Bronchiolitis Obliterans Syndrome Patients Research Articles

Identification of Particles in Exhaled Air Using Mass Spectrometry

Bronchiolitis obliterans syndrome (BOS) as a form of chronic lung allograft dysfunction is a limiting factor to the survival of lung transplant recipients. Detection and monitoring of chronic rejection is hampered by a lack of clinically available markers. Particles in exhaled air (PExA) is proposed as a noninvasive means of potentially identifying and observing BOS patients. This pilot study aims to capture the range of exhaled particles expected in human samples and identify possible candidate markers. The PExA device collects exhaled air using a two-way valve that allows subjects to inhale filtered air and exhale air into a reservoir from which an optical particle counter counts and sizes entering particles. Particles are collected according to their inertia and those above a threshold impact onto a plate, collecting onto a membrane of polytetrafluoroethylene. Total accumulated mass can be measured by the PExA device. Samples were obtained from two healthy subjects (1 combined 600 ng sample and 200 ng from each) and from five patients with lung pathology (1 grouped sample of 600 ng from three patients and 200 ng samples from two unique patients). The samples were analyzed via liquid chromatography-mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer coupled to an easy-nLC1200 liquid chromatography system. The fifty most abundant proteins identified by mass spectrometry represented a breadth of biological function and a variety of localizations. Ten could be traced to immunological system processes and included a number of immunoglobulin constant regions. Sixteen of these most abundant proteins could be found in the extracellular region. A number are components of the plasma, including serotransferrin and annexin proteins. Fifteen proteins were found in greater abundance in the healthy subjects compared to patients while two (haptoglobin and gasdermin-A) were notable for a lower abundance in the patients. The PExA device presents as a novel method for non-invasive analysis of lung transplant patients with the potential to monitor developing chronic rejection. This pilot study characterizes the range of proteins identified from samples across subjects and serves as a basis for the exploration of markers in chronic rejection patients. This work establishes a method for the ongoing effort to use PExA to identify and monitor BOS in lung transplant recipients.

Transition from BOS to RAS Impairs Prognosis after Lung Transplantation - CLAD Subtype Analysis by CT-Volumetry

Chronic lung allograft dysfunction (CLAD) is the most important reason for poor patient survival after lung transplantation. Of the two CLAD subtypes, restrictive allograft syndrome (RAS) has worse prognosis than bronchiolitis obliterans syndrome (BOS), but only limited information is available on the incidence and clinical effect of CLAD subtype change after CLAD onset. We set out to characterize CLAD subtype development by computed tomography (CT) volumetry after CLAD onset. 167 adult patients who underwent de novo lung transplantation in a nationwide transplant centre between 2003-2015 were retrospectively analyzed. CLAD subtype was determined by CT volumetry at CLAD onset. Lung volume decrease of <15% from baseline resulted in BOS and >15% in RAS classification. Patients were re-classified to BOS->RAS or RAS->BOS if persistent lung volume change was detected, and no other specific reasons for lung volume change were present. Radiologic findings of air trapping and fibrosis were linked to lung volume change. Seventy-one (43%) patients were diagnosed with CLAD which was classified as BOS in 63 (89%) and RAS in 8 (11%) patients. After CLAD onset, 6 (9.5%) of BOS patients were re-classified as BOS->RAS, and one (12.5%) RAS patient was re-classified as RAS->BOS whereas other patients remained in their original CLAD subtype. Class change from BOS to RAS occurred at a median of 515.5 days after CLAD onset. Recipient (Figure A, p= 0.002) and graft survival (Figure B, p= 0.003) after CLAD diagnosis was impaired in BOS->RAS group compared to patients remaining in the BOS category. Radiological evidence of both air trapping and fibrosis was seen in 83% of BOS->RAS patients, and fibrotic changes preceded the subtype change in 67% of patients. A significant proportion of BOS patients transform to RAS after CLAD diagnosis, which has an adverse effect on recipient and graft survival.

Pulmonary Markers of Epithelial Cell Activation and Injury in Chronic Lung Allograft Dysfunction

Airway epithelial injury is thought to be a key event in the pathogenesis of CLAD. We investigated whether markers of epithelial activation and injury in bronchoalveolar lavage (BAL) correlate with CLAD diagnosis and the major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS) / mixed phenotype. This was a retrospective, cohort study of all consecutive adult, first, bilateral lung transplants performed 2010-2015. CLAD status and phenotypes were retrospectively determined based on 2019 ISHLT definitions. All patients with RAS/mixed phenotype were included and 1:1 matched with BOS patients based on time from transplant to CLAD-onset. CLAD-free controls were randomly selected. 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 epithelial cell 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. Forty-five CLAD (27 BOS, 11 RAS, 7 mixed) and 26 CLAD-free controls were included. BAL levels of M30, MUC1 and MUC16 were significantly higher in patients with CLAD compared to CLAD-free controls. When comparing CLAD phenotypes, only M30 demonstrated a difference between RAS/mixed and BOS with a significantly higher M30 level in RAS/mixed patients. 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.

