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Definitions for chronic lung allograft dysfunction (CLAD) phenotypes were recently revised (2019 ISHLT consensus). Post-CLAD onset phenotype transition may occur as a result of change in obstruction, restriction, or RAS-like opacities (RLO). We aimed to assess the prevalence and prognostic implications of these transitions. This was a single-center, retrospective cohort study of bilateral lung transplants performed in 2009-2015. CLAD phenotypes were determined per ISHLT guidelines. CLAD phenotype transition was defined as a sustained change in obstruction, restriction or RLO. We specifically focused on phenotype changes based on RLO emergence. Association of RLO development with time to death or retransplant were assessed using Kaplan-Meier and Cox proportional hazards models. Among 211 patients with CLAD, 47 (22.2%) experienced a phenotype transition. Nineteen patients developed RLO. Development of RLO phenotype after CLAD onset was associated with a shorter time to death/retransplant when considering the entire CLAD patient cohort (HR = 4.00, CI 2.74-5.83, P

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and extracellular matrix in the lung. Connective-tissue growth factor (CTGF) is known to exacerbate pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we show the upregulation of CTGF from a rat lung fibrosis model induced by adenovirus vector encoding active TGF-β1 (AdTGF-β1), and also in patients with IPF. The expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on days 7 or 14 as well as endothelial cells sorted from fibrotic lungs on day 14 or 28 respectively. These findings suggest the role of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases pro-fibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, fibrotic extracellular matrix upregulated the expression of CTGF, as compared to normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for the treatment of pulmonary fibrosis.

Background Development of primary lung cancer in donor lung post-lung transplantation is very rare, with few described cases. The safety of stereotactic ablative radiotherapy (SABR) for early-stage lung cancer arising from donor lung is unclear. Case presentation Herein, we present a case of a patient with a Stage IB adenocarcinoma arising from donor lung 8 years post-double lung transplantation, which was performed due to advanced emphysema. The patient was ineligible for surgical management due to chronic lung allograft dysfunction, which significantly compromised pulmonary function. Full dose SABR was delivered with curative intent after a discussion with the patient. The patient tolerated the treatment well, with one episode of subacute toxicity that resolved with treatment. There was no evidence of recurrence at 15 months post-treatment and the patient’s pulmonary function did not deviate from the pre-SABR baseline. Conclusions SABR appears feasible for medically-inoperable early-stage primary lung adenocarcinoma in the setting of previous double-lung transplantation.

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

Background Ex-vivo lung perfusion (EVLP), a technique for donor lung assessment, also represents a platform for donor lung repair and immunomodulation. α 1 -Anti-trypsin (A1AT), a medication used to treat emphysema in A1AT-deficient patients, has anti-inflammatory properties and has been shown to attenuate ischemia–reperfusion injury in rat and pig lung transplants . The objective of this study was to determine whether administration of A1AT during EVLP can improve donor lung quality after prolonged hypothermic preservation. Methods Pig donor lungs were retrieved, preserved at 4°C for 24 hours, and then subjected to normothermic EVLP for 12 hours using the Toronto protocol. The treatment group (n = 6) received 3 mg/ml A1AT (Zemaira) in the EVLP perfusate, acellular Steen solution. The control group (n = 6) was perfused with Steen solution only. Physiologic functions and gas exchange were measured hourly. Pulmonary edema, lung injury, apoptosis and inflammatory mediators were evaluated in lung tissues and perfusate. Results A1AT treatment significantly reduced pulmonary arterial pressure , pulmonary vascular resistance and airway pressure changes from the baseline when compared with controls. A1AT treatment significantly improved both dynamic and static pulmonary compliance , and change in partial pressure of oxygen (ΔPO 2 ) between the left atrium and the pulmonary artery. Furthermore, A1AT treatment also significantly reduced pulmonary edema (wet-to-dry ratio), pulmonary cell apoptosis and pro-inflammatory cytokine levels (interleukin-1α and -8) in the perfusate. Conclusion Treatment of 24-hour-preserved pig donor lungs with A1AT during EVLP resulted in improved physiologic function, reduced pulmonary edema and inflammation and decreased cell death. Our findings suggest that treatment of donor lungs during EVLP with A1AT is a promising strategy to attenuate early lung injury and improve donor lung function before lung transplantation.

Purpose The majority of potential donor lungs are not being used. The primary reason for underutilization is the concern for primary graft dysfunction (PGD). There is currently no clinically available therapy for PGD. Alpha 1 antitrypsin (A1AT) is a serine protease inhibitor with anti-inflammatory and cytoprotective properties. We and others have shown benefits of A1AT in small and large animal lung transplant studies. Before performing clinical trials, evidence of therapeutic efficacy in human lungs would be valuable. We tested the effect of A1AT given during ex vivo lung perfusion (EVLP) to human lungs rejected for transplantation. Methods Double lung blocks rejected for transplantation (n=8) were divided and placed on separate EVLP circuits for 12h. Lungs were randomly assigned to receive A1AT or placebo, with the contralateral lung serving as control for the treated lung. Outcome measures included: hourly physiologic lung function, perfusate loss, wet-dry weight ratio, inflammatory mediators, endothelin-1 (ET-1), and zonula occludens tight junction protein‐1 (ZO‐1, immunofluorescence staining). Results The A1AT-treated group demonstrated significantly better lung function: higher pO2 and compliance (Fig A), lower pulmonary artery pressure (-0.4 mmHg p= 0.04) and vascular resistance (-30.4 dynes·s cm−5 p= 0.01). Perfusate loss, a surrogate for lung permeability, was lower in the A1AT group, as was the wet-dry ratio (Fig B). A1AT also decreased ET-1 levels in perfusate (Fig C) and increased ZO-1 expression on endothelial cells (Fig D). Conclusion In this study we demonstrated that human A1AT was able to improve the quality of severly injuried human lungs, likely through endothelial cell protection. The encouraging results justify a clinical trial, in order to improve donor lung quality and clinical outcomes in lung transplantation. Our study also illustrates that testing selected drugs on injured human lungs on EVLP is a viable strategy prior to clinical application.

