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

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

Prognostic Value of Chest CT Findings at BOS Diagnosis in Lung Transplant Recipients.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) after lung transplantation is characterized by fibrotic small airway remodeling, recognizable on high-resolution computed tomography (HRCT). We studied the prognostic value of key HRCT features at BOS diagnosis after lung transplantation. METHODS: The presence and severity of bronchiectasis, mucous plugging, peribronchial thickening, parenchymal anomalies, and air trapping, summarized in a total severity score, were assessed using a simplified Brody II scoring system on HRCT at BOS diagnosis, in a cohort of 106 bilateral lung transplant recipients transplanted between January 2004 and January 2016. Obtained scores were subsequently evaluated regarding post-BOS graft survival, spirometric parameters, and preceding airway infections. RESULTS: A high total Brody II severity score at BOS diagnosis (P = 0.046) and high subscores for mucous plugging (P = 0.0018), peribronchial thickening (P = 0.0004), or parenchymal involvement (P = 0.0121) are related to worse graft survival. A high total Brody II score was associated with a shorter time to BOS onset (P = 0.0058), lower forced expiratory volume in 1 s (P = 0.0006) forced vital capacity (0.0418), more preceding airway infections (P = 0.004), specifically with Pseudomonas aeruginosa (P = 0.002), and increased airway inflammation (P = 0.032). CONCLUSIONS: HRCT findings at BOS diagnosis after lung transplantation provide additional information regarding its underlying pathophysiology and for future prognosis of graft survival.

COVID-19 progression in hospitalized patients using follow-up in vivo CT and ex vivo microCT.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19) which can lead to acute respiratory distress syndrome (ARDS) and evolve to pulmonary fibrosis. Computed tomography (CT) is used to study disease progression and describe radiological patterns in COVID-19 patients. This study aimed to assess disease progression regarding lung volume and density over time on follow-up in vivo chest CT and give a unique look at parenchymal and morphological airway changes in "end-stage" COVID-19 lungs using ex vivo microCT. Methods: Volumes and densities of the lung/lobes of three COVID-19 patients were assessed using follow-up in vivo CT and ex vivo whole lung microCT scans. Airways were quantified by airway segmentations on whole lung microCT and small-partition microCT. As controls, three discarded healthy donor lungs were used. Histology was performed in differently affected regions in the COVID-19 lungs. Results: In vivo, COVID-19 lung volumes decreased while density increased over time, mainly in lower lobes as previously shown. Ex vivo COVID-19 lung volumes decreased by 60% and all lobes were smaller compared to controls. Airways were more visible on ex vivo microCT in COVID-19, probably due to fibrosis and increased airway diameter. In addition, small-partition microCT showed more deformation of (small) airway morphology and fibrotic organization in severely affected regions with heterogeneous distributions within the same lung which was confirmed by histology. Conclusions: COVID-19-ARDS and subsequent pulmonary fibrosis alters lung architecture and airway morphology which is described using in vivo CT, ex vivo microCT, and histology.

Aspergillus-Specific IgG Antibodies are Associated With Fungal-Related Complications and Chronic Lung Allograft Dysfunction After Lung Transplantation.

Fungal exposure and sensitization negatively affect outcomes in various respiratory diseases, however, the effect of fungal sensitization in lung transplant (LTx) recipients is still unknown. We performed a retrospective cohort study of prospectively collected data on circulating fungal specific IgG/IgE antibodies, and their correlation with fungal isolation, chronic lung allograft dysfunction (CLAD) and overall survival after LTx. 311 patients transplanted between 2014 and 2019 were included. Patients with elevated Aspergillus fumigatus or Aspergillus flavus IgG (10%) had more mold and Aspergillus species isolation (p = 0.0068 and p = 0.0047). Aspergillus fumigatus IgG was specifically associated with Aspergillus fumigatus isolation in the previous or consecutive year (AUC 0.60, p = 0.004 and AUC 0.63, p = 0.022, respectively). Elevated Aspergillus fumigatus or Aspergillus flavus IgG was associated with CLAD (p = 0.0355), but not with death. Aspergillus fumigatus, Aspergillus flavus or Aspergillus niger IgE was elevated in 19.3% of patients, but not associated with fungal isolation, CLAD or death. Mold isolation and Aspergillus species isolation from respiratory cultures were associated with CLAD occurrence (p = 0.0011 and p = 0.0005, respectively), and Aspergillus species isolation was also associated with impaired survival (p = 0.0424). Fungus-specific IgG could be useful in long-term follow-up post-LTx, as a non-invasive marker for fungal exposure, and thus a diagnostic tool for identifying patients at risk for fungal-related complications and CLAD.