VEGF synthesis and VEGF receptor 2 expression in patients with bronchiolitis obliterans syndrome after lung transplantation.

Chronic lung allograft dysfunction including Bronchiolitis obliterans syndrome (BOS) is common after lung transplantation. Histologically, BOS is recognized as fibrotic lesions with accumulated extracellular matrix (ECM) in small airways. Lung fibroblasts are major producers of ECM and vascular endothelial growth factor (VEGF). In this study we hypothesize that VEGF is involved in BOS development after lung transplantation. We investigated the effect of profibrotic transforming growth factor (TGF-β) on VEGF synthesis in lung fibroblasts isolated from distal lung tissue biopsies taken from patients at 3, 6 and 12 months after lung transplantation (n=14). Co-expression of VEGF receptor (VEGFR) 2 and collagen marker prolyl4-hydroxylase (p4OH) were analyzed in lung tissue from patients with BOS (n=11). VEGF synthesis from distal derived lung fibroblasts were significantly lower 3 months after lung transplantation (168.6±133.7; n=7) compared to non-transplanted subjects (451.8±185.9; n=9; p=0.0033) and increased over time at 6 months (584.1±264.9; n=9; p=0.0033) and 12 months (451.1±207.5; n=8; p=0.0065) post transplantation. TGF-β significantly induced VEGF synthesis at all time points. At 12 months post transplantation there was significantly less VEGF synthesis after TGF-β stimulation in fibroblasts obtained from BOS patients (1170±450.2; n=4) compared to patients without any chronic rejection process (1980±417.9; n=4; p<0.039). The numbers of cells expressing VEGFR2/p4OH were increased in patients with BOS (33.2±10.9; n=11) compared to control subjects (10.1±9.9; n=11; p<0.001). Our results support that changes in VEGF/VEGFR2 axis could be involved in BOS development and marker of poor outcome.

Lung Transplantation Has a Strong Impact on the Distribution and Phenotype of Monocyte Subsets

BackgroundLung transplantation (LTx) is a last treatment option for patients with an end-stage pulmonary disease. Chronic lung allograft dysfunction, which generally manifests as bronchiolitis obliterans syndrome (BOS), is a major long-term survival limitation. During injury, inflammation and BOS monocytes are recruited. We determined whether changes in count, subset distribution, and functionality by surface marker expression coincided with BOS development. MethodsFresh whole-blood samples were analyzed from 44 LTx patients, including 17 patients diagnosed with BOS, and compared with 10 age-matched healthy controls and 9 sarcoidosis patients as positive controls. Monocytes were quantified and analyzed using flow cytometry. Based on surface marker expression, classical, intermediate, and nonclassical subsets were determined, and functional phenotypes were investigated. ResultsThe absolute count of monocytes was decreased in LTx and slightly increased in BOS patients. The relative count shifted toward classical monocytes at the expense of nonclassical monocytes in LTx and BOS. Surface marker expression was highest on intermediate monocytes. The expression of both CD36 and CD163 was significantly increased in the LTx and BOS cohort. The difference between the BOS cohort and the LTx cohort was only subtle, with a significant decrease in HLA-DR expression on nonclassical monocytes in BOS. ConclusionsMonocyte subsets and surface marker expression changed significantly in transplantation patients, while BOS-specific changes were understated. More research is needed to determine whether and how monocytes influence the disease process and how current immunosuppressants affect their normal function in vivo.

Use of serum KL-6 level for detecting patients with restrictive allograft syndrome after lung transplantation.