Purpose Previous studies suggest Chronic Lung Allograft Dysfunction (CLAD) subtypes with RAS-like opacities (RLO) (i.e. persistent parenchymal or pleural fibrosis) at CLAD onset to have worse survival. However, the significance of transitions between CLAD subtypes remains obscure. Methods This was a single center, retrospective cohort study of consecutive adult, first bilateral lung transplants from 2010-2015. Patients with CLAD were classified into ISHLT phenotypes BOS, RAS, Mixed, undefined and some remained unclassified. We defined phenotype transition as a change of at least one of three determinants: obstruction, restriction or RLO. Association of phenotype transition with time to death or retransplant starting at CLAD onset or transition date was assessed using Cox PH models. Multivariable models included age and CMV mismatch. Results Of 173 patients with CLAD, 108 patients with no RLO and 28 with RLO at CLAD onset did not change phenotype. 37 patients (21.3%) had a phenotype transition at a median time of 329 days (IQR 166-591) post CLAD onset. 15 patients without RLO (12 BOS, 2 undefined, 1 unclassified) developed RLO and changed phenotype to RAS (1), mixed (7), or undefined (7). 22 patients had a phenotype transition owing to a change in the ventilatory defect pattern (10 with RLO and 12 without RLO). Patients who developed RLO after CLAD onset had a non-significant trend towards worse survival from CLAD onset (HR=1.29, p=0.7) and also from transition date (HR=3.45, p=0.1) compared to patients who transitioned to a phenotype without RLO (Fig 1A), probably due to small number of patients. Survival after RLO emergence did not differ between patients with RLO at CLAD onset and patients who developed RLO post CLAD onset (HR=1.69, p=0.17 (Fig 1B). Conclusion Our study shows that phenotype transition is a relatively common phenomenon, although a transition to RAS-like opacities occurred in only 9% of CLAD patients. The poor survival from emergence of RAS-like opacities appears to be similar regardless of timing relative to CLAD onset.

Long-term outcomes after lung transplantation remain inferior to those of other solid organ groups. The significance of eosinophils detected on transbronchial biopsies (TBBx) after lung transplantation and their relationship to long-term outcomes remain unknown. A retrospective single-center cohort study was performed of patients transplanted between January 01, 2001, and July 31, 2018, who had at least 1 TBBx with evaluable parenchymal tissue. Multivariable Cox proportional hazard models were used to assess the associations between eosinophil detection and: all-cause mortality and Chronic Lung Allograft Dysfunction (CLAD). 8887 TBBx reports from 1440 patients were reviewed for the mention of eosinophils in the pathology report. 112 (7.8%) patients were identified with eosinophils on at least one TBBx. The median (95% CI) survival time for all patients was 8.28 (7.32-9.31) years. Multivariable analysis, adjusted for clinical variables known to affect post-transplant outcomes, showed that the detection of eosinophils was independently associated with an increased risk of death (HR 1.51, 95% CI 1.24-1.85, p < 0.01) and CLAD (HR 1.35, 95% CI 1.07-1.70, P = 0.01). Eosinophils detected in TBBx are associated with an increased risk of CLAD and death. There may be benefit in specifically reporting the presence of eosinophils in TBBx reports and incorporating their presence in clinical decision-making.

The mortality rate for individuals on the wait list for lung transplant is 15% to 25%, and still only 20% of lungs from multiorgan donors are used for lung transplant. The lung donor pool may be increased by assessing and reconditioning high-risk extended criteria donor lungs with ex vivo lung perfusion (EVLP), with similar short-term outcomes.To assess the long-term outcomes of transplant recipients of donor lungs treated with EVLP.This retrospective cohort single-center study was conducted from August 1, 2008, to February 28, 2017, among 706 recipients of donor lungs not undergoing EVLP and 230 recipients of donor lungs undergoing EVLP.Donor lungs undergoing EVLP.The incidence of chronic lung allograft dysfunction and allograft survival during the 10-year EVLP era were the primary outcome measures. Secondary outcomes included donor characteristics, maximum predicted percentage of forced expiratory volume in 1 second, acute cellular rejection, and de novo donor-specific antibody development.This study included 706 patients (311 women and 395 men; median age, 50 years [interquartile range, 34-61 years]) in the non-EVLP group and 230 patients (85 women and 145 men; median age, 46 years [interquartile range, 32-55 years]) in the EVLP group. The EVLP group donors had a significantly lower mean (SD) Pao2:fraction of inspired oxygen ratio than the non-EVLP group donors (348 [108] vs 422 [88] mm Hg; P .001), higher prevalence of abnormal chest radiography results (135 of 230 [58.7%] vs 349 of 706 [49.4%]; P = .02), and higher proportion of smoking history (125 of 204 [61.3%] vs 322 of 650 [49.5%]; P = .007). More recipients in the EVLP group received single-lung transplants (62 of 230 [27.0%] vs 100 of 706 [14.2%]; P .001). There was no significant difference in time to chronic lung allograft dysfunction between the EVLP and non-EVLP group (70% vs 72% at 3 years; 56% vs 56% at 5 years; and 53% vs 36% at 9 years; log-rank P = .68) or allograft survival between the EVLP and non-EVLP groups (73% vs 72% at 3 years; 62% vs 58% at 5 years; and 50% vs 44% at 9 years; log-rank P = .97) between the 2 groups. All secondary outcomes were similar between the 2 groups.Since 2008, 230 of 936 lung transplants (24.6%) in the Toronto Lung Transplant Program were performed after EVLP assessment and treatment. Use of EVLP-treated lungs led to an increase in the number of patients undergoing transplantation, with comparable long-term outcomes.

BACKGROUND Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the new International Society for Heart and Lung Transplantation (ISHLT) CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. METHODS We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed from 2010 to 2015. Patients with CLAD were classified on the basis of the 2019 ISHLT consensus document. CLAD phenotypes and other potential predictors of survival after CLAD onset were assessed using Kaplan–Meier and Cox proportional hazards models. RESULTS Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed, and 19 (10.9%) undefined phenotype. A total of 26 patients (14.9%) did not match any of these 4 categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The 45 patients (26.8%) with undefined/unclassified phenotype were combined and recategorized on the basis of the presence or absence of characteristic RAS-like opacities on chest imaging; those with RAS-like opacities had significantly worse allograft survival than patients with BOS (hazard ratio, 2.14; 95% confidence interval, 1.17–3.93; p = 0.014) and similar survival to RAS or mixed phenotype. CONCLUSIONS The new ISHLT CLAD phenotype classification is informative with regards to post-CLAD outcomes. Chest imaging demonstrating persistent parenchymal or pleural fibrosis may be used for risk-stratification of patients who do not match the major CLAD phenotypes.

Chronic lung allograft dysfunction (CLAD) remains the leading cause of late death after lung transplantation. Epithelial injury is thought to be a key event in the pathogenesis of CLAD. M30 and M65 are fragments of cytokeratin-18 released specifically during epithelial cell apoptosis and total cell death, respectively. We investigated whether M30 and M65 levels in bronchoalveolar lavage (BAL) correlate with CLAD subtypes: restrictive allograft syndrome (RAS) versus bronchiolitis obliterans syndrome (BOS). BALs were obtained from 26 patients with established CLAD (10 RAS, 16 BOS) and 19 long-term CLAD-free controls. Samples with concurrent infection or acute rejection were excluded. Protein levels were measured by ELISA. Variables were compared using Kruskal-Wallis, Mann-Whitney U test and Chi-squared tests. Association of M30 and M65 levels with post-CLAD survival was assessed using a Cox PH models. M65 levels were significantly higher in RAS compared to BOS and long-term CLAD-free controls and correlated with worse post-CLAD survival. Lung epithelial cell death is enhanced in patients with RAS. Detection of BAL M65 may be used to differentiate CLAD subtypes and as a prognostic marker in patients with established CLAD. Understanding the role of epithelial cell death in CLAD pathogenesis may help identify new therapeutic targets to improve outcome.

