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The family of RNA-binding proteins (RBP) functions as a crucial regulator of multiple biological processes and diseases. However, RBP function in the clinical setting of idiopathic pulmonary fibrosis (IPF) is still unknown. We developed a practical in silico screening approach for the characterization of RBPs using multi-sources data information and comparative molecular network bioinformatics followed by wet-lab validation studies. Data mining of bulk RNA-Sequencing data of tissues of patients with IPF identified Quaking (QKI) as a significant downregulated RBP. Cell-type specific expression was confirmed by single-cell RNA-Sequencing analysis of IPF patient data. We systematically analyzed the molecular interaction network around QKI and its functional interplay with microRNAs (miRs) in human lung fibroblasts and discovered a novel regulatory miR-506-QKI axis contributing to the pathogenesis of IPF. The in silico results were validated by in-house experiments applying model systems of miR and lung biology. This study supports an understanding of the intrinsic molecular mechanisms of IPF regulated by the miR-506-QKI axis. Initially applied to human lung disease, the herein presented integrative in silico data mining approach can be adapted to other disease entities, underlining its practical relevance in RBP research.

Background. Idiopathic pulmonary fibrosis (IPF) is a progressive disease for which two antifibrotic drugs recently were approved. However, an unmet need exists to predict responses to antifibrotic treatment, such as pirfenidone. Recent data suggest that upregulated expression of CXCR4 is indicative of outcomes in IPF. Research Question. Can quantitative, molecular imaging of pulmonary CXCR4 expression as a biomarker for disease activity predict response to the targeted treatment pirfenidone and prognosis in patients with IPF? Study Design and Methods. CXCR4 expression was analyzed by immunohistochemistry examination of lung tissues and reverse-transcriptase polymerase chain reaction analysis of BAL. PET-CT scanning with the specific CXCR4 ligand 68Ga-pentixafor was performed in 28 IPF patients and compared with baseline clinical characteristics. In 16 patients, a follow-up scan was obtained 6 to 12 weeks after initiation of treatment with pirfenidone. Patients were followed up in our outpatient clinic for > 12 month. Results. Immunohistochemistry analysis showed high CXCR4 staining of epithelial cells and macrophages in areas with vast fibrotic remodeling. Targeted PET scanning revealed CXCR4 upregulation in fibrotic areas of the lungs, particularly in zones with subpleural honeycombing. Baseline CXCR4 signal demonstrated a significant correlation with Gender Age Physiology stage (r = 0.44; P = .02) and with high-resolution CT scan score (r = 0.38; P = .04). Early changes in CXCR4 signal after initiation of pirfenidone treatment correlated with the long-term course of FVC after 12 months (r = −0.75; P = .0008). Moreover, patients with a high pulmonary CXCR4 signal on follow-up PET scan after 6 weeks into treatment demonstrated a statistically significant worse outcome at 12 months (P = .002). In multiple regression analysis, pulmonary CXCR4 signal on follow-up PET scan emerged as the only independent predictor of long-term outcome (P = .0226). Interpretation. CXCR4-targeted PET imaging identified disease activity and predicted outcome of IPF patients treated with pirfenidone. It may serve as a future biomarker for personalized guidance of antifibrotic treatment.

Cigarette smoke (Cs) inhalation is a main reason to develop chronic obstructive pulmonary disease (COPD), characterised by degradation of alveoli, mucus hypersecretion, emphysema development and inflammation [1]. The pathophysiology of COPD is not well understood so the mechanisms that underlie various components of COPD need to be modelled in vitro, specifically using Cs. We assessed the pathophysiological effects of Cs and Cs condensate (Csc) on fresh human lung tissue. Human Precision Cut Lung Slices (PCLS) were exposed to Csc submerged or whole Cs in an Air-Liquid Interface using the in vitro exposure device P.R.I.T.® ExpoCube®. Cytotoxicity, release of cytokines and extracellular matrix (ECM) proteins were measured and gene expression analysis upon RNA isolation from PCLS was performed. PCLS exposed to Csc or Cs showed concentration-dependent loss of tissue viability. Gene expression analysis upon RNA isolated from lung tissue exposed to nontoxic doses of Csc of literature-based COPD-relevant genes indicate damage of epithelium by upregulation of genes involved in tissue injury (e.g. mmp) and specific metabolic activity (e.g. cyp1a1). Samples exposed to control substance LPS did not show increased expression in emphysema associated genes but in genes indicating inflammation (e.g. il-6). These data can be supported by increased production of proteins related to the extracellular matrix (e.g. MMP9, RAGE) and inhibited expression in pro-inflammatory cytokines after Cs exposure in an air-liquid culture. Supportive results were presented in BAL samples from COPD subjects showing markers of apoptosis [2]. These results indicate that early stages of Cs induced lung tissue changes are primarily not provoked by immunological processes but by apoptosis, increased specific metabolic activity and damage of the epithelium. [1] Hodge, S. et al. (2005). ERJ 25 (3), S. 447-454. [2] Shaykhiev, R.; Crystal, R. (2014). AnnalsATS 11 Suppl 5, S252-8.

