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Background: For endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) under general anaesthesia, both rigid bronchoscopy and laryngeal masks (LMAs) with superimposed high-frequency jet ventilation can be used. Despite the fact that in Europe rigid bronchoscopy for EBUS-TBNA is still widely used, an increasing number of centres use jet ventilation via the LMA for this procedure. To our knowledge no clinical trials have ever been made to compare these two methods. This trial aimed to evaluate whether patients recover from the procedure more quickly when a LMA is used for ventilation compared with rigid bronchoscopy where muscle relaxants and deep anaesthesia are required. Objectives: We wanted to test the hypothesis that there is no difference in the postoperative recovery of patients in the postanaesthesia care unit (PACU) after EBUS-TBNA with jet ventilation via a rigid bronchoscope and a LMA. Secondary outcomes were the difference of duration of anaesthesia, the diagnostic outcome of the procedure and drug quantities for both groups. Design: Prospective randomised single blinded two centre controlled trial. Setting: Two centres in Austria participated. Patients were enrolled from December 2016 until January 2018. Patients: Ninety patients for elective EBUS-TBNA were enrolled and assigned to one of two intervention groups. Two patients were excluded before and eleven patients were excluded after EBUS-TBNA. Seventy-seven were analysed. Interventions: Patients assigned to group 1 were ventilated with a LMA; those assigned to group 2 were ventilated via a rigid bronchoscope. Vital signs, drug dosage, duration of anaesthesia, recovery, PACU stay and Aldrete score at the PACU were recorded. Main outcome measures: The primary endpoint was an integral over time of a modified Aldrete score. Secondary endpoints were the durations of the interventions, the recovery from anaesthesia and PACU stay, initial and mean Aldrete values at PACU, the effect site concentration of Propofol according to the Schnider pharmacokinetic model, the peak ultiva rates and the diagnostic outcome. Results: We were not able to show any significant difference regarding the postoperative recovery criteria based on the Aldrete score, the durations measured and the diagnostic outcomes. Vital signs remained stable and in an equal range in both groups. There were no differences in the mean effect site propofol concentration and the peak ultiva rates. Conclusion: EBUS-TBNA under general anaesthesia using a LMA with SHJV is equal to rigid bronchoscopy with superimposed high-frequency jet ventilation for the variables analysed. Trial registration: ISRCTN (ISRCTN58911367).

BackgroundAlthough the number of lung transplantations (LTx) performed worldwide for coronavirus disease 2019 (COVID-19)-induced acute respiratory distress syndrome (ARDS) is still low, there is general agreement that this treatment can save a subgroup of the most severely ill patients with irreversible lung damage. However, the true proportion of patients eligible for LTx, the overall outcome and the impact of LTx on the pandemic are unknown.MethodsA retrospective analysis was performed using a nationwide registry of hospitalised patients with confirmed severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection admitted between 1 January 2020 and 30 May 2021 in Austria. Patients referred to one of the two Austrian LTx centres were analysed, and grouped into patients accepted and rejected for LTx. Detailed outcome analysis was performed for all patients who received a LTx for post-COVID-19 ARDS and compared with patients who underwent LTx for other indications.ResultsBetween 1 January 2020 and 30 May 2021, 39 485 patients were hospitalised for COVID-19 in Austria. 2323 required mechanical ventilation and 183 received extracorporeal membrane oxygenation (ECMO) support. 106 patients with severe COVID-19 ARDS were referred for LTx. Of these, 19 (18%) underwent LTx. 30-day mortality after LTx was 0% for COVID-19 ARDS transplant recipients. At a median follow-up of 134 (47–450) days, 14 out of 19 patients were alive.ConclusionsEarly referral of ECMO patients to a LTx centre is pivotal in order to select patients eligible for LTx. Transplantation offers excellent midterm outcomes and should be incorporated in the treatment algorithm of post-COVID-19 ARDS.

Introduction: The 6-minute walking distance (6MWD) is a well-established predictor for mortality in patients with interstitial lung disease (ILD). In this study, we aimed to investigate the 6MWD as a possible predictor for long-term survival after double lung transplantation (DLuTx) in patients with ILD. Methods: A single-center, retrospective data analysis in patients with ILD undergoing DLuTx was conducted (EC: 1251/2019). All ILD patients transplanted between 2013 and 2018 having a 6MWD measurement were included. Patients were divided into quintiles according to their 6MWD (QI: 0-10m; QII: ≥10-70m; QIII: 71-230m; QIV: 231-360m; QV: 361-600m) and compared to short- and long-term survival after DLuTx. Statistical calculations included Kaplan-Meier survival and Cox proportional hazard models. Results: A total of 115 patients were included and 61.7% (n=71) were diagnosed with IPF. The median age was 56 years and 67.8% (n=78) were males. The survival probability after 1 year was 78.1% and after 5 years 71.3%, with significant differences between the quintiles (log-rank test p=0.037). The 5-year survival probability for the five groups was: QI: 50.2% (95% CI 29%-71%); QII: 81.8% (95% CI 66%-98%); QIII: 64% (95% CI 44%-84%); QIV: 78.3% (95% CI 61%-95%); QV: 82.4% (95% CI 67%-98%). In contrast, lung function parameters and lung allocation score could not be identified as predictors for survival. Conclusion: The collected 6MWD of patients with ILD at the time of listing showed a significant association with the survival after DLuTx. These findings may impact the pre-transplant management of patients with ILD.