BACKGROUND Low-level CMV DNAemia is common and in the absence of treatment may either progress to higher viral loads that require therapy, or may spontaneously resolve. The clinical predictors of progression and spontaneous viral clearance are not well defined. METHODS We performed a retrospective cohort study of organ transplant recipients who had untreated low-level CMV DNAemia (

RationaleThe diffusing capacity of the lung for carbon monoxide corrected for haemoglobin (DLCOcor) measures gas movement across the alveolar–capillary interface. We hypothesised that DLCOcor is a sensitive measure of injurious allograft processes disrupting this interface.ObjectivesTo determine the prognostic significance of the DLCOcor trajectory on chronic lung allograft dysfunction (CLAD) and survival.MethodsA retrospective analysis was conducted of all bilateral lung transplant recipients at a single centre, between January 1998 and January 2018, with one or more DLCOcor measurements. Low baseline DLCOcor was defined as the failure to achieve a DLCOcor >75% predicted. Drops in DLCOcor were defined as >15% below recent baseline.Results1259 out of 1492 lung transplant recipients were included. The median (range) time to peak DLCOcor was 354 (181–737) days and the mean±sdDLCOcor was 80.2±21.2% pred. Multivariable analysis demonstrated that low baseline DLCOcor was significantly associated with death (hazrd ratio (HR) 1.68, 95% CI 1.27–2.20; pDLCOcor was not independently associated with CLAD after adjustment for low baseline forced expiratory volume in 1 s or forced vital capacity. Any DLCOcor declines ≥15% were significantly associated with death, independent of concurrent spirometric decline. Lower percentage predicted DLCOcor values at CLAD onset were associated with shorter post-CLAD survival (HR 0.75 per 10%-unit change, pConclusionLow baseline DLCOcor and post-transplant declines in DLCOcor were significantly associated with survival, independent of spirometric measurements. We propose that DLCOcor testing may allow identification of a subphenotype of baseline and chronic allograft dysfunction not captured by spirometry. There may be benefit in routine monitoring of DLCOcor after lung transplantation to identify patients at risk of poor outcomes.

Purpose Chronic Lung Allograft Dysfunction (CLAD) is a heterogeneous disease. Three major phenotypes: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and mixed phenotype, are defined based on combinations of lung physiology and chest imaging. Total lung capacity (TLC)≤90% of baseline is used to define RAS/Mixed; however, many centers do not routinely obtain TLC and use instead forced vital capacity (FVC)≤80%. The objective of the current study was to assess the diagnostic accuracy of FVC≤80% for determining RAS/Mixed in a large center that routinely monitors both TLC and FVC. Methods This was a retrospective cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with CLAD were included. Spirometry, lung volumes, and chest imaging were available. RAS/mixed were determined based on 2019 ISHLT definitions using TLC≤90% and the presence of RAS-like opacities. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic accuracy of FVC≤80% combined with RAS-like opacities for diagnosis of RAS/Mixed were computed. Results A total of 174 patients developed CLAD: 25(14.4%) met criteria for RAS/mixed by TLC. The sensitivity, specificity, PLR, NLR and diagnostic accuracy for FVC to determine RAS/Mixed was 84.0% (95%CI 63.9-95.5), 95.3% (95%CI 90.6-98.1), 17.9 (95%CI 8.5-37.6), 0.17 (95%CI 0.07-0.41) and 93.7% (95%CI 89.0-96.8). Conclusion Low FVC has an overall good diagnostic value for estimating RAS/mixed phenotype. Given the high specificity, it is possible to rule in RAS/mixed phenotype with spirometry. However, it cannot be ruled out confidently based on spirometry alone. In CLAD patients with a clinical suspicion of RAS/mixed phenotype and a preserved FVC, measurement of TLC is important for a more accurate diagnosis.

Purpose Lung and heart-lung transplant candidates and recipients are susceptible to mycobacterial infections that can cause morbidity and mortality. We characterized the incidence, clinical characteristics and treatment of mycobacterial findings and assessed their impact on patient outcomes. Methods We conducted a retrospective review of patients who underwent lung or heart-lung transplantation in our center between June 1988 and May 2020 and identified patients with mycobacterial findings, in most cases positive sputum or bronchoscopy sample cultures. Pre-transplant patients are screened for mycobacterial infections with bronchoscopy and sputum samples. Post-transplant patients undergo surveillance bronchoscopies several times during the first post-transplant year, annually thereafter, and when clinically warranted. The decision to initiate anti-mycobacterial treatment is based on clinical, radiologic and mycobacterial sample results, with the aim to treat mycobacterial disease and monitor colonization. Results In a cohort of 355 patients, 15/355 (4.2%) patients had a pre-transplant mycobacterial finding and 9/15 received anti-mycobacterial treatment. In one patient, mycobacterium tuberculosis infection recurred post-transplant. No deaths were related to mycobacterial infections in patients with a pre-transplant mycobacterial finding. In post-transplant patients, 27/355 (7.6%) patients had a mycobacterial finding and 5/27 received anti-mycobacterial treatment. One patient died due to disseminated mycobacterium abscessus infection. Mycobacterial findings pre- or post-transplant did not affect overall patient survival compared to non-infected patients (Log Rank p = 0.312). Conclusion Mycobacterial findings are rare in patients undergoing lung or heart-lung transplantation, and do not seem to affect overall survival of patients. Most post-transplant mycobacterial findings represent colonization, but disseminated mycobacterial infection may be fatal.