The MUC5B Promoter Polymorphism is Not Associated With Non-ILD Chronic Respiratory Diseases or Post-transplant Outcome.

The MUC5B promoter polymorphism (rs35705950) has been associated with interstitial lung disease (ILD) and with prolonged pre-transplant survival in idiopathic pulmonary fibrosis (IPF), but no information is available regarding its prevalence in other respiratory diseases and its influence on post-transplant outcome. We included the Leuven lung transplantation cohort between 1991 and 2015 (n = 801). We assessed the minor allele frequency (MAF) of the MUC5B variant in the entire study cohort and investigated the influence of recipient MUC5B promoter polymorphism on post-transplant outcome in patients who were transplanted after 2004. MUC5B was successfully genotyped in 746 patients. The MAF was significantly higher in ILD (17.6%) compared to chronic obstructive pulmonary disease (COPD)/emphysema (9.3%), cystic fibrosis (CF)/bronchiectasis (BRECT) (7.5%) and pulmonary hypertension (PHT) (7.4%) (p < 0.001). No association was observed between rs35705950 and chronic lung allograft dysfunction (CLAD)/graft loss in the ILD population [CLAD: HR 1.37 95% CI (0.70-2.68); graft loss: HR 1.02 95% CI (0.55-1.89)], nor the entire study cohort [CLAD: HR 0.96 95% CI (0.69-1.34); graft loss: HR 0.97 95% CI (0.70-1.35)]. The MUC5B promoter polymorphism is a very specific predictive factor for the presence of pulmonary fibrosis as it is only associated with pulmonary fibrosis and not with other chronic respiratory diseases. While the MUC5B promoter variant is associated with better pre-transplant survival among IPF patients, recipient MUC5B promoter variant does not play a role in post-transplant outcome.

Pulmonary graft-versus-host disease and chronic lung allograft dysfunction: two sides of the same coin?

Allogeneic haematopoietic stem-cell transplantation (HSCT) and lung transplantation are both established life-saving treatment options for carefully selected patients with various haematological disorders or end-stage lung diseases, respectively. However, long-term survival after allogeneic HSCT is severely limited by chronic graft-versus-host disease (cGVHD)-of which pulmonary cGVHD in particular has a very poor prognosis-and long-term survival after lung transplantation is hampered by chronic lung allograft dysfunction (CLAD). Both pulmonary cGVHD and CLAD are characterised by similar underlying immunopathology, which results in transplant-related pulmonary fibrosis and structural lung remodelling, leading to respiratory dysfunction. Accurate clinical identification and appropriate management is of utmost importance to allow for timely diagnosis, to further optimise current preventive and treatment strategies of pulmonary cGVHD and CLAD, and to ameliorate quality of life and long-term outcomes after allogeneic HSCT and lung transplantation. In this Review, we provide a unique state-of-the-art perspective of both entities for respiratory care practitioners.

Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

Lung Microenvironments and Disease Progression in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis (fHP) is an interstitial lung disease caused by sensitization to an inhaled allergen. Objectives: To identify the molecular determinants associated with progression of fibrosis. Methods: Nine fHP explant lungs and six unused donor lungs (as controls) were systematically sampled (4 samples/lung). According to microcomputed tomography measures, fHP cores were clustered into mild, moderate, and severe fibrosis groups. Gene expression profiles were assessed using weighted gene co-expression network analysis, xCell, gene ontology, and structure enrichment analysis. Gene expression of the prevailing molecular traits was also compared with idiopathic pulmonary fibrosis (IPF). The explant lung findings were evaluated in separate clinical fHP cohorts using tissue, BAL samples, and computed tomography scans. Measurements and Main Results: We found six molecular traits that associated with differential lung involvement. In fHP, extracellular matrix and antigen presentation/sensitization transcriptomic signatures characterized lung zones with only mild structural and histological changes, whereas signatures involved in honeycombing and B cells dominated the transcriptome in the most severely affected lung zones. With increasing disease severity, endothelial function was progressively lost, and progressive disruption in normal cellular homeostatic processes emerged. All six were also found in IPF, with largely similar associations with disease microenvironments. The molecular traits correlated with in vivo disease behavior in a separate clinical fHP cohort. Conclusions: We identified six molecular traits that characterize the morphological progression of fHP and associate with in vivo clinical behavior. Comparing IPF with fHP, the transcriptome landscape was determined considerably by local disease extent rather than by diagnosis alone.

Antifibrotic drugs in lung transplantation and chronic lung allograft dysfunction: a review.

This review aims to provide an overview of pre-transplant antifibrotic therapy on peri-transplant outcomes and to address the possible role of antifibrotics in lung transplant recipients with chronic lung allograft dysfunction.Lung transplantation is an established treatment modality for patients with various end-stage lung diseases, of which idiopathic pulmonary fibrosis and other progressive fibrosing interstitial lung diseases are growing indications. Theoretically, widespread use of antifibrotics prior to lung transplantation may increase the risk of bronchial anastomotic complications and impaired wound healing.Long-term graft and patient survival are still hampered by development of chronic lung allograft dysfunction, on which antifibrotics may have a beneficial impact.Antifibrotics until the moment of lung transplantation proved to be safe, without increasing peri-transplant complications. Currently, best practice is to continue antifibrotics until time of transplantation. In a large multicentre randomised trial, pirfenidone did not appear to have a beneficial effect on lung function decline in established bronchiolitis obliterans syndrome. The results of antifibrotic therapy in restrictive allograft syndrome are eagerly awaited, but nonrandomised data from small case reports/series are promising.

Connective Tissue Growth Factor Is Overexpressed in Explant Lung Tissue and Broncho-Alveolar Lavage in Transplant-Related Pulmonary Fibrosis.

Background: Connective tissue growth factor (CTGF) is an important mediator in several fibrotic diseases, including lung fibrosis. We investigated CTGF-expression in chronic lung allograft dysfunction (CLAD) and pulmonary graft-versus-host disease (GVHD). Materials and Methods: CTGF expression was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry in end-stage CLAD explant lung tissue (bronchiolitis obliterans syndrome (BOS), n=20; restrictive allograft syndrome (RAS), n=20), pulmonary GHVD (n=9). Unused donor lungs served as control group (n=20). Next, 60 matched lung transplant recipients (BOS, n=20; RAS, n=20; stable lung transplant recipients, n=20) were included for analysis of CTGF protein levels in plasma and broncho-alveolar lavage (BAL) fluid at 3 months post-transplant, 1 year post-transplant, at CLAD diagnosis or 2 years post-transplant in stable patients. Results: qPCR revealed an overall significant difference in the relative content of CTGF mRNA in BOS, RAS and pulmonary GVHD vs. controls (p=0.014). Immunohistochemistry showed a significant higher percentage and intensity of CTGF-positive respiratory epithelial cells in BOS, RAS and pulmonary GVHD patients vs. controls (p<0.0001). BAL CTGF protein levels were significantly higher at 3 months post-transplant in future RAS vs. stable or BOS (p=0.028). At CLAD diagnosis, BAL protein content was significantly increased in RAS patients vs. stable (p=0.0007) and BOS patients (p=0.042). CTGF plasma values were similar in BOS, RAS, and stable patients (p=0.74). Conclusions: Lung CTGF-expression is increased in end-stage CLAD and pulmonary GVHD; and higher CTGF-levels are present in BAL of RAS patients at CLAD diagnosis. Our results suggest a potential role for CTGF in CLAD, especially RAS, and pulmonary GVHD.