Background The use of organs from polytrauma donors for lung transplantation is controversial in the literature. For many centers, the radiologic manifestation of lung contusions is a clear reason to reject an organ offer. This results in the loss of potentially viable organs for the donor pool. Methods We analyzed 1152 donor lungs procured by our transplant center between January 2010 and June 2018. These included 118 lungs with a history of polytrauma involving the chest. Sixteen polytrauma donor lungs were rejected after procurement. A total of 102 lungs were transplanted, divided into 2 groups: the polytrauma contusion group (n = 44), comprising polytrauma donors with radiologic signs of lung contusion at the time of offer, and the polytrauma clear group (n = 58), comprising polytrauma donors without lung contusion. Nontrauma donor lungs transplanted during the study period were assigned to a polytrauma control group (n = 650). Short- and long-term outcomes of the 3 groups were compared. Results Basic demographic data and preoperative factors were similar in the 3 groups. Rates of primary graft dysfunction grade 3 at 72 hours did not differ among the 3 groups (0.0% vs 3.4% vs 3.9%; P = .409). The duration of ventilation was similar the 3 groups: 45 hours (interquartile range [IQR], 28-94 hours), 37 hours (IQR, 22-71 hours), and 42 hours (IQR, 22-96 hours), respectively (P = .674). Long-term graft survival was not impaired in the trauma groups compared with controls. One-year survival rates were 84.1% for the polytrauma contusion group, 93.1% for the polytrauma clear group, and 83.1% for the no polytrauma group. Five-year graft survival in the 3 groups was 74.7%, 87.2%, and 70.0%, respectively. Conclusions Lung transplantation using organs from polytrauma donors is associated with similar short- and long-term results as transplantation from nontrauma donors. The presence or absence of radiologic signs of lung contusion at the time of offer has no impact on primary graft function and long-term survival.

Purpose Allograft adaptation after lung transplantation is a multi-factor process. We hypothesized that early adaptation can influence the risk of matrix remodeling and CLAD development by shaping the inflammatory state of the lung environment. In a longitudinal study, we followed a defined patient cohort in year 1 p.t. and detected ordered cell composition changes with repercussions in the microbiome, small molecular composition and lipidome in BAL samples. We delimited clusters of pro-inflammatory factors and identified long-chain lipid species from the lung environment that modulated inflammatory response in vitro. Methods We used FACS to identify cell populations and 16S sequencing, metabolomics, lipidomics to characterize the lung environment. With correlations and hierarchical clustering we extracted pro- and anti-inflammatory clusters around well-characterized cell populations. For detection of inflammation drivers, we quantified feature differential abundances in samples with more or less neutrophilia. Predicted candidates were tested to modify inflammatory responses in vitro by screening IL-6 production of LPS-triggered macrophages. Results Early after surgery, the allograft experienced neutrophilia, a lack of resident macrophages and a gradual influx of host-derived macrophage-precursors directly resonating with changes in the pulmonary microenvironment. The computational analysis revealed features that clustered either with neutrophils or alveolar macrophages. From these, 30% showed significant abundance differences in high and low neutrophilia conditions. Consistently, certain ceramide species displayed antagonistic behavior to several phosphatidylcholines. We tested their capability of modifying macrophage response and found the ceramide to enhance, and PCs to reduce IL-6 production. Conclusion Our study suggests that the lung microenvironment plays a key role in allograft adaptation and specific lipid species are capable of modulating cellular inflammatory response in a differentiated manner early after transplantation. Our results provide first fundamental concepts for developing concrete precision therapies based on reprogramming tissue homoeostasis and inflammation management.

Idiopathic pulmonary fibrosis (IPF) is a progressive terminal lung disease, and therapies aim to block fibrosis. Fibroblast proliferation is controlled by C/EBP-β, microRNA cluster 17-92 (miR17-92), and Erk1/2 mitogen-activated protein kinase. This study assessed the role of miR17-92 in IPF-fibroblast proliferation and its modification by treprostinil. Fibroblasts were isolated from eight IPF patients, five interstitial lung fibrosis patients, and seven control lungs. Fibroblasts were stimulated with TGF-β1 over 24 h. The miR17-92 expression was analyzed by RT-qPCR, and protein expression by Western blotting. TGF-β1 upregulated C/EBP-β in all fibroblasts, which was reduced by treprostinil in control-fibroblasts, but not in IPF-fibroblasts. Compared to controls, the guide strands miR-19a-3p, miR-19b-3p, miR-20a-5p, and miR-92a-3p, as well as the passenger strands miR-17-3p, miR-18-3p, miR-19a-1-5p, and miR-92a-5p were significantly increased in IPF-fibroblasts. In controls, TGF-β1 and treprostinil significantly reduced specific miR17-92 members. IPF-fibroblast proliferation was inhibited by treprostinil through increased expression of the Erk1/2 inhibitor DUSP1. These data suggest that proliferation control via miR17-92 and C/EBP-β is disrupted in IPF-fibroblasts. Therefore, the inhibition of early stages of signaling cascades or specific mitogen receptors might be less effective. However, the increased proliferation is sensitive to Erk1/2 inhibition by treprostinil-induced DUSP1.

RationaleLung transplantation is the ultimate treatment option for patients with end-stage respiratory diseases but bears the highest mortality rate among all solid organ transplantations due to chronic lung allograft dysfunction (CLAD). The mechanisms leading to CLAD remain elusive due to an insufficient understanding of the complex post-transplant adaptation processes.ObjectivesTo better understand these lung adaptation processes after transplantation and to investigate their association with future changes in allograft function.MethodsWe performed an exploratory cohort study of bronchoalveolar lavage samples from 78 lung recipients and donors. We analysed the alveolar microbiome using 16S rRNA sequencing, the cellular composition using flow cytometry, as well as metabolome and lipidome profiling.Measurements and main resultsWe established distinct temporal dynamics for each of the analysed data sets. Comparing matched donor and recipient samples, we revealed that recipient-specific as well as environmental factors, rather than the donor microbiome, shape the long-term lung microbiome. We further discovered that the abundance of certain bacterial strains correlated with underlying lung diseases even after transplantation. A decline in forced expiratory volume during the first second (FEV1) is a major characteristic of lung allograft dysfunction in transplant recipients. By using a machine learning approach, we could accurately predict future changes in FEV1 from our multi-omics data, whereby microbial profiles showed a particularly high predictive power.ConclusionBronchoalveolar microbiome, cellular composition, metabolome and lipidome show specific temporal dynamics after lung transplantation. The lung microbiome can predict future changes in lung function with high precision.