KL-6 is an antigen produced mainly by damaged type II pneumocytes that is involved in interstitial lung disease. Chronic lung allograft dysfunction (CLAD) after lung transplantation (LT) is a major concern for LT clinicians, especially in patients with restrictive allograft syndrome (RAS). We investigated KL-6 levels in serum and bronchoalveolar lavage fluid (BALF) as a potential biomarker of the RAS phenotype. Levels of KL-6 in serum and BALF were measured in 73 bilateral LT recipients, and patients were categorized into 4 groups: stable (ST), infection (LTI), bronchiolitis obliterans syndrome (BOS), and RAS. We also studied a healthy cohort to determine reference values for serum KL-6. The highest levels of KL-6 were found in the serum of patients with RAS (918 [487.8–1638] U/mL). No differences were found for levels of KL-6 in BALF. Using a cut-off value of 465 U/mL serum KL-6 levels was able to differentiate RAS patients from BOS patients with a sensitivity of 100% and a specificity of 75%. Furthermore, higher serum KL-6 levels were associated with a decline in Forced Vital Capacity (FVC) at 6 months after sample collection. Therefore, KL-6 in serum may well be a potential biomarker for differentiating between the BOS and RAS phenotypes of CLAD in LT recipients.

Economic Burden of Bronchiolitis Obliterans Syndrome (BOS) Following Allogeneic Hematopoietic Stem Cell Transplant (alloHSCT) in Patients with Commercial Insurance in the United States

Background Bronchiolitis obliterans syndrome (BOS), also known as respiratory chronic Graft versus Host Disease (cGvHD), is an obstructive airway disease of the lungs following alloHSCT that has significant morbidity and mortality. BOS is characterized by T-cell mediated inflammation and fibrosis of bronchiolar walls that reduces the diameter of the bronchioles resulting in progressive and irreversible airflow obstruction. BOS is a well described complication following lung transplant and can also be a complication following alloHSCT, with similar histopathology and clinical symptoms. There is currently no approved therapy for the treatment of BOS. While data on alloHSCT costs are available (Milliman 2017; Broder et al., 2017), little is known about the impact of BOS on healthcare resource use (HRU) and costs in alloHSCT. The aim of this study is to quantify the economic burden of BOS in alloHSCT patients. Methods We performed a retrospective analysis of commercial claims data from the IQVIA PharMetrics Plus™ database for patients aged 0-64 who were treated with alloHSCT from 1/1/2007 to 9/30/2017. Patients were observable 12 months before and after index alloHSCT. Those who developed BOS were identified using International Classification of Disease (ICD) diagnosis codes (ICD-9: 491.8, 491.9, 515, 516.34, 561.8; ICD-10: J41.8, J42, J84.09, J84.89, J84.115) and propensity score matched to identify patients who did not develop BOS after alloHSCT. We calculated mean annual per patient rates of hospitalizations, tests and treatments for BOS, as well as costs in the follow-up year for inpatient admissions, Emergency Department (ED) and non-ED outpatient visits, and prescription medications. Costs for the index HSCT were included in the first follow-up year costs. All analyses were repeated for the subset of patients observable in the second post-alloHSCT year. The effect of BOS was estimated as the difference in HRU and costs between the matched BOS and non-BOS alloHSCT patients. Results We identified 161 alloHSCT patients with commercial insurance coverage who developed BOS. A majority were male (60%). Mean age was 51.0 yrs (+/- SD: 12.13); a small proportion (2%) were under age 18. We also identified 161 matched alloHSCT patients who did not develop BOS. More BOS patients were diagnosed with leukemias or lymphomas compared with non-BOS patients (94% vs. 84%) in the pre-alloHSCT year, and more had pulmonary disease (asthma or COPD; 24% vs. 16%). No other differences were statistically significant at p&amp;gt;0.05. In the year following alloHSCT, BOS patients had higher mean per patient annual numbers of inpatient admissions (3.9 [+/- SD: 3.3] vs. 2.6 [+/- SD: 2.5]). More BOS patients received lung function testing, including spirometry (60% vs. 42%), and more were treated with ventilation, oxygen therapy or pulmonary rehabilitation (61% vs. 39%). Costs were higher for BOS patients ($560,048 vs. $408,764), with inpatient costs responsible for most of this difference ($446,622 vs. $300,146). Lung function test costs were over 3 times as high for BOS patients ($35,744 vs. $10,192) and lung function treatment costs 2.5 times as high ($31,761 vs. $7,994). Costs for patients observable in the second post-alloHSCT year were considerably lower, as the costs for the initial alloHSCT costs were reflected in the first year expenditures. Nonetheless, costs remained higher for BOS patients ($72,829 vs. $43,665), with higher inpatient, outpatient and prescription drug costs and HRU (Table, Figure). Conclusion AlloHSCT patients who develop BOS in the U.S. have higher rates of hospitalization and have more lung function tests and treatments, compared with alloHSCT patients with no diagnosis of BOS in the first 1-2 years post-alloHSCT. These higher rates of healthcare service use are accompanied by mean annual per patient costs that are $151,000 higher in the first post-alloHSCT year. Disclosures Sacks: Breath Therapeutics Inc.: Other: Employee of Precision Health Economics (PHE). PHE received funding from Breath Therapeutics for this research. . Healey:Breath Therapeutics Inc.: Other: Employee of Precision Health Economics (PHE). PHE received funding from Breath Therapeutics for this research. . Cyr:Breath Therapeutics Inc.: Other: Employee of Precision Health Economics (PHE). PHE received funding from Breath Therapeutics for this research. . Batt:Breath Therapeutics Inc.: Other: Scientific Adviser to Precision Health Economics (PHE). PHE receieved funding from Breath Therapeutics for this research.. Henig:Breath Therapeutics Inc.: Employment.