Free Airway C4d after Lung Transplantation - A Quantitative Analysis of Bronchoalveolar Lavage Fluid.

In recent years, the utility of vascular complement factor 4d (C4d) deposition as diagnostic tool for antibody mediated rejection (AMR) after lung transplantation, has become a controversial issue. We aimed to pinpoint the problematic nature of C4d as biomarker with a simple experiment. We quantified C4d in broncho-alveolar lavage (BAL) of lung transplant patients with diverse post-transplant complications in 3 different settings of clinically clear cases of: 1/ chronic lung allograft dysfunction (CLAD); 2/ acute complications acute rejection (AR), lymphocytic bronchiolitis (LB), antibody-mediated rejection (AMR) and respiratory infection (INF); 3/ patients with parallel C4d immunostaining and Anti-HLA. All groups were compared to BAL of stable patients. C4d was measured via standard ELISA. C4d was increased in CLAD, predominantly in RAS (p = 0.0026) but not in BOS (p = 0.89). C4d was increased in all acute events, AR (p = 0.0025), LB (p < 0.0001), AMR (p = 0.0034), infections (p < 0.0001). In patients with parallel C4d immunostaining and serum HLA antibodies, C4d was increased in C4d-/HLA- (p = 0.0011); C4d-/HLA+ (p = 0.013); HLA+/C4d + (p = 0.0081). A correlation of systemic C-reactive protein (CRP) with C4d was found in all patients (r = 0.49; p < 0.0001). We hypothesize that free C4d in BAL may only be representative of a general immune response in the transplanted lung.

Flow-controlled ventilation during EVLP improves oxygenation and preserves alveolar recruitment.

BACKGROUND: Ex vivo lung perfusion (EVLP) is a widespread accepted platform for preservation and evaluation of donor lungs prior to lung transplantation (LTx). Standard lungs are ventilated using volume-controlled ventilation (VCV). We investigated the effects of flow-controlled ventilation (FCV) in a large animal EVLP model. Fourteen porcine lungs were mounted on EVLP after a warm ischemic interval of 2 h and randomized in two groups (n = 7/group). In VCV, 7 grafts were conventionally ventilated and in FCV, 7 grafts were ventilated by flow-controlled ventilation. EVLP physiologic parameters (compliance, pulmonary vascular resistance and oxygenation) were recorded hourly. After 6 h of EVLP, broncho-alveolar lavage (BAL) was performed and biopsies for wet-to-dry weight (W/D) ratio and histology were taken. The left lung was inflated, frozen in liquid nitrogen vapors and scanned with computed tomography (CT) to assess regional distribution of Hounsfield units (HU). RESULTS: All lungs endured 6 h of EVLP. Oxygenation was better in FCV compared to VCV (p = 0.01) and the decrease in lung compliance was less in FCV (p = 0.03). W/D ratio, pathology and BAL samples did not differ between both groups (p = 0.16, p = 0.55 and p = 0.62). Overall, CT densities tended to be less pronounced in FCV (p = 0.05). Distribution of CT densities revealed a higher proportion of well-aerated lung parts in FCV compared to VCV (p = 0.01). CONCLUSIONS: FCV in pulmonary grafts mounted on EVLP is feasible and leads to improved oxygenation and alveolar recruitment. This ventilation strategy might prolong EVLP over time, with less risk for volutrauma and atelectrauma.