Purpose The diagnosis and treatment of antibody-mediated rejection (AMR) after LTx has gained increasing recognition within the transplant community. Extracorporeal photopheresis (ECP) modulates various pathways of the immune system and is currently used to treat CLAD. In AMR, adding ECP to established AMR treatments could potentially prevent rebound of DSA. This study aimed to analyze the role of ECP as adjunct treatment of AMR. Methods Patients who developed antibody-mediated rejection between 2009 and 2019 were included in this single-center retrospective analysis. The following parameters were evaluated: DSAs, C4d deposition and lung histology. Results A total of 39 patients developed clinical AMR during follow-up. Nineteen (48%) patients had positive DSA against HLA Class I and eighteen (46%) HLA Class II. Median time to diagnosis of AMR was 180 days (IQR: 2-2477). First line treatment was immunoadsorption (IA) in 34 (87%) patients, intravenous immunoglobulins with concomitant immunoadsorption in 3 (8%) patients and in 2 cases ATG. Twenty (51%) patients primarily treated with IA and the two patients treated with ATG received ECP. Among the patients treated with ECP, 12 had DSA against HLA Class I and 13 against HLA Class II. ECP treatment was initially performed twice a week. After stabilization of lung function and improvement of clinical situation, ECP was then performed once a month for a minimum of 6 months. Median length of treatment was 12 months. Patients who received ECP after first line treatment had 1-year survival rate of 62% compared to 27% of other AMR patients. In all patients treated with IA followed by ECP, DSA disappeared or MFI reduced below our institutional threshold of 5000. Conclusion Extracorporeal photopheresis is associated with a reduction of de novo DSA after antibody-mediated rejection. Prospective studies are necessary to confirm the beneficial effect of ECP as an add-on therapy after AMR.

Background Lung transplantation has evolved to a routinely performed surgical procedure in patients with end-stage pulmonary disease. Bronchial healing problems are rare but represent a potential life-threatening complication. Herein, we aimed to define the incidence, classification, and treatment of bronchial complications after lung transplantation. Material and Methods All patients receiving lung transplantation between January 1999 and December 2017 were included in this retrospective study. All bronchial anastomoses were performed in a standardized technique using a single, polydioxanone running suture. The rate of anastomotic complications requiring an intervention, type of complication according the 2018 International Society for Heart and Lung Transplantation classification, and the clinical management were retrospectively analyzed. Results A total of 2941 anastomoses were performed in 1555 patients. The overall incidence of relevant anastomotic complications was 1.56%, 0.68% for left anastomoses, and 2.44% for right anastomoses. In 6 patients, a surgical revision or retransplantation was performed, whereas endoscopic treatment alone was sufficient in 39 patients. One patient underwent right-sided retransplantation 6 months after the first lung transplantation after failed endoscopic treatment attempts. International Society for Heart and Lung Transplantation grade “S Lc Ec” was the most common type of anastomotic complication. The overall incidence decreased within the study period from 2.4% in the era 1999 to 2003 to 0.8% in the era 2014 to 2017. We found no significant difference in overall survival of patients with and without anastomotic complications (P = .995; hazard ratio, 0.99; 95% confidence interval, 0.63-1.58). Conclusions The single running suture technique is associated with a very low rate of true anastomotic complications. Close follow-up and early endoscopic treatment of patients with anastomotic complications result in excellent long-term outcomes.

Remodelling of the peripheral lung tissue and fibrotic foci are the main pathologies of idiopathic pulmonary fibrosis (IPF), a disease that is difficult to treat. TGF-β activation of peripheral lung fibroblasts is indicated as the major cause of tissue remodelling in IPF and is resulting in fibroblast hyperplasia and deposition of extracellular matrix. Soluble guanylate cyclase (sGC) stimulators combined with cyclic AMP (cAMP) activators have been reported to reduce proliferation and matrix deposition in other conditions than IPF. Therefore, this drug combination may present a novel therapeutic concept for IPF. This study investigated the effect of BAY 41-2272 and forskolin on remodelling parameters in primary human lung fibroblasts. The study determined TGF-β induced proliferation by direct cell counts after 3 days; and deposition of collagen type-I, type III, and fibronectin. BAY 41-2272 significantly reduced TGF-β induced fibroblast proliferation, but did not reduce viability. This inhibitory effect was further supported by forskolin. Both BAY 41-2272 and forskolin alone reduced TGF-β induced collagen and fibronectin de novo synthesis as well as deposition. This effect was significantly stronger when the two compounds were combined. Furthermore, the TGF-β induced expression of fibrilar α-smooth muscle actin was reduced by BAY 41-2272 and this effect was strengthened by forskolin. In addition, BAY 41-2272 and forskolin reduced TGF-β induced β-catenin. All effects of BAY 41-2272 were concentration dependent. The findings suggest that BAY 41-2272 in combination with cAMP stimulation may present a novel therapeutic strategy to reduce tissue remodelling in IPF.