Prevalence of Bronchiolitis Obliterans Syndrome (BOS) Following Allogeneic Hematopoietic Stem Cell Transplant (alloHSCT) in the USA, Europe and Japan

Background: Bronchiolitis obliterans syndrome (BOS), also known as respiratory chronic Graft versus Host Disease (cGvHD) is an obstructive airway disease of the lungs mainly associated with alloHSCT or lung transplantation. BOS is characterized by T-cell mediated inflammation and fibrosis of bronchiolar walls that reduces the diameter of the bronchioles resulting in progressive and irreversible airflow obstruction. BOS is a well described complication following lung transplant with a 5-year prevalence rate of 50%. It can also be a complication following alloHSCT with similar histopathology and clinical symptoms. Currently, there are no approved treatments for BOS. The aim of this study is to describe the prevalence of BOS following alloHSCT. A review and analysis of the following data sources was performed: AlloHSCT activity reports from The European Society for Blood and Marrow Transplant 2016 the Center for International Blood and Marrow Transplant Research 2016, and the Japanese Data Center Hematopoeitic Stem Cell Transplantation 2015 in order to identify the number of procedures performed. Further a PubMED literature search was also conducted and identified publications with &gt; 100 patients using the key terms "bronchiolitis obliterans and haematopoietic stem cell", and "bronchiolitis obliterans after stem cell transplant" from 2011 to 2017. Case reports and redundant publications were excluded from the analysis. Results Approximately 30,000 alloHSCTs were performed each year in the USA (8766 in 2016), Europe (17,617 in 2016) and Japan (3700 in 2015). Based on seven eligible studies published between 2011 and 2016, the reported prevalence of BOS in patients who underwent alloHSCT ranged from 3.4% to 10%. Median time to diagnosis of BOS following alloHSCT was 273 to 547 days post-transplant. Those with routine Forced Expiratory Volume at 1 second (FEV1) monitoring had a higher likelihood of being diagnosed with BOS and a shorter time to diagnosis. Conclusion Based on estimated prevalence, up to 3000 patients following alloHSCT per year could be diagnosed with BOS. Routine monitoring may positively impact early and shorten time to diagnosis and allow treating such a population. Figure Disclosures Hofstetter: Breath Therapeutics GmbH: Consultancy. Henig:Breath Therapeutics Inc.: Employment. Kappeler:Breath Therapeutics GmbH: Consultancy.

Efficacy of Mesenchymal Stem Cells in Bronchiolitis Obliterans Syndrome after Allogeneic HSCT: A Multicenter Prospective Cohort Study

Background: Bronchiolitis obliterans syndrome (BOS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a devastating complication with limited therapeutic options. We aimed to assess the efficacy and safety of mesenchymal stem cells (MSCs) in BOS after allo-HSCT. Methods: Eighty-one allo-HSCT recipients with BOS diagnosed within 6 months were enrolled in this multicenter prospective cohort study. The choice of prednisone and azithromycin combined with or without MSCs was based on patient preferences (MSC n=49, non-MSC n=32). The primary endpoint was response rate at 3 months, defined as the proportion of patients achieving FEV1 improvement or steroid sparing. This trial was registered at ClinicalTrials.gov, NCT02543073. Results: Response rate was 35/49 patients (71%, 95% confidence intervals [CI] 59 to 84%) (FEV1 improvement n=10, steroid sparing n=25) and 14/32 (44%, 95% CI 27 to 61%) (FEV1 improvement n=3, steroid sparing n=11) in the MSC and non-MSC group, respectively (p=0.013). The adjusted odds ratio of response in the MSC group compared with the non-MSC group was 3.32 (95% CI 1.21-9.11; p=0.02). The addition of MSCs was associated with a better difference for change in FEV1 rate of decline, compared to non-MSC group (53 ml/months, 2 to 103; p=0.040). The 3-year overall survival post-diagnosis was 70.6% (95% CI 55.9 to 85.3%) and 58.2% (95% CI 36.1 to 78.5%) in the MSC and non-MSC group, respectively (p=0.21). Clinical improvement was accompanied by a significant increase of interleukin (IL)-10-producing CD5+ B cells. The incidences of infection and leukemia relapse were no significant difference between two groups. Multiple infusions of MSCs were well-tolerated with no serious adverse events. Interpretation: MSCs might be an effective and safe therapy for BOS patients after allo-HSCT. Our study strengthens evidence for clinical practice of MSC therapy in BOS. These data also offer insight into potential biological mechanisms of MSC treatment and support further investigation in larger randomized controlled trials. Disclosures No relevant conflicts of interest to declare.

