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Background: Insults to the airway epithelium play a key role in constrictive bronchiolitis after lung transplantation, the typical hallmark of chronic rejection. Our hypothesis is that immunosuppressives might affect airway integrity., Methods: A biculture of human bronchial epithelial cells and lung microvascular endothelial cells was exposed to immunosuppressives (serum through levels) for 24 hours or 4 days. Cytotoxicity, transepithelial electrical resistance (TEER), and permeability was measured after exposure to monotherapies and combination therapies. Apoptosis, oxidative stress, inflammation (IL-8), real-time polymerase chain reaction for epithelial-to-mesenchymal transition and tight junction proteins were assessed in exposed cells., Results: Mycophenolate mofetil (MMF) and combination therapies including MMF, at serum trough levels and higher, are toxic for the human bronchial epithelial cells after 4-day exposure. Moreover, already after 24 hours, TEER of cells exposed to MMF decreases and permeability increases. MMF did not induce apoptosis, oxidative stress, loss of tight junctions or production of IL-8 after 24 hours, but possibly induces epithelial-to-mesenchymal transition in epithelial cells. MMF was detectable at both sides of the biculture and was also present in bronchoalveolar lavage of lung transplantation patients. Other immunosuppressives were not toxic, neither changed TEER or permeability., Conclusions: Our findings suggest that MMF is present in the airways of lung transplant patients and might affect the structural integrity of the airway, which needs further investigation and validation in the clinical setting.

The single most important cause of late mortality after lung transplantation is chronic lung allograft dysfunction (CLAD). However, the pathological development of CLAD was not as simple as previously presumed and subclassification phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD), have been introduced. We want to re-investigate how CLAD manifests in the murine orthotopic lung transplant model and investigate the role of interleukin 17A (IL-17A) within this model. Orthotopic LTx was performed in CB57BL/6, IL-17 WT and IL-17 KO mice. In a first experiment, CB57BL/6 mice receiving an isograft (CB57BL/6) or allograft (BALB/C) were compared. In a second experiment IL-17 WT and IL-17 KO mice (both CB57BL/6 background) received an allograft (BALB/C). Mice received daily immunosuppression with steroids and cyclosporine and were sacrificed 10weeks after transplantation for histopathological analysis by an experienced lung pathologist. After murine orthotopic lung transplantation, the allograft histopathologically presented features of human rCLAD (i.e. overt inflammation, pleural/parenchymal fibrosis and obliterative bronchiolitis). In the IL-17A KO group, less inflammation in the bronchovascular axis (p=0.03) was observed and a non-significant trend towards less bronchovascular fibrosis, pleural/septal inflammation and fibrosis, and parenchymal inflammation and fibrosis when compared to WT mice. The major mismatch orthotopic lung transplant model resembles features of human rCLAD. IL-17A mediated immunity is involved in the inflammatory component, but had little influence on the degree of fibrosis. Further mechanistic and therapeutic studies in this mouse model are needed to fully understand the mechanisms in rCLAD., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bronchiolitis obliterans syndrome (BOS) remains a major complication after lung transplantation. Air trapping and mosaic attenuation are typical radiological features of BOS; however, quantitative evaluation remains troublesome. We evaluated parametric response mapping (PRM, voxel-to-voxel comparison of inspiratory and expiratory computed tomography [CT] scans) in lung transplant recipients diagnosed with BOS (n = 20) and time-matched stable lung transplant recipients (n = 20). Serial PRM measurements were performed prediagnosis, at time of BOS diagnosis, and postdiagnosis (T pre , T 0 , and T post , respectively), or at a postoperatively matched time in stable patients. PRM results were correlated with pulmonary function and confirmed by microCT analysis of end-stage explanted lung tissue. Using PRM, we observed an increase in functional small airway disease (fSAD), from T pre to T 0 (p = 0.006) and a concurrent decrease in healthy parenchyma (p = 0.02) in the BOS group. This change in PRM continued to T post , which was significantly different compared to the stable patients (p = 0.0002). At BOS diagnosis, the increase in fSAD was strongly associated with a decrease in forced expiratory volume in 1 s (p = 0.011). Micro-CT confirmed the presence of airway obliteration in a sample of a BOS patient identified with 67% fSAD by PRM. We demonstrated the