Purpose Spirometric lung function is one of the central clinical parameters to assess allograft function after lung transplantation (LTX). Reduction in FEV1 can be the result of infections, rejections episodes and most importantly development of CLAD. Currently, there is no means to predict short-term changes in lung function after LTX. Methods Bronchoalveolar samples of patients after standard double LTX were taken during follow-up bronchoscopy at multiple different time points. Lipidomic, metabolomic, flow cytometric and bacterial 16S rRNA gene sequencing (microbiome) data were analyzed. We then used a machine learning approach to predict future changes in FEV1 from these sample data to characterize patient lung function trajectories. We trained support vector machine (SVM) regressors on the collected lipidomic, metabolomic, microbiome and flow cytometry datasets. Changes in FEV1 within 30, 60 or 90 days after lavage sample collection were used as response variables. To train hyper-parameters and evaluate model accuracy, a nested leave-one-out cross validation scheme was used. Model accuracy was benchmarked against a model trained on clinical metadata. Results At a prediction timeframe of 30 days, lipidomic data were available from 34 samples of 20 patients. Lipidomics showed the highest predictive power for short-term changes 30 and 60 days after sample collection. Prediction accuracy (R2) of intra-alveolar lipid composition for a 30-day projection was 0.24, meaning that BAL lipid profiles could explain more than 20 percent of total variation in relative change in FEV1 for this time span. R² for clinical metadata alone was only 0.1, and metabolomics, microbiome and FACS analysis of BAL showed no predictive accuracy at this time point. Conclusion Our results suggest that the intra-alveolar lipid composition is a powerful predictor of short-term changes in lung allograft function. This could potentially facilitate pre-emptive therapeutic interventions.

Purpose The pulmonary microbiome after lung transplantation has recently come into the focus of research. Especially, host-microbiome interactions are thought to be an important factor in graft-related immunological processes. In lung transplantation, temporal changes of the pulmonary microbiome have not yet been elucidated. Methods In a total cohort of 80 patients receiving standard double lung transplantation, 50 bronchioalveolar lavage (BAL) samples from donors were collected prior to cold ischemia. After transplantation, 128 BAL samples were collected at various time points of routine follow-up bronchoscopies between 0 and 400 days post-transplant. Bacterial 16S rRNA gene sequencing was conducted to analyze the composition of the pulmonary microbiome in these samples. Results The lung microbiome showed significant temporal dynamics after lung transplantation. Recipient-donor similarity between matched samples decreased after the first week post transplantation. Underlying transplant indications were significantly associated with microbial profiles even after transplantation. Shannon diversity and Chao1 richness at the species level showed an increasing diversity of the microbiome over time. Conclusion Our data provides new insights into the dynamics of microbial profiles after transplantation. We observed that recipient-associated factors, rather than the donor microbiome, shape the lung microbiome after transplantation.

Purpose New biomarkers reflecting the degree of immunosuppression in transplant recipients are needed to provide an optimal balance between risk for rejection and risk of severe infections. Methods We prospectively measured plasma TTV-DNA dynamics in 365 lung transplant recipients (LuTR) transplanted between 1/2013 and 12/2020. Severe infections, acute cellular and humoral rejection, incidence of CLAD and malignancies were recorded and correlated with dynamic changes in TTV levels. Results Patient demographics are summarized in Table 1. 95% of patients had detectable TTV levels pre-transplant with a mean log value of 4.0±1.6. Post-transplant, all LuTR became TTV positive with a peak value of log9.6±0.6 (range 5.9-10.9). TTV peaked at a mean of 143±109 days after transplantation. Mean TTV PCR values after 3, 4 and 5 years were log6.9±1,4, log6,4±1,4 and log6.0±1.2, respectively. TTV loads were significantly lower in LuTR who had episodes of acute cellular rejection (TTV PCR Conclusion TTV PCR measurement is a potent tool to predict acute cellular rejection episodes, infectious complications and CLAD. It represents a promising tool to monitor individualized immunosuppressive therapy in near future.

Aim: To investigate international medical students’ attitudes toward the impact of 6-year longitudinal course, Fundamentals of Medical Skills (FMS), at Medical Studies in English at the University of Zagreb on the development of their practical, clinical, and communication skills. Methods: This cross-sectional study used a 23-item online survey to collect data from five generations of students attending the FMS course from January 31 to February 3, 2017. First-year students were not included. Invitations and reminders were sent to 202 FMS students by e-mail, SMS, and in closed groups in social networks. Results: The response rate was 69.8% (141/202 students). The majority of students found the course useful (83.7%); favored practical over communication skills (92.9%); found practical skills more useful in higher years (82.3%); thought more time was needed to practice by simulation on mannequins (75.2%); preferred Objective Structured Clinical Examination (OSCE) stations to traditional oral exams (78%); and would recommend a course like FMS to future students or students at other universities (79.4%). Significantly more women than men favored practical over communication skills (P = 0.044). Significantly more 5th and 6th students than students at lower years preferred OSCE stations to traditional learning (P = 0.025) and would recommend a course like FMS to future students or students at other universities (P = 0.001). Conclusion: Students positively evaluated the FMS course, but underestimated the communication skills aspect.

Torque teno viruses (TTV) are small DNA-viruses, of the genus Alphatorquevirus, whose replication is linked to immune status. TTV load may be an indicator for efficacy of IS in lung transplant recipients (LTRs). In a prospective single-center-study 143 LTRs were followed up and tested by quantitative TTV-DNA PCR. Using multivariate Cox-regression contribution of TTV-load to the occurrence of severe infections, chronic lung allograft dysfunction (CLAD), acute cellular rejection (ACR), and death was assessed. During follow-up 28 (20%) patients developed infections with a rate of 7.7 per 100 patient-years (PY). The hazard-ratio (HR) associated with a one-log10 increase of TTV-load before the event was 5.05. CLAD occurred with a rate of 6.0%-PY. HR for a 1 log10 increase of the lowest TTV level before the event was 0.71 (CI: 0.54-0.93). TTV-load predicts clinical events and may be useful to optimize IS during the first years of follow-up of LTRs.