Hyaluronic Acid-Decorated Liposomes as Innovative Targeted Delivery System for Lung Fibrotic Cells.

Collagen Tissue Disease–associated Interstitial Lung Fibrosis (CTD-ILDs) and Bronchiolitis Obliterans Syndrome (BOS) represent severe lung fibrogenic disorders, characterized by fibro-proliferation with uncontrolled extracellular matrix deposition. Hyaluronic acid (HA) plays a key role in fibrosis with its specific receptor, CD44, overexpressed by CTD-ILD and BOS cells. The aim is to use HA-liposomes to develop an inhalatory treatment for these diseases. Liposomes with HA of two molecular weights were prepared and characterized. Targeting efficiency was assessed toward CTD-ILD and BOS cells by flow cytometry and confocal microscopy and immune modulation by RT-PCR and ELISA techniques. HA-liposomes were internalized by CTD-ILD and BOS cells expressing CD44, and this effect increased with higher HA MW. In THP-1 cells, HA-liposomes decreased pro-inflammatory cytokines IL-1β, IL-12, and anti-fibrotic VEGF transcripts but increased TGF-β mRNA. However, upon analyzing TGF-β release from healthy donors-derived monocytes, we found liposomes did not alter the release of active pro-fibrotic cytokine. All liposomes induced mild activation of neutrophils regardless of the presence of HA. HA liposomes could be also applied for lung fibrotic diseases, being endowed with low pro-inflammatory activity, and results confirmed that higher MW HA are associated to an increased targeting efficiency for CD44 expressing LFs-derived from BOS and CTD-ILD patients.

Diagnostic value of ventilation/perfusion single-photon emission computed tomography/computed tomography for bronchiolitis obliterans syndrome in patients after lung transplantation.

To evaluate the diagnostic value of function volume/morphological volume ratio calculated from ventilation/perfusion single-photon emission computed tomography/computed tomography in distinguishing the lungs with bronchiolitis obliterans syndrome (BOS) from the lungs without this syndrome after lung transplantation and to assess its relationship with spirometry parameters. We retrospectively identified 84 consecutive lung transplant recipients and 13 donors who underwent ventilation/perfusion single-photon emission computed tomography/computed tomography. Differences in the function volume/morphological volume ratio of unilateral lungs were tested for significance between the lungs with and without BOS. Receiver operating characteristics and correlations between function volume/morphological volume ratios of bilateral lungs and forced expiratory volume in 1 s, forced vital capacity, and total lung capacity were analyzed. The function volume/morphological volume ratios of ventilation and perfusion images of unilateral lungs were significantly lower in lungs with BOS (each P<0.0001). The area under the curve values of ventilation and perfusion images were 0.97 and 0.99, respectively. Significant correlations were identified between the function volume/morphological volume ratios of ventilation and perfusion images and forced expiratory volume in 1 s (r=0.54, P<0.0001 and r=0.45, P<0.0001, respectively). The function volume/morphological volume ratio of ventilation image had a significantly weak correlation with forced vital capacity. The function volume/morphological volume ratio enables a semiquantitative assessment of ventilation and perfusion lung functions and is useful for diagnosing BOS after lung transplantation.

Factors Associated With Mortality and Response to Extracorporeal Photopheresis in Lung Allograft Recipients With Bronchiolitis Obliterans Syndrome.