Purpose Airway epithelial injury is thought to be a key event in the pathogenesis of Chronic lung allograft dysfunction (CLAD). We investigated whether markers of epithelial activity and injury in bronchoalveolar lavage (BAL) correlate with CLAD diagnosis and major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS)-related phenotypes (including RAS, mixed phenotype, and all other patients with RAS-like opacities). Methods CLAD status and phenotypes were retrospectively determined in a cohort of all consecutive adult, first, bilateral lung transplants performed 2010-2015. All patients with RAS-related phenotypes were included and 1:1 matched with BOS patients based on time from transplant to CLAD-onset. CLAD-free subjects were used as controls. Proteins that maintain the barrier function of the airway epithelial mucosa (club cell secretory protein (CCSP), surfactant protein-D (SP-D) and epithelial mucins: MUC1, MUC5AC, MUC5B, MUC16), as well as epithelial cell death markers (M30&M65 representing apoptosis and overall death, respectively), were measured in BAL obtained within 6-months from CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using Kruskal-Wallis and Mann-Whitney-U-test. Association between protein levels and graft survival was assessed using Cox proportional hazards models, adjusted for CMV serology mismatch status and phenotype. Results Fifty-four CLAD (27 BOS, 11 RAS, 7 mixed, 9 others with RAS-like opacities) patients and 26 CLAD-free controls were included. Median BAL levels were significantly higher in patients with CLAD compared to CLAD-free controls for M30 (124.5 vs. 88.9), MUC1 (6.8 vs. 3.28) and MUC16 (121.0 vs. 31.1). When comparing CLAD phenotypes, M30 was significantly higher in patients with RAS-related phenotypes compared to BOS (160.9 vs. 114.6). In multivariable models, levels of M30 and MUC5B were associated with allograft survival after CLAD onset independent of phenotype (P Conclusion Airway epithelial mucin and cell death markers are enhanced in the BAL fluid of patients with CLAD and can assist in differentiating between CLAD phenotypes. Abnormal airway mucin expression and epithelial cell death may be involved in the airway inflammatory response of CLAD and thus aid in future selection of targeted therapies.

Acute cellular rejection (ACR) is a significant risk factor for chronic lung allograft dysfunction (CLAD). Although clinically manifest and higher grade (≥A2) ACR is generally treated with augmented immunosuppression, management of minimal (grade A1) ACR remains controversial. In our program, patients with subclinical and spirometrically stable A1 rejection (StA1R) are routinely not treated with augmented immunosuppression. We hypothesized that an untreated first StA1R does not increase the risk of CLAD or death compared to episodes of spirometrically stable no ACR (StNAR). The cohort was drawn from all consecutive adult, first, bilateral lung transplantations performed between 1999 and 2017. Biopsies obtained in the first-year posttransplant were paired with (forced expiratory volume in 1 second FEV1 ). The first occurrence of StA1R was compared to a time-matched StNAR. The risk of CLAD or death was assessed using univariable and multivariable Cox proportional hazards models. The analyses demonstrated no significant difference in risk of CLAD or death in patients with a first StA1R compared to StNAR. This largest study to date shows that, in clinically stable patients, an untreated first A1 ACR in the first-year posttransplant is not significantly associated with an increased risk for CLAD or death. Watchful-waiting approach may be an acceptable tactic for stable A1 episodes in lung transplant recipients.

Purpose Allograft infection after lung transplant (LTx) has a significant impact on outcome and has been associated with chronic lung allograft dysfunction (CLAD). Commensal bacteria (CB) are commonly isolated from bronchoalveolar lavages (BAL) in LTx recipients but the clinical significance is unknown. Our objective was to evaluate the association of high growth of CB (≥106 CFU/L) in BAL cultures with subsequent infection or CLAD. Methods We conducted a single-center, retrospective, cohort study of consecutive adult, first bilateral LTx performed 2010-2017. Patients who had no evidence of BAL infection and were alive and CLAD-free throughout the first 6-months post-transplant were included. Infection was defined as the presence of treatment-requiring pathogens in BAL. Outcomes of patients with ≥1CB within the first 6-months post-transplant were compared to patients with no CB isolation episodes within the same time interval. Association of CB with subsequent clinically significant BAL infections using logistic regression models and with time to CLAD was assessed using Cox Proportional Hazards models. Results The study cohort consisted of 202 patients with a median of 7 (IQR 7, 8) bronchoscopies per patient. Seventy of the 202 (34.7%) had subsequent BAL infection at some point post-transplant. Median time to CLAD was 497 days (IQR 131, 1043). Fifty-four (26.7%) patients had at least one episode of CB within the first 6-months post-transplant. In a multivariable analysis accounting for age, sex, primary lung disease, acute rejection score and CMV-mismatch, there was no difference in risk for subsequent infections (OR=0.83, 95% CI 0.40-1.69, P=0.60) or CLAD (HR=0.97, 95% CI 0.50-1.87, P=0.92) between patients with ≥1CB episode versus none. Conclusion We found that in the absence of specific infectious pathogens, isolation of commensal bacteria from BAL of LTx recipients during the first 6-months is not associated with an increased risk of subsequent infections or development of CLAD.

Purpose Chronic lung allograft dysfunction (CLAD) is a heterogeneous condition. Characterization of CLAD phenotypes is essential to enhance the understanding of pathogenesis and guide new therapies. The study objective was to validate the 2019 ISHLT CLAD classification system and further explore patients who do not fall into the defined CLAD sub-categories. Methods We performed a single-center, retrospective cohort study of adult, first, bilateral lung transplants performed 2010-2015. Classification of CLAD phenotypes was based on the 2019 ISHLT consensus document. Survival after CLAD onset was assessed using Kaplan-Meier and Cox proportional hazards models. Results Among the 174 subjects with CLAD, 104 (59.8%) had bronchiolitis obliterans syndrome (BOS), 16 (9.2%) restrictive allograft syndrome (RAS), 9 (5.2%) mixed and 19 (10.9%) undefined phenotype. Twenty-six patients (14.9%) did not match any of these four categories and remained unclassified. Allograft survival post-CLAD onset was longer for patients with BOS (median, 500 days) than patients with RAS (median, 372 days) or mixed (median, 328 days). The remaining 45 (25.9%) patients were combined and re-categorized based on the presence or absence of characteristic RAS-like opacities on chest imaging: those with RAS-like opacities at CLAD onset had significantly worse allograft survival compared to patients with BOS (HR=2.14, 95% CI 1.17-3.93, P=0.014) and similar survival to RAS or mixed phenotype. Conclusion The new ISHLT CLAD phenotype classification is informative with regards to post-CLAD outcomes. Chest imaging demonstrating persistent parenchymal or pleural fibrosis may be used for the risk-stratification of patients who do not match the major CLAD phenotypes.

Purpose There are contradictory reports on outcomes after bilateral lung transplant (BLT) vs. single lung transplant (SLT) in patients with COPD, which may be confounded by differences in allocation preferences and patient care between programs. We report outcomes of SLT vs. BLT for COPD at our center. Methods We retrospectively analyzed COPD patients who underwent SLT and BLT between Jan 2008 and Dec 2018. Pre- and post-transplant characteristics were compared between groups with chi‐square and t-tests. Unadjusted Kaplan‐Meier survival estimates were used to compare differences in freedom from CLAD (2019 ISHLT criteria) and allograft survival with log‐rank tests. Results 276 patients with COPD underwent LT (233 BLT and 43 SLT). SLTs were older than BLTs [median 63y (IQR 59-69) vs. 61y (IQR 57-67) p=0.04]. There were no other differences in baseline characteristics. SLTs had younger donors [median 27y (IQR 23-49) vs. 36y (IQR 22-53) p=0.01] and more often underwent EVLP [49% vs. 29% p=0.01]. BLTs required more intraoperative ECMO [14% vs. 2.3% p=0.03] and blood transfusion [40% vs. 19% p=0.02]. Mean post-transplant hospital and ICU length of stay was similar between groups [BLT 29.1d (95%CI 27.1-34.9) vs. SLT 23.5d (95% CI 19.6-27.5) p=0.11], and [BLT 6.8d (95%CI 5.3-8.1) vs. SLT 5.4d (95% CI 3.7-7.1) p=0.36]. Postoperative FEV1 and FVC were better in BLT [2.75L (95% CI 2.2-3.39) vs. 1.63L (95% CI 1.39-1.92) p Conclusion BLT for COPD resulted in better postoperative lung function and exercise capacity, but no significant differences in freedom from CLAD and allograft survival. There was a trend toward improved longer-term survival with BLT for COPD.