Treprostinil is applied for pulmonary arterial hypertension (PAH) therapy. However, the mechanism by which the drug achieves its beneficial effects in PAH vessels is not fully understood. This study investigated the effects of treprostinil on PDGF-BB induced remodelling parameters in isolated human pulmonary arterial smooth muscle cells (PASMC) of four PAH patients. The production of TGF-β1, CTGF, collagen type-I and -IV, and of fibronectin were determined by ELISA and PCR. The role of cAMP was determined by ELISA and di-deoxyadenosine treatment. Proliferation was determined by direct cell count. Treprostinil increased cAMP levels dose and time dependently, which was not affected by PDGF-BB. Treprostinil significantly reduced PDGF-BB induced secretion of TGF-β1 and CTGF, both was counteracted when cAMP generation was blocked. Similarly, the PDGF-BB induced proliferation of PASMC was dose dependently reduced by treprostinil through signalling via cAMP-C/EBP-α p42 -p21(WAf1/Cip1). In regards to extracellular matrix remodelling, treprostinil significantly reduced PDGF-BB-TGF-β1-CTGF induced synthesis and deposition of collagen type I and fibronectin, in a cAMP sensitive manner. In contrast, the deposition of collagen IV was not affected. The data suggest that this action of treprostinil in vessel wall remodelling may benefit patients with PAH and may reduce arterial wall remodelling.

Idiopathic pulmonary fibrosis (IPF) is characterized by peripheral lung fibrosis and increased interstitial extracellular matrix (ECM) deposition. In IPF, tumor growth factor (TGF)-β1 which is the major stimulus of ECM deposition, and platelet derived growth factor (PDGF)-BB is a potent stimulus of fibrosis. Thus, the effect of Treprostinil on TGF-ß1 and PDGF-induced fibroblast proliferation and ECM deposition was investigated. Human peripheral lung fibroblasts of seven IPF patients and five lung donors were stimulated by PDGF, or TGF-β1, or the combination. Cells were pre-incubated (30 min) with either Treprostinil, forskolin, di-deoxyadenosine (DDA), or vehicle. Treprostinil time dependently activated cAMP thereby preventing PDGF-BB induced proliferation and TGF-β1 secretion. Cell counts indicated proliferation; α-smooth muscle actin (α-SMA) indicted differentiation, and collagen type-1 or fibronectin deposition remodeling. Myo-fibroblast indicating α-SMA expression was significantly reduced and its formation was altered by Treprostinil. Collagen type-I and fibronectin deposition were also reduced by Treprostinil. The effect of Treprostinil on collagen type-I deposition was cAMP sensitive as it was counteracted by DDA, while the effect on fibronectin was not cAMP mediated. Treprostinil antagonized the pro-fibrotic effects of both PDGF-BB and TGF-β1 in primary human lung fibroblasts. The data presented propose a therapeutic relevant anti-fibrotic effect of Treprostinil in IPF.

Purpose Within the last years increasing attention has been given to antibody-mediated rejection (AMR) due to its poor long-term outcomes. The impact of alemtuzumab on donor-specific antibodies (DSAs) and AMR in lung transplantation is currently unclear and most evidence comes from kidney transplantation. This study aimed to analyze the impact of alemtuzumab induction on the incidence of DSAs and AMR. Methods All patients transplanted between 2007 and 2017, who received alemtuzumab as induction therapy, were included in this retrospective single-center analysis. The following parameters were evaluated: donor-specific antibodies, C4d deposition, lung histology and occurrence of antibody-mediated rejection. Results Within the study period 525 patients received alemtuzumab as induction therapy. Pre-transplant DSAs were present in 64 (12.2%) recipients (53 anti-HLA class I and 40 anti-HLA class II). De novo (dn) DSAs developed in 93 (17.7%) recipients after transplantation (78 anti-HLA class I and 113 anti-HLA class II). Median time to development of dnDSA was 104 days (42-165) and incidence rates at 1 and 5-years after transplantation were 12.1% and 21.4%, respectively. In 20 (3.8%) recipients clinical AMR was diagnosed. Among them, 7 (35%) patients had a definite diagnosis, 9 (45%) a probable one and 4 (20%) a possible one as defined by the latest ISHLT AMR grading system. Deposition of C4d was only positive in 4 (20%) clinical AMR patients. Capillaritis was observed in 4 (20%) cases and neutrophilic septal margination in 3 cases (15%). Anti-HLA class I and class II were positive in 11 (55%) and 13 (65%) recipients, respectively. Long-term survival of recipients, who developed AMR, was significantly worse than other recipients, with a 5-years rate of 45% vs 75% (p Conclusion Alemtuzumab induction therapy is associated with a low incidence of dnDSAs and AMR. Long-term outcome of these patients remains poor.

Long acting β₂-agonists (LABA) have been reported to modify the extracellular matrix (ECM) composition in the airway wall. Based on our earlier studies we here investigated the mechanism underlying the control of ECM modification by LABA in primary human airway smooth muscle cells. Cells were treated with formoterol or salmeterol (30 min) before TGF-β₁ stimulation (2-3 days) Using RT-PCT, immuno-blotting and ELISA the de novo synthesis and deposition of collagen type-I, -III, -IV and fibronectin were determined. Matrix metalloproteinases (MMP)-2 and -9 were analyzed by zymography. Both LABA activated cAMP and its corresponding transcription factor CREB within 60 min and thus partly reduced TGF-β₁-induced gene transcription of collagen type-I, -III, fibronectin and connective tissue growth factor (CTGF). The inhibitory effect of both LABA on collagen type-I and -III deposition involved a cAMP dependent mechanism, while the inhibitory effect of the two drugs on TGF-β1-induced fibronectin deposition and on CTGF secretion was independent of cAMP. Interestingly, none of the two LABA reduced CTGF-induced synthesis of collagen type-I or type-III deposition. In addition, none of the two LABA modified collagen type-IV deposition or the expression and activity of MMP-2 or MMP-9. Our results show that LABA can prevent de novo deposition of specific ECM components through cAMP dependent and independent signaling. However, they do not reduce all ECM components by the same mechanism and they do not reduce existing collagen deposits. This might explain some of the controversial reports on the anti-remodeling effect of LABA in chronic inflammatory lung diseases.

