DZL Annual Meeting 2017
DZL Annual Meeting 2017 Munich
Abstract Book 25.01.2017
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DZL Annual Meeting 2017
Moderators & Moderated Poster Discussion
Disease Area Asthma & Allergies
Moderators: Heinz Fehrenbach, Oliver Fuchs, Ulrich Zissler Moderated Poster Discussion: Abstracts # 68, 126, 152, 159, 168, 193, 200, 220, 273, 282
Disease Area Cystic Fibrosis
Moderators: Antje Munder, Mirjam Stahl, Mark Wielpütz Moderated Poster Discussion: Abstracts # 11, 27, 31, 57, 89, 95, 110, 201, 226, 263, 280, 286, 293
Disease Area COPD
Moderators: Mariola Bednorz, Anne Hilgendorff, Stefan Karrasch, Benjamin Waschki Moderated Poster Discussion: Abstracts # 29, 47, 52, 54, 63, 116, 139, 181, 267, 270
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DZL Annual Meeting 2017
Disease Area Diffuse Parenchymal Lung Disease
Moderators: Ellie El Agha, Michael Kreuter, Isis Fernandez Moderated Poster Discussion: Abstracts # 6, 17, 42, 59, 76, 86, 97, 109, 125, 130, 174, 237, 257
Disease Area End Stage Lung Disease
Moderators: Sotiris Korossis, Bettina Wiegmann Moderated Poster Discussion: Abstracts # 8, 73, 121, 131, 145, 170, 178, 180, 186, 187, 205, 216, 228, 289
Disease Area Pulmonary Hypertension
Moderators: Bakytbek Kojonazarov, Soni Pullamsetti, Natascha Sommer
Moderated Poster Discussion: Abstracts # 41, 55, 148, 162, 176, 179, 197, 208, 214, 262
Disease Area Pneumonia & Acute Lung Injury
Moderators: Jürgen Lohmeyer, Ulrich Maus Moderated Poster Discussion: Abstracts # 44, 45, 70, 75, 85, 96, 98, 101, 108, 149, 154, 156, 165, 206, 207, 255, 276, 296 25.01.2017
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DZL Annual Meeting 2017
Disease Area Lung Cancer
Moderators: Heiko Golpon, Martin Rech, Thorsten Stiewe Moderated Poster Discussion: Abstracts # 1, 33, 34, 62, 74, 80, 153, 161, 203, 227, 243, 295
Platforms
Moderators: tba
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DZL Annual Meeting 2017
Table of Contents #
Topic
Page
Disease Area Asthma and Allergies 007
Physical activity, airway resistance and small airway dysfunction in severe 1 asthma Thomas Bahmer
048
Relative roles of reduced mucus clearance and mucus hypersecretion in the pathogenesis of airway mucus plugging in mice Sandra Christochowitz
2
053
Associations of early-life events, and current environmental and lifestyle factors with lung function in adolescents (GINIplus & LISAplus studies) Agnes Luzak
3
056
6q12 and 11p14 variants are associated with postnatal exhaled nitric oxide and respiratory symptoms Oliver Fuchs
4
064
Characterization of peanut defensins as potential candidates for in-vitro diagnostic Skadi Kull
5
068
RV-infection modifies DNA-methylation in nasal airway epithelium cells in asthmatics and non-asthmatics children Martin Pech
6
069
Systemic and Bronchial Inflammation entail regulatory compartments of regulatory B and T cells in grass-pollen allergic rhinitis and asthma patients Ulrich Zissler
7
071
Regulatory B cells and Shift in Th17 cell populations raise in Allergenspecific Immunotherapy Ulrich Zissler
8
078
Innate immune network in asthma: studies on dendritic cell interaction with airway epithelium Karina Stein
9
081
Neutrophil-epithelial cell interactions in allergic asthma Brigiite Kasper
10
083
Mast cells and mast cell chymase regulate airway hyperreactivity in chronic experimental asthma Marjan Ahmadi
11
088
The influence of the microbiome on asthma susceptibility - project outline of a doctoral thesis Joni Valeska Lund
12
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DZL Annual Meeting 2017 102
Glycolipids of house dust mites – studying the impact of allergen-lipid association Nestor Gonzalez Roldan
13
106
A possible role for microRNA-17 and -21 in regulatory T cell (Treg) function in allergic asthma Sabine Bartel
14
115
Looking for the anti-inflammatory mechanism of the probiotic compound D-Tryptophan – does it influence T cell differentiation? Gregor Jatzlauk
15
119
Effects of prenatal cigarette smoke on thymic T cell composition and lung function in offspring Barbara Hammer
16
126
Clinical and data-driven phenotype definitions in the pediatric arm of the DZL All Age Asthma Cohort (ALLIANCE) Alexander Hose
17
128
Development of atopic sensitization in Finnish and Estonian children – a latent class analysis in a multicenter cohort Markus Ege
18
129
A pathway analysis of severe atopy, childhood asthma, and impaired lung function Alexander Hose
19
138
Airway-liquid interface cultures of human bronchial epithelial cells for the investigation of biomarkers for asthma exacerbations Johanna C. Ehlers
20
150
The Search for Early Biomarkers of Asthma and its Clinical Course across 21 Child-and Adulthood, the DZL All Age Asthma Cohort (ALLIANCE) Oliver Fuchs
151
Measurement of exhaled volatile organic compounds in children by gas chromatography-mass spectrometry in the DZL ALL Age Asthma Cohort (ALLIANCE) Maximilian Ehrmann
22
152
Physiological phenotyping by lung function and assessment of airway inflammation in children of the DZL All Age Asthma Cohort (ALLIANCE) Oliver Fuchs
23
159
Rhinovirus infection of HDM sensitized lung tissue ex vivo proves inadequate anti-viral immune response in small airways Olga Danov
24
166
Measurement of volatile organic compounds (VOCs) in exhaled air in children with an electronic nose (e-nose) as part of the DZL ALL Age Asthma Cohort Stefan Tümmers*
25
168
Influence of differently glycosylated IgG subclass antibodies on allergic reactions Alexandra Epp
26
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DZL Annual Meeting 2017 173
Are metabolomic markers associated with spirometric lung function indices? Results of the KORA-F4 Study. Claudia Flexeder
27
175
Studying the pathophysiological role of the asthma susceptibility genes STAT3/6 and ORMDL3 using the fruit fly Drosophila melanogaster as a model Christine Fink
28
193
Comparison of diagnostic tools for detection of allergen-specific IgE in the pediatric arm of the DZL All Age Asthma Cohort (ALLIANCE) Anna-Maria Dittrich
29
200
The role of B-cells and B-cell activating factor of the TNF family (BAFF) in a mouse model of house dust mite-induced allergic airway inflammation Anika Habener
30
209
Anatomical features and functions of mast cell-C-fibers interactions are comparable in human and non-human primate airways Elaine Cabral Serrao
31
210
Identification of new IgE targets via One-Bead-One-Compound (OBOC) libraries for diagnosis and treatment of allergic asthma Thorsten Krause
32
219
Species comparison of anti-viral and inflammatory host response to rhinovirus infection in fresh lung tissue Helena Obernolte
33
220
Chipcytometry based comprehensive immunophenotyping of peripheral blood mononuclear cells of the DZL All Age Asthma Cohort (ALLIANCE) Adan Chari Jirmo
34
225
Drosophila melanogaster as model organism for investigations on transgenerational effects of nicotine exposure Stephanie Papenmeier
35
231
Drosophila melanogaster - A promising model to study transgenerational effects of parental smoking Karolina-Theresa Neumann
36
238
Development of asthma phenotypes: Predictors and Mechanisms – Aims of WP1 in the Asthma and Allergy (AA) Disease Area in DZL2.0 Bianca Schaub
37
242
T-cell dependency of HDM-induced neutrophil airway inflammation Stefanie Hagner
38
246
IRF-1 SNPs influence the risk for childhood allergic asthma in the CLARA-study: a critical role for pro-inflammatory immune regulation Katja Landgraf-Rauf
39
252
Nuclear localization of Suppressor of Cytokine Signaling (SOCS)-1 regulates local immunity in the lung Jana Zimmer
40
264
Characterization of cellular sources for non-quantal ACh release in mouse 41 airways Lucas Delventhal
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DZL Annual Meeting 2017 272
B-cell subsets are modulated during OVA-induced allergic asthma but are not required for the development of respiratory tolerance in a murine model Anika Habener
42
273
Extracellular microRNAs in pediatric asthma, the DZL All Age Asthma Cohort (ALLIANCE) Christine Happle
43
282
THE CROSS-TALK BETWEEN AIRWAY EPITHELIUM AND IMMUNE CELLS IN ALLERGY RESEARCH Ulrich Zissler
44
283
Towards Standardized Airway Hyperresponsiveness Measurements in Mice Winfried Möller
45
288
Analysis of cytokine levels using the Bio-Plex multiplex system in the pediatric arm of the ALLIANCE cohort Markus Weckmann
46
290
Attenuated AHR and migration of dendritic cells to the lungs and mediastinal lymph nodes in absence of IL-17 in experimental asthma Adan Chari Jirmo
47
Disease Area COPD 010
Neutrophil Extracellular Traps are Regulated by Chemokine Receptor CXCR2 in Chronic Obstructive Pulmonary Disease Frauke Pedersen
48
012
Changes of cardiac function, morphology and wall stress in COPD during 1.5 years of COSYCONET follow up Peter Alter
49
026
An epigenetic cell-of-origin approach to chronic obstructive pulmonary disease Reinhard Liebers
50
029
Identification of target genes in COPD-tissues: a molecular-clinicalhistopathologic puzzle Lena Heinbockel
51
046
microRNAs constitute a negative feedback loop in Streptococcus pneumoniae induced macrophage activation Kathrin Griss
52
047
Global profiling of bronchial epithelial cell response to Streptococcus pneumoniae infection André Wesener
53
052
Costs and health-related quality of life in COPD patients with alpha-1antitrypsin deficiency: results of the COSYCONET COPD cohort Florian M. Karl
54
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DZL Annual Meeting 2017 054
The real life data registry BeoNet - Leadoff results of the first use case: COPD Heidrun Lingner
55
060
Transfer factor for carbon monoxide in patients with COPD and diabetes: Results from the German COSYCONET cohort Kathrin Kahnert
56
061
The effect of voluntary activity on obesity-associated pathological changes of the lung Julia Schipke
57
063
Breath volatile organic compounds (VOC) in COPD – first results from a large validation trial Olaf Holz
58
087
Chromatin modulator HMGN5 regulates cellular apoptosis of lung epithelial cells in pathogenesis of COPD Rim Sabrina Jahan Sarker
59
103
NON-CANONICAL NOTCH PATHWAY PARTIALLY REGULATES MACROPHAGES IMMUNE RESPONSE IN COPD Carolina Ballester López
60
116
Insights into COPD pathogenesis by 2D cross-omics enrichment analysis of mice and human lung. Thomas Conlon
61
123
A common coding variant in SERPINA1 increases the risk for large artery stroke Rainer Malik
62
134
Targeting the mTOR signaling pathway to inhibit lung cell senescence in chronic obstructive pulmonary disease (COPD) Amal HOUSSAINI
63
137
Dependence of observed lung T1 relaxation time on echo time and oxygen concentration - a preparatory study Bertram Jobst
64
139
Repetitive exposure to cigarette smoke alters the airway structure of the fruit fly Drosophila melanogaster Marcus Thiedmann
65
143
Natural history and therapy are associated with distinct phenotypes of alpha-1-antitrypsin deficiency Sebastian Fähndrich
66
158
The influence of cigarette smoke on the lung microbiota and its interaction with the bronchial epithelium Draginja Kovacevic
67
164
YACTA as Tool for Big Data Analysis in Large Lung MDCT Data Sets – Preliminary Results of the COSYCONET study Oliver Weinheimer
68
167
The phenotypic characterisation of patients with severe COPD Sandhya Matthes
69
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DZL Annual Meeting 2017 181
The role of mitochondria in biological aging of the lung Claudia Fernanda Garcia Castro
70
183
Prevalence of Dyslipidemia in the COSYCONET cohort and its relationship to comorbidities and lung function Kathrin Kahnert
71
192
Cigarette smoke-induced emphysema and pulmonary hypertension – The role of FGF10 Stefan Hadzic
72
194
Impact of different spirometric criteria on the prevalence of spirometrically defined COPD and its comorbidities in middle and advanced age Stefan Karrasch
73
195
Effects of electronic cigarette vapour extract exposure on primary isolated mouse lung cells Elsa T. Roxlau
74
196
Innate immune cells in a murine model of COPD-like inflammation Martin Wolff
75
199
Elastin microfibril interfacers in chronic obstructive pulmonary disease decrease lung repair via inhibition of WNT/β-catenin signaling Rita Costa
76
202
Identification of novel compounds for WNT/β-catenin induced lung repair in Chronic Obstructive Pulmonary Disease Rita Costa
77
211
Superior anti-inflammatory effects of Narrow spectrum kinase inhibitors in 78 airway smooth muscle cells from subjects with COPD Juergen Knobloch
212
Reduced Frizzled receptor 4 expression prevents Wnt/β-catenin-driven alveolar lung repair in COPD Wioletta Skronska-Wasek
79
233
Does urinary peptide content differ between COPD patients with and without inherited alpha1-antitrypsin deficiency? Alfonso Carleo
80
247
Plasminogen activator inhibitor-1 is elevated in patients with COPD independent of metabolic and cardiovascular function Benjamin Waschki
81
256
Cigarette smoke causes acute airways disease and exacerbates chronic obstructive lung disease in neonatal mice Thomas Conlon
82
259
The minimal important difference for target lobe volume reduction following endoscopic valve therapy in patients with advanced emphysema Daniela Gompelmann
83
260
Impact of fissure integrity on emphysema distribution in patients with severe chronic obstructive pulmonary disease Daniela Gompelmann
84
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DZL Annual Meeting 2017 265
Lung volume reduction coil (LVR-coil) treatment of patients with advanced emphysema. Effectiveness and complications after 1 year Konstantina Kontogianni
85
266
Quantitative CT (QCT) analysis of emphysema and air trapping in Coilbased Lung Volume Reduction (LVRC) treatment Konstantina Kontogianni
86
267
Assessment of Airway Instability and Respiratory Dynamics with LowDose 4D-CT in Chronic Obstructive Pulmonary Disease – Preliminary Results Anja Dutschke
87
270
Reversal of established cigarette smoke (CS)-induced pulmonary hypertension and emphysema in mice by sGC stimulation Mariola Bednorz
88
Disease Area Cystic fibrosis 009
Randomized, double-blind, controlled pilot study on safety of hypertonic saline as preventive inhalation therapy in newborn patients with cystic fibrosis – follow-up after LOP Mirjam Stahl
89
011
PYOCYANIN FROM P. AERUGINOSA INDUCES DYSBIOSIS AND INFLUENCES ESTABLISHMENT OF CHRONIC INFECTION Sebastien Boutin
90
014
The cystic fibrosis upper and lower airways microbial metagenome of patients with cystic fibrosis or immune deficiency Lutz Wiehlmann
91
019
Non-contrast Enhanced Ventilation and Perfusion Magnetic Resonance Imaging in Early Cystic Fibrosis Lung Disease Mark Wielpütz
92
020
Characterization of genetic modifiers that alter the CFTR-mediated chloride conductance in Cystic Fibrosis epithelia Ellen Raddatz
93
024
Absence of T cells reduces structural lung damage in mice with cystic fibrosis-like lung disease Matthias Hagner
94
027
Altered Airway Macrophage Phenotype and Function in Mice with Mucociliary Clearance Dysfunction Michelle Paulsen
95
031
Pseudomonas aeruginosa microevolution during chronic infection of cystic fibrosis airways Nina Cramer
96
039
Role of free versus membrane-associated neutrophil elastase activity in cystic fibrosis lung disease A. Susanne Dittrich
97
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DZL Annual Meeting 2017 057
Specific IgE production against Staphylococcus aureus, Escherichia coli, Hemophilus influenzae and Pneumococci in cystic fibrosis patients Anna-Maria Dittrich
98
072
Visualization of channel-activating protease (CAP) activity by peptidebased FRET probes Verena Rickert-Zacharias
99
079
Lack of Slc26a9 Cl- channel produced neonatal mortality due to airway mucus plugging Pamela Millar-Büchner
100
089
Altered epithelial ion transport across cultured nasal epithelia Johannna Salomon
101
090
Assay development and high-throughput screen installation for identifying SLC26A9 chloride channel activators Anita Balázs