Forced expiratory volume in 1 second (FEV1 ) from spirometry is the most commonly used parameter to detect early allograft dysfunction after lung transplantation (LTx). There are concerns regarding its sensitivity. Nitrogen-multiple breath washout (N2 -MBW) is sensitive at detecting early global (lung clearance index [LCI]) and acinar (Sacin ) airway inhomogeneity. We investigated whether N2 -MBW indices indicate small airways pathology after LTx in children with stable spirometry. Thirty-seven children without bronchiolitis obliterans syndrome [BOS] at a median of 1.6 (0.6-3.0) years after LTx underwent N2 -MBW and spirometry, 28 of those on 2 occasions (≤6 months apart) during clinically stable periods. Additional longitudinal data (11 and 8 measurements, respectively) are provided from 2 patients with BOS. In patients without BOS, LCI and Sacin were significantly elevated compared with healthy controls. LCI was abnormal at the 2 test occasions in 81% and 71% of patients, respectively, compared with 30% and 39% of patients with abnormal FEV1 /forced vital capacity (FVC). Correlations of LCI with FEV1 /FVC (r = 0.1, P = .4) and FEV1 (r = -0.1, P = .6) were poor. N2 -MBW represents a sensitive and reproducible tool for the early detection of airways pathology in stable transplant recipients. Moreover, indices were highly elevated in both patients with BOS. Spirometry and LCI showed poor correlation, indicating distinct and complementary physiologic measures.

Background: Human Respiratory Syncytial virus (HRSV) is a major cause of respiratory disease young children and is known as a risk factor for asthma. HRSV and the viral mimic Poly I:C activate toll‐like receptor 3 (TLR3), initiating an innate response with release of immune mediators, e.g. interferon gamma‐induced protein 10 (IP‐10). In this work, we aimed to perform an ex vivo HRSV infection in precision‐cut lung slices (PCLS) from human, rhesus, and cynomolgus macaques, comparing whenever possible the response with the viral surrogate poly I:C. Method: PCLS containing airways were prepared from lung sections of human, rhesus, and cynomolgus macaques. The slices were inoculated with HRSV‐A2 106 IU/mL, UV‐inactivated HRSV, or vehicle control for 48 hours. Macaque slices were also incubated with Poly I:C 100 mg/mL with and without the immunosuppressive dexamethasone 50 mg/mL. Viral replication, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices. Results: The inoculum infectivity of 106 IU/mL as well the UV‐inactivation were confirmed by plaque‐assay on Hep‐2 cells Immunofluorescence staining using a FITC‐labeled anti‐RSV showed the presence of infected macrophages in PCLS, but not in mock infected samples. HRSV stimulation slightly decreased tissue viability, as seen by Live/DEAD staining and LDH assay. The viral infection increased IP‐10 production in PCLS of human, rhesus, and cynomolgus macaques, reaching respectively 13.3, 3.4, and 1.7 fold‐increase in comparison to the vehicle controls. Poly I:C stimulation caused IP10 response comparable to HRSV in rhesus and cynomolgus PCLS. The IP‐10 production ratio comparing HRSV/Poly I:C was 1.1 in rhesus and 0.9 in cynomolgus PCLS. Conclusion: HRSV infects ex vivo PCLS of human and non‐human primates, inducing the release of the pro‐inflammatory chemokine IP‐10. This response is comparable to the viral surrogate poly I:C. In the future, these systems can be used to further investigate host response to HRSV, especially in the context of asthma development.