Purpose According to ISHLT increasing number of lung recipients receive induction therapy. Long-term data on the use of alemtuzumab are scarce, therefore, we aimed to review our experience and analyzed the impact of alemtuzumab. Methods Patients transplanted between 2007 and 2017, who received alemtuzumab were included in this retrospective single-center analysis. The following parameters were evaluated: survival, ACR, CLAD, malignancies and kidney function, infections incidence. Results Within the study period 525 patients received alemtuzumab as induction therapy. An episode of infection within the first year after LTx was reported in 358 patients (68%), however, only 216 of them (22%) needed hospitalization. In the same period 39 recipients (7.4%) died from infection complications . As one of the main advantages of induction therapy is the reduction of CNI, we analyzed rates of kidney insufficiency. During follow-up 94 patients (17.9%) developed kidney insufficiency (eGFR> 60 ml/min). Only two patients required chronic dialysis and one a kidney transplantation . At 1- and 5-years 98% and 97% patients were free from ACR. Three- and 5-years freedom rates from CLAD were 78.7% and 71.8%. Overall survival rates at 3- and 5-years were 79% and 74%, respectively. Conclusion This study shows that alemtuzumab is an effective induction agent with low incidence of severe infections and good survival rates. The associated reduction of CNI doses led to low incidence of kidney insufficiency and long freedom rates from severe CLAD.

Background Incidentally discovered lung cancers in lung transplant (LuTX) recipients are rare but may affect outcome. We aim to report our single center experience with incidence, management, and survival of patients with previously unverified primary lung cancer discovered at the time of LuTX. Methods A total of 1262 patients undergoing LuTX between 1989 and 2012 were retrospectively analyzed in our prospective database. Results Patients identified were six men and five women with a mean age of 54.4±9.9 years. The indication for LuTX was COPD (n=9), pulmonary fibrosis (n=1), and cystic fibrosis (n=1). Histological diagnosis of the explanted lung revealed adenocarcinoma in six, squamous cell carcinoma in three, and lepidic predominant adenocarcinoma in two cases, respectively. Staging revealed stage IA (pT1a/b pN0) in eight and IB (pT2a pN0) in two cases and one patient in stage IIA (pT1b pN1). Subsequent cancer staging after LuTX revealed no metastasis. Immunosuppression was adjusted and no adjuvant chemo- or radiotherapy was administered. The 5-year survival rate was 90.5% with no detection of recurrence. Conclusion In patients with incidentally detected early stage lung cancer at the time of LuTX, rates of recurrence and survival based on this sample appear to be acceptable.

Background Pulmonary alveolar microlithiasis (PAM) is a rare lung disease caused by calcifications within the alveolar space. The only known effective treatment for an end-stage PAM is lung transplantation (LuTX). Methods We performed a retrospective chart review of all individuals that underwent lung transplantation at our center between 1989 and 2013. Five consecutive patients with PAM were identified. Results Four females and one male with a mean age of 46.3 yr were identified. Extracorporeal membrane oxygenation (ECMO) support was required intraoperatively in four cases and post-operatively in one case. Mean post-operative intubation time was 3.3 (range, 2-5) d and mean intensive care unit (ICU) stay was 8.3 (range, 4-12) d. No intraoperative complications were observed. One early patient (operated in 1995) underwent acute re-transplantation on the second post-operative day (POD) and died from sepsis on the 11 POD. In one patient reperfusion edema was observed requiring a prolonged weaning process. No other severe perioperative complications were observed. Four of five patients are currently still alive with normal follow-up parameters. No recurrence of PAM was observed. Conclusions Lung transplantation is a feasible therapy option in patients with end-stage PAM showing good post-operative results comparable to other indications for LuTX.

IntroductionLung cancer represents a major healthcare problem. Accordingly, there is an urgent need to identify serum biomarkers for early diagnosis of lung pathology. We have recently described that patients with manifest COPD evidence elevated levels of heat shock proteins (HSPs). Based on these data, we speculated whether HSPs are also increased in patients with diagnosed lung cancer.MethodsSerum levels of HSP27, phospho-HSP27 (pHSP27) and HSP70 in patients with non-small cell lung cancer (NSCLC) diagnosed at an early (stages I–II, n=37) or advanced (stages IIIA–IV, n=72) stage were determined by using ELISA. Healthy smokers (n=24), healthy never-smoker volunteers (n=33) and COPD patients (n=34) according to GOLD classification served as control population.ResultsSerum levels of HSP27 were elevated in patients with NSCLC diagnosed at an early or advanced stage when compared with both healthy control groups (P