102
095
Characterization of myeloid cells after transplantation in a mouse model of cystic fibrosis Kerstin Brinkert
103
110
The effect of the anti-inflammatory IL-1R antagonist anakinra in mice with CF-like lung disease and acute Pseudomonas infection André Schütte
104
111
Role of the IL-1 signaling pathway in the development of type 2 airway inflammation in juvenile Scnn1b-Tg mice Ryan Brown
105
113
Genetic deletion of MMP-9 does not reduce chronic neutrophilic inflammation and structural lung damage in mice with cystic fibrosis-like lung disease Claudius Wagner
106
201
Impact of age at diagnosis on development of lung disease in children with cystic fibrosis Eva Steinke
107
218
Bicarbonate (HCO3-) improves mucus properties in β-ENaCoverexpressing mice with CF-like lung disease Mario Pieper
108
226
Characterization of Cystic Fibrosis-like Lung Disease in Mice: Preliminary Results of an intermodal ex vivo micro CT study Willi Wagner
109
229
Development of enzyme-cleavable FRET reporters to quantify MMP-9 activity in lung diseases. Victoria Halls
110
251
Pseudomonas aeruginosa modulates the antiviral response of airway epithelial cells Julia Kantorek
111
263
Magnetic Resonance Imaging Detects Mosaic Perfusion in Early Cystic Fibrosis Lung Disease Patricia Leutz
112
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DZL Annual Meeting 2017 280
Early Development of Paranasal Sinusitis in Cystic Fibrosis Olaf Sommerburg
113
286
Infection of fresh human lung tissue with Pseudomonas aeruginosa Laura Boge
114
292
Epigenetic modulation of NRF2 and BACH1 in Cystic Fibrosis Bronchial Epithelial Cells by Curcumin and Histone Deactylase Inhibitors Rescues Heme Oxygenase-1 expression. Virajith Garapati
115
293
Regulation of Ferroportin in Cystic Fibrosis Bronchial Epithelial Cells: Possible Role of TGF-β1, ENaC and Hypoxia. Gaurav Sarode
116
Disease Area DPLD 003
Origin and fate of the lipofibroblasts during lung development and disease Saverio Bellusci
117
006
Prognosis and Longitudinal Changes of Physical Activity in Idiopathic Pulmonary Fibrosis Thomas Bahmer
118
015
Analysis of Serum Metabolome in Idiopathic Pulmonary Fibrosis Alfonso Carleo
119
017
Improved alveolar dynamics and structure after alveolar epithelial type II cell transplantation in bleomycin induced lung fibrosis Elena Lopez-Rodriguez
120
021
Using Electron Microscopes to look into the Lung Jan Hegermann
121
022
Functional proteomics of cellular mechanosensing mechanisms Anita A. Wasik
122
025
Monocyte immunophenotyping reflects aberrant activation patterns in Interstitial Lung Disease patients Flavia Greiffo
123
028
CDCP1/TGFβ1 cross-talk regulates (myo)fibroblast differentiation Nina Noskovicova
124
032
Proteasome activator 200 (PA200) is dysregulated in fibrotic tissue remodeling Vanessa Welk
125
036
Interim Analysis of the EXCITING-ILD registry (Registry for Exploring Clinical and Epidemiological Characteristics of Interstitial Lung Diseases) Michael Kreuter
126
038
Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo Mareike Lehmann
127
042
CURRENT PRACTICE OF DRUG TREATMENT IN CHILDREN WITH ILD: FIRST INSIGHTS FROM THE CHILD-EU REGISTRY Boglárka Szentes
128
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DZL Annual Meeting 2017 049
High-throughput single cell mRNA sequencing reveals cell identity programs in the murine lung Maximilian Strunz
129
050
Deep proteome profiling reveals common and distinct features of human lung and skin fibrosis Herbert Schiller
130
058
FK506-BINDING PROTEIN 11, A PLASMA CELL-SPECIFIC PROTEIN FOLDING CATALYST, IS INCREASED IN PULMONARY FIBROSIS Stefan Preisendörfer
131
059
ABERRANT COLLAGEN SYNTHESIS AND PROLYL-3HYDROXYLATION VIA INDUCTION OF PROLYL-3-HYDROXYLASE 1 MAY LEAD TO DYSREGULATED HOMEOSTASIS OF THE BRONCHIAL EPITHELIUM IN IDIOPATHIC PULMONARY FIBROSIS Claudia Staab-Weijnitz
132
066
WISP1 mediates IL6-dependent proliferation in healthy and IPF-derived primary human lung fibroblasts Stephan Klee
133
076
Pirfenidone exerts anti-fibrotic effects through inhibition of GLI transcription factors Miroslava Didiasova
134
084
Nuclear miRNA/exosome-mediated transcriptional silencing within the context of TGFB1 signaling and Idiopathic Pulmonary Fibrosis. Indrabahadur Singh
135
086
Development of Pulmonary Fibrosis in Adult Nedd4-2 Deficient Mice Dominik Leitz
136
093
LRP1 controls the transcriptional activity of SMAD3 in idiopathic pulmonary fibrosis Jennifer Schnieder
137
097
The European IPF Registry: Clinical Data from a European Registry for Patients with Idiopathic Pulmonary Fibrosis and other Interstitial Lung Diseases Jasmin Wagner
138
099
Exploring Efficacy and Safety of Oral Pirfenidone for Progressive, non-IPF 139 Lung Fibrosis (RELIEF-Study) Jürgen Behr
105
Antifibrotic effects of Nintedanib and Pirfenidone on alveolar epithelial cells in 2D and 3D culture Lara Buhl
140
107
Bleomycin Induced Pulmonary Fibrosis in Rats and Mice Show Similar Progression in Lung Function, Biochemical and Histological Analyses Dorothee Walter
141
109
Regulatory role of dendritic cells in mice with pulmonary fibrosis Meritxell Tort Tarrés
142
114
PDGF dependant alveolar pathology following postnatal lung injury Prajakta Oak
143
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DZL Annual Meeting 2017 124
Monocyte-centred inflammatory response and consecutive TGF-β signalling as central drivers of neonatal interstitial lung disease Prajakta Oak
144
125
Diagnosis of Bronchopulmonary Dysplasia using novel biomarkers and MR imaging analysis, The AIRR study (attention to infants with respiratory risk) Kai Förster
145
127
A dual role of SFRP1 in lung fibrosis Arunima Seal
146
130
Distinct niches in the extracellular matrices of decellularized 3D lung tissue cultures (3D-LTCs) instruct cellular behavior Gerald Burgstaller
147
133
The role of Notch signaling pathway in the etiology and pathogenesis of idiopathic lung fibrosis Roxana Wasnick
148
136
Dissecting the involvement of the autophagy marker protein, MAP1LC3B in the development of lung fibrosis Vidya sagar Kesireddy
149
155
Comparative Analysis of the Effects of Nintedanib and Pirfenidone on Collagen Synthesis and Maturation in Donor and IPF Fibroblasts Larissa Knüppel
150
163
Regulation and role of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) in Idiopathic Pulmonary Fibrosis. Oleksiy Klymenko
151
174
Species comparison of pro-fibrotic biomarkers in ex vivo lung tissue slices Christina Hesse
152
182
An ex vivo human Model of Idiopathic Pulmonary Fibrosis using Precision Cut Lung Slices Hani Alsafadi
153
188
Cultivating bronchial epithelial cells collected through bronchoscopic microsampling in patients with interstitial lung disease Nicolas Kahn
154
191
Autophagy in lung fibrosis: Exploring the mitophagy pathways Jennifer Arndt
155
198
Stereological characterization of progressive pulmonary remodeling in conditional Nedd4-2 deficient mice Theresa Engelmann
156
204
Modulated deposition of extracellular matrix proteins in a high content imaging assay Michael Gerckens
157
213
CD82 is a novel mediator of fibroblast proliferation Katharina Heinzelmann
158
221
The role of p50ATF6 and sXBP1 in the pathogenesis of lung fibrosis Irina Shalashova
159
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DZL Annual Meeting 2017 230
Surfactant replacement therapy reduces acute lung injury and collapse induration related lung remodeling in the bleomycin model Lilian Steffen
160
236
Quantitative Proteomics Reveals Novel Fibrotic Networks of MyeloidDerived Suppressor Cell and Monocytes in IPF Isis E. Fernandez
161
237
Pro-fibrotic role of Histone Deacetylase 9 isoforms in pulmonary fibrosis Pouya Sarvari
162
239
WNT5A is secreted by extracellular vesicles in pulmonary fibrosis and increases fibroblasts proliferation Aina Martin Medina
163
249
Nanoparticle exposure of persistently herpesvirus-infected cells reactivates latent virus and restores features of an acute virus infection Christine Sattler
164
254
Disruption of the hepcidin/ferroportin regulatory circuitry causes pulmonary iron overload and restrictive lung disease Joana Neves
165
257
Rescue of mutant ABCA3 by small molecular correctors Susanna Kinting
166
274
iPSC-derived Pulmonary Macrophage Transplantation Therapy in a murine model of hereditary PAP Christine Happle
167
277
Classical Transient Receptor Potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis Susanne Fiedler
168
285
3T MRI in infants without medical sedation – a novel and gentle way to achive MRI imaging Andreas Pomschar
169
294
Temporal Subtraction Method for detection of ILD progression on successive thoracic CT Hoen-oh Shin
170
Disease Area Endstage Lung Disease 008
Cytokine expression in humanized mice reflects development of primary graft dysfunction in lung transplant recipients Ann-Kathrin Knöfel
051
Breath VOC patterns of lung transplant recipients with and without chronic 172 lung allograft dysfunction (CLAD) Lucas Küppers
067
Cooperation between nicotinic acetylcholine receptor subunits α7, α9 and α10 is mandatory for cholinergic inhibition of IL-1β release in monocytes Katrin Richter
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171
173
DZL Annual Meeting 2017 073
Lung volumes predict survival in patients with chronic lung allograft dysfunction Nikolaus Kneidinger
174
121
Assessment of Flow Dynamics in a Blood Oxygenator - Implications for Endothelialisation Strategies Daniele Dipresa
175
131
Alternatively activated naive human T cells exhibit strong suppressive capacity in a humanized transplant arteriosclerosis model Linda Pauksch
176
145
STEROID PULSE THERAPY FOR LOSS OF LUNG FUNCTION IN LUNG TRANSPLANT RECIPIENTS AFTER EXCLUSION OF ACUTE CELLULAR REJECTION Dieter Munker
177
170
Generation of disease-specific iPSCs and development of transgenic reporter cell lines for cystic fibrosis disease modelling and drug screening Madline Schubert
178
178
Lung transplant under current therapy with pirfenidon or nintedanib: a case series Gabriela Leuschner
179
180
Stepwise Generation of CFTR-expressing Airway Epithelial Cells from Human Pluripotent Stem Cells Saskia Ulrich
180
186
Investigation of the Effect of Oxidative Stress on the behaviour of ECs Seeded onto ECMO Membranes Kanchan Chauhan
181
187
ORTHOTOPIC LUNG TRANSPLANTATION IN A SINGLE-MISMATCHBASED MOUSE MODEL SHOWS SIGNS OF CHRONIC LUNG ALLOGRAFT DYSFUNCTION ASSOCIATED TO iBALT FORMATION Natalia Smirnova
182
205
Efficient generation of expandable and genetically stable endothelial cells from human pluripotent stem cells in scalable suspension culture Ruth Olmer
183
215
Hypothermic preservation of endothelialised surfaces Hayan Merhej
184
216
Generation of a NKX2.1/p63 knockin human induced pluripotent stem cell reporter line for monitoring the generation of respiratory cells Sandra Baus
185
228
Reduced Preoperative Treg levels Remain Suppressed Following Lung Transplantation and Represent a Risk factor for Increased Acute Rejection Rates Nicole Strobl
186
289
Reliability and validity of the Transplant Evaluation Rating Scale (TERS) in lung transplant candidates Sarah Weusthoff
187
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DZL Annual Meeting 2017
Disease Area Lung Cancer 001
Mutation analysis of circulating DNA to monitor non-small cell lung cancer Steffen Dietz
188
002
p38 MAPK builds a hyaluronan niche to drive lung tumorigenesis Anna Brichkina
189
004
Prevalence of Somatic Mitochondrial Mutations and Spatial Distribution of Mitochondria in Non-Small Cell Lung Cancer Daniel Kazdal
190
005
Widespread epigenetic activation of ΔNp73 in small cell lung cancer causes vulnerability to Tip60-p400 inhibition Andrea Nist
191
016
Regulation of Glycodelin Expression – an Immunomodulatory and Pregnancy associated Protein in NSCLC Rebecca Weber
192
023
The influence of EGF/HGF receptor stoichiometry on therapy resistance in NSCLC cell lines Florian Salopiata
193
033
Chemoresistant NSCLC cells are hypersensitive to metabolic drugs due to mTOR-mediated inhibition of autophagy Michael Wanzel
194
034
Contribution of CD4+ T cell subpopulations to lung carcinogenesis Ylia Salazar
195
035
Prognostic impact of CT-quantified muscle and fat distribution before and after firstline-chemotherapy in lung cancer patients. Johanna Nattenmueller
196
037
Re-education of Tumor-Associated Macrophages by Modulating Histone Deacetylases in Lung Cancer Xiang Zheng
197
040
Comparison of costs and care of lung cancer patients at the end-of-life in Germany depending on the time of survival after diagnosis Julia Walter
198
043
Development of a high-throughput Drosophila model for lung cancer Judith Bossen
199
062
Non-invasive lung cancer diagnosis by detection of GATA6 and NKX2-1 isoforms in exhaled breath condensate. Aditi Mehta
200
065
Targeting the TGFβ pathway in lung cancer: Impact of Pirfenidone on non-small cell lung cancer Sebastian Marwitz
201
074
Study of angiogenesis-related biomarkers in patients with resected lung adenocarcinomas Helen Pasternack
202
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Aberrant DNA methylation in the diagnostics of non-small cell lung cancer Swetlana Scheufele
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Expression of TGFbeta-inducible Myosin-X predicts survival and chemotherapy resistance in squamous cell lung cancer Dmytro Dvornikov
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Immunoprofiling of patients with squamous NSCLC undergoing anti-PD-1 mAb maintenance treatment Jochen Behrends
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Microsimulation model for an introduction of a population-based lung cancer screening program in Germany Ines Aumann
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microRNAs control of cancer cell – macrophage communication: role in tumor progression and metastasis Kati Turkowski
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Reversal of chemoresistance by targeting phosphodiesterase 5/10 mediated signaling in lung cancer Prema Subbarayal
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Erythropoietin Exhibit Angiogenic Potential And The Role of Erythropoietin Receptor In Lung Cancer Cells Xiaoqing Liu
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Investigating the dynamics of tumor–stroma interactions in lung cancer Magdalena Szczygiel
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Progastrin-releasing peptide (ProGRP) as a tool for response evaluation in patients with small-cell lung carcinoma (SCLC) Thomas Muley
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Inhalable nanoparticles for in vivo genome editing mediated by crisprcas9 delivery for undruggable KRAS driven lung cancers Aditi Mehta
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Ultra-early response capturing in the treatment of NSCLC using diffusionweighted MRI: a prospective multicenter study (ERT) Nadja V. Batora
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Interferon Regulatory Factor 9 mediated regulation of lung cancer progression and metastasis David Brunn
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Lipopolysaccharides Induce Radiotherapy Resistance in Non-Small Cell Lung Cancer Cell Lines - the Role of Protein Kinase-Activation Mira Yasemin Gökyildirim
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Tumor infiltration by CD8+-cells correlates with better post-operative survival in stage IA-IIIA non-small cell lung cancer (NSCLC) Julia Stump
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Modulation of tumor growth by bevacizumab and cisplatin within its dynamic human ex vivo microenvironment Sebastian Konzok
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Reprogramming of Tumor Associated Macrophages by modulating Wnt/βcatenin signalling in Lung Cancer Poonam Sarode
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Circulating fibrocytes plays a key role in lung tumor progression by modulating macrophage phenotype, angiogenesis and endothelin system Alina Asafova