Purpose Surveillance bronchoscopies with bronchoalveolar lavage (BAL) and transbronchial biopsies (TBB) are used in the evaluation of patients after lung transplant to detect acute cellular rejection (ACR) or infection. We have previously shown that a focused BAL protein signature early post-transplant may predict CLAD or death in a subset of clinically-stable patients with A1-grade (minimal) ACR. The aim of the current study was to determine whether similar BAL markers would have similar predictive characteristics irrespective of ACR grade. Methods The cohort consisted of all adult, first, bilateral lung transplants performed 2010-2017. Clinical status was categorized as unstable or stable based on presence or absence of ≥10% concurrent drop in FEV1. Clinically stable patients with grade AX TBB (inadequate biopsies, which are not routinely treated at our center, thus avoiding the confounding effect of ACR treatment) during the first-year post-transplant, not preceded by ACR (grade A≥1 or grade B≥1) were included. IL6, S100A8, IL10, TNF-receptor1, IL-1α, pentraxin3 and CXCL10, previously shown to be associated with worse outcomes, were measured in the BAL using a multiplex bead assay. Association with subsequent CLAD or death was assessed using Cox proportional hazards models adjusted for age, sex, native lung disease, and CMV-mismatch. Results 107 patients matched the inclusion criteria with a stable AX occurring at a median time of 188 days (IQR 96-279) post-transplant. Median time from AX TBB to CLAD or death was 462 (IQR 330, 928) and 582 days (IQR 251-1410), respectively. In multivariable models, levels of CXCL10 and IL10 were associated with both CLAD development and death while IL6, S100A8 and pentraxin3 were only associated with death (P Conclusion A focused BAL protein signature in clinically stable patients with ungradable TBB, early post-transplant, may be informative in determining increased risk for worse outcome and may benefit from a more aggressive management strategy.

Summary of Objectives Chronic lung allograft dysfunction (CLAD) affects up to 70% of lung transplant (LTx) recipients and is the major impediment to long-term survival. CLAD is a retrospective diagnosis of exclusion when other causes for sustained drop of ≥20% of forced expiratory volume in 1s or forced vital capacity have been ruled out. No effective treatments exist, likely due to irreversible lung damage at diagnosis. Oscillometry (Osc) is a promising pulmonary function test that is particularly sensitive to changes in small airways, the site of CLAD onset. Our aim is to characterize Osc in patients with early CLAD. Methods In a prospective study that began in December 2017, all new double LTx recipients are enrolled for Osc prior to routine spirometry. Endpoints CLAD will be diagnosed according to ISHLT guidelines. By March 2020 we anticipate enrolment of 300 patients and 30 to have CLAD. We will compare Osc in CLAD patients with those who remain CLAD free after ≥6 mths follow-up. Univariate and mixed models will be used to examine spirometry vs Osc parameters of small airway disease: namely, X5 (reactance at 5Hz), Ax (area of reactance) and R5-19 (difference of resistance at 5 and 19 Hz). Receiver operating characteristic area under the curve (AUC) will be used to assess the predictive power of each. In interim analysis of patients enrolled by 31 July 2019, 13/197 had CLAD [all with bronchiolitis obliterans syndrome (BOS)], with median time of diagnosis at 24 wks post-LTx (range 10-59). Osc was abnormal and significantly different in CLAD vs. 87 CLAD-free recipients who had follow-up of ≥ 6 mths (median 40 wks). The mean Osc measurements at time of CLAD diagnosis vs last follow-up of CLAD-free patients are: X5 = -3.7 vs -2.1 cmH20.s/L, Ax = 31.6 vs 15.9 cmH20/L and R5-19 = 1.5 vs 0.8 cmH20.s/L (p

The growing demand for suitable lungs for transplantation drives the quest for alternative strategies to expand the donor pool. The aim of this study is to evaluate the outcomes of lung transplantation (LTx) with donation after circulatory determination of death (DCDD) and the impact of selective ex vivo lung perfusion (EVLP). From 2007 to 2013, 673 LTx were performed, with 62 (9.2%) of them using DCDDs (seven bridged cases). Cases bridged with mechanical ventilation/extracorporeal life support were excluded. From 55 DCDDs, 28 (51%) underwent EVLP. Outcomes for LTx using DCDDs and donation after neurological determination of death (DNDD) donors were similar, with 1 and 5-year survivals of 85% and 54% versus 86% and 62%, respectively (p = 0.43). Although comparison of survival curves between DCDD + EVLP versus DCDD-no EVLP showed no significant difference, DCDD + EVLP cases presented shorter hospital stay (median 18 vs. 23 days, p = 0.047) and a trend toward shorter length of mechanical ventilation (2 vs. 3 days, p = 0.059). DCDDs represent a valuable source of lungs for transplantation, providing similar results to DNDDs. EVLP seems an important technique in the armamentarium to safely increase lung utilization from DCDDs; however, further studies are necessary to better define the role of EVLP in this context.

Intragraft DSA can be found in explanted lungs of patients with chronic lung allograft dysfunction in the absence of circulating DSA. New diagnostic approaches to assess for intragraft DSA may add to our diagnostic arsenal in treating AMR.http://bit.ly/31kS00b

BACKGROUND: Obliterative bronchiolitis after lung transplantation is characterized by chronic airway inflammation leading to the obliteration of small airways. Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular responses to hypoxia and inflammation. The Von Hippel-Lindau protein (pVHL) drives the degradation of oxygen-sensitive subunit HIF-1 alpha that controls the activity of HIF-1. We investigated the effect of myeloid cell targeted gene deletion of HIF-1 alpha or its negative regulator pVHL on the development of obliterative airway disease (OAD) in the recipients of tracheal allografts, a mouse model for obliterative bronchiolitis after lung transplantation. METHODS: Tracheal allografts were heterotopically transplanted from BALB/c donor mice to fully major histocompatibility complex mismatched recipient mice with HIF-1 alpha or VHL gene deletion in myeloid cells. The recipients were left non-immunosuppressed or received tacrolimus daily. Histologic, immunohistochemical, and real-time reverse transcription polymerase chain reaction analyses were performed at 3, 10, and 30 days. RESULTS: In the absence of immunosuppression, myeloid cell-specific VHL deficiency of the recipient mice improved epithelial recovery, decreased inflammatory cell infiltration and expression of pro-inflammatory cytokines, increased regulatory forkhead box P3 messenger RNA expression, and reduced OAD development in tracheal allografts. In the presence of tacrolimus immunosuppression, loss of HIF-1 alpha activity in myeloid cells of the recipient by HIF-1 alpha gene deletion accelerated OAD development in mouse tracheal allografts. CONCLUSIONS: Activity of the HIF-pathway affects the development of allograft rejection, and our results suggest that myeloid cell-specific VHL-deficiency that potentially increases HIF-activity decreases allograft inflammation and the subsequent development of OAD in mouse tracheal allografts. (C) 2016 International Society for Heart and Lung Transplantation. All rights reserved.