SummaryPathogenetic mechanisms leading to chronic obstructive pulmonary disease (COPD) remain poorly understood. Because clonogenic T cells (CD4+CD28null) were shown to be increased in autoimmune diseases we hypothesized that CD4+CD28null T cells play a role in COPD. Here we describe that enhanced presence of CD4+CD28null cells is associated with impaired lung function. Sixty-four patients and controls were included. T cell phenotype was analysed using flow cytometry. Enzyme-linked immunosorbent assays were utilized to determine cytokines. Statistical evaluations were performed using non-parametric group comparisons and correlations. A logistic regression model was used to determine predictive values of CD4+CD28null in the diagnosis of COPD. Populations of CD4+ T cells lacking surface co-stimulatory CD28 were enlarged significantly in evaluated patients when compared with controls. Natural killer (NK)-like T cell receptors (CD94, 158) and intracellular perforin, granzyme B were increased in CD4+CD28null cells. Cytokine production after triggering of peripheral blood mononuclear cells (PBMCs) was elevated in patients at early disease stages. Receiver operating characteristic curve plotting revealed that presence of CD4+CD28null T cells has a diagnostic value. These CD4+CD28null T cells show increased expression of NK-like T cell receptors (CD94, 158) and intracellular perforin and granzyme B. Furthermore, triggering of PBMCs obtained from patients with mild COPD led to increased interferon-γ and tumour necrosis factor-α production in vitro compared with controls. Our finding of increased CD4+CD28null T cells in COPD indicates that chronic antigen exposure, e.g. through contents of smoke, leads to loss of CD28 and up-regulation of NK cell receptors expression on T cells in susceptible patients.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Collagen and hyaluronic acid (HA) are main components of the extracellular matrix and their enhanced accumulation is associated with tissue fibrosis. Pirfenidone is a pyridone that exhibits anti-fibrotic effects. Its beneficial effects were demonstrated in patients with IPF by reducing disease progression. The inhibition of TGF-β-induced effects has been suggested to be part of pirfenidone9s anti-fibrotic properties. Aim: To study the effect of pirfenidone on secretion of collagen and HA in primary human lung fibroblasts. Methods: Primary human fibroblasts were isolated from lungs derived from patients with IPF. After incubation with pirfenidone (0.5 - 4 mM) and TGF-β1 (5 ng/ml), secretion of collagen and HA was measured by enzyme-linked immunosorbent assay, and down-stream signalling pathways of TGF-β were studied by western blotting. Results: TGF-β1 significantly induced the secretion of collagen type-III and HA. Pirfenidone significantly reduced the TGF-β1-induced secretion of collagen type-III (36% reduction at 1 mM [p=0.02], 40% reduction at 2 mM [p=0.01], 55% reduction at 4 mM pirfenidone [p=0.002]), and significantly antagonized TGF-β1-induced HA secretion (57% reduction at 2 mM [p=0.01], 65% reduction at 4 mM pirfenidone [p=0.006]). TGF-β1-induced phosphorylation of ERK1/2 MAP kinase was prevented in IPF fibroblasts by pirfenidone. Conclusion: Our data demonstrate that pirfenidone counteracts TGF-β-induced pro-fibrotic effects and suggest that the antagonistic effect of pirfenidone on TGF-β-induced effects is associated with the inhibition of ERK1/2.

We have read with great interest the work by Cui et al. (2015) and the letter to the Editor by Cappello et al. (2015). Historical knowledge of data acquisition and interpretation is paramount in the field of applied medical research. Our group was the first to describe increased HSP27 serum concentrations in patients with incipient and manifest COPD, in patients with lung cancer (Hacker et al. 2009; Ankersmit et al. 2012; Zimmermann et al. 2012) and patients with end-stage kidney disease (Lebherz-Eichinger et al. 2008). All these information originated from our research dating back to 2006. In this work, our group (a) investigated serum HSP27/60/70/90 content in patients that underwent coronary artery bypass grafting (CABG) surgery (Szerafin et al. 2008) and (b) identified COPD as an autoimmune disease (Lambers et al. 2009). How was this serum HSP27 insight generated in our laboratory? (a) We have collected serum samples of mild and severe COPD and these were stored at our biobank; (b) A Croatian research group had investigated for the first time the role of HSP27/70 in peripheral blood mononuclear cells (PBMC) derived from COPD patients (Lada et al., 2008); (c) Serendipity was in play since the right R&D HSP27/70 ELISA kit, as “left over” from a prior CABG study and was present in our fridge. All these coincidences led to granted patents stating that HSP27 has the potential to be a serum biomarker for early and manifest COPD and lung cancer (EP2141499, EP2652504). By interpreting the report by Cui et al. (2015) and its comment by Cappello et al. (2015), we want to allude to a recent publication by our research group (Zimmermann et al. 2014). It has to be accepted by the community that an ELISA detection kit for protein XY is sometimes not detecting protein XY (Mueller et al. 2012). In order to investigate this question, we have selected lung cancer patients with known increased serum HSP27 and evaluated five commercially available assays for HSP27 measurement with respect to their capabilities to differentiate non-small cell lung cancer (NSCLC) patients from healthy controls. The following HSP27 ELISA kits were utilized: RD Enzo Life Sciences, 0.823; Invitrogen, 0.780; Abcam 0.642; and MyBioSource, 0.523. From these results, we concluded that it was pure coincidence that we have unintentionally picked the “right” ELISA kit for our investigations in our study period (2007–2015). Abcam or MyBioSource commercial ELISA system would have completely failed to show any difference in our laboratory investigations. We conclude that the results of our clinical method comparison study revealed that commercially available HSP27 assays are not equally useful in detecting increased serum HSP27. By looking at Cui et al. (2015), we observed that a Stressgen Bioreagent HSP27 ELISA was utilized. Since we have not included this commercially available ELISA system in our study, we have difficulty in interpreting the presented results. Since our R&D HSP27 ELISA has repetitively corroborated our primary data set in COPD/lung cancer (>400 patients), it might be possible that Stressgen is detecting another moiety of HSP27 as compared to the R&D HSP27 system. As far as we are concerned, we believe that scientific statements, discussions and publications dealing with serum HSP27 and COPD/lung cancer are important when comparative methodologies are utilized. In conclusion, from an epistemological standpoint, it is of great interest to accept that non-hypothesis-driven “luck factor” has helped to gain a better understanding in COPD genesis. The future will show whether the scientific community will be able to present a biomarker to the public that is able to identify patients with risk to develop COPD or lung cancer. We are deeply convinced that only commercial interest will bring this important knowledge into the realm of clinics.