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Mechanism of resistance of pemetrexed and vinorelbine in adenocardinoma Fei Tian
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Frequency and clinical relevance of EGFR-mutations and EML4-ALKtranslocations in octagenarians with NSCLC Amanda Tufman
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Clinical and histological factors associated with SUV in PET-CT in patients with adenocarcinoma of the lung Amanda Tufman
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IL-17C mediates the recruitment of tumor-associated neutrophils and lung tumor growth Christoph Beisswenger
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Hypoxia and soluble mediators from H1975 Lung Adenocarcinoma Cells affect Na-Transport in Human Alveolar Epithelium Ezgi Ermis-Kaya
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Heme and iron shape the phenotype of macrophages in the tumor microenvironment of non-small cell lung cancer Milene Costa da Silva
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Progression patterns and prognostic factors of ALK-driven lung cancer treated with targeted therapies and conventional chemotherapy Petros Christopoulos
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Immunohistochemical and molecular characterization of large cell lung cancer Alexander Harms
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Mathematical model for the optimal treatment of lung cancer-associated anemia. Agustin Rodriguez-Gonzalez
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Inflammation-related DNA methylation changes in blood lymphocytes as biomarkers for lung cancer risk Esther Schamschula
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Platforms 094
Alveolar fibroelastosis and bronchiolitis obliterans after lung and stem cell transplantation: morphological and molecular motifs Danny Jonigk
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Implementation of a Data Management Workflow: Data Import, Cohort Management and Data Export for Statistical and Data Mining Analyses Daniel Firnkorn
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DZL Annual Meeting 2017 117
Using the DZL central data warehouse for queries and submitting data Raphael W. Majeed
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Structured reporting of HR-CT examinations in post-lung transplantation patients Nina Hesse
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Case and sample management of the Lung Biobank Heidelberg – one year experience with STARLIMS Marc A. Schneider
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Visualizing perfusion and ventilation signal progression from non-contrast enhanced 2D-proton MRI measurements by use of phase maps David Bondesson
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Biobanks of the DZL-Platform Biobanking Clemens Ruppert
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Development and Implementation of a harmonized DZL-phenotype and specimen classification system Clemens Ruppert
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Regional gas-uptake measurements in the lung using hyperpolarized 129Xe magnetic resonance imaging and spectroscopy Agilo Luitger Kern
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Diagnostic accuracy of MRI for the detection of pulmonary nodules using a chest phantom Olga Solyanik
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Computational imaging for assessing airway remodeling in Drososophila melanogaster Hinnerk Schulz-Hildebrandt
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DZL Biobanking at BREATH Inga Bernemann
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Biobanking at CPC-M Katharina Heinzelmann
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Evaluation of biventricular systolic and diastolic cardiac function in experimental pulmonary arterial hypertension by micro computed tomography Baktybek Kojonazarov
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A systematic comparison of lung ventilation between Fourier Decomposition and Jacobian Determinant method Filip Klimeš
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Depiction of pneumothoraces in a large animal model using x-ray darkfield radiography Katharina Hellbach
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X-Ray Dark-field Imaging to Monitor the Development of Acute Lung Injury in Mice Katharina Hellbach
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3D Lung Ventilation 1H Imaging using a Pseudo 3D Approach or Alternatively a Self-Navigated Sequence in Comparison with 2D Lung Fourier Decomposition
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DZL Annual Meeting 2017 Andreas Voskrebenzev 284
Comparison of real-time dynamic fluorinated gas MRI in free breathing with clinical lung function test in patients with COPD Marcel Gutberlet
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The Image eVAluation Service (IVA) – Software as a Service (SaaS) for the German Center for Lung Research (DZL) Iven Fellhauer
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Disease Area Pneumonia and Acute Lung Injury 018
C-type lectin Mincle recognizes glucosyl-diacylglycerol of Streptococcus pneumoniae and plays a protective role in pneumococcal pneumonia Friederike Behler-Janbeck
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Macrophages render alveolar epithelial cells hypo-responsive to Legionella pneumophila Christine Schulz
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Genome-wide chromatin profiling of Legionella pneumophila-infected human macrophages reveals activation of the pro-bacterial host factor TNFAIP2 Ilona Du Bois
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A Bacterial Signal Peptide Increases Mucociliary Clearance in Explanted Mouse Trachea Alexander Perniß
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Optogenetic mouse strains for studying airway innervation Amir Rafiq
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Modelling alveolar micromechanics in progressing bleomycin-induced lung injury Lars Knudsen
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Deficiency of Immunoproteasome Subunits Increases Alternative Polarization of Alveolar Macrophages Ilona Kammerl
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Protein arginine methylation facilitates LPS-triggered enolase-1 exteriorization Dariusz Zakrzewicz
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Bone morphogenetic protein-2 regulates expression of protease-activated receptor-2 via Smad and PI3K/Akt signaling pathways in lung epithelial cells Tobias Mawassii
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Nasopharyngeal pneumococcal colonization triggers dendritic celldependent adaptive immunity against invasive pneumococcal disease in mice Anne Dommaschk
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Role of TRAIL in the pathogenesis of Bronchopulmonary Dysplasia (BPD) Tayyab Shahzad
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DZL Annual Meeting 2017 154
Cleavage and activation of the influenza virus hemagglutinin by nonhuman primate orthologues of TMPRSS2 Pawel Zmora
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Hypercapnia induces endoplasmic reticulum-associated degradation of the Na,K-ATPase β-subunit Vitalii Kryvenko
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TGF-β reduces megalin cell surface stability by promoting shedding and transcriptional downregulation of the receptor in alveolar epithelial cells Luciana Mazzocchi
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Pulmonary aging exacerbates pro-inflammatory response in acute lung injury Christina Brandenberger
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Chemosensory cholinergic signaling network in the thymic medullary epithelium A. Soultanova
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Hypercapnia induces c-Jun N-terminal kinase activation altering AMPK/Nedd4-2-mediated trafficking of alveolar epithelial ENaC Paulina Gwozdzinska
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A protective role of TRPV4 in ischemia/reperfusion-induced edema formation in the lung Jonas Weber
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PROGRESS – Prospective Observational Study on Hospitalized Community Acquired Pneumonia Peter Ahnert
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Disease Area Pulmonary hypertension 030
Vascular RASSF1A: HIF-1 feed-forward loop triggers hypoxia induced pulmonary hypertension Swati Dabral
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Applicability of the SU5416/hypoxia rat model to study human pulmonary hypertension Lavinia Maegel
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MicroRNAs are regulators of PDGFRβ expression in pulmonary arterial hypertension Astrid Weiss
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The role of mitophagy in development of pulmonary hypertension Alireza Saraji
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The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling Oleg Pak
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Vascular cell-specific epigenome-wide profiling uncovers novel gene regulatory networks in human pulmonary arterial hypertension Prakash Chelladurai
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DZL Annual Meeting 2017 176
Proangiogenic and wound healing molecular and histological fingerprint of chronic thromboembolic pulmonary hypertension Dijana Iloska
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Role and Regulation of Jumonji C domain-containing histone demethylases 1A and 2B in Pulmonary Hypertension Christian Mücke
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Wnt-Signaling Pathway drives right ventricular remodeling Alexandra tretyn
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The response of the pulmonary vasculature to hypoxia in isolated lungs of mice expressing the alternative oxidase (AOX) from Ciona intestinalis Nasim Alebrahimdehkordi
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Histone Deacetylase 7 regulates master transcription factors through modulation of mitochondrial function Elisabetta Gamen
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The role of BDNF in chronic hypoxia-induced pulmonary hypertension Katharina Schäfer
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Gas6/Axl Axis: a Potential Therapeutic Target for Pulmonary Arterial Hypertension Tatyana Novoyatleva
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The physiological significance of GPCRs in the development and treatment of PAH: the role of the P2Y2 receptor Mazen Shihan
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Non-invasive prediction of pulmonary hypertension based on automated 3D volumetry of pulmonary CT angiography. Claudius Melzig
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Epigenetic changes might be induced by exercise training in a hypoxia pulmonary hypertension mouse model Christina Eichstaedt
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Plasma drug-concentrations in patients with pulmonary arterial hypertension on combination treatment Ekkehard Grünig
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Influence of CYP2c gene deletion on hypoxic pulmonary vasoconstriction in isolated, ventilated and perfused mouse lungs Alexandra Erb
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DZL Annual Meeting 2017
Abstract No. 007 Physical activity, airway resistance and small airway dysfunction in severe asthma Thomas Bahmer 1,* , Benajmin Waschki 1, Fee Schatz 2, Christian Herzmann 3, Peter Zabel 3 , Anne-Marie Kirsten 2, Klaus F. Rabe 1, and Henrik Watz 2 1
LungenClinic Grosshansdorf Pulmonary Research Institute at LungenClinic Grosshansdorf 3 Research Center Borstel, Medical Clinic *Presenting author 2
RATIONALE: While physical activity (PA) has been extensively investigated in COPD, curiously there are very limited data for asthma. We aimed to investigate levels of PA in adult asthmatics, and identify associations between PA and lung function. METHODS: We measured PA (steps per day and minutes of moderate activity) in 146 stable asthma patients (severe asthma, n=63; mild-to-moderate asthma, n=83) and 29 healthy controls by accelerometry (SenseWear Armband) for one week. We assessed lung function by spirometry, bodyplethysmography and impulse oscillometry (IOS), indicating airflow limitation (FEV1, PEF), airway resistance (sReff, R5Hz) and small airway dysfunction (FDRabs=R5Hz-R20Hz), respectively. RESULTS: Severe asthma showed significantly lower steps (median 6174 [4822-9277]) and minutes of moderate activity (125 [68-172]) compared to mild-to-moderate asthma and healthy controls (steps reduced by 21% and 31%; moderate activity reduced by 17% and 23%, respectively; p<0.05). In multivariate linear regression analyses adjusting for potential confounders, R5Hz and FDRabs were the best lung functional predictors for daily steps in asthma (p<0.05). CONCLUSIONS: Physical activity is reduced in patients with severe asthma. Impulse oscillometric measures of airway resistance and small airway dysfunction are better predictors of physical activity compared to airflow limitation, questioning the clinical predominance of this parameter in asthma care.
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DZL Annual Meeting 2017
Abstract No. 048 Relative roles of reduced mucus clearance and mucus hypersecretion in the pathogenesis of airway mucus plugging in mice Sandra Christochowitz 1,* , Simone Schmidt 1, Jolanthe Schatterny 1, and Marcus A. Mall 1 1
Translational Lung Research Center Heidelberg, University of Heidelberg *Presenting author
Background: Reduced mucociliary clearance (MCC) and type 2 mediated airway inflammation triggering goblet cell metaplasia (GCM) and mucus hypersecretion are key features of chronic obstructive airways diseases including asthma, cystic fibrosis (CF) and COPD. However, the relative roles of increased mucin/mucus production vs. reduced mucus clearance in the pathogenesis of airway mucus obstruction characteristic of these diseases remain poorly understood. Methods: To determine the relative roles of reduced MCC vs. type 2-mediated mucus hypersecretion, we generated i) βENaC-Tg mice lacking IL-13 (βENaC-Tg/IL-13-/-) to study the impact of airway surface dehydration and impaired MCC in the absence of this key type 2 cytokine; ii) mice with lung-specific overexpression of IL-13 (IL-13-Tg/IL-13-/-) to determine the impact of IL-13 mediated GCM and mucus hypersecretion alone; and iii) βENaC-Tg mice with lung-specific overexpression of IL-13 (βENaC-Tg/IL-13-Tg/IL-13-/-) to study combined effects of reduced MCC and mucus hypersecretion. Results: We found that reduced MCC due to airway surface dehydration is sufficient to induce chronic airway inflammation and airway mucus plugging in juvenile βENaC-Tg/IL-13/- mice. However, expression of Muc5ac, Muc5b and GCM were reduced. In IL-13-Tg/IL-13/- mice, eosinophilic inflammation, Muc5ac and Muc5b transcript levels and GCM were increased, but mucus plugging was reduced compared to βENaC-Tg/IL-13-/- mice. In neonatal βENaC-Tg/IL-13-Tg/IL-13-/- mice, Muc5ac and Muc5b transcript levels and GCM were elevated to similar levels compared to IL-13-Tg/IL-13-/- mice, but all mice died in the first week of life due to severe airway mucus plugging. Conclusions: We demonstrate that reduced MCC is sufficient to produce mucus plugging in the absence of IL-13. However, IL-13 mediated GCM and elevated mucin expression aggravate mucus plugging leading to invariable death in neonatal βENaC-Tg mice with reduced MCC. These results indicate that impaired MCC and mucus hypersecretion act synergistically in the in vivo pathogenesis of airway mucus plugging.