Peripheral Blood Eosinophilia Is Associated with Poor Outcome Post-Lung Transplantation.

Eosinophils play a role in many chronic lung diseases. In lung transplantation (LTx), increased eosinophils in bronchoalveolar lavage (BAL) was associated with worse outcomes. However, the effect of peripheral blood eosinophilia after LTx has not been investigated thoroughly. A retrospective study was performed including all LTx patients between 2011-2016. Chronic lung allograft dysfunction (CLAD)-free and graft survival were compared between patients with high and low blood eosinophils using an 8% threshold ever during follow-up. A total of 102 patients (27.1%) had high blood eosinophils (≥8%) (45 before CLAD and 17 after, 40 had no CLAD) and 274 (72.9%) had low eosinophils (<8%). Patients with high blood eosinophils demonstrated worse graft survival (p = 0.0001) and CLAD-free survival (p = 0.003) compared to low eosinophils. Patients with both high blood and high BAL (≥2%) eosinophils ever during follow-up had the worst outcomes. Within the high blood eosinophil group, 23.5% had RAS compared to 3% in the group with low eosinophils (p < 0.0001). After multivariate analysis, the association between high blood eosinophils and graft and CLAD-free survival remained significant (p = 0.036, p = 0.013) independent of high BAL eosinophils and infection at peak blood eosinophilia, among others. LTx recipients with ever ≥8% blood eosinophils demonstrate inferior graft and CLAD-free survival, specifically RAS, which requires further prospective research.

Successful Pseudomonas aeruginosa eradication improves outcomes after lung transplantation: a retrospective cohort analysis.

Long-term survival after lung transplantation (LTx) is hampered by development of chronic lung allograft dysfunction (CLAD). Pseudomonas aeruginosa is an established risk factor for CLAD. Therefore, we investigated the effect of P. aeruginosa eradication on CLAD-free and graft survival.Patients who underwent first LTx between July, 1991, and February, 2016, and were free from CLAD, were retrospectively classified according to P. aeruginosa presence in respiratory samples between September, 2011, and September, 2016. P. aeruginosa-positive patients were subsequently stratified according to success of P. aeruginosa eradication following targeted antibiotic treatment. CLAD-free and graft survival were compared between P. aeruginosa-positive and P. aeruginosa-negative patients; and between patients with or without successful P. aeruginosa eradication. In addition, pulmonary function was assessed during the first year following P. aeruginosa isolation in both groups.CLAD-free survival of P. aeruginosa-negative patients (n=443) was longer compared with P. aeruginosa-positive patients (n=95) (p=0.045). Graft survival of P. aeruginosa-negative patients (n=443, 82%) was better compared with P. aeruginosa-positive patients (n=95, 18%) (p<0.0001). Similarly, P. aeruginosa-eradicated patients demonstrated longer CLAD-free and graft survival compared with patients with persistent P. aeruginosa Pulmonary function was higher in successfully P. aeruginosa-eradicated patients compared with unsuccessfully eradicated patients (p=0.035).P. aeruginosa eradication after LTx improves CLAD-free and graft survival and maintains pulmonary function. Therefore, early P. aeruginosa detection and eradication should be pursued.

Early protein expression profile in bronchoalveolar lavage fluid and clinical outcomes in primary graft dysfunction after lung transplantation.