Background Investigation of basic chronic inflammatory mechanisms and development of new therapeutics targeting the respiratory tract requires appropriate testing systems, including those to monitor long- persistence. Human precision-cut lung slices (PCLS) have been demonstrated to mimic the human respiratory tract and have potential of an alternative, ex-vivo system to replace or augment in-vitro testing and animal models. So far, most research on PCLS has been conducted for short cultivation periods (≤72 h), while analyses of slowly metabolized therapeutics require long-term survival of PCLS in culture. In the present study, we evaluated viability, physiology and structural integrity of PCLS cultured for up to 15 days. Methods PCLS were cultured for 15 days and various parameters were assessed at different time points. Results Structural integrity and viability of cultured PCLS remained constant for 15 days. Moreover, bronchoconstriction was inducible over the whole period of cultivation, though with decreased sensitivity (EC501d = 4 × 10−8 M vs. EC5015d = 4 × 10−6 M) and reduced maximum of initial airway area (1d = 0.5% vs. 15d = 18.7%). In contrast, even though still clearly inducible compared to medium control, LPS-induced TNF-α secretion decreased significantly from day 1 to day 15 of culture. Conclusions Overall, though long-term cultivation of PCLS need further investigation for cytokine secretion, possibly on a cellular level, PCLS are feasible for bronchoconstriction studies and toxicity assays. Electronic supplementary material The online version of this article (doi:10.1186/s12995-017-0158-5) contains supplementary material, which is available to authorized users.

Toxicity of chemicals is traditionally assessed using animal studies. For reasons of ethics, economy and legislation predictive alternatives have to be used, whenever possible. Nevertheless, the development of alternatives to animal testing has lagged behind. One alternative in risk assessment of chemicals is read-across. Here, toxicity data of a tested compound is used to predict the toxicity of a "similar" non-tested compound. As one of the main routes of exposure to chemicals is the inhalation route, our project combines the read-across approach with genomic data of in vitro and ex vivo respiratory assays. First, we selected three categories of chemicals (vicinale halogenide, aromates, and ester) and defined one lead compound and two read-across in each category. Categories were chosen based on potentially different mode of action. Secondly, repeated exposure of chemicals was performed on three days for three hours daily in rat and human precision-cut lung slices (PCLS). The cytotoxicity of chemicals was assessed in PCLS by LDH and WST-1 assay. At least one substance in each category showed cytotoxic effect in dose dependent manner. The chemicals did not show increased sensitivity upon repeated exposure. Human PCLS were less sensitive to chemicals compared to rat PCLS maybe due to differences in metabolic activity. This study shows differences of rodent and human metabolism. Although toxicity profiling is based on rodent exposure data, species diversity can be studied ex vivo for better predictively in human. Moreover, results of the project will be used to evaluate the read-across approach based on the tested chemicals.

Zielsetzung: Mit der Lungentransplantation (LTX) sind spezifische Herausforderungen assoziiert. Der zunehmende Organmangel, die vergleichsweise geringe Ischämietoleranz, sowie die Effizienz der kalten Organkonservierung stehen in direktem Zusammenhang mit dem Outcome nach Transplantation. Das m[for full text, please go to the a.m. URL], 20. Jahrestagung der Deutschen Gesellschaft für Thoraxchirurgie

Objective: Experiments were designed to compare large animal models for lung ischemia-reperfusion injury using either 90 minutes' warm or 24 hours' cold ischemia. Methods: In 6 pigs, the left lung was perfused in situ with cold LPD solution. Reperfusion was started after 90 min of warm ischemia. The left lung of 6 donor pigs was perfused with LPD solution and the lung transplanted after 24 hours of cold storage into 6 recipient pigs. In both groups the right pulmonary artery and main bronchus were clamped after reperfusion of the left lung. Bronchoalveolar lavage (BAL) was obtained before ischemia and after 2 h of reperfusion. Hemodynamic and respiratory parameters were monitored. Surfactant activity was determined from the BAL in a pulsating bubble surfactometer. Results: Moderate lung injury was evident after reperfusion, but without any significant differences between groups. Surfactant composition and function were mildly impaired after reperfusion in both groups. Conclusions: Our results suggest that short periods of warm ischemia might be accepted as an accelerated model for ischemia-reperfusion injury of the lung after cold storage.

Objective: Improvement of organ preservation is essential to facilitate acceptance of marginal donor lungs for transplantation. Thus, recruiting non-heart-beating donors (NHBD) may be one reasonable strategy to augment the organ-pool especially in the field of pulmonary transplantation. Topical cooling (TC) of donor lungs could provide fast organ-protection and is an available procedure even in smaller centers. In this study transplanted lung function and surfactant activity in same lungs, which were preserved by TC, were assessed following transplantation. Methods: Twelve porcine allogeneic single lung transplants were performed. Six lungs that were flush preserved through the antegrade route served as controls. The other six lungs were preserved by TC for 30 min after induction of cardiac arrest by repeated application of cold saline (8 °C) to both pleural cavities. Lungs of both groups were stored in LPD solution for 24 h at 8 °C. After transplantation, the recipient's right bronchus and right pulmonary artery were clamped. Major endpoints included early graft function over a period of 7 h. Hemodynamic measures and respiratory functions were recorded in 30-min intervals. Surfactant function was determined before transplantation and 2 h after reperfusion by broncho-alveolar lavage fluid analysis. Results: Only four animals of the control-group survived the 7 h reperfusion period. Right heart failure occurred in two animals after 150 and 240 min of reperfusion. All six animals in the TC group survived the observation period. Pulmonary vascular resistance (p