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DZL Annual Meeting 2017
Abstract No. 053 Associations of early-life events, and current environmental and lifestyle factors with lung function in adolescents (GINIplus & LISAplus studies) Agnes Luzak 1,* , Elaine Fuertes 2, Claudia Flexeder 1, Marie Standl 1, Andrea von Berg 3, Dietrich Berdel 3, Sibylle Koletzko 4, Joachim Heinrich 5, Dennis Nowak 6, and Holger Schulz 7
1
Institute of Epidemiology I, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany 2 Institute of Epidemiology I, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany; ISGlobal, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain 3 Research Institute, Department of Pediatrics, Marien-Hospital Wesel, Wesel, Germany 4 Dr von Hauner Children’s Hospital, Ludwig-Maximilians-University of Munich, Munich, Germany 5 Institute of Epidemiology I, Helmholtz Zentrum München - German Research Center for Environmental Health; Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, University Hospital of Munich (LMU), Munich, Germany; Comprehensive Pneumology Center Munich (CPC-M), Member of the German Center for Lung Research 6 Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, University Hospital of Munich (LMU), Munich, Germany; Comprehensive Pneumology Center Munich (CPC-M), Member of the German Center for Lung Research 7 Institute of Epidemiology I, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany; Comprehensive Pneumology Center Munich (CPC-M), Member of the German Center for Lung Research *Presenting author
Background: Throughout lung development and growth, various early-life and current lifestyle factors may influence lung function and increase susceptibility to lung diseases in adulthood. We aimed to identify factors associated with lung function in 15-year olds and to investigate the relative importance of early-life events, current environmental/lifestyle factors, and allergic diseases. Methods: Data from 1326 participants from the German GINIplus and LISAplus cohorts with valid spirometric indices (forced expiratory volume in 1s (FEV1), forced vital capacity (FVC) and flow rates) were analysed. Best subset selection was performed for linear regression models to investigate associations of early-life (e.g. parental atopy and education, birthweight, breastfeeding, as well as peak weight velocity (PWV) and pulmonary infections, within the first 2 and 3 years of life, respectively), current lifestyle/environmental factors (e.g. air pollution, active smoking, serum vitamin D concentration, body mass index (BMI)), and allergic diseases (e.g. rhinitis, asthma) with spirometric parameters. For each parameter, a thousand replication analyses were performed in randomly selected subpopulations (N=884) to assess which factors remained in >70% of the replication models. Results: Of the early-life factors considered, early lung infections and higher PWV were negatively associated with airway function. Negative associations with FEV1/FVC were found for environmental/lifestyle factors at 15 years, such as indoor secondhand smoke exposure, serum vitamin D concentration, BMI, and for asthma. Only BMI and vitamin D
DZL Annual Meeting 2017 concentration were positively associated with FEV1 and FVC. Sex, height and BMI captured the majority of the explained variance, while early-life factors contributed less (median<4.5%). Conclusion: In addition to the well-established influence of sex, height and asthma status on lung function, our results indicate that current BMI, as well as PWV and pulmonary infections in young age were associated with lung function at age 15 years. Our findings highlight factors that studies on adolescent lung health should consider.
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DZL Annual Meeting 2017
Abstract No. 056 6q12 and 11p14 variants are associated with postnatal exhaled nitric oxide and respiratory symptoms Oliver Fuchs 1,* , Olga Gorlanova 2, Philipp Latzin 3, Anne Schmidt 2, Maximilian Schieck 4, Antoaneta A Toncheva 5, Sven Michel 5, Vincent D Gaertner 5, Michael Kabesch 5, and Urs Frey 2 1
Dr von Hauner Children’s Hospital, Ludwig Maximilians University, Munich, Germany, and Comprehensive Pneumology Center Munich (CPC-M), Germany; Member of the German Center for Lung Research (DZL) 2 University Children’s Hospital (UKBB), University of Basel, Basel, Switzerland 3 Inselspital, University of Bern, Bern, Switzerland 4 Institute of Human Genetics, Hannover, Germany 5 University Children’s Hospital Regensburg (KUNO), Regensburg, Germany *Presenting author
Rationale: Exhaled nitric oxide (eNO) is a biomarker of airway inflammation and seems to precede respiratory symptoms, such as asthma in childhood. Identifying genetic determinants of postnatal eNO may aid in unraveling the role of eNO in epithelial function or airway inflammation and disease. We aimed at identifying genetic determinants of early postnatal eNO and subsequent respiratory symptoms during the first year of life. Methods: Within a population-based birth cohort, eNO was measured in healthy term infants aged 5 weeks during quiet tidal breathing in unsedated sleep. We assessed associations of single-nucleotide polymorphisms with eNO in a genome-wide association study, and subsequent symptoms of lower respiratory tract infections during the first year of life; and asked if this was modified by prenatal and early-life environmental factors. Results: We identified so far unknown determinants of infant eNO: rs208515 (p=3.3 x 10-8) located at 6q12, probably acting in “trans”, and explaining 10.3% of eNO variance; and furthermore rs1441519 (p=1.6 x 10-6) at 11p14, potentially impacting NO synthase 3 (NOS3) expression as shown by in-vitro functional analyses. Moreover, the 6q12 locus was inversely associated with subsequent respiratory symptoms (p<0.05) and time to recovery after first respiratory symptoms during the first year of life (p<0.05). Conclusions: The identification of novel genetic determinants of infant eNO may implicate that postnatal eNO metabolism in healthy infants prior to first viral infections and sensitization is related to mechanisms other than those associated with asthma, atopy or increased risk thereof later in life.
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DZL Annual Meeting 2017
Abstract No. 064 Characterization of peanut defensins as potential candidates for in-vitro diagnostic Skadi Kull 1,* , Christian Schwager 1, Marisa Böttger 1, Jochen Behrends 2, and Uta Jappe 3 1
Division of Clinical and Molecular Allergology, Research Center Borstel, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany 2 Core Facility Fluorescence Cytometry, Research Center Borstel, Borstel, Germany 3 Division of Clinical and Molecular Allergology, Research Center Borstel, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany and Interdisciplinary Allergy Outpatient Clinic, MK III, University of Lübeck, Lübeck, Germany *Presenting author
Peanut allergy is the most common cause of life-threating anaphylaxis in children and adolescents in Germany. Besides accidental ingestion of peanuts, inhalation of peanut particles is a primary cause for severe allergic reactions, including allergic asthma. Recently, we identified two novel peanut allergens, the defensins, named Arah12 and Arah13. Interestingly, the only other known defensin allergen, Glym2 from soy, has been associated with allergic asthma. Therefore, we aimed to investigate a potential correlation between defensin sensitization, symptom severity and organ specificity of peanut-allergic patients. Furthermore, we analysed the impact of thermal processing of peanuts on the IgE reactivity of peanut defensins as food processing (e.g. roasting) has been shown to increase the allergenicity of diverse peanut allergens. Peanut defensins of raw and in-shell roasted peanuts were isolated by lipophilic extraction and subsequent chromatographic separation techniques. The isolated proteins were verified by mass spectrometry and N-terminal sequencing. Sera of peanut-allergic patients with severe symptoms, sensitized but peanut-tolerant patients and non-allergic individuals were screened by immunoblot analysis for IgE binding to defensins. Additionally, the ability of defensins to trigger allergic reactions was assessed by basophil activation test. The majority of peanut-allergic patients sensitized to defensins displayed more severe allergic symptoms. Remarkably, defensins from in-shell roasted peanuts had a higher IgE binding capacity in western blot analysis and led to an increased basophil activation compared to peanut defensins from raw peanuts. In the course of defensin purification, we encountered a third and so far novel peanut defensin which also showed an IgE binding in immunoblot and in vitro stimulation of basophils. Our data suggests that IgE binding to peanut defensins correlates with the severity of allergic symptoms. Hence, the addition of peanut defensins to the established routine diagnostic might help to identify patients who are potentially at risk for anaphylactic reactions to peanuts.
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Abstract No. 068 RV-infection modifies DNA-methylation in nasal airway epithelium cells in asthmatics and non-asthmatics children Martin Pech 1,* , Markus Weckmann 1, Femke-Anouska Heinsen 2, Andre Franke 2, Brain G. Oliver 3, Gesine Hansen 4, Erika v. Mutius 5, and Matthias Kopp 1 1
University Medical Center Schleswig-Holstein Lübeck Christian-Albrechts-University of Kiel 3 Woolcock Institute of Medical Research Sydney 4 Hannover Medical School 5 Ludwig-Maximilians-University Munich *Presenting author 2
In children, asthma is one of the most common chronic diseases with increasing prevalence. Human rhinovirus infection (HRVI) plays an important role in asthma exacerbations and is thought to be involved in the asthma development during childhood. McErlean et al. reported that HRVI changes the epigenetic gene regulatory mechanism of DNA methylation in nasal epithelium cells. We hypothesized that HRVI in the epithelium initiate a different methylation pattern in asthmatic and non-asthmatic children. The analyzed nasal epithelium cells were collected by a nasal brushing of asthmatic (AST) and non- asthmatic (non-AST) children during a KIRA (Kinder-Register Asthma) cohort visit. The cells were cultured up to passage 2 in BEGM (Lonza) and infected for 48 h with RV-16 using MOI 10. The genome wide DNA-methylation was analyzed using the HumanMethylation450 BeadChip Kit (Illumina). Flow (Partek), Partek Genomics Suite (Partek) and Prism (graphPad) were used for statistical data analysis. The overlap of viral modified DNA methylation sites in AST and non-AST is with 211 CpGs (p<0.05) very limited. In the AST we detected 6922 (p<0.05) viral altered CpGs which were not modified after HRVI in non-AST. Furthermore 6569 CpGs (p<0.05) showed a viral induced DNA methylation modification in non-AST but not in AST subjects. Vanin-1 showed a decreased DNA methylation after HRVI specifically in AST (p =0.012 ). The in-vitro results show a specific effect of HRVI on the genome wide DNA methylation in nasal epithelium. We confirmed the decreased DNA methylation of Vanin-1 as reported by Xiao et al., involved in corticosteroid therapy success. This data suggests that HRVI induced methylation may influence the asthma pathogenesis respectively the treatment.
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DZL Annual Meeting 2017
Abstract No. 069 Systemic and Bronchial Inflammation entail regulatory compartments of regulatory B and T cells in grass-pollen allergic rhinitis and asthma patients Ulrich Zissler 1,* , Sandra Rothkirch 1, Larissa Lewitan 1, Constanze Jakwerth 1, Carsten Schmidt-Weber 1, and Adam Chaker 2 1
Center of Allergy & Environment (ZAUM), Technical University and Helmholtz Center Munich 2 Department of Otorhinolaryngology and Head and Neck Surgery, Klinikum rechts der Isar, Technical University Munich *Presenting author
Background: Allergic airway inflammation is characterized by activation and expansion of Th2 cells, IgE production and eosinophilia. Frequently, this process is related to an inappropriate T cell response to environmental allergens, whereas other T cell compartments such as Th17 and Th22 cells may also be involved. Regulatory T cells (Tregs) are T cells that suppress potentially harmful immune responses. Recent evidence shows that certain B-cell subsets can also inhibit T-cell mediated immune responses. Like regulatory Tregs, these regulatory B cells (Bregs) appear to comprise several subpopulations. Methods: Induced sputum was collected of 40 symptomatic pollen allergic (n=19 rhinitis and n= 21 allergic asthma) and 12 non-allergic subjects during in and out of the grass pollen season. Phenotypic detection of T and B cells was performed by multicolour flow cytometry. Results: Allergic patients showed an increased frequency of allergen-specific CD5+CD27CD38+ effector T-cells which were terminally differentiated and activated. Furthermore, rhinitis and asthma patients showed higher frequencies of CD25+ Tregs in and out of season than controls. Out of the season, allergic patients showed significantly increased levels of CD19+/CD27+ memory B-cells compared to pollen season. In addition, we were able to identify CD19+CD5+ B-cells in induced sputa. We were able to confirm substantial frequencies of different regulatory B- cell phenotypes such as CD19+CD5+CD1d+, CD19+CD24+CD38+, and CD19+CD27+CD24+. Interestingly, allergic patients showed increased frequencies of CD19+CD5+CD1d+ during rather than off season. Moreover, allergic subjects showed an overall higher proportion of CD19+CD27+CD24+ B-cells. Conclusion: This study is an essential step toward a better understanding of allergic processes in the lower airways in vivo. Allergic patients showed increased pulmonary infiltrate of memory B cells, confirming the presence of regulatory T and B cells in induced sputum samples. Nevertheless, future studies need to address the functional role and relevance of Breg subsets in allergic airway disease.
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DZL Annual Meeting 2017
Abstract No. 071 Regulatory B cells and Shift in Th17 cell populations raise in Allergen-specific Immunotherapy Ulrich Zissler 1,* , Constanze Jakwerth 1, Ferdinand Gürth 1, Zuzana Hajdu 2, Carsten Schmidt-Weber 1, and Adam Chaker 2 1
Center of Allergy & Environment (ZAUM), Technical University and Helmholtz Center Munich 2 Department of Otorhinolaryngology and Head and Neck Surgery, Klinikum rechts der Isar, Technical University Munich *Presenting author
Background: Regulatory immune cells are under current investigation in several autoimmune and allergic disease entities. IL-10 produced by B cells is important for controlling inflammation to maintain regulatory capacities of T cells while curtailing Th1 and Th17 differentiation. Recently, it has been shown that regulatory T cells (Tregs) can not only differentiate into effector Th17 cells via an intermediate subset expressing FoxP3 and IL-17 simultaneously, but also, that Th17 cells carry the ability to transdifferentiate back into Tregs. Methods: In this study, we isolated peripheral blood monocytes (PBMCs) from 27 allergic patients at several time points during an standard up-dosing period of grass-pollen specific immunotherapy (SIT) and analyzed immune cell populations using flow cytometry. We prospectively monitored these patients over three years and collected further samples in and out of respective grass pollen season. Results: Systemic T- and B-cell subsets were compared at baseline and six hours after the last maintenance top dose of SIT. We found a significant increase in IL-10-producing Bregs shortly after the last maintenance injection. On top, the IL-10+/TNF-α+ ratio in B cells was significantly increased, which has been postulated to indicate their regulatory function even stronger than IL-10 production alone. The increased Breg population in the blood coincided with a significant decrease of effector Th17 cells and, notably, with a decrease in the IL-17expressing CD4+FoxP3+ Treg population. Conclusion: We propose that the SIT-triggered induction of Bregs leads to a shift of Th17 cells towards a rather regulatory phenotype, as not only the Th17 population, but also the intermediate IL-17+FoxP3+ Treg subset was significantly decreased shortly after the last maintenance injection. In conclusion, the frequency of Th17 cells and IL-17-producing regulatory T cells in the peripheral blood may represent early biomarkers of immunotherapy efficacy.