Despite increasing evidence about the role of donor-specific human leukocyte antigen (HLA) antibodies in transplant outcomes, the incidence and impact of de novo donor-specific antibodies (dnDSA) after lung transplantation remains unclear.To describe the incidence, characteristics, and impact of dnDSA after lung transplantation.We investigated a single-center cohort of 340 lung transplant recipients undergoing transplant during 2008 to 2011. All patients underwent HLA-antibody testing quarterly pretransplant and at regular intervals over the first 24 months after transplant. The patients received modified immunosuppression depending on their pretransplant sensitization status. Risk factors for dnDSA development, as well as the associations of dnDSA with patient survival and chronic lung allograft dysfunction (CLAD), were determined using multivariable analysis.The cumulative incidence of dnDSA was 47% at a median of 86 days (range, 44-185 d) after lung transplantation. Seventy-six percent of recipients with dnDSA had DQ-DSA. Male sex and the use of ex vivo lung perfusion were associated with an increased risk of dnDSA, whereas increased HLA-DQB1 matching was protective. DQ-dnDSA preceded or coincided with the diagnosis of CLAD in all cases. Developing dnDSA (vs. no dnDSA) was associated with a twofold increased risk of CLAD (hazard ratio, 2.04; 95% confidence interval, 1.13-3.69). This association appeared to be driven by the development of DQ-dnDSA.dnDSA are common after lung transplantation, with the majority being DQ DSA. DQ-dnDSA are associated with an increased risk of CLAD. Strategies to prevent or treat DQ-dnDSA may improve outcomes for lung transplant recipients.

Background Obliterative bronchiolitis after lung transplantation is characterized by airway inflammation leading to obliteration of small airways. Statins are known to have lipid-independent immunomodulatory properties. We investigated the effect of simvastatin treatment on innate and adaptive immune responses and the development of obliterative airway disease (OAD). Methods In fully MHC-mismatched rat tracheal allograft recipients, we used simvastatin at different doses (0.1 to 20 mg/kg/day orally) to assess its effect on OAD development. No immunosuppressive treatment was administered. Histologic, immunohistochemical and real-time RT-PCR analyses were performed 3, 10 and 30 days after transplantation. Results Simvastatin treatment with doses ranging from 0.5 to 20 mg/kg/day significantly enhanced early epithelial recovery and reduced the development of OAD. No dose response was observed. Simvastatin treatment markedly reduced IL-23 mRNA and lymphocyte chemokine CCL20 production, and the infiltration of CD4 + and CD8 + T cells into allografts already at 3 days. At 10 days, simvastatin significantly attenuated the production of pro-inflammatory cytokines, IL-1β, TNF-α, MCP-1 and IP-10, and Th17-polarizing cytokines, IL-6 and IL-17e, and inhibited allograft infiltration by inflammatory cells. The protective effects of simvastatin on inflammation and OAD were partially mediated through nitric oxide synthase. Conclusions Simvastatin treatment inhibited adaptive T-cell alloimmune activation as depicted by reduced expression of lymphocyte chemokine and pro-inflammatory cytokine mRNA and reduced allograft infiltration by inflammatory cells. Importantly, simvastatin inhibits the development of OAD and this effect is partially mediated by increased nitric oxide activity. These results suggest a role for simvastatin in the prevention of obliterative bronchiolitis.

Background Obliterative bronchiolitis (OB) is the most prominent cause of morbidity and mortality among lung transplant patients. No effective treatment for OB exists, but preliminary clinical studies have suggested that a calcineurin inhibitor, tacrolimus, may delay the development of OB when compared with standard cyclosporine-based immunosuppression. Methods Using a murine tracheal transplantation model, we examined the effects of tacrolimus prophylaxis and treatment on the development of obliterative airway disease (OAD). Tracheal allografts were transplanted heterotopically from BALB/c to C57 black mice into a subcutaneous pouch. The mice received different doses of tacrolimus monotherapy, ranging from 0 to 3 mg/kg/day, subcutaneously initiated at 0 (prophylaxis), 7 (early treatment) or 14 (late treatment) days. We harvested the grafts 30 days after transplantation for histologic and immunohistochemical analyses. Results We found that tacrolimus prophylaxis dose-dependently inhibited OAD, and that early treatment halts OAD progression and that late treatment delays progression. Syngeneic grafts showed no obliterative changes. Tacrolimus prophylaxis was associated with inhibition of recruitment of CD4 + , CD8 + and interleukin-2R + inflammatory cells into the allografts, suggesting a central role for interleukin-2 in the development of OAD. In addition, a dose-dependent correlation between epithelial necrosis and tracheal occlusion was observed, suggesting that epithelial injury is required for the development of OAD. When tacrolimus treatment was initiated at the time the obliterative lesion had already started to develop, it inhibited the progression of OAD significantly. Conclusions The findings from this study suggest that tacrolimus therapy is effective during the early stages of clinical OB.

T cell activation is a proximal event in the initiation of chronic rejection that may ultimately lead to obliterative bronchiolitis (OB) after lung transplantation. In addition to primary signals generated by the T cell receptor, T cell activation relies on costimulatory signals, of which the most important are mediated via interaction between CD28 and its ligands B7-1 and B7-2. In nontreated rat tracheal allografts, B7-2, but not B7-1, expression peaked 10 d after transplantation, unlike in syngeneic grafts, where no B7-2 upregulation was observed. Selective blockade of the CD28/B7-1 T cell costimulatory pathway by a mutant form of CTLA4Ig (cytotoxic T lymphocyte antigen 4 immunoglobulin), CTLA4IgY100F, did not affect epithelial injury or degree of luminal occlusion in rat tracheal allografts. Treatment with CTLA4Ig fusion protein, which blocks both CD28/B7-1 and CD28/B7-2 interaction, significantly delayed the development of epithelial injury and airway occlusion. Immunohistochemical analyses of allografts showed that selective inhibition of the CD28/B7-1 pathway did not affect cytokine expression. In contrast, treatment with CTLA4Ig was associated with a significant decrease in the intragraft production of tumor necrosis factor alpha, interleukin 2, and interferon gamma, as well as slightly increased intragraft expression of interleukin 10. In conclusion, CTLA4Ig-mediated costimulatory blockade delays epithelial injury and attenuates obliterative changes and is associated with marked suppression of helper T cell type 1 (Th1)-dominated cytokine response. These observations emphasize the role of the CD28/B7-2 costimulatory pathway in regulating proinflammatory and Th1 cytokine responses and thereby in the development of epithelial and graft injury gradually leading to obliteration of the airway lumen.