Proliferation of vascular smooth muscle cells (VSMC) contributes to the progression of atherosclerotic plaques. Calcium channel blockers have been shown to reduce VSMC proliferation, but the underlying molecular mechanism remains unclear. p21(Waf1/Cip1) is a potent inhibitor of cell cycle progression. Here, we demonstrate that amlodipine (10(-6) to 10(-8) M) activates de novo synthesis of p21(Waf1/Cip1) in vitro. We show that amlodipine-dependent activation of p21(Waf1/Cip1) involves the action of the glucocorticoid receptor (GR) and C/EBP-alpha. The underlying pathway apparently involves the action of mitogen-activated protein kinase or protein kinase C, but not of extracellular signal-related kinase or changes of intracellular calcium. Amlodipine-induced p21(Waf1/Cip1) promoter activity and expression were abrogated by C/EBP-alpha antisense oligonucleotide or by the GR antagonist RU486. Amlodipine-dependent inhibition of cell proliferation was partially reversed by RU486 at 10(-8) M (58%+/-29%), antisense oligonucleotides targeting C/EBP-alpha (91%+/-26%), or antisense mRNAs targeting p21(Waf1/Cip1) (96%+/-32%, n=6); scrambled antisense oligonucleotides or those directed against C/EBP-beta were ineffective. The data suggest that the anti-proliferative action of amlodipine is achieved by induction of the p21 (Waf1/Cip1) gene, which may explain beneficial covert effects of this widely used cardiovascular therapeutic drug beyond a more limited role as a vascular relaxant.

Combined muscarinic receptor antagonists and long acting β2-agonists improve symptom control in chronic obstructive pulmonary disease (COPD) significantly. In clinical studies aclidinium bromide achieved better beneficial effects than other bronchodilators; however, the underlying molecular mechanisms are unknown. This study assessed the effect of aclidinium bromide combined with formoterol on COPD lung (n=20) and non-COPD lung (n=10) derived epithelial cells stimulated with TGF-β1+carbachol on: (i) the generation of mesenchymal cells in relation to epithelial cells, (II) extracellular matrix (ECM) deposition, and (iii) the interaction of ECM on the generation of epithelial and mesenchymal cells. TGF-β1+carbachol enhanced the generation of mesenchymal cells, which was significantly reduced by aclidinium bromide or formoterol. The effect of combined drugs was additive. Inhibition of p38 MAP kinase and Smad by specific inhibitors or aclidinium bromide reduced the generation of mesenchymal cells. In mesenchymal cells, TGF-β1+carbachol induced the deposition of collagen-I and fibronectin which was prevented by both drugs dose-dependently. Formoterol alone reduced collagen-I deposition via cAMP, this however, was overruled by TGF-β1+carbachol and rescued by aclidinium bromide. Inhibition of fibronectin was cAMP independent, but involved p38 MAP kinase and Smad. Seeding epithelial cells on ECM collagen-I and fibronectin induced mesenchymal cell generation, which was reduced by aclidinium bromide and formoterol. Our results suggest that the beneficial effect of aclidinium bromide and formoterol involves cAMP affecting both, the accumulation of mesenchymal cells and ECM remodeling, which may explain the beneficial effect of the drugs on lung function in COPD.

Long acting β₂-agonists (LABA) have been reported to modify the extracellular matrix (ECM) composition in the airway wall. Based on our earlier studies we here investigated the mechanism underlying the control of ECM modification by LABA in primary human airway smooth muscle cells. Cells were treated with formoterol or salmeterol (30 min) before TGF-β₁ stimulation (2-3 days) Using RT-PCT, immuno-blotting and ELISA the de novo synthesis and deposition of collagen type-I, -III, -IV and fibronectin were determined. Matrix metalloproteinases (MMP)-2 and -9 were analyzed by zymography. Both LABA activated cAMP and its corresponding transcription factor CREB within 60 min and thus partly reduced TGF-β₁-induced gene transcription of collagen type-I, -III, fibronectin and connective tissue growth factor (CTGF). The inhibitory effect of both LABA on collagen type-I and -III deposition involved a cAMP dependent mechanism, while the inhibitory effect of the two drugs on TGF-β1-induced fibronectin deposition and on CTGF secretion was independent of cAMP. Interestingly, none of the two LABA reduced CTGF-induced synthesis of collagen type-I or type-III deposition. In addition, none of the two LABA modified collagen type-IV deposition or the expression and activity of MMP-2 or MMP-9. Our results show that LABA can prevent de novo deposition of specific ECM components through cAMP dependent and independent signaling. However, they do not reduce all ECM components by the same mechanism and they do not reduce existing collagen deposits. This might explain some of the controversial reports on the anti-remodeling effect of LABA in chronic inflammatory lung diseases.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo. Aim To determine the in vitro effect of nintedanib on primary human lung fibroblasts. Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen. Results IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib. Conclusion Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug’s anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

Background: Although chronic obstructive pulmonary disease (COPD) is amongst the leading causes of morbidity and mortality, no biomarkers for its early detection are known. We have recently demonstrated that COPD is accompanied by elevated serum heat shock protein (HSP) 27 levels as compared to a control population. Objectives: In an open prospective study, we investigated whether elevated HSP27 levels are associated with the early radiological signs of COPD, i.e. air trapping (AT), emphysema (E) and impaired lung function. Methods: In total, 120 apparently healthy smokers underwent lung function testing and serum sampling. Serum levels of HSP27, phospho-HSP27, CXCR2 chemokines and proteins related to inflammation, tissue remodeling and apoptosis were evaluated by ELISA. Of these 120 subjects, 94 voluntarily underwent a high-resolution computed tomography scan. Results: AT or AT and E were detected in 57.45%. Subjects with AT and E (n = 23) showed significantly higher HSP27 levels than those without any pathology [i.e. nothing abnormal detected (NAD)] (4,618 ± 1,677 vs. 3,282 ± 1,607 pg/ml; p = 0.0081). In a univariate logistic regression model including NAD and AT and E, the area under the curve of HSP27 in the receiver-operating-characteristic curve was 0.724, (0.594–0.854, 95% CI; p = 0.0033). Interestingly, proinflammatory IL-8 was elevated in those subjects with evidence of AT and E compared to those with AT and NAD. Lung function did not correlate with increased HSP27 levels or pathological radiological findings. Conclusions: HSP27 serum levels correlated with the early radiological signs of COPD, whereas lung function did not match with radiological findings or HSP27 serum levels. Serum HSP27 levels may serve as a potential marker to identify the early signs of COPD independent of lung function in young smokers.