OBJECTIVES: Primary graft dysfunction (PGD) remains a major post-transplant complication and is associated with increased morbidity and mortality. Mechanisms evoking PGD are not completely clear, but inflammation plays a central role. We investigated the association between PGD and inflammatory proteins present in immediate postoperative bronchoalveolar lavage. METHODS: All double-lung recipients transplanted at our institution from 2002 to 2018 were included in our study. We retrospectively selected 80 consecutive lung transplant recipients with different PGD grades (n = 20 for each PGD grades 0-1 to 2-3). In bronchoalveolar lavage performed within the first 24 h after donor aortic cross-clamping following lung transplantation, concentrations of 30 cytokines, chemokines and growth factors were assessed by enzyme-linked immunosorbent assay (ELISA) and correlated with donor and recipient demographics and outcomes. For analysis, 2 groups were defined: 'mild' PGD (grade 0-1) and 'severe' PGD (grades 2-3). RESULTS: Significant differences between mild and severe PGD were found in 8 biomarkers [interleukin (IL)-6, IL-10, IL-13, eotaxin, granulocyte colony-stimulating factor, interferon γ, macrophage inflammatory protein 1α, surfactant protein D (SP-D); P < 0.05]. Increased IL-10 and IL-13, but none of the other proteins, were associated with short-term outcome (longer time to extubation; P = 0.005 and P < 0.0001; increased intensive care unit stay; P = 0.012 and P < 0.0001; and hospital stay; P = 0.041 and P = 0.002). There were no significant differences in donor and recipient characteristics between the groups. CONCLUSIONS: Expression profiles of key inflammatory mediators in bronchoalveolar lavage fluid differed significantly between lung transplant recipients with severe versus mild PGD and correlated with clinical outcome variables. Further research should focus on the early mechanisms leading to PGD.

Vitamin D Modulates the Response of Bronchial Epithelial Cells Exposed to Cigarette Smoke Extract.

In chronic obstructive pulmonary disease (COPD), the bronchial epithelium is the first immune barrier that is triggered by cigarette smoke. Although vitamin D (vitD) has proven anti-inflammatory and antimicrobial effects in alveolar macrophages, little is known about the direct role of vitD on cigarette smoke-exposed bronchial epithelial cells. We examined the effects of vitD on a human bronchial epithelial cell line (16HBE) and on air-liquid culture of primary bronchial epithelial cells (PBEC) of COPD patients and controls exposed for 24 h to cigarette smoke extract (CSE). VitD decreased CSE-induced IL-8 secretion by 16HBE cells, but not by PBEC. VitD significantly increased the expression of the antimicrobial peptide cathelicidin in 16HBE and PBEC of both COPD subjects and controls. VitD did not affect epithelial to mesenchymal transition or epithelial MMP-9 expression and was not able to restore impaired wound healing by CSE in 16HBE cells. VitD increased the expression of its own catabolic enzyme CYP24A1 thereby maintaining its negative feedback. In conclusion, vitD supplementation may potentially reduce infectious exacerbations in COPD by the upregulation of cathelicidin in the bronchial epithelium.

Intragraft donor-specific anti-HLA antibodies in phenotypes of chronic lung allograft dysfunction.

INTRODUCTION: Circulating anti-human leukocyte antigen (HLA) serum donor-specific antibodies (sDSAs) increase the risk of chronic lung allograft dysfunction (CLAD) and mortality. Discrepancies between serological and pathological/clinical findings are common. Therefore, we aimed to assess the presence of tissue-bound graft DSAs (gDSAs) in CLAD explant tissue compared with sDSAs. METHODS: Tissue cores, obtained from explant lungs of unused donors (n=10) and patients with bronchiolitis obliterans syndrome (BOS; n=18) and restrictive allograft syndrome (RAS; n=18), were scanned with micro-computed tomography before elution of antibodies. Total IgG levels were measured via ELISA. Anti-HLA class I and II IgG gDSAs were identified using Luminex single antigen beads and compared with DSAs found in serum samples. RESULTS: Overall, mean fluorescence intensity was higher in RAS eluates compared with BOS and controls (p<0.0001). In BOS, two patients were sDSA+/gDSA+ and two patients were sDSA-/gDSA+. In RAS, four patients were sDSA+/gDSA+, one patient was sDSA+/gDSA- and five patients were sDSA-/gDSA+. Serum and graft results combined, DSAs were more prevalent in RAS compared with BOS (56% versus 22%; p=0.04). There was spatial variability in gDSA detection in one BOS patient and three RAS patients, who were all sDSA-. Total graft IgG levels were higher in RAS than BOS (p<0.0001) and in gDSA+ versus gDSA- (p=0.0008), but not in sDSA+ versus sDSA- (p=0.33). In RAS, total IgG levels correlated with fibrosis (r= -0.39; p=0.02). CONCLUSIONS: This study underlines the potential of gDSA assessment as complementary information to sDSA findings. The relevance and applications of gDSAs need further investigation.