Background Ex vivo lung perfusion (EVLP) is an effective method to assess and improve the function of otherwise unacceptable lungs, alleviating the shortage of donor lungs. The early results with EVLP have been encouraging, but longer-term results, including functional and patient-reported outcomes, are not well characterized. Methods This retrospective single-center study included all lung transplants performed between September 2008 and December 2012. We investigated whether survival or rate of chronic lung allograft dysfunction (CLAD) differed in recipients of EVLP-treated lungs compared with contemporaneous recipients of conventional donor lungs. We also studied functional (highest forced expiratory volume in 1 second predicted, change in 6-minute walk distance, number of acute rejection episodes) and quality of life outcomes. Results Of 403 lung transplants that were performed, 63 patients (15.6%) received EVLP-treated allografts. Allograft survival for EVLP and conventional donor lung recipients was 79% vs 85%, 71% vs 73%, and 58% vs 57% at 1, 3, and 5 years after transplant, respectively (log-rank p = not significant). Freedom from CLAD was also similar (log-rank p = 0.53). There were no significant differences in functional outcomes such as highest forced expiratory volume in 1 second predicted (76.5% ± 23.8% vs 75.8% ± 22.8%, p = 0.85), change in 6-minute walk distance (194 ± 108 meters vs 183 ± 126 meters, p = 0.57), or the number of acute rejection episodes (1.5 ± 1.4 vs 1.3 ± 1.3, p = 0.36). The EVLP and conventional donor groups both reported a significantly improved quality of life after transplantation, but there was no intergroup difference. Conclusion EVLP is a safe and effective method of assessing and using high-risk donor lungs before transplantation and leads to acceptable long-term survival, graft function, and improvements of quality of life that are comparable with conventionally selected donor lungs.

Study objectives: Acute rejection and cytomegalovirus (CMV) infection are important complications after lung and heart--lung transplantation. We sought to investigate whether acute rejection and CMV infection demonstrated as CMV antigenemia had an effect on the cell profiles of peripheral blood (PB), bronchoalveolar lavage fluid (BAL-F), or TBB histology. Patients and design: In this prospective study, composition of cells in PB, BAL-F, and TBB samples from 20 lung or heart-lung transplantation patients were analyzed during episodes of acute rejection or CMV antigenemia. Rejection was graded according to the International Society for Heart and Lung Transplantation criteria. As controls, samples with no evidence of rejection or infection were used. To evaluate the effect of time on cellular findings, samples were divided into three groups according to time after transplantation: 1-30, 31-180, and more than 180 d after transplantation. Results: Acute rejection was associated with mild blood basophilia (p < 0.05; specificity 94%, sensitivity 42%). In BAL-F during rejection, the number of basophils (p < 0.05), eosinophils (p < 0.05), and lymphocytes (p < 0.05; specificity 77%, sensitivity 64%) was increased compared to controls during the post-operative month 1. Later-occurring rejections were associated with increased amounts of neutrophils in BAL-F (p < 0.05; specificity 82%, sensitivity 74%). In TBB histology, acute rejections were associated with perivascular and/or peribronchial infiltration of lymphocytes (p < 0.001) and plasma cells (p < 0.05) compared to controls. In our patients receiving gancyclovir prophylaxis, CMV antigenemia did not significantly alter the cell profiles in PB and BAL-F nor the inflammatory cell picture in TBB histology. Conclusion: TBB histology remains the gold standard' for diagnosing rejection in lung and heart-lung transplantation patients, as the inflammatory cell findings in TBB specimens are highly specific for rejection. The cellular changes associated with rejection, mild PB basophilia and increased proportions of lymphocytes in early- and neutrophils in later-occurring rejection, observed in BAL-F cannot be considered specific for rejection, but may warrant clinical suspicion of rejection.

There is a wealth of clinical and experimental evidence indicating the interaction of cytomegalovirus (CMV) infection and rejection in cardiac and other solid organ allografts. A plausible explanation for this association comes from data showing that therapy with biologicals, sepsis, and rejection, all lead to the release of TNF-alpha which, upon binding to its receptor, activates NF-kB. TNF-alpha is also able to stimulate the activity of the CMV-IE enhancer/promoter region. CMV infection of several cell lines leads to NF-kB activation. NF-kB binding sites are present in regulatory regions of various cellular and viral genes, including the IE enhancer region of CMV. In a reciprocal situation, CMV infection, most likely via gamma-interferon, leads to upregulation of MHC antigens in the transplant and, thereby, to increased transplant immunogenicity. Thus, a vicious circle is induced. We have investigated in detail the pathobiology of CMV and allograft vasculopathy (chronic rejection) in experimental animals, using aortic and cardiac allografts as well as a trachea model. The results may be summarized as follows: Infection of the recipient with rat CMV results in an early inflammatory response in the aortic and cardiac allograft vascular adventitia and intima (endothelialitis) and in the airway wall of tracheal allografts. This early inflammatory response leads to enhanced intimal thickness in aortic and cardiac allografts and enhanced luminal occlusion of tracheal allografts. Timewise, this coincides with early activation of intragraft inflammatory leukocytes and increased mRNA of various growth factors and cytokines. When the recipients receive gancyclovir, the enhanced intimal response in aortic and cardiac allografts and luminal occlusion in tracheal allografts is entirely abolished. Gancyclovir treatment dramatically reduces the inflammatory response in the allograft, and thereby growth factor synthesis in response to injury. However, gancyclovir does not prevent the expression of IE antigen of CMV, suggested to inactivate tumor suppressor protein p53 predisposing smooth muscle cells to increased growth. Taken together, the effect of CMV infection on cardiac allograft dysfunction is bidirectional and biphasic. The bidirectional nature emerges from the observations that acute CMV infection may accelerate acute rejection, and, on the other hand, acute alloimmune response-associated cytokine response may activate latent CMV infection. The biphasic effect of CMV on allograft dysfunction refers to its early and late detrimental effects, i.e. during the time of acute and chronic rejection. These two effects of CMV on allograft dysfunction emphasize the need for precise diagnosis of CMV infection in transplant recipients and pre-emptive or prophylactic anti-viral therapy. The benefits of this strategy may not be evident during the early post-transplant period, but 5-10 years after transplantation they manifest as better graft survival.