This study was designed to identify factors associated with clinical response to extracorporeal photopheresis (ECP) and mortality after ECP in lung allograft recipients with bronchiolitis obliterans. Forced expiratory volume in 1 second (FEV1) values obtained 6 months before (baseline) and 6 months after initiation of ECP were used to plot the linear relationship between FEV1 versus time before and after ECP. Response to ECP was assigned when a positive integer was derived after subtracting the baseline rate of decline from the rate of decline 6 months after ECP. Univariate and multivariate logistic regression analyses were used to identify demographic, treatment-related factors or spirometric parameters that may be associated with response to ECP or mortality at either 6 or 16 months after initiation of ECP. Forced expiratory volume in 1 second just before ECP was associated with mortality (P = 0.007) at 16 months after ECP initiation. An FEV1 of 1.50 L or less had a sensitivity of 87% and specificity of 60% to identify patients who died within 16 months after ECP initiation. Patients whose FEV1 decline exceeded 40 mL/month were 12 times more likely to have a response to ECP (P = 0.0001). Patients whose decline in FEV1 before ECP was statistically significant (P < 0.05) were nearly 10 times (P = 0.008) more likely to respond to ECP. Forced expiratory volume in 1 second is an important predictor of mortality, and the response to ECP is influenced by both the extent (>40 mL/mo) and statistical significance of the relationship between FEV1 versus time before ECP initiation. Therefore, earlier bronchiolitis obliterans detection and more timely implementation of ECP (ie, when FEV1 values >1.5 L) should be considered especially in patients with a more aggressive rate of decline of lung function.

Clinical analysis of bronchiolitis obliterans syndrome after haplo- hematopoietic stem cell transplantation

目的探讨单倍型造血干细胞移植（haplo-HSCT）后闭塞性细支气管炎综合征（BOS）的临床特征、危险因素以及预后。方法回顾性分析苏州大学附属第一医院2013年1月至2015年12月444例行haplo-HSCT且总生存（OS）期>100 d患者的临床资料。结果至2018年1月1日随访结束，共计25例（5.63%）患者发生BOS（BOS组），中位发病时间为移植后448（165~845）d，移植后1、2、3年BOS累积发生率分别为1.6%（95%CI 1.5%~1.6%）、4.8%（95%CI 4.7%~4.8%）、5.8%（95%CI 5.7%~5.8%）；在慢性移植物抗宿主病（cGVHD）患者中，移植后1、2、3年BOS累积发生率分别为2.8%（95%CI 2.7%~2.8%）、9.5%（95%CI 9.4%~9.5%）、11.5%（95%CI 11.4%~11.6%）。多因素分析结果显示疾病高危、Ⅱ~Ⅳ度急性移植物抗宿主病（aGVHD）、BOS发生前存在其他部位cGVHD是BOS发生的独立危险因素。BOS组、非BOS组移植后3年OS率分别为71.8%（95%CI 53.9%~89.6%）、72.4%（95%CI 68.1%~76.7%）（P=0.400）；然而，半标志性分析结果显示，BOS组从BOS诊断至随访截止1、3年OS率分别为78.4%（95%CI 61.5%~95.3%）、37.0%（95%CI 2.5%~71.5%），非BOS组从移植后448 d（标志点）至随访截止1、3年OS率分别为93.9%（95%CI 91.3%~96.4%）、89.3%（95%CI 84.8%~93.0%）（P<0.001）。BOS组3年移植相关死亡率（TRM）显著高于非BOS组（28.2%对10.9%，P<0.001）。重度BOS患者OS率显著低于轻、中度BOS患者（P=0.049）。结论BOS是haplo-HSCT后重要并发症，疾病高危、Ⅱ~Ⅳ度aGVHD、BOS发生前存在其他部位cGVHD是BOS发生的独立危险因素。BOS患者预后较差。

Impact of mir21 Expression in Chronic Lung Allograft Dysfunction after Lung Transplantation

Purpose Long-term survival after lung transplantation is still limited by the development of chronic lung allograft dysfunction (CLAD), presenting either as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Epithelial-to-mesenchymal transition (EMT) is recognized as one of the pathogenic processes contributing to CLAD. MiR-21 is a post-translational regulator of several pathways of fibrosis and has recently been related to EMT. In this study we aimed to characterize miR-21 expression and other markers of EMT in lung tissue of BOS and RAS patients. Methods Formalin-fixed paraffin-embedded tissue of 17 BOS patients and 7 RAS patients undergoing a lung retransplantation due to CLAD and 5 normal lung tissue samples (control) were evaluated for the expression following markers: vimentin, notch intracellular domain (NICD), p-SMAD 2/3, β-catenin, and E-cadherin. Slides were additionally stained for miR-21 by in situ hybridization (ISH). Expression of respective markers were graded in a semi-quantified manner by two blinded pathologists. Results Vimentin, NICD, p-SMAD 2/3 were positive in 13 (77%), 12 (71%), and 7 (42%) BOS patients, and in 5 (71%), 4 (57%) and 4 (57%) RAS patients, respectively. In contrast, β-catenin and E-cadherin staining were consistently negative in both groups. In comparision, no significant difference was found between BOS and RAS regarding these markers. MiR-21 expression was positive in 5 (30%) BOS cases and 6 (86%) RAS cases. The rate of miR-21 expression was significantly higher in RAS than in BOS (p=0.01). MiR-21 expression was associated with significant shorter freedom to BOS (11.5 vs 46.0 months, p=0.001, Figure 1), but had no impact on the development of RAS. Conclusion This first preliminary study could identify miR-21 as a possible biomarker and therapeutic target of CLAD and mirR-21 expression was related to worse outcomes in BOS cases. Further research is necessary to validate these findings in a larger cohort of patients.