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Abstract No. 078 Innate immune network in asthma: studies on dendritic cell interaction with airway epithelium Karina Stein 1,* , André Jenckel 1, Uta Jappe 1, and Holger Heine 1 1
Research Center Borstel *Presenting author
Background: The interaction of various types of innate and adaptive immune cells during an ongoing allergic asthma is pivotal for the outcome and the resolve of the disease. Dendritic cells (DCs) are key regulators of this network, however, the airway epithelium is the first entry site for airborne allergens and signals derived from these cells can affect DC function decisively. Methods: A 3D co-culture model involving the airway epithelium cell line Calu-3, cultured under air-liquid interface (ALI) conditions, and monocyte-derived DC was used to enable studies on the interaction mode of these cell types when challenged with airborne allergens. Results: Stimulation experiments of Calu-3 cells with house dust mite extract (HDM) or the major allergen of Betula pendula Bet v1 resulted in minor activation of these epithelial cells whilst allergen-treatment of DC led to diverging induction of immune stimulatory cytokines such as IL-6, CXCL8 and CCL22. The ALI co-culture system further demonstrated that the cytokine induction is markedly changed following allergen challenge. This was already observed under conditions where direct epithelium/DC contact was prevented but it was at maximum when contact was enabled, indicating the importance of soluble factors together with direct cell interaction. Further, allergen-stimulation of ALI co-cultures under the polarizing influence of the Th1-cytokine IFN-γ or the Th2-cytokines IL-13 and IL-4 resulted in opposing regulation of asthma-associated molecules, e.g. IL-1 superfamily members. Conclusion: The establishment of cell culture systems that enable allergen challenge studies on DC/airway epithelial cell co-cultures reveals the complexity and importance of the cellular interplay in this context and will provide further insights into the nature of the DC-epithelium interaction.
Submitted as: Presentation 265 words of 300 possible words
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Abstract No. 081 Neutrophil-epithelial cell interactions in allergic asthma Brigiite Kasper 1,* , Reza Akbarzadeh Najar 1, Xinhua Yu 1, and Frank Petersen 1 1
Forschungszentrum Borstel *Presenting author
Background: Neutrophil activation, an essential branch of the first line in host defense, represents an essential driver of the pathogenesis of many chronic disorders. Surprisingly, although neutrophils are observed in asthma especially during late phase reaction and in uncontrolled disease, their basic pathological role remains largely unclear. In an experimental asthma model we found that beside airway hyperreactivity (AHR), goblet cell hyperplasia and mucus production were significantly reduced in the absence of neutrophils indicating a pathogenic impact of these cells on the airway epithelium. Objective: We sought to define pathomechanisms of neutrophil-epithelial cell interactions in allergic asthma. Methods: Neutrophil-epithelial cell interactions were investigated by using human neutrophils, human epithelial cell lines, and human primary epithelial cells isolated from bronchial tissue. By the use of Real Time Cell Analyzer (xCELLigence system), flow cytometry, and real-time PCR we analyzed the effect of neutrophils on epithelial cell barrier integrity, survival, and gene expression, and neutrophil elastase activity was analyzed by FRET analysis. Results: Neutrophils provoke a dose-dependent loss of epithelial cell integrity which became visible in a reduction of cell adhesion (detachment) and changes in epithelial cell morphology. These effects were dependent on cell-cell contact, indicating the involvement of neutrophil proteases. Although elastase activity in supernatants from neutrophil-epithelial cell co-cultures was only slightly increased, an increased elastase activity could be detected at neutrophil-epithelial cell contacts by FRET-analysis of fluorescence-conjugated elastase substrates. Interestingly, epithelial cell survival was not affected by neutrophils. Furthermore, gene expression in epithelial cells was differentially regulated by neutrophils dependent from whether epithelial cells were grown in submerged or air-liquid interface (ALI) culture. Conclusion: These results provide evidence for a dual role for neutrophils in asthma identifying them as essential promotors of the disease pathology and regulators of epithelial cell gene expression.
Submitted as: Presentation 291 words of 300 possible words
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Abstract No. 083 Mast cells and mast cell chymase regulate airway hyperreactivity in chronic experimental asthma Marjan Ahmadi 1, Simon Hogan 2, Xinhua Yu 1, Brigitte Kasper 1, and Frank Petersen 1,* 1
Forschungszentrum Borstel Cincinnati Children's Hospital *Presenting author 2
Mast cells (MC) have been shown to be essentially involved in allergic diseases by releasing a variety of mediators upon activation. Among these, human MC chymase and its murine homologue Mcpt4 have been proven to drive several inflammatory processes like tissue damage or proteolytical processing of cytokines. Surprisingly, recent studies suggest a potent protective role of Mcpt4 in a model of acute asthma. In this study, we examined the role of MC and Mcpt4 in a chronic model of experimental allergic asthma. Mice of the strains B6, B6-Mcpt4-/-, MC-deficient KitW-sh as well as KitW-sh reconstituted either with wild type MC or Mcpt4-/--MC were sensitized with OVA alum-free i.p. followed by 8 fold weekly repeated OVA challenge i.t. up to week 10. In this model, airway hyperreactivity (AHR) to metacholine was unexpectedly higher in MC-deficient KitW-sh mice than in the corresponding WT controls. This effect could be reverted to control levels by reconstitution of KitW-sh mice with WT MC indicating a protective role of MC in our chronic model. Moreover, Mcpt4-/- mice as well as KitW-sh mice reconstituted with Mcpt4-/--MC were found to be resistant to an induction of AHR in experimental asthma demonstrating the essential role of Mcpt4 in the disease. Further analysis revealed that goblet cell hyperplasia and mucus production was increased in all sensitized groups irrespective whether MC or Mcpt4 were present or not. Furthermore, Th2 cytokine production of lung cells did not differ between MC-sufficient and -deficient groups. Our data provide evidence that MC selectively regulate AHR by two antagonistic pathways. While MC-derived Mcpt4 represents a specific promoter of AHR in chronic experimental asthma, a corresponding protective mechanism remains to be elucidated. Targeting MC chymase in chronic asthma could be a promising new strategy to reduce airway limitations and to prevent asthma exacerbation.
Submitted as: Presentation 296 words of 300 possible words
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Abstract No. 088 The influence of the microbiome on asthma susceptibility - project outline of a doctoral thesis Joni Valeska Lund 1,* , Sabine Bartel 1, Sebastian Reuter 1, and Susanne Krauss-Etschmann 1
1
Research Center Borstel *Presenting author
Background: Only a few years ago new analytical methods as 16S rRNA analysis and metagenomics became available. Since then they have been used intensively to investigate the function of the microbiome in health and disease. The functionality and diversity of microorganism colonizing the human body is determined by host genetics and environmental influences. Genetic factors are also already known to increase the susceptibility for asthma [Toncheva et al., 2015], and it is speculated that the microbiome composition could also influence asthma development or even prevent it. Objective: The overall aim of this study is to identify bacterial species within the microbiome that have the potential to promote health irrespective of the host’s genetic makeup. Concept: Initially, four different mouse strains with distinct susceptibility for allergic airway inflammation (AAI) will be analysed for their microbial composition using 16S rRNA analysis directly after delivery. To assess whether microbial differences between the strains remain stable after housing in our facilities, mice will be retested one month later. Next, the reported susceptibility for AAI in the different strains (Shinagawa et al., 2003) will be confirmed by inducing the condition with a house dust mite treatment (HDM) (Dermatophagoides pteronyssinus, Nr. 218234; Greer, Lenoir, USA). The severity of the developing AAI will be analysed by lung function, histology and immune status. Finally, AAI will be assessed in germ-free mice, colonized with microbiota of selected highly or low susceptible strains, and in offspring of these highly or low susceptible strains after cross-fostering with low or respectively highly susceptible mothers. Expectations: This project aims to gain insight into the link between asthma susceptibility and a certain microbiome composition independent of the genetic background. Thus, this project creates the prerequisites to identify ‘AAI protective’ bacterial species that could be further investigated for the prevention of allergic airway diseases.
Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 102 Glycolipids of house dust mites – studying the impact of allergen-lipid association Nestor Gonzalez Roldan 1,* , Uta Jappe 2, and Katarzyna Duda 1 1
Research Center Borstel, DZL Junior Group Allergobiochemistry Research Center Borstel, Interdisciplinary Allergy Outpatient Clinic, MK III, University of Lübeck *Presenting author 2
Introduction: House dust mites (HDM) are important inductors of allergic asthma. HDM and its fecal pellets consist of glycoproteins and a wide range of chemically distinct substances, including lipids, which potentially initiate and/or modulate allergic reactions. Lipids can associate to allergens and protect them from degradation and enhance their cellular uptake. Several major allergens from different sources bear hydrophobic domains, thus enabling them to interact with lipids. Methods: In order to examine the role of HDM-derived lipids on the initiation of immune responses, lipid-containing molecules were obtained from Dermatophagoides pteronyssinus (D p) bodies (Greer) and from D p culture medium (Allergopharma) utilizing chloroform/methanol/water extraction. The organic compounds were further fractionated on silica gel and by HPLC; and chemically characterized by HPTLC, GC and GC/MS. The total organic fraction and the subsequent lipid-containing fractions were screened for biological activity on mouse (bone marrow-derived mast and dendritic cells) and human (PBMC) systems by flow cytometry using a drug-discovery approach. Results: With regard to polarity and chemical composition, the analysis of HDM bodies and their feces revealed the presence of a broad spectrum of lipid-containing molecules. Our high throughput flow cytometry analysis, enable us to identify various lipid compounds able to induce and/or enhance mast cell degranulation, as well as dendritic cell maturation. Additionally, we developed a multiparametric panel for human PBMC, which allowed the detection and analysis of innate-like lipid-reactive lymphocytes (NKT and subpopulations of gamma/delta cells) recognizing HDM-derived glycolipids. Discussion: The full chemical characterization of HDM-derived lipids, and the further analysis of the effector responses (cytokine release, co-stimulatory signals) induced on innate lipid-reactive immune cells, may provide the rational base to understand the structure-function relationship behind the influence of lipids (as adjuvants) on the modulation of allergic responses towards otherwise (innocuous) allergens.
Submitted as: Presentation 291 words of 300 possible words
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Abstract No. 106 A possible role for microRNA-17 and -21 in regulatory T cell (Treg) function in allergic asthma Sabine Bartel 1,* , Katja Landgraf-Rauf 2, Andreas Böck 2, Bianca Schaub 2, and Susanne Krauss-Etschmann 1 1
Research Center Borstel, Member of the German Center for Lung Research (DZL) LMU Munich, Children's Hospital, Member of the German Center for Lung Research (DZL) *Presenting author 2
Background: The immunological balance is maintained by regulatory T cells (Tregs) and needs to be tightly controlled. Recently, we observed that microRNA (miR)-17, miR-146a and miR-21 are increased in murine experimental asthma. Transfection of primary murine CD4+ T cells with miR-17 and miR-21 reduced Treg differentiation and function in vitro. Therefore, we aimed to investigate if miR-17 and miR-21 are relevant for Treg function in paediatric allergic asthma. Methods: Pulmonary blood mononuclear cells (PBMC) were isolated from 7 healthy control children (HC), 7 with allergic asthma (AA), and 7 with non-allergic asthma (NA) (Clinical Asthma Research Association cohort study (Raedler et al., JACI, 2015)) and stimulated with lipid A (LpA), anti-CD3/CD28 or none. Frequencies of Treg in blood were quantified via flow cytometry. microRNA levels in PBMCs were assessed via qRT-PCR (Exiqon, Vedbaek, Denmark; normalized to snoRRA66). Results: miR-17 and miR-21 levels were significantly increased in PBMCs after stimulation with anti-CD3/CD28, but did not differ significantly among HCs, NA or AA overall. NA showed increased expression of miR-146a compared to HC after anti-CD3/CD28 (FC=2.14, p=0.0263) and LpA (FC=2.68; p=0.0263). However, we observed a positive correlation of PBMC miR-17 (r=-0.519, p=0.023), miR-21 (r=-0.519, p=0.023) and miR-146a (r=-0.761; p<0.001) expression with percentages of CD4+CD25+Foxp3+CD49d- T cells, while they were negatively correlated with the frequency of CD4+IL17+ T cells (miR-17: r=0.555, p=0.014; miR-21: r=0.621, p= 0.005, miR-146a: r=0.565, p=0.012). Conclusion: miRNA levels in PBMCs seem to be influenced by different stimulations and their expression correlates with distinct cell types in the blood. Thus, we speculate that a dysregulation of miRNAs during the development of allergic asthma may contribute to the development of chronic airway inflammation. Next, whole miRNA profiles of PBMCs from AA vs NA vs HC will be analyzed and dysregulated miRNAs will be functionally validated for their role in Tregs.
Submitted as: Presentation 303 words of 300 possible words Page 14 of 286
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Abstract No. 115 Looking for the anti-inflammatory mechanism of the probiotic compound DTryptophan – does it influence T cell differentiation? Gregor Jatzlauk 1,* , Sabine Bartel 1, Sebastian Reuter 1, and Susanne Krauss-Etschmann 2 1
Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany, Member of the Airway Research Center North (ARCN), German Center for Lung Research (DZL) 2 Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany, Member of the Airway Research Center North (ARCN), German Center for Lung Research (DZL) and Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany *Presenting author
Introduction: Maternal or infant supplementation with probiotic bacteria has been proposed for the prevention of allergic diseases, such as asthma. However, clinical studies have shown contradictory findings, which is possibly explained by the individual and highly complex interaction between probiotics and the host. To overcome this complexity, we are investigating the use of isolated probiotic compounds, instead of living bacteria, for potential disease prevention. Recently, we showed that the probiotic compound D-Tryptophan (DTrp) ameliorates allergic airway inflammation (AAI) in mice (Kepert et al, JACI, 2016). However, the underlying mechanisms remained unclear. Goal: We now aim to investigate which particular (immune) cells are sensitive for D-Trp, starting with the question whether D-Trp affects T-cell differentiations in vitro. Methods: Isolated naïve CD4+ T-cells from murine spleen were stimulated with IL-2 and anti-CD3/CD28. T-cells were further differentiated into subtypes with the following cytokine mixtures: Th0 (-), Th1 (Il-12, anti-Il-4), Th2 (Il-4, anti-Ifnγ), and iTreg (Tgf-β, all Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured for 6 days in presence or absence of 10 µM or 50 µM D-Trp (Sigma-Aldrich, Steinheim, Germany). Differentiation was examined by qRT-PCR, flow cytometry and cytometric bead array. Results: D-Trp slightly decreased differentiation into Th2-cells, as indicated by lowered protein levels of Il-5 and Il-13, and down-regulated mRNA levels of Il-4 and Il-5. D-Trp treatment also resulted in minimal increase of Foxp3+ Treg cells. In these cells Il-10 mRNA levels were elevated. A consistent effect of D-Trp on Th1- and Th0-cells was not observed. Discussion: Our results indicate that D-Trp slightly affects T-cell differentiation by suppressing Th2 and promoting Treg differentiation. However, this effect was not as pronounced as indicated by the amelioration of AAI observed in vivo. Therefore, we will further investigate the influence of D-Trp on the interaction between dendritic cells and Tcells in a co-culture system.