Background: Activins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation. Methods: We used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis. Results: Activin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed. Conclusions: The upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF. peerReviewed

Background— Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. Methods and Results— Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell–endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury–induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N -nitro- l -arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-β1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-β1–induced microvascular endothelial-to-mesenchymal transition. Conclusions— Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

Background We assessed cellular innate and adaptive immune responses in a rat heterotopic tracheal allograft model during the development of obliterative airway disease. Methods Syngeneic tracheal grafts were transplanted heterotopically from DA to DA rats and fully MHC-mismatched allografts from DA to WF rats. The recipients received either no immunosuppression or two different doses of cyclosporine and were euthanized at 3, 10 and 30 days. Non-transplanted DA tracheas served as controls. Histologic, immunohistochemical and real-time RT-PCR analyses were performed. Results The syngrafts had normal epithelium at 10 days and no tracheal occlusion was seen at 30 days. In non-immunosuppressed allografts, almost total loss of epithelium was observed at 10 days, culminating in tracheal occlusion at 30 days. The activation of innate immune response was observed during the ischemic period at 3 days in both groups. Influx of the infiltrating inflammatory cells was more prominent in the allografts. In syngrafts, mRNA expression of pro-inflammatory, but also tolerogenic, cytokines was significantly upregulated, whereas Th1 and Th17 priming factors were significantly downregulated. In allografts, prominent mRNA expression of pro-inflammatory cytokines was seen and adaptive Th1 and Th17 alloresponses were increased. Cyclosporine treatment reduced tracheal occlusion and inhibited both tolerogenic and pro-inflammatory T-cell responses in allografts. Conclusions Ischemia induced a self-limiting, alloantigen-independent innate immune response in syngrafts. In allografts, the predominant pro-inflammatory milieu and alloantigen-dependent Th1 and Th17 responses were linked to the development of obliterative airway disease and were inhibited by cyclosporine treatment.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. The exact molecular and cellular mechanisms contributing to obliterative lesion formation are unknown. Pathological characteristics of OB are epithelial damage, peribronchial inflammation, and increasing obliteration of bronchioli. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that exerts proinflammatory effects by increasing endothelial permeability and inducing expression of endothelial adhesion molecules. We investigated the role of VEGF in the development of OB in rat tracheal allografts and the role of VEGF receptors (VEGFR)-1 and -2 in the development of OB in mouse tracheal allografts. In nontreated allografts, with increasing loss of epithelium and airway occlusion, VEGF messenger RNA (mRNA) and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene led to a decrease in epithelial necrosis but increased luminal occlusion by >50% compared with AdLacZ-treated rat tracheal allografts. When compared with the control immunoglobulin (Ig)G group, simultaneous treatment with antibodies against VEGFR-1 and -2 significantly lowered the degree of luminal occlusion of mouse tracheal allografts.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. We investigated the role of vascular endothelial growth factor (VEGF) in the development of OB in rat tracheal allografts. In nonimmunosuppressed allografts, VEGF mRNA and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells with progressive loss of epithelium and airway occlusion compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF(164) gene increased early epithelial cell proliferation and regeneration but increased microvascular remodeling and lymphangiogenesis and luminal occlusion by more than 50% compared with AdlacZ-treated allografts. Although VEGF receptor inhibition decreased early epithelial regeneration in noninfected allografts, it reduced microvascular remodeling, lymphangiogenesis, intragraft traffic of CD4(+) and CD8(+) T cells, and the degree of luminal occlusion. Simultaneous VEGF gene transfer and platelet-derived growth factor receptor inhibition with imatinib preserved respiratory epithelium and totally prevented luminal occlusion. In conclusion, our findings indicate that VEGF has a dual role in transplant OB. Our results suggest that VEGF may protect epithelial integrity. On the other hand, VEGF may enhance luminal occlusion by increasing the recruitment of mononuclear inflammatory cells with platelet-derived growth factor acting as a final effector molecule in this process.

Transplant coronary artery disease (TxCAD) as a manifestation of chronic rejection is a major limitation to long-term survival of heart transplant recipients. Although the exact molecular and cellular mechanisms contributing to neointimal formation are unknown, it has been generally believed that smooth muscle cells (SMC) of donor origin migrate from the media into the subendothelial layer of the vascular wall, where SMC proliferate and synthesize extracellular matrix resulting in intimal thickening. However, recent observations indicate that hematopoietic and vascular progenitor cells derived from recipient bone marrow may contribute to the arteriosclerotic lesion formation in the coronary arteries of the transplant. On the other hand, studies on postnatal hematopoiesis indicate that angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) may regulate the recruitment of these cells into distant organs. Furthermore, embryonic VEGFR-2 /CD34+ stem cells may serve as vascular progenitor cells and their differentiation into endothelial cells and SMC may be regulated by VEGF and platelet-derived growth factor (PDGF), respectively. In this review, we discuss the role of angiogenic growth factors such as VEGF, Ang, and PDGF in the recruitment of hematopoietic and vascular progenitor cells in TxCAD and suggest novel therapies targeted at homing, differentiation and proliferation of these cells in the allograft.

Purpose: The molecular mechanisms of BO are not fully understood. Matrix metalloprotease (MMP)-9 broncho-alveolar lavage levels increase in BO. Interleukin (IL)-17 plays a role in lung graft rejection; and azithromycin (AZI) downregulates IL-17 production. This study examined the effect of broad spectrum MMP inhibition and AZI treatment on BO development. Methods and Materials: BO was induced by transplantation of Fisher F344 rat lungs to Wistar Kyoto (WKY) rats. Five groups of n 5 were studied for 8 weeks: 1. WKY-WKY isografts, 2. F344-WKY control allografts, 3. F344-WKY with MMP inhibitor (tanomastat, Bayer) treatment, 4. F344-WKY with AZI treatment, and 5. F344-WKY with tanomastat and AZI combined treatment. Tanomastat was administered daily and orally starting on the first post-transplant day. AZI was administered i.p. daily for the first 3 days following transplantation and thereafter 3 times per week. Explanted lung grafts were analyzed histologically and by Western blotting. Results: Allografts showed BO characteristics histologically, while isografts did not. MMP-9 and IL-17 protein levels increased in allografts versus isografts (P 0.05). Of all treated groups, the tanomastat/ AZI combination had the best histological outcome evidenced by reduced extracellular matrix deposition and better lung parenchymal preservation compared to control allografts. MMP-9 was not significantly changed in treatment groups versus control allografts. IL-17 protein expression was significantly decreased in both groups receiving AZI treatment versus control allografts (P 0.05). Conclusions: Our results indicate that IL-17 is involved in chronic lung allograft rejection, and that AZI reduces IL-17 protein expression in vivo. MMP inhibition is effective in ameliorating BO when combined with AZI.