use of PRM as an adequate output to monitor BOS progression in lung transplant recipients., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Recently, antibody mediated rejection (AMR) has been associated with a higher incidence of chronic lung allograft dysfunction (CLAD) and mortality after lung transplantation (LTx). We investigated markers related to AMR and matrix remodeling in CLAD, with special attention for its two phenotypes being bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD)., Methods: Immunoglobulins (IgA, IgE, IgG1-IgG4, total IgG and IgM) and complement (C4d and C1q) were quantified in lung lavage samples at the moment of BOS (n=15) or RAS (n=16) diagnosis; and were compared to stable transplant patients who served as control (n=14). Also, airway remodeling and metalloproteinases (MMPs) were investigated via zymography and gelatin degradation. The presence of DSA was additionally assessed in blood., Results: Total IgG, IgG1-IgG4 and IgM were increased in rCLAD versus control (p<0.001) and BOS patients (p<0.01). IgA and IgE were increased in rCLAD compared to control (respectively p<0.05 and p<0.01), but not to BOS. Total IgG and IgE were increased in BOS versus control (respectively p<0.01 and p<0.05). Complement proteins were exclusively present in rCLAD and correlated positively with immunoglobulins. Additionally, in blood, DSA were more present in rCLAD (p=0.041). MMP-9 levels increased in RAS and BOS versus control (p<0.001) and MMP-9 induced gelatin degradation was only increased in BOS compared to control (p<0.01)., Conclusion: We demonstrated increased levels of immunoglobulins and complement proteins dominantly present in rCLAD. This leads to the belief that antibodies and AMR might play a more important role in rCLAD compared to BOS. Therefore, anti B-cell therapy could offer beneficial therapeutic effects in patients diagnosed with rCLAD, which needs further research., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Prophylactic azithromycin treatment has been demonstrated to improve freedom from bronchiolitis obliterans syndrome (BOS) 2 years after lung transplantation (LTx). In the current study, we re-evaluated the long-term effects of this prophylactic approach in view of the updated classification system for chronic lung allograft dysfunction (CLAD). A retrospective, intention-to-treat analysis of a randomized controlled trial comparing prophylactic treatment with placebo (n = 43) versus azithromycin (n = 40) after LTx was performed. Graft dysfunction (CLAD), graft loss (retransplantation, mortality), evolution of pulmonary function and functional exercise capacity were analyzed 7 years after inclusion of the last study subject. Following LTx, 22/43 (51%) patients of the placebo group and 11/40 (28%) patients of the azithromycin group ever developed CLAD (p = 0.043). CLAD-free survival was significantly longer in the azithromycin group (p = 0.024). No difference was present in proportion of obstructive versus restrictive CLAD between both groups. Graft loss was similar in both groups: 23/43 (53%) versus 16/40 (40%) patients (p = 0.27). Long-term pulmonary function and functional exercise capacity were significantly better in the azithromycin group (p < 0.05). Prophylactic azithromycin therapy reduces long-term CLAD prevalence and improves CLAD-free survival, pulmonary function, and functional exercise capacity after LTx., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Chronic lung allograft dysfunction (CLAD) remains the major barrier to long-term success after lung transplantation. This report compares gross and microscopic features of lungs removed from patients receiving a redo-transplant as treatment for CLAD. Lungs donated by patients with either the bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS) phenotype of CLAD and appropriate control lungs (eight per group) were air-inflated, frozen solid and kept frozen while a multi-detector computed tomography (MDCT) was obtained. The lung was then cut into 2-cm thick transverse slices and sampled for micro-CT and histopathology. The MDCT showed reduced lung volume with increased lung weight and density in RAS versus BOS and control (p<0.05). Although pre-terminal bronchioles were obstructed in both phenotypes, RAS lungs showed a reduction of pre-terminal bronchioles (p<0.01). Micro-CT and matched histopathology showed that RAS was associated with reduced numbers of terminal bronchioles/lung compared to BOS and controls (p<0.01), with expansion of the interstitial compartment and obliteration of the alveolar airspaces by fibrous connective tissue. RAS is associated with greater destruction of both pre-terminal and terminal bronchioles. Additionally, the interstitial compartments are expanded and alveolar airspaces are obliterated by accumulation of fibrous connective tissue., (Copyright ©ERS 2015.)