BACKGROUND: Pulmonary preservation with high potassium/low oncotic pressure Euro-Collins (EC) solution is associated with endothelial dysfunction and reduced surfactant function. We compared two low potassium solutions, histidine-tryptophane-ketoglutarate (HTK) and Celsior, to EC in lung ischemia-reperfusion injury. METHODS: In 19 minipigs, the left lung was perfused in situ with cold preservation solution (EC, n=6; HTK, n=6; Celsior, n=7). Reperfusion was started after 90 min of warm ischemia. The right pulmonary artery and main bronchus were clamped. Bronchoalveolar lavage (BAL) was obtained before ischemia and after 2 h of reperfusion. Surfactant activity was determined from the BAL in a pulsating bubble surfactometer. RESULTS: Animals in the EC group survived 3.7+/-1.4 h. Six Celsior and five HTK treated animals survived the observation period of 7 h (P

OBJECTIVE: Surfactant function was shown to be impaired in clinical and experimental lung transplantation. This study was designed to define the impact of retrograde flush perfusion on graft and surfactant function after an extended period of ischemia. METHODS: Left lung transplantation was performed after 24 hours of graft ischemia in 12 pigs. In half of the grafts antegrade cold flush perfusion (Perfadex) was used for preservation. In the second group grafts were flushed in a retrograde fashion via the left atrium. Graft function was monitored for 7 hours after transplantation. Before transplantation (basal) and after 2 hours of reperfusion, bronchoalveolar lavage fluid was obtained. Minimal surface tension of bronchoalveolar lavage fluid was determined and the ratio of small and large surfactant aggregates was calculated. Lung water content was analyzed online in the reperfusion period. RESULTS: Right-sided heart failure developed in 2 animals of group 1 (antegrade perfusion) within 2 and 4.5 hours of reperfusion, respectively. All other pigs survived the observation period. PO(2)/FIO(2) (P =.001) and dynamic lung compliance (P =.001) were superior in retrogradely flushed grafts. A comparable increase of minimal surface tension was found after reperfusion in both groups. Small/large surfactant aggregate ratio after reperfusion (P =.03), as well as extravascular lung water content, was higher in the antegrade perfusion group. CONCLUSION: Retrograde flush perfusion for 24-hour lung preservation with low-potassium dextran (Perfadex) solution led to better initial graft function than the standard antegrade perfusion technique. A moderate impairment of surfactant function was found in both groups, which was more pronounced in the antegrade perfusion group.

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Polarforschung

Die Autoren geben eine Stellungnahme zu dem Beitrag von H. Roer (Beitr. Ent., 11, 594-613; 1961). Sie betrachten die Schlußfolgerung Roer's, Vanessa atalanta L. überwintere in Mitteleuropa, sei also hier ständig heimisch, als Fehlschluß auf Grund ungenügender Angaben und unrichtiger Voraussetzungen., Beiträge zur Entomologie = Contributions to Entomology, Bd. 14 Nr. 1-2 (1964)

Polarforschung

Polarforschung

Polarforschung

Polarforschung

Polarforschung

This is a short review of the statistical mechanical definition of entropy production for systems composed of a large number of interacting components. Emphasis is on open systems driven away from equilibrium where the entropy production can be identified with a logarithmic ratio of microstate multiplicities of the original macrostate with respect to the time-reversed state. A special role is taken by Gibbs measures for the stationary spatio-temporal distribution of trajectories. The mean entropy production is always non-negative and it is zero only when the system is in equilibrium. The fluctuations of the entropy production satisfy a (Gallavotti-Cohen)symmetry first observed and then derived for the phase space contraction rate in a class of strongly chaotic dynamical systems. Aspects of the general framework are illustrated via a bulk driven diffusive lattice gas. ispartof: pages:251-267 ispartof: Entropy pages:251-267 ispartof: Symposium on Entropy location:GERMANY, Max Planck Inst Phys Complex Syst, Dresden date:25 Jun - 28 Jun 2000 status: published