Myeloid-Derived Suppressor Cells in Lung Transplantation.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immune cells from the myeloid lineage. MDSCs expand in pathological situations, such as chronic infection, cancer, autoimmunity, and allograft rejection. As chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation (LTx), MDSCs may play a role in its pathophysiology. We assessed phenotype and frequency of MDSCs in peripheral blood from lung transplant recipients and its relationship to post-transplant complications and immunosuppression. Granulocytic (G)-MDSC were identified and quantified by flow cytometry of blood from 4 control subjects and 20 lung transplant patients (stable n = 6, infection n = 5; CLAD n = 9). G-MDSC functionality was assessed in vitro by their capability to block CD4 and CD8 T cell proliferation. More G-MDSC could be assessed using EDTA tubes compared to heparin tubes (p = 0.004). G-MDSC were increased in stable lung transplant recipients vs. non-transplant controls (52.1% vs. 9.4%; p = 0.0095). The infection or CLAD groups had lower G-MDSCs vs. stable recipients (28.2%p = 0.041 and 33.0%; p = 0.088, respectively), but were not different among CLAD phenotypes. G-MDSC tended to correlate with cyclosporine A and tacrolimus levels (r2 = 0.18; r2 = 0.17). CD4 and CD8 cells proliferation decreased by 50 and 80% if co-cultured with MDSCs (1:6 and 1:2 MDSC:T-cell ratio, respectively). In conclusion, circulating MDSCs are measurable, functional and have a G-MDSC phenotype in lung transplant patients. Their frequency is increased in stable patients, decreased during post-transplant complications, and related to level of immunosuppression. This study may pave the way for further investigations of MDSC in the context of lung transplantation.

The pleural mesothelium and transforming growth factor-ß1 pathways in restrictive allograft syndrome: A pre-clinical investigation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) hampers long-term survival after lung transplantation. Common fibrosis-related mechanisms in idiopathic pulmonary fibrosis and CLAD instigated the consideration of investigating the differential regulation of pleural mesothelium and transforming growth factor-ß1 (TGF-ß1) in restrictive allograft syndrome (RAS). METHODS: TGF-ß1 was assessed in bronchoalveolar lavage (BAL) fluid using enzyme-linked immunoassay and via immune staining of explant biopsies. To assess the role of the pleura, explanted bronchiolitis obliterans syndrome (BOS) and RAS lungs were compared using computed tomography scans, calretinin stainings, Western blot, and quantititative real-time PCR. Last, a pleural mesothelial cell line was used to assess mesothelial-to-mesenchymal transition and its inhibition. RESULTS: TGF-ß1 was increased in BAL of RAS patients (p = 0.035), and was present in the (sub)pleural area of biopsies. Explanted RAS lungs demonstrated an increased volume fraction of pleura (p = 0.0004), a higher proportion of calretinin-positive stainings (p = 0.0032), and decreased E-cadherin (p = 0.019) and increased α-smooth muscle actin (p = 0.0089) mRNA expression and protein levels in isolated pleural tissue. Moreover, TGF-ß1 stimulation of pleural mesothelial cells led to a phenotypical switch to mesenchymal cells, accompanied with an increased migratory capacity. Interleukin-1α was able to accentuate TGF-ß1‒induced mesothelial-to-mesenchymal transition. None of the tested drugs could inhibit mesothelial-to-mesenchymal transition at the used concentrations. CONCLUSIONS: Our results support an interplay between TGF-ß1 and the pleural mesothelium in the pathophysiology of RAS.