Background: Chronic lung allograft dysfunction is a heterogeneous entity limiting long-term survival after lung transplantation. Different clinical phenotypes (bronchiolitis obliterans syndrome [BOS]-neutrophilic BOS-restrictive allograft syndrome [RAS]) have been identified but the mechanisms remain elusive., Methods: In this study, we measured 34 different cytokines, chemokines, and growth factors in bronchoalveolar lavage fluid of 20 stable patients, 20 patients suffering from non-neutrophilic BOS, 17 from neutrophilic BOS, and 20 from RAS using classic enzyme-linked immunosorbent assay and multiplex technology., Results: Total cell count and % neutrophils were elevated in neutrophilic BOS and RAS compared to stable and non-neutrophilic BOS patients, whereas also the % eosinophils was elevated at diagnosis of RAS. Levels of interleukin (IL)-1β (P<0.01), IL-1Rα (P<0.001), IL-6 (P<0.001), IL-8/CXCL8 (P<0.001), IP-10/CXCL10 (P<0.05), MCP-1/CCL2 (P<0.05), macrophage inflammatory protein (MIP)-1α/CCL3 (P<0.001), MIP-1β/CCL4, and vascular endothelial growth factor (VEGF; P<0.05) were differentially regulated in RAS compared to stable, whereas in neutrophilic BOS IL-1β (P<0.001), IL-1Rα (P<0.01), IL-7 (P<0.05), IL-8/CXCL8 (P<0.001), MCP-3/CXCCL7 (P<0.05) and MIP-1α/CCL-3 (P<0.05) were significantly upregulated compared to stable patients. We could not detect any differences between non-neutrophilic BOS and stable patients. Interestingly, bronchoalveolar lavage IL-6, interferon gamma-induced protein (IP)-10/CXCL10 and interferon-inducible T-cell alpha chemoattractant/chemokine (C-X-C motif) ligand 11 (ITAC/CXCL10) were associated with survival after diagnosis in RAS patients., Conclusion: There were major differences in cytokine and chemokine expression in our different study groups. Especially IL-6, but also IP-10/CXCL10, and VEGF may be interesting mediators in RAS.

Distinct phenotypes of chronic lung allograft dysfunction (CLAD) after lung transplantation are emerging with lymphocytic bronchiolitis (LB)/azithromycin reversible allograft dysfunction (ARAD), classical or fibrotic bronchiolitis obliterans syndrome (BOS), and restrictive allograft syndrome (RAS) proposed as separate entities. We have additionally identified lung transplant recipients with prior LB, demonstrating persistent airway neutrophilia (PAN) despite azithromycin treatment. The aim of this study was to evaluate differences in the airway microenvironment in different phenotypes of CLAD. Bronchoalveolar lavage (BAL) from recipients identified as stable (control), LB/ARAD, PAN, BOS, and RAS were evaluated for differential cell counts and concentrations of IL-1α, IL-1β, IL-6, IL-8, and TNF-α. Primary human bronchial epithelial cells were exposed to BAL supernatants from different phenotypes and their viability measured. BOS and RAS showed increased BAL neutrophilia but no change in cytokine concentrations compared with prediagnosis. In both LB/ARAD and PAN, significant increases in IL-1α, IL-1β, and IL-8 were present. BAL IL-6 and TNF-α concentrations were increased in PAN and only this phenotype demonstrated decreased epithelial cell viability after exposure to BAL fluid. This study demonstrates clear differences in the airway microenvironment between different CLAD phenotypes. Systematic phenotyping of CLAD may help the development of more personalized approaches to treatment., (© 2014 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.)