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Abstract No. 119 Effects of prenatal cigarette smoke on thymic T cell composition and lung function in offspring Barbara Hammer 1,* , Martin Wolff 1, Sabine Bartel 1, Sebastian Reuter 1, and Susanne Krauss-Etschmann 1 1
Research Center Borstel *Presenting author
Introduction: Epidemiological studies demonstrate that in utero cigarette smoke exposure negatively affects lung development, thereby increasing susceptibility for asthma and COPD (Svanes, Thorax 2010). Transgenerational animal models could help to understand the mechanisms how disease risks are shaped. T cells are continually generated in the thymus. A disturbed T cell development could predispose to respiratory diseases. Objective: To analyze the composition of thymic T cells in prenatally smoke-exposed mice. Methods: Female C57BL/6 mice were exposed to mainstream smoke produced from research cigarettes 3R4F (CTRP, University of Kentucky) by using the inexpose exposure system (SCIREQ, Canada). Before (4 days) and after onset of pregnancy until delivery, female mice were exposed to cigarette smoke for one hour daily. Thymic T cells were analyzed by flow cytometry on postnatal day (PND) 21. Lung function was assessed by invasive measurement using a FlexiVent system (SCIREQ, Canada). Results: In utero smoke-exposed offspring (PND21) had a normal lung function both at baseline measurements and after methacholine provocation. However, thymic CD3hi T cells populations were increased in in utero smoke-exposed offspring compared to air control (females: p=0.01; males: p=0.0003; n=15/group, Mann-Whitney test). This affected CD4+ T cells (female: p<0.0001; male: p=0.003; n=15/group, Mann-Whitney test) but not CD8+ T cells. Regulatory T cells (Treg) were reduced in male offspring (p=0.02; n=15/group, MannWhitney test). Conclusion: Despite normal lung function, our data reveal altered T cell composition in the thymus in a model of mild maternal smoking. This could be a first indication of disturbed T cell development in in utero smoke-exposed offspring. Further, this could lead to altered immune responses and a higher asthma susceptibility observed in children exposed in utero to cigarette smoke. Functional analyses of these Treg are required.
Submitted as: Presentation 282 words of 300 possible words
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Abstract No. 126 Clinical and data-driven phenotype definitions in the pediatric arm of the DZL All Age Asthma Cohort (ALLIANCE) Alexander Hose 1, Oliver Fuchs 1, Erika von Mutius 1, Gesine Hansen 2, Matthias Kopp 3, Markus Ege 1,* , and the ALLIANCE Study Group 4 1
Dr. von Hauner Children's Hospital University Hospital of the Medizinische Hochschule Hannover (MHH) 3 University Hospital Schleswig Holstein Campus Luebeck (UKSH) 4 ARCN, BREATH, CPC-M *Presenting author 2
Rationale: In the ALLIANCE study clinical phenotypes of asthma, wheeze, and atopy have previously been defined. The aim of this study was to compare clinical phenotypes with data-driven phenotype definitions to corroborate or amend them for subsequent analyses. Methods: Individuals aged 3 to 18 years from the ALLIANCE study were grouped by a latent class analysis (LCA) based on clinical symptoms at recruitment reflecting severity of asthma, e.g. by frequency of asthma symptoms or an asthma control score according to GINA. The resulting classes were related to lung function parameters and atopic sensitization as determined by measurement of sIgE. Results: Of all 146 individuals with available sIgE values at recruitment and lung function parameters the majority of individuals (86 %) belonged to the asthma phenotype and served as the basis for subsequent analyses. The LCA for clinical symptoms revealed the best model fit for the 3-class solution as determined by the Bayesian information criterion. Classes 2 and 3 were characterized by atopic symptoms such as itchy eyes or a runny nose. Class 3 differed from the two other classes by an enrichment of individuals with high symptom scores for asthma severity and asthma control. Class 1 was neither atopic nor affected by severe asthma symptoms. Levels of total sIgE values did not differ between the 2 clinically atopic classes but strongly between them and the non-atopic class (geometric means ratio = 1.7, p = 0.0002). With respect to lung function parameters, the non-atopic class ranged in between the two atopic classes, thereby explaining the absent association between atopy and lung function in the entire asthma phenotype. Conclusions: For subsequent laboratory analyses, the differentiation by the LCA of clinical symptoms might suitably complement the dichotomy of atopic and non-atopic asthma patients.
Submitted as: Presentation 290 words of 300 possible words
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Abstract No. 128 Development of atopic sensitization in Finnish and Estonian children – a latent class analysis in a multicenter cohort Markus Ege 1,* , Felicitas Schmidt 1, Erika von Mutius 1, Mikael Knip 2, and the DIABIMMUNE study group 3 1
Dr. von Hauner Children's Hospital University of Helsinki
2 3
*Presenting author
Background: The prevalence of atopy is associated with a Westernized lifestyle as illustrated by the Karelia studies, which compared two neighboring regions with different socioeconomic backgrounds. However, the distribution of the underlying atopy phenotypes and their association with environmental factors and disease development remain unclear. Objective: To define phenotypes of atopic sensitization in early childhood and to examine their association with allergic diseases in Finland and Estonia Methods: The analysis included 1603 Finnish and 1657 Estonian children from the DIABIMMUNE multicenter young children cohort. Specific IgE levels were measured at age 3, 4 and 5 respectively, and categorized into three CAP classes. Latent Class Analysis (LCA) was performed with the statistic software package poLCA in R. Results: Both populations differed in terms of socioeconomic status, environmental determinants, and prevalence of allergic diseases. Nevertheless, we found similar latent classes (LC) in both populations: an unsensitized LC, a food LC, two inhalant LCs, differentiating between seasonal and perennial aero-allergens, and a severe atopy class. The latter was characterized by high total and sIgE levels and particularly strong associations with wheeze (odds ratio 5.64 [3.07-10.52] and 4.56 [2.35-8.52]), allergic rhinitis (22.4 [11.67-44.54] and 13.97 [7.33-26.4]) and atopic eczema (9.39 [4.9-19.3] and 9.5 [5.217.5], for Finland and Estonia, respectively). The seasonal inhalant class was additionally related to genetic risk of type 1 diabetes in Estonia as determined by HLA-DQ genotypes (2.5 [1.2-5.1]). Conclusion: LCA revealed similar patterns of atopic sensitization in countries with different environmental determinants. Atopic disease was related to a different type of atopy than the genetic risk of type 1 diabetes.
Submitted as: Presentation 260 words of 300 possible words
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Abstract No. 129 A pathway analysis of severe atopy, childhood asthma, and impaired lung function Alexander Hose 1, Oliver Fuchs 1, Erika von Mutius 1, Markus Ege 1,* , and the MAS and PASTURE study groups 2 1
Dr. von Hauner Children's Hospital
2
*Presenting author
Background: For childhood asthma various pheno- or endotypes have been defined. For atopy the situation is less clear. The aim of the current study was the definition of atopy phenotypes by a latent class analysis (LCA) of sIgE values and a subsequent path analysis explaining the atopy features relevant for asthma and impaired lung function. Methods: Based on sIgE data from 680 children of the MAS study and 766 children of PASTURE birth cohort, two three-dimensional LCAs were performed including allergen specificity, sIgE levels, and time course of sIgE over the first 6 years of life. The resulting latent classes were related to atopic diseases. A path analysis including asthma diagnosis, lung function, and the latent classes and other features of atopy was applied to atopic children. Results: The 5 class solution in both cohorts showed the best model fit based on the Akaike information criterion. In both studies, a severe atopy class was identified with high risk of asthma, hay fever, eczema, and impaired lung function. Asthma was determined by severe atopy through an excessive increment in production of sIgE to seasonal allergens in early childhood resulting in high sIgE levels at 6 years. Early sensitization to food allergens, TH2/TH1 ratio, and polysensitization were similarly determined directly or indirectly by severe atopy but not related to asthma. The inverse association of sIgE levels and FEV1 was completely explained by severe atopy (change in estimate, 104%), as held partially true for the association of asthma and FEV1 (change in estimate, 38%). Conclusions: Severe atopy as a latent phenomenon might underlie both childhood asthma and sensitization patterns. The path analysis performed in atopic subjects now suggests a link between severe atopy and asthma through excessive sIgE production, particularly to seasonal allergens early in life.
Hose et al., JACI 2016, doi: 10.1016/j.jaci.2016.08.046 Submitted as: Presentation 293 words of 300 possible words
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Abstract No. 138 Airway-liquid interface cultures of human bronchial epithelial cells for the investigation of biomarkers for asthma exacerbations Johanna C. Ehlers 1,* , Sabine Bartel 1, Christina Vock 1, and Heinz Fehrenbach 1 1
Research Center Borstel, Airway Research Center North (ARCN), German Center for Lung Research *Presenting author
Asthma is one of the most frequent chronic respiratory diseases. Although it can be managed quite well, bacterial or viral infections can lead to an acute worsening of the disease. Such acute exacerbations require the use of systemic corticosteroids, hospitalization of the patient, or even both. To date, early reliable prediction of acute exacerbations is not feasible, so that patients cannot be effectively treated before the onset of worsening of the symptoms. To identify potential biomarkers of an asthma exacerbation, primary human bronchial epithelial cells from healthy as well as asthmatic patients were cultured for 29 days at airliquid interface (ALI). These ALI-cultures were treated with the human recombinant T helper cell type 2 cytokine interleukin-13 (IL-13) for seven days and/or poly(I:C), a surrogate for replicating RNA-viruses, to mimic an exacerbation. Healthy and asthmatic ALI-cultures showed a pseudostratified epithelium consisting of basal, ciliated and secretory cells resembling a differentiated human airway epithelium. In both ALI-cultures treated with IL-13, paraffin sections stained with periodic acid Schiff revealed increased number and size of goblet cells. Furthermore, gene expression of the mucin Muc5AC was strongly induced especially in ALI-cultures from asthmatic patients. Thus, stimulation with IL-13 resulted in an asthma-like phenotype of the epithelium. Stimulation with poly(I:C) alone resulted in an increased expression of proinflammatory mediators, such as TNF-α, IL-8 and CCL-26. However, the expression of proinflammatory mediators was most prominent in ALI-cultures from asthmatic patients stimulated with IL-13 followed by poly(I:C). We suggest that this in vitro model might help to identify early events during induction of asthma exacerbations.
Supported by the BMBF (DZL and joint Leibniz research project EXASENS)
Submitted as: Presentation 268 words of 300 possible words
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Abstract No. 150 The Search for Early Biomarkers of Asthma and its Clinical Course across Child-and Adulthood, the DZL All Age Asthma Cohort (ALLIANCE) Oliver Fuchs 1,* , Thomas Bahmer 2, Barbara Rösler 1, Anna-Maria Dittrich 3, Malik Aydin 3, Isabell Baumann 4, Laila Sultansei 4, Markus Weckmann 4, Michael Zemlin 5, Ernst Rietschel 6 , Erika von Mutius 1, Klaus F Rabe 2, Gesine Hansen 3, and Matthias Kopp 4 1
Dr. von Hauner Children’s Hospital, Ludwig Maximilians University, Munich, CPC-M, Member of the German Center for Lung Research (DZL) 2 LungenClinic Grosshansdorf GmbH, Grosshansdorf, Germany, ARCN, Member of the German Center for Lung Research (DZL) 3 University Hospital of the Medizinische Hochschule Hannover (MHH), BREATH, Member of the German Center for Lung Research (DZL) 4 University Hospital Schleswig Holstein Campus Luebeck (UKSH), ARCN, Member of the German Center for Lung Research (DZL) 5 University Children’s Hospital Marburg, Germany, UGMLC, Member of the German Center for Lung Research (DZL) 6 University Children’s Hospital Cologne, Germany *Presenting author
Rationale: Clinical and genetic studies suggest that asthma is a syndrome. Currently, no predictors of distinct phenotypes or future disease course across age exist. We aim to decode underlying mechanisms for distinct wheeze/asthma phenotypes in child- and adulthood and to discover predictors for distinct phenotypes and their clinical course. Methods: A multi-center (ARCN, BREATH, CPC-M, GMLC, University of Cologne), All Age Asthma Cohort (ALLIANCE) of wheezing or asthmatic children and of adult asthmatics is recruiting (1, pediatric arm)) children with (1a) established diagnoses and (1b) new-onset disease (steroid- and leukotriene receptor antagonist-naïve), (2, adult arm) adults with asthma, and healthy controls (HC) for either study arm. Deep phenotyping is performed for numerous biosamples in ‘omics’ analyses plus comprehensive clinical assessment and lung function measurements. Episodic viral wheeze (EVW) was defined as discrete wheeze episodes in association with viral colds, multiple-trigger wheeze (MTW) was defined as both discrete wheeze episodes in association with viral colds and symptoms in between episodes due to other triggers. Asthma was generally defined by diagnosis per guidelines. Results: Currently, the pediatric arm (1) includes n=210 preschool wheezers (n=126 EVW, n=80/126 new-onset, n=74/126; n=84 MTW, n=40/84 new-onset; n=208 childhood asthmatics, n=38 new-onset; so far n=248 cases under follow-up (FU) after 12 months, n=156 after 24 months, and n= 17 after 36 months with low attrition each, as well as n=120 HC. (2) The adult arm includes n=209 asthmatics and n=43 HC. A biobank for either arm now contains >17,000 samples for deep phenotyping (genomics, epigenomics, transcriptomics, metabolomics, microbiome, virome, inflammasome). Response rates in children were continuing to be >90% for most biomaterials; response rates for adults were almost always > 90%. Conclusions: Recruitment and response in our cohorts are still excellent and allow in depth characterization of wheeze/asthma phenotypes and identification of relevant biomarkers and predictors.
DZL Annual Meeting 2017
Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 151 Measurement of exhaled volatile organic compounds in children by gas chromatography-mass spectrometry in the DZL ALL Age Asthma Cohort (ALLIANCE) Maximilian Ehrmann 1,* , Erika von Mutius 1, Gesine Hansen 2, Matthias Kopp 3, Jens Hohlfeld 4, Olaf Holz 4, and Oliver Fuchs 1 1
Dr. von Hauner Children’s Hospital, Ludwig Maximilians University, Munich, CPC-M, Member of the German Center for Lung Research (DZL) 2 University Hospital of the Medizinische Hochschule Hannover (MHH), BREATH, Member of the German Center for Lung Research (DZL) 3 University Hospital Schleswig Holstein Campus Luebeck (UKSH), ARCN, Member of the German Center for Lung Research (DZL) 4 Fraunhofer ITEM, Hannover, Germany, BREATH, Member of the German Center for Lung Research (DZL) *Presenting author
Rationale: Recent studies suggest that asthma is a syndrome. Currently, no predictors of distinct phenotypes and future disease course across age exist. We aim to decode underlying mechanisms for distinct phenotypes in childhood and adulthood and to discover biomarkers for phenotypes and future course by measurements of volatile organic compounds (VOCs) in exhaled air by gas chromatography-mass spectrometry (GC-MS). Methods: As part of the multi-center DZL All Age Asthma Cohort (ALLIANCE) of wheezing or asthmatic children and asthmatic adults, deep phenotyping is performed in ‘omics’ analyses plus comprehensive clinical assessment. This setup now includes measurements of the ‘breathome’ by analysis of VOCs in exhaled air with GC-MS in children. As a crossdisease area cooperation (AA and COPD), this measurement has been established in one recruiting center (CPC-M) so far. Episodic viral wheeze (EVW), multiple-trigger wheeze (MTW) and asthma were generally defined per guidelines, atopy was clinically defined. Preliminary evaluations of pediatric data by univariate regression analysis have been performed. Results: So far, GC-MS measurements have been successfully performed in n=15 pediatric cases (n=6/15 preschool wheezers (n=5 EVW, n=4/5 non-atopics; n=1 MTW (atopic); n=9 asthmatics, n=1/9 non-atopic), and n=1 HC (non-atopic). Preliminary analyses show typical VOC compositions for breath (major compounds isoprene and acetone) and room air (disinfection compounds propanols and ethanol). With yet limited numbers we do not find VOCs to be significantly related to assessed phenotypes or atopy, but we observed some VOCs to be associated with age and gender. Conclusions: Measurements of VOCs in exhaled air by GC-MS as part of the deep phenotyping approach in the DZL ALLIANCE Cohort has been successfully established in children. The addition of this non-invasive technique is an excellent complementary measure to allow in depth characterization of wheeze/asthma phenotypes and identification of relevant biomarkers and predictors.