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.

Development of a New Local Therapeutic Approach for BOS: Efficacy of Imatinib Loaded -antiCD44 Coated Gold Nanoparticles In Vitro and In Vivo

Purpose The use of gold nanoparticles (GNPs) as local targeted drug delivery system are a promising issue for several orphan diseases. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction limiting long term survival after lung transplant, and occurs also as chronic pulmonary involvement in the context of GVHD. To now treatment strategies in BOS are scarce and poorly effective. In the present work, we investigated the effect of GNP decorated with the half chain of monoclonal antibody against CD44 (GNP-HC), surface receptor overexpressed by primary BOS derived mesenchymal cells (MCs) that cause the partial or total occlusion of alveolar tract. We loaded GNP-HC with imatinib (GNP-HCim), an cAbl inhibitor used in the management of chronic GVHD. Methods In vitro experiments were performed on MCs derived from BOS patients evaluating cell cytotoxicity after incubation with GNP-HC, GNP-HCim and Imatinib alone. We further performed a mouse heterotopic trachea transplantation model modified by the subcutaneous placement of Alzet Model 2004 miniosmotic pumps used to deliver into the trachea different solutions at a constant rate for 28 days. This was performed in order to assess the efficacy of the local intratracheal administration of our nanovectors. Results Thanks to MTT and apoptosis assay we observed that GNP-HCim decrease MCs viability more than GNP-HC and Imatinib alone, due to an induction of apoptosis (50 ± 0.5 % for GNP-HCim in contrast to GNP-HC 40 ± 0.35 % and Imatinib alone 30 ± 1.2 %). Moreover, animal model data confirmed the in vitro results: GNP-HCim significantly reduced the percentage of tracheal obliterative area (12.99 ± 2.5%) respect to GNP-HC (46.79± 5.7%) and vehicle treated mice (30.92 ± 5.8%). Conclusion We conclude that this approach by newly developed nano-based strategies for BOS treatment and especially targeted imatinib gold nanoparticles are promising candidates for further development.

Rapid versus gradual lung function decline in bronchiolitis obliterans syndrome after haematopoietic stem cell transplantation is associated with survival outcome.

Bronchiolitis obliterans syndrome (BOS) after haematopoietic stem cell transplantation (HSCT) presents with lung function decline. The pattern of lung function decline after BOS diagnosis could impact prognostication of BOS as a complication after HSCT. The aim of this study was to assess the impact of lung function decline on overall survival (OS) in BOS subjects. Subjects with BOS were compared to those without BOS and matched for age, gender, primary diagnoses, conditioning regimes and chronic graft versus host disease. Lung function tests at baseline, at BOS diagnosis and every 3 months after HSCT were evaluated. Of the 1461 subjects undergoing allogeneic HSCT (allo-HSCT) between 1998 and 2015, 95 (6.5%) were diagnosed with BOS. A total of 159 matched HSCT recipients without BOS were identified. A 25% decline in FEV1 within the first 3 months after BOS diagnosis would separate BOS subjects into a subgroup with initial rapid decline and another subgroup with initial gradual decline in lung function. The rapid decline group showed lower subsequent lung function parameters and significantly worse OS compared to the gradual decline group (P = 0.013). Post-HSCT BOS subjects with initial rapid lung function decline within 3 months after BOS diagnosis will have significantly poorer lung function and worse OS compared to those with initial gradual decline in lung function after BOS diagnosis. HSCT BOS patients with rapid initial decline in lung function warrant closer monitoring for the development of other post-HSCT complications that could affect their survival.

Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.

