The orphan nuclear receptor Nr5a2 is a transcriptional factor, and a regulator of stemness of embryonic stem cells and induced pluripotent stem cells. However, whether Nr5a2 regulates the self‐renewal of lung CSCs is unknown. Here, we showed the diagnostic and prognostic values of elevated Nr5a2 expression in human lung cancer. We generated the mouse LLC‐SD lung carcinoma CSC cellular model in which Nr5a2 expression was enhanced. Using the LLC‐SD model, through transient and stable siRNA interference of Nr5a2 expression, we provided convincing evidence for a regulatory role of Nr5a2 in the maintenance of lung CSC self‐renewal and stem cell properties in vitro. Further, using the syngeneic and orthotopic lung transplantation model, we elucidated augmented cancer biological properties associated with Nr5a2 promotion of LLC‐SD self‐renewal. More importantly, we revealed that Nr5a2’s regulatory role in promoting LLC‐SD self‐renewal is mediated by transcriptional activation of its direct target Nanog. Taken together, in this study, we have provided convincing evidence in vitro and in vivo demonstrating that Nr5a2 can induce lung CSC properties and promote tumorigenesis and progression through transcriptional up‐regulation of Nanog.

The Amide Local Anesthetic Ropivacaine Attenuates Acute Rejection After Allogeneic Mouse Lung Transplantation.

Acute allograft rejection after lung transplantation remains an unsolved hurdle. The pathogenesis includes an inflammatory response during and after transplantation. Ropivacaine, an amide-linked local anesthetic, has been shown to attenuate lung injury due to its anti-inflammatory effects. We hypothesized that the drug would also be able to attenuate acute rejection (AR) after allogeneic lung transplantation. Allogeneic, orthotopic, single left lung transplantation was performed between BALB/c (donors) and C57BL/6 (recipients) mice. Prior to explantation, lungs were flushed with normal saline with or without ropivacaine (final concentration 1µM). Plasma levels of tumor necrosis factor-α and interleukins - 6 and - 10 were measured 3h after transplantation by ELISA. Lung function was assessed on postoperative day five and transplanted lungs were analyzed using histology (AR), immunohistochemistry (infiltrating leukocytes) and Western blot (phosphorylation and expression of Src and caveolin-1). Ropivacaine pre-treatment significantly reduced AR scores (median 3 [minimum-maximum 2-4] for control vs. 2 [1-2] for ropivacaine, p < 0.001) and plasma levels of tumor necrosis factor-α (p = 0.01) compared to control, whereas plasma concentrations of interleukin - 6 (p = 0.008) and - 10 (p < 0.001) were increased by ropivacaine. The number of T-lymphocytes infiltrating the transplanted lung was attenuated (p = 0.02), while no differences in macrophage or B-lymphocyte numbers could be observed after ropivacaine pre-treatment. Caveolin-1 phosphorylation in ropivacaine-treated lungs was diminished (p = 0.004). Pre-treatment of donor lungs with the local anesthetic ropivacaine diminished histological signs of AR after orthotopic left lung transplantation in mice, most likely due to reduced infiltration of T-lymphocytes into the graft.

Synergism of imatinib, vatalanib and everolimus in the prevention of chronic lung allograft rejection after lung transplantation (LTx) in rats.

Chronic lung allograft dysfunction (CLAD) still remains a major drawback in the outcome following lung transplantation (LTx). New therapeutic strategies are warranted. Growth factors and their receptors like platelet-derived growth factor-receptor (PDGFR) and vascular endothelial growth factor-receptor (VEGFR), may play a crucial role in the development of CLAD, especially bronchiolitis obliterans (BO) and vasculopathy. In this study, we used an orthotopic left lung transplantation model from Fischer (F344) to Wystar Kyoto (WKY) rats to investigate the effect of the receptor tyrosine kinase inhibitor (RTKI) vatalanib alone, the dual combination of vatalanib and imatinib and a triple therapy consisting of vatalanib, imatinib and the mammalian target of rapamycin inhibitor (mTORI) everolimus on the development of CLAD after LTx in rats. With this trial we demonstrated that monotherapy with vatalanib attenuated mild and severe chronic vascular rejection, whereas dual therapy (vatalanib and imatinib) after LTx also showed a significant reduction of chronic bronchiolar rejection and interstitial fibrosis. By adding everolimus, the effect of vatalanib and imatinib could additionally be increased. In conclusion, the combination of mTORI and RTKIs might be a possible strategy in the prevention of CLAD and BO.