Submitted as: Presentation 294 words of 300 possible words Page 22 of 286
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Abstract No. 152 Physiological phenotyping by lung function and assessment of airway inflammation in children of the DZL All Age Asthma Cohort (ALLIANCE) Oliver Fuchs 1,* , Johanna Kurz 1, Anna-Maria Dittrich 2, Isabell Baumann 3, Leila Sultansei 3, Gesine Hansen 2, Matthias Kopp 3, and Erika von Mutius 1 1
Dr. von Hauner Children’s Hospital, Ludwig Maximilians University, Munich, CPC-M, Member of the German Center for Lung Research (DZL) 2 University Hospital of the Medizinische Hochschule Hannover (MHH), BREATH, Member of the German Center for Lung Research (DZL) 3 University Hospital Schleswig Holstein Campus Luebeck (UKSH), ARCN, Member of the German Center for Lung Research (DZL) *Presenting author
Rationale: Multiple-breath washout (MBW) has been shown to be a sensitive measure for small airway function across distinct wheeze/asthma phenotypes in childhood. Within the DZL All Age Asthma Cohort (ALLIANCE) of children with wheeze/asthma, we aim to decode underlying mechanisms for distinct phenotypes across age and to discover predictors for them as well as their future clinical course by a comprehensive physiological phenotyping approach comprising MBW in addition to spirometry or body plethysmography, complemented by exhaled nitric oxide (eNO) as a marker of airway inflammation. Methods: Deep phenotyping in ALLIANCE is performed by ‘omics’ analyses plus clinical assessments and lung function measurements including analyses of small airway function in cases and healthy controls (HC). Preschool wheeze (PW) and asthma (A) were defined per international recommendations, atopy was defined clinically. Lung function as well as eNO measurements were performed according to guidelines. Results: As yet, valid measurements of spirometry, body plethysmography, and eNO have been performed in n=294, 259, and 221 children, respectively, demonstrating airway function abnormalities in cases beyond preschool age and allergic airway inflammation in the case of atopy independent of age. In addition, MBW has been performed in another n=252 children (n=75 HC, n=159 A, n=18 PW). In contrast to spirometry and despite low numbers so far, MBW was much more sensitive for detection of small airway function in PW, with S(cond) being the most sensitive parameter in multivariable models adjusted for age and atopy (multitrigger-wheeze [MTW] vs. HC, β=0.14, 95%-CI 0.003-0.024, p=0.012). Distinction between different wheeze phenotypes by S(cond) was not possible yet. Conclusions: Among different approaches for physiological phenotyping, MBW is a sensitive tool to detect small airway function abnormalities in PW. Particularly, MTW is associated with ventilation inhomogeneity independent of atopy, with [S(cond)] being promising for a further distinction between different PW phenotypes.
Submitted as: Presentation 298 words of 300 possible words
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DZL Annual Meeting 2017
Abstract No. 159 Rhinovirus infection of HDM sensitized lung tissue ex vivo proves inadequate antiviral immune response in small airways Olga Danov 1,* , Lisa Laßwitz 1, Helena Obernolte 1, Christina Hesse 1, Armin Braun 1, Sabine Wronski 1, and Katherina Sewald 1 1
Fraunhofer ITEM *Presenting author
Annually millions of asthmatic patients suffer from virus induced exacerbation. Mainly human rhinovirus (HRV) triggers worsening of the disease by infecting and replicating in airway epithelial cells. Inadequate immune response leading to insufficient virus elimination is assumed to be responsible for exacerbation. The impact of viral infection on the anti-viral and pro-inflammatory immune response was studied in asthmatic tissue ex vivo using precision-cut lung slices (PCLS) of HDM sensitized mice. Balb/c mice were sensitized and challenged with HDM or saline intranasally for 28 days. After the last challenge PCLS were prepared and infected with HRV1B (105 TCID50/mL) or UV-inactivated HRV1B for 48 h. The cytokines IL-4, IL-5, IL-13, IFN-, IL-10, MCP-1, IL-6, IP-10 and IL-17A were assessed by ELISA or MSD and normalized to the internal untreated control. HDM sensitization resulted in a TH-2 dominated microenvironment in lung tissue ex vivo as shown by significant upregulation of IL-4, IL-5, IL-6, IL-10, IL-17A and IP-10. HRV1B infection of healthy control PCLS resulted in induction of an anti-viral and pro-inflammatory immune response as indicated by a significantly higher secretion of IFN- (480 %), IP-10 (1625 %), IL-17A (1300%) or TNF-α (200 %). Contrary to this, anti-viral and proinflammatory response was impaired in sensitized compared to sham-sensitized lung tissue resulting in significantly attenuated secretion of IFN-γ (206%). IP-10 (133 %), IL-17A (140 %) were also reduced whereas MCP-1, TNF-α, IL-5 or IL-13 were comparable. However IL4 was further induced upon HRV1B infection in PCLS of HDM sensitized mice. Sensitization significantly reduces the epithelial response to virus infection ex vivo. The role of immune modulation in virus exacerbating chronic asthma has been discussed recently. We provide evidence that the host response to virus infection is also dysregulated ex vivo. The knowledge can be used to reveal pathways involved in virus infection and immune modulation.
Submitted as: Presentation 300 words of 300 possible words
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DZL Annual Meeting 2017
Abstract No. 166 Measurement of volatile organic compounds (VOCs) in exhaled air in children with an electronic nose (e-nose) as part of the DZL ALL Age Asthma Cohort Stefan Tümmers* 1, Lea Samija* 2, Erika von Mutius 2, Gesine Hansen 3, Matthias V. Kopp 1, Paul Brinkmann 4, Peter J. Sterk 4, Markus Weckmann** 1, Oliver Fuchs** 5, and ALLIANCE Study Group 6 1
University Hospital Schleswig Holstein Campus Luebeck (UKSH) Dr. von Hauner Children’s Hospital, Ludwig Maximilians University 3 University Hospital of the Medizinische Hochschule Hannover (MHH), 4 University of Amsterdam Medical Center (AMC), Amsterdam, The Netherlands 5 Dr. von Hauner Children’s Hospital, Ludwig Maximilians University, Munich 6 Member of the German Center for Lung Research (DZL) 2
Rationale: Clinical and genetic studies suggest that asthma is a syndrome. Currently, no predictors of distinct phenotypes and the future disease course across child- and adulthood exist. We aim to decode underlying mechanisms for distinct wheeze/asthma phenotypes and to discover predictors for future clinical course by measurements of volatile organic compounds (VOCs) in exhaled air as part of the ‘breathome’ by an electronic nose (e-nose). Methods: As part of the multi-center DZL All Age Asthma Cohort (ALLIANCE) of wheezing or asthmatic children and asthmatic adults, deep phenotyping is performed for numerous biosamples in ‘omics’ analyses plus comprehensive clinical assessment. This setup now includes measurements of the ‘breathome’ in children by analysis of VOCs in exhaled air with an e-nose. In cooperation with the University of Amsterdam Medical Center (AMC), this measurement has been established in pediatric cases and controls in two recruiting centers (ARCN and CPC-M). Preliminary evaluations by principal component (PCA) have been performed. Results: So far, e-nose measurements have been successfully performed in n=42 pediatric cases (mean age 9.26 years; n=10 preschool wheezers [n=6 EVW; n=4 MTW]; n=31 asthmatics, and n=17 HC (mean age 10.45 years). Preliminary analyses by PCA show that sensors sensitive to water vapor contamination (sensors number 5,6,23, and 31) impacted loading of PC1 significantly less compared to the other sensors (p<0.001), indicating minimal influence from ambient air and exhaled air humidity. Inter-operator variability assessed in one center (ARCN) did not show adverse effects on data variance. Conclusions: Measurements of VOCs in exhaled air by an e-nose as part of the deep phenotyping approach in the DZL ALLIANCE Cohort has been successfully established. The addition of this non-invasive technique is an excellent complementary measure to allow in depth characterization of wheeze/asthma phenotypes and identification of relevant biomarkers and predictors. */** equal contribution.
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Abstract No. 168 Influence of differently glycosylated IgG subclass antibodies on allergic reactions Alexandra Epp 1, Juliane Hobusch 1, Yannic Bartsch 1, Wolfgang Pfützner 2, Uta Jappe 3, Fred Finkelman 4, and Marc Ehlers 1,* 1
University of Luebeck Philipps University Marburg 3 Research Center Borstel 4 Cincinnati Children's Hospital Medical Center, USA *Presenting author 2
The increasing prevalence of allergies in westernized countries brings therapeutic approaches into focus. Allergen-specific immunotherapies (AIT) are established for several allergens, which induce tolerance and regulatory T cells and often also neutralizing IgG antibodies (Abs) that block the interaction of the allergen with IgE Abs. Companies now try to improve these AID protocols e.g. by using other adjuvants to increase also the induced IgG Ab titers. However, the role of AIT-induced IgG Abs is not well understood because IgG Abs can also induce effector functions and even have the potential to induce IgG-mediated anaphylaxis. The effector function of IgG Abs is dependent on their subclass and also on their type of IgG Fc glycosylation. Our research focuses on the development and function of differently glycosylated IgG subclass Abs (Oefner et al, 2012; Hess et al, 2013). Here, we investigated the role of differently glycosylated IgG subclass Abs in IgE- and IgGmediated allergic reactions in mice and humans. To assess the role and function of differentially glycosylated murine IgG subclass Abs, we established mouse models of IgEand IgG-induced anaphylaxis. Further, we investigated the role of IgG Fc glycosylation in AIT-treated birch pollen allergic patients. Our observations suggest that adjuvants used to promote IgG production during AIT should be selected for their ability to induce a certain IgG subclass with a certain type of IgG Fc glycosylation.
Oefner CM, Winkler A, Hess C, …, and Ehlers M. Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs. J Allergy and Clinic Immunol 2012; 129:1647-1655. Hess C, Winkler A, Lorenz AK, …, and Ehlers M. T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies. J Clinical Invest 2013; 123:3788– 3796. Submitted as: Presentation 226 words of 300 possible words Page 26 of 286
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Abstract No. 173 Are metabolomic markers associated with spirometric lung function indices? Results of the KORA-F4 Study. Claudia Flexeder 1,* , Stefan Karrasch 2, Gabi Kastenmüller 1, Christa Meisinger 1, AnnKristin Petersen 1, Elisabeth Thiering 2, Cornelia Prehn 1, Rui Wang-Sattler 1, Stephan Weidinger 3, Christian Gieger 1, Joachim Heinrich 4, Rolf Holle 1, Annette Peters 1, Thomas Illig 5, Jerzy Adamski 6, Karsten Suhre 7, and Holger Schulz 8 1
Helmholtz Zentrum München - German Research Center for Environmental Health Helmholtz Zentrum München - German Research Center for Environmental Health; Ludwig Maximilians University of Munich 3 University Hospital Schleswig-Holstein - Campus Kiel 4 Helmholtz Zentrum München - German Research Center for Environmental Health; Ludwig Maximilians University of Munich; Comprehensive Pneumology Center Munich (CPC-M) Member of the German Center for Lung Research 5 Helmholtz Zentrum München - German Research Center for Environmental Health; Hannover Medical School 6 Helmholtz Zentrum München - German Research Center for Environmental Health; Technical University of Munich 7 Helmholtz Zentrum München - German Research Center for Environmental Health; Weill Cornell Medical College in Qatar 8 Helmholtz Zentrum München - German Research Center for Environmental Health; Comprehensive Pneumology Center Munich (CPC-M) - Member of the German Center for Lung Research *Presenting author 2
Background: Respiratory function shows a large interindividual variability which is related to genetic, developmental and environmental factors. These determinants influence an individual’s metabolism, but little is known about metabolic processes and lung function. Therefore, this study investigates the association between lung function indices and concentrations of metabolomic markers in adults from a population-based study. Methods: Spirometry was performed in a subpopulation of the KORA-F4 cohort (Augsburg, Germany) comprising 1321 subjects aged 41 to 62 years. In fasting blood samples, over 650 metabolites were determined on two different (a targeted and a non-targeted) mass spectrometry based metabolomics platforms. Linear regression models were calculated for each metabolite after adjustment for age, sex, smoking, body mass index (BMI) and batch effects, and the residuals of these models were used to assess associations between metabolites and percent predicted values for forced expiratory volume in 1 second (FEV1pp) and forced vital capacity (FVCpp), respectively. Results: We identified 30 metabolites that were significantly associated with lung function indices after correction for multiple testing (q-values ranged from 4.9∙10-2 to 3.3∙10-4). Of these metabolites, 29 were associated with FVCpp and 23 were associated with FEV1pp. These indices are linked to different metabolites involved in lipid metabolism, tocopherol metabolism and tyrosine metabolism. An association with the xenobiotic pathway was also observed. Conclusion: Our results suggest that metabolites from different pathways are associated
DZL Annual Meeting 2017 with spirometric measures of lung function. The lipid metabolites identified suggest pulmonary surfactant is involved in lung function, as surfactant is mainly composed of different phospholipids and is vital for the facilitation of peripheral air spaces. The observed association with tocopherol metabolism may suggest an importance of antioxidant or antiinflammatory defence for respiratory function.
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Abstract No. 175 Studying the pathophysiological role of the asthma susceptibility genes STAT3/6 and ORMDL3 using the fruit fly Drosophila melanogaster as a model Christine Fink 1,* , Kimberly Kallsen 2, Ruben Prange 1, Holger Heine 2, and Thomas Roeder 1
1
CAU Kiel, Zoological Institute Research Center Borstel *Presenting author 2
Chronic respiratory diseases like Asthma are characterized by periodic or permanent inflammation of the airway system, hypersecretion of mucus and a remodeling of the airway epithelium resulting in a diminished airflow. Many risk factors for developing Asthma are known (genetic predisposition, allergens, air pollution or smoke exposure) but the signaling events and key players for Asthma pathogenesis are not understood at all. We are mainly interested in the pathophysiological roles of two susceptibility genes that are already in discussion to influence airway remodeling in Asthma patients, ORMDL3 and the transcription factor STAT (3/6). ORMDL3 is an ER transmembrane protein that has previously been associated with sphingolipid metabolism [1], the unfolded protein response, Ca2+ homeostasis [2] and T cell activation [3]. Moreover, the evolutionary highly conserved JAK/STAT pathway was found to play a role for asthma susceptibility because it effects epithelial integrity and homeostatic stability [4]. We could mirror the influence of both genes using the airway system of Drosophila melanogaster as a model. By enhancing expression the fly’s homologue to ORMDL3 (dORMDL) we were able to detect substantially higher stress sensitivity of the airway epithelium and regulation of highly important signaling pathways including JAK/STAT signaling. We could observe structural changes by ectopic activation of JAK/STAT signaling in the airways comprising epithelial thickening and metaas well a hyperplasia.
Submitted as: Presentation 218 words of 300 possible words
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Abstract No. 193 Comparison of diagnostic tools for detection of allergen-specific IgE in the pediatric arm of the DZL All Age Asthma Cohort (ALLIANCE) Anna-Maria Dittrich 1,* , Oliver Fuchs 2, Chrysanthi Skevaki 3, Markus Weckmann 4, David S. DeLuca 5, Harald Renz 3, Matthias Kopp 4, Erika von Mutius 2, Gesine Hansen 1, and All Age Asthma Cohort (ALLIANCE) study group 6 1
Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany 2 Department of Pediatrics, Dr von Hauner Children's Hospital of Ludwig Maximilian University of Munich, Munich, Germany 3 Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps University Marburg, University Hospital Giessen and Marburg GmbH Baldingerstr, Marburg, Germany 4 Department of Pediatric Allergy and Pulmonology, Clinic of Pediatrics UKSH, University of Luebeck, Luebeck, Germany 5 Bioinformatics Core, BREATH, Hannover Medical School, Hannover, Germany 6 Disease Area Asthma *Presenting author
Background: At BREATH, specific IgE (sIgE) measurements were performed via three different analytic tools: ImmunoCAP® sx1 and fx5 (ICAP), Euroimmun® (EI) and ImmunoCAP ISAC 112® (ISAC) for participants of the pediatric arm of DZL’s ALLIANCE. Methods: ICAP provides extract-based measurements of sIgE to 16 allergens via a sandwich immunoassay. EI utilizes an immunoblot-based methodology for 37 different allergens. ISAC determines sIgE to 112 molecular allergen components by a microarray. Thirteen allergens are covered by all three analysis tools. Samples of 97 children included in ALLIANCE have been measured across all platforms and were included in an initial comparison. Results: Comparison of detection rates for all assessed sIgE revealed lower detection rates for EI and ISAC as compared to ICAP. Using a threshold of > 0,7 kU/l, we iteratively designated each method as a reference and assessed the performance of the remaining two methods. In the case of ICAP as reference, the performance metrics of EI and ISAC are respectively: sensitivity, 0.43, 0.64; specificity 0.97, 0.93; positive predictive value, 0.89, 0.83; and negative predictive value 0.75, 0.82. For the specific allergens Dermatophagoides pteronyssinus, cat and dog dander, Cladosporium herbarum, birch pollen, mugwort, hens egg, cow’s milk and wheat ICAP also had higher detection rates than EI or ISAC. For sIgE to peanut and grass pollen, ISAC displayed the highest detection rates. SIgE to soy was detected equally among the ICAP and ISAC. Conclusions: We observed striking differences in detection rates with regards to detection of common sIgE overall with ICAP providing the highest detection rates. For single allergens, performance of the three tools was dependent on the allergen in question.
DZL Annual Meeting 2017
Our next step is to compare the performance of the three tools in terms of disease phenotyping and prognosis including disease persistence.
Submitted as: Presentation 291 words of 300 possible words
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Abstract No. 200 The role of B-cells and B-cell activating factor of the TNF family (BAFF) in a mouse model of house dust mite-induced allergic airway inflammation Anika Habener 1,* , Christine Happle 1, Jelena Skuljec 1, Kathleen Dalüge 1, Helena Obernolte 2, Armin Braun 2, Katherina Sewald 2, Almut Meyer-Bahlburg 1, and Gesine Hansen 1 1
Hannover Medical School Fraunhofer Institute for Toxicology and Experimental Medicine *Presenting author 2
Objective: Allergic asthma is a widespread chronic inflammatory disease of the airways but the influence of different B-cell subsets and the cytokine B-cell activating factor of the TNF family (BAFF), which is crucial for peripheral B-cell survival and maturation, are not wellknown. To better understand the impact of B-cells and BAFF in allergic asthma we analyzed these factors in a model of house dust mite (HDM)-induced allergic airway inflammation using wildtype (WT) and B cell deficient (µMT) mice. Methods: Airway hyperreactivity (AHR) of mice and bronchoconstriction of precision cut lung slices (PCLS) were measured after methacholine provocation by invasive lung function and determination of the airway area respectively. Cytokine production was determined by ELISA, B-cells and T-cells were characterized by flow cytometry. To directly test the B-cell impact, naïve B-cells were transferred intravenously into µMT mice before start of the HDM protocol. Results: HDM-treated µMT mice showed an increased AHR, diminished CD4+ T-cells, regulatory T-cells (Tregs) and Th2 cytokine levels, especially IL-10, compared to HDMtreated WT mice. Furthermore, allergic µMT mice developed elevated serum BAFF levels compared to control µMT and HDM-treated WT mice. Kinetic studies revealed that BAFF levels in serum and especially in lung and BALF increase the more often HDM is administered intranasally. Transfer of WT B-cells from unmanipulated mice attenuated AHR, reduced BAFF level, increased numbers of regulatory B-cells, CD4+ T-cells, Tregs and elevated Th2 cytokines, especially IL-10, in HDM-treated µMT mice. Moreover, bronchoconstriction and cytokine secretion from PCLS of HDM-treated and untreated mice reflect our results generated by lung function and restimulation of isolated cells. Conclusions: Our data indicate that B-cells and BAFF may play a critical role in HDMinduced allergic airway inflammation, which might be modulated by regulatory B-cells, Tcells and BAFF. To further determine specific stimuli and mechanisms PCLS will be used.
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Abstract No. 209 Anatomical features and functions of mast cell-C-fibers interactions are comparable in human and non-human primate airways Elaine Cabral Serrao 1,* , Susann Dehmel 2, Sharon Jiménez Delgado 2, Franziska Dahlmann 1, Katherina Sewald 2, and Armin Braun 2 1
Fraunhofer ITEM, German Primate Center Fraunhofer ITEM *Presenting author
2
The transient receptor potential vanilloid type 1 receptor (TRPV1) is distributed in sensory nerve endings of human airways and contributes to chemical hypersensitivity in irritantinduced asthma. Stimulation of peripheral sensory nerves with the TRPV1 agonist capsaicin causes a release of neuropeptides, which were shown to induce mast cell degranulation, and lead to bronchoconstriction. Here we aimed to analyse the anatomy and function of mast cell-C-fiber interactions using the model of precision-cut lung slices (PCLS) of humans in comparison to non-human primates (NHP). PCLS of both species were passively sensitized and incubated +/- the histamine 1 receptor antagonist Ceterizine®. Capsaicin was added and airway area was determined. Confocal images of PCLS derived from humans and NHP were analysed regarding their localisation of pan-neuronal marker PGP9.5 and neuropeptides. Mast cell degranulation and substance P release were determined in PCLS after capsaicin stimulation. Humans and NHP show a similar response of about 30 % airway area reduction in response to capsaicin. Bronchoconstriction was completely blocked by 10 µM Ceterizine®. Volumetric analysis showed reduced substance P content from 4.4x105µm3 to 2.1x105µm3 per mm3 airway after capsaicin treatment. The overall distribution of immune-reactive nerve fibers seems to be comparable, whereas only intraepithelial substance P positive nerve fibers in NHP distinguish clearly from humans. Confocal pictures depict the release of tryptase and substance P from mast cells in response to capsaicin. Blocking of the H1 receptor leads to complete inhibition of capsaicin-induced bronchoconstriction in pre-sensitized PCLS, showing that mast cell derived mediators are involved in the process of capsaicin-induced bronchoconstriction. NHP derived lung sections reflect the human situation regarding co-localisation and neuropeptide composition of tissue resident mast cells and peripheral sensory nerve fibers. Since substance P release following bronchoconstriction was detected in response to capsaicin, C fibers are functional in PCLS of both species.
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Abstract No. 210 Identification of new IgE targets via One-Bead-One-Compound (OBOC) libraries for diagnosis and treatment of allergic asthma Thorsten Krause 1,* , Niels Röckendorf 1, Christian Schwager 1, Heike Sinnecker 1, Oliver Fuchs 2, Uta Jappe 1, Erika von Mutius 2, and Andreas Frey 1 1
Research Center Borstel LMU *Presenting author 2
Allergic asthma - in terms of chronic inflammatory airway diseases - and systemic allergies from a broader perspective - are global health burdens affecting hundreds of millions of people worldwide. Both have in common a pivotal role of IgE in disease development, manifestation and persistence. To date, treatment mainly focusses on alleviation of the allergic symptoms by employing corticosteroids and anti-IgE therapies – both procedures which often are impaired by adverse side effects. The only curative treatment available at present is an immunotherapy where the IgE-based immune reaction is redirected into protective IgG/IgA-based immunity. This approach requires knowledge of the exact targets of the prevailing IgE species. However, in most patients with allergic asthma the specificities of the majority of the respective immunoglobulins are unknown, precluding their being used as potential immunotherapeutic targets. To identify a multitude of these unknown IgE specificities with relevance to allergic asthma, we make use of so called one-bead-one-compound (OBOC) libraries with which we can screen millions of peptides within one assay for their ability to serve as IgE targets when tested with sera from asthmatic patients. By optimizing our methodology in terms of the OBOC microparticles used as well as IgE detection reagents applied, we are currently able to detect amounts < 10 IU/ml of specific IgE bound to their target peptide epitopes on single beads out of a 10,000-bead-library. We are now in the process of setting up a semi-automatized procedure to detect, capture and analyze single positive beads within an OBOC library, which will eventually lead us to finding new IgE targets. These targets could then be used to diagnose and potentially cure patients with allergic asthma and other IgE-related chronic inflammatory disorders.
Submitted as: Presentation 282 words of 300 possible words
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Abstract No. 219 Species comparison of anti-viral and inflammatory host response to rhinovirus infection in fresh lung tissue Helena Obernolte 1,* , Olga Danov 1, Peter Braubach 2, Danny Jonigk 2, Marcus Krüger 2, Gregor Warnecke 2, Olaf Pfennig 3, Hans-Gerd Fieguth 3, Armin Braun 1, Sabine Wronski 1, and Katherina Sewald 1 1
Fraunhofer Institute for Toxicology and Experimental Medicine Medical School Hannover 3 KRH clinics Hannover *Presenting author 2
Human rhinovirus (HRV) is a main cause of airway infections and a major risk factor of exacerbations in patients suffering from asthma or COPD. However, investigations of HRV pathogenesis have been hampered by the lack of readily available experimental models that closely represent the human respiratory system. We hypothesize that HRV infects airway epithelial cells in fresh human lung tissue ex vivo and anti-viral pathways for minor serotypes are activated comparable in murine precision-cut lung slices (PCLS), mimicking the human situation. Aim of this study was to compare immune responses in murine and human PCLS to HRV1B infection. Prevention of anti-viral responses by intervention with Rupintrivir and Pleconaril was assessed. Murine and human PCLS were prepared from fresh lung tissue. PCLS were inoculated with HRV1B, UV-inactivated HRV in absence or presence of inhibitors. Tissue was cultured for 24h to 72h. Cytotoxic effects were evaluated using LIVE/DEAD staining. Pro-inflammatory and anti-viral cytokine profiles were determined by ELISA. Infection of murine and human PCLS ex vivo with HRV1B induced significant expression of pro-inflammatory cytokine IL-6 in murine and human PCLS after 24h and 48h or 72h, respectively. In both species, anti-viral cytokines IP-10 and interferons were induced, as indicated by upregulation of IP-10 (6-fold in mouse and 25-fold in human PCLS). In both systems, treatment with Rupintrivir reduced release of all measured cytokines in murine PCLS (24h) and significantly anti-viral cytokines in human PCLS (24h, 72h), up to a reduction in IP-10 of 5-fold in human and 1.3-fold in murine PCLS. HRV1B infects lung tissue ex vivo and induces anti-viral and pro-inflammatory immune responses with comparable profile in human and mouse and serves as readily available ex vivo viral infection model. Anti-viral treatment protects lung tissue from infection. Human lung tissue showed a stronger anti-viral immune response compared to murine PCLS.
Katherina Sewald and Sabine Wronski contributed equally to the study Olga Danov and Helena Obernolte contributed equally to the study Submitted as: Presentation 299 words of 300 possible words Page 33 of 286
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Abstract No. 220 Chipcytometry based comprehensive immunophenotyping of peripheral blood mononuclear cells of the DZL All Age Asthma Cohort (ALLIANCE) Adan Chari Jirmo 1,* , Christine Happle 1, David DeLuca 1, Christian Dopfer 1, Mareike Price 1 , Malik Aydin 1, Anna Maria Dittrich 1, Marcus Weckmann 2, Bianca Schaub 3, Oliver Fuchs 3 , Matthias Kopp 2, Erika von Mutius 3, Gesine Hansen 1, and ALLIANCE STUDY GROUP 4 1
DZL (BREATH), Hannover Medical School DZL (ARCN), University Clinic, Schleswig-Holstein, Lübeck 3 DZL (CPC-M), University Children's Hospital, Munich 4 Others *Presenting author 2
Introduction: Elucidation of specific immunological alterations in childhood asthma can disclose novel diagnostic markers and therapeutic options. We use chipcytometry, a powerful novel cell phenotyping technique to elucidate specific ‘immuno-endotypes’ within both the pediatric and adult arm of the multi-center DZL ALL Age Asthma Cohort (ALLIANCE). Chipcytometry allows for high content analysis of a virtually unlimited number of biomarkers on single cell level without loss of sample stability for more than one year. Methods: Chipcytometry phenoyping was optimized to obtain valid cytometry data due to the multi-center sampling design. For leucocyte characterization (T-, B-, NK-, dendritic cells, monocytes), n=16 biomarkers were measured (CD123, CD14, CD11c, CD141, CD303, HLA-DR, CD3, CD19, CD172α, CD44, FceR1, CD56, CD16, CD45, MDC-1, and CD1c). Chipcytometry data from the initial 104 children is subjected to multi-dimensional analysis including support vector machines to differentiate potential asthma endo-/phenotypes. Results: Successful and reproducible, chipcytometry identification of specific PBMCs could be achieved. Preliminary analyses showed differential expression of functionally relevant biomarkers such as HLA-DR, FceR1, CD44 and CD172 on myeloid DC subsets. Analysis of these markers on myeloid dendritic cells can be used for diagnostic discrimination between different phenotypes. Implementation of this data into support vector machine analyses allowed for prediction of “healthy” versus asthma phenotype with >80% accuracy in n=21 asthmatic probands (aged 6-18 years) or preschool wheezers (n=60 probands) versus healthy controls (n=25 children). We are validating this initial results using a large group with n=305 probands . Conclusion: We show that chipcytometry can be used in large clinical studies to phenotype specific leukocyte subsets and their immune activation. Employing support vector machine approaches, asthma phenotypes can be predicted with >80% accuracy. Next steps include validation this approach in a larger set of ALLIANCE study participants and elucidation of functional role of identified immune patterns in asthma.
Submitted as: Presentation 299 words of 300 possible words Page 34 of 286
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Abstract No. 225 Drosophila melanogaster as model organism for investigations on transgenerational effects of nicotine exposure Stephanie Papenmeier 1,* , Arne Krüger 1, and Susanne Krauss-