Chronic lung allograft dysfunction (CLAD) is a fatal condition that limits survival after lung transplantation (LTx). The pathological hallmark of CLAD is obliterative bronchiolitis (OB). A subset of patients present with a more aggressive CLAD phenotype, called restrictive allograft syndrome (RAS), characterized by lung parenchymal fibrosis (PF). The mouse orthotopic single LTx model has proven relevant to the mechanistic study of allograft injury. The minor-alloantigen-mismatched strain combination using C57BL/10(B10) donors and C57BL/6(B6) recipients reportedly leads to OB. Recognizing that OB severity is a spectrum that may coexist with other pathologies, including PF, we aimed to characterize and quantify pathologic features of CLAD in this model. Left LTx was performed in the following combinations: B10→B6, B6→B10, B6→B6. Four weeks posttransplant, blinded pathologic semi-quantitative assessment showed that OB was present in 66% of B10→B6 and 30% of B6→B10 grafts. Most mice with OB also had PF with a pattern of pleuroparenchymal fibroelastosis, reminiscent of human RAS-related pathology. Grading of pathologic changes demonstrated variable severity of airway fibrosis, PF, acute rejection, vascular fibrosis, and epithelial changes, similar to those seen in human CLAD. These assessments can make the murine LTx model a more useful tool for further mechanistic studies of CLAD pathogenesis.

Montelukast for bronchiolitis obliterans syndrome after lung transplantation: A randomized controlled trial.

Bronchiolitis obliterans syndrome (BOS) remains the major problem which precludes long-term survival after lung transplantation. Previously, an open label pilot study from our group demonstrated a possible beneficial effect of montelukast in progressive BOS patients with low airway neutrophilia (<15%), and already on azithromycin treatment, in whom the further decline in pulmonary function was attenuated. This was, however, a non-randomized and non-placebo controlled trial. The study design is a single center, prospective, interventional, randomized, double blind, placebo-controlled trial, with a two arm parallel group design and an allocation ratio of 1:1. Randomization to additional montelukast (10 mg/day, n = 15) or placebo (n = 15) was performed from 2010 to 2014 at the University Hospitals Leuven (Leuven, Belgium) in all consecutive patients with late-onset (>2years posttransplant) BOS ≥1. Primary end-point was freedom from graft loss 1 year after randomization; secondary end-points were acute rejection, lymphocytic bronchiolitis, respiratory infection rate; and change in FEV1, airway and systemic inflammation during the study period. Graft loss at 1 y and 2y was similar in both groups (respectively p = 0. 981 and p = 0.230). Montelukast had no effect on lung function decline in the overall cohort. However, in a post-hoc subanalysis of BOS stage 1 patients, montelukast attenuated further decline of FEV1 during the study period, both in absolute (L) (p = 0.008) and % predicted value (p = 0.0180). A linear mixed model confirmed this association. Acute rejection, lymphocytic bronchiolitis, respiratory infections, systemic and airway inflammation were comparable between groups over the study period. This randomized controlled trial showed no additional survival benefit with montelukast compared to placebo, although the study was underpowered. The administration of montelukast was associated with an attenuation of the rate of FEV1 decline, however, only in recipients with late-onset BOS stage 1.

High circulating CD4+CD25hiFOXP3+ T-cell sub-population early after lung transplantation is associated with development of bronchiolitis obliterans syndrome

Chronic bronchiolitis obliterans syndrome (BOS) remains a major limitation for long-term survival after lung transplantation. The immune mechanisms involved and predictive biomarkers have yet to be identified. The purpose of this study was to determine whether peripheral blood T-lymphocyte profile could predict BOS in lung transplant recipients. An in-depth profiling of CD4+ and CD8+ T cells was prospectively performed on blood cells from stable (STA) and BOS patients with a longitudinal follow-up. Samples were analyzed at 1 and 6 months after transplantation, at the time of BOS diagnosis, and at an intermediate time-point at 6 to 12 months before BOS diagnosis. Although no significant difference was found for T-cell compartments at BOS diagnosis or several months beforehand, we identified an increase in the CD4+CD25hiFoxP3+ T-cell sub-population in BOS patients at 1 and 6 months after transplantation (3.39 ± 0.40% vs 1.67 ± 0.22% in STA, p < 0.001). A CD4+CD25hiFoxP3+ T-cell threshold of 2.4% discriminated BOS and stable patients at 1 month post-transplantation. This was validated on a second set of patients at 6 months post-transplantation. Patients with a proportion of CD4+CD25hiFoxP3+ T cells up to 2.4% in the 6 months after transplantation had a 2-fold higher risk of developing BOS. This study is the first to report an increased proportion of circulating CD4+CD25hiFoxP3+ T cells early post-transplantation in lung recipients who proceed to develop BOS within 3 years, which supports its use as a BOS predictive biomarker.