Rationale: The chronic rejection of lung allografts is attributable to progressive small airway obstruction., Objectives: To determine precisely the site and nature of this type of airway obstruction., Methods: Lungs from patients with rejected lung allografts treated by a second transplant (n = 7) were compared with unused donor (control) lungs (n = 7) using multidetector computed tomography (MDCT) to determine the percentage of visible airways obstructed in each airway generation, micro-computed tomography (microCT) to visualize the site of obstruction, and histology to determine the nature of this obstruction., Measurements and Main Results: The number of airways visible with MDCT was not different between rejected and control lungs. However, 10 ± 7% of observed airways greater than 2 mm in diameter, 50 ± 22% of airways between 1 and 2 mm in diameter, and 73 ± 10% of airways less than 1 mm in diameter were obstructed in the rejected lungs. MicroCT confirmed that the mean lumen diameter of obstructed airways was 647 ± 317 μm but showed no difference in either total number and cross-sectional area of the terminal bronchioles or in alveolar dimensions (mean linear intercept) between groups (P > 0.05). In addition, microCT demonstrated that only segments of the airways are obstructed. Histology confirmed a constrictive form of bronchiolitis caused by expansion of microvascular-rich granulation tissue in some locations and collagen-rich scar tissue in others., Conclusions: Chronic lung allograft rejection is associated with a progressive form of constrictive bronchiolitis that targets conducting airways while sparing larger airways as well as terminal bronchioles and the alveolar surface.

Lung transplantation is currently considered as an ultimate live-saving treatment for selected patients suffering from end-stage pulmonary disease. Long-term survival, however, is hampered by chronic rejection, or chronic lung allograft dysfunction (CLAD). Recently, various phenotypes within CLAD have been identified, challenging the established clinical definition of bronchiolitis obliterans syndrome (BOS). Some patients with presumed BOS, for instance, demonstrate an important improvement in forced expiratory volume in the first second of expiration (FEV1) after treatment with azithromycin. These patients are characterized by the presence of excess (≥ 15%) bronchoalveolar lavage (BAL) neutrophils, in absence of concurrent infection. This phenotype of CLAD has been redefined as neutrophilic reversible allograft dysfunction (NRAD), and these patients generally have a very good prognosis after diagnosis. Another group of patients with CLAD develop a restrictive rather than an obstructive pulmonary function defect (defined as a decline in total lung capacity of at least 10%) and demonstrate persistent interstitial and ground-glass opacities on chest computed tomographic (CT) scan. This phenotype is called restrictive allograft syndrome (RAS), and patients with RAS have a much worse prognosis after diagnosis. This review further discusses both of these CLAD phenotypes that do not fit the classical definition of BOS. Potential pathophysiological mechanisms, etiology, diagnosis, prognosis, and treatments are discussed., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Chronic rejection is the major problem hampering long-term survival after lung transplantation. Recently, it became clear that patients may develop an obstructive (bronchiolitis obliterans syndrome [BOS]) or a restrictive lung function defect (restrictive allograft syndrome [RAS]), for which specific risk factors are unknown., Methods: A retrospective analysis of our lung transplantation cohort was performed (n=380). Patients with an irreversible decline in forced expiratory volume in 1 second were identified and classified as BOS or RAS. Patient characteristics, bronchoalveolar lavage (BAL) cellularity, rates of respiratory tract infection, colonization, acute rejection, and lymphocytic bronchiolitis were compared between BOS, RAS, and stable patients., Results: There were 103 patients suffering from chronic rejection, of which 79 had BOS and 24 were diagnosed with RAS. There were more patients with infection and pseudomonal colonizations in BOS and RAS compared with control (P=0.0090 and P=0.0034, respectively). More patients ever experienced acute and severe acute rejections (A≥2; both P<0.0001) and lymphocytic bronchiolitis (P=0.0006) in BOS and RAS versus control. There were more patients experiencing severe lymphocytic bronchiolitis in RAS compared with BOS (P=0.031). BAL neutrophilia in BOS and RAS were elevated at days 360, 540, and 720 versus control. BOS, but especially RAS patients, experienced more frequent episodes of increased BAL eosinophilia (≥2%; P<0.0001)., Conclusion: Acute rejection, lymphocytic bronchiolitis, colonization with pseudomonas, infection, and BAL eosinophilia and neutrophilia are risk factors for the later development not only of RAS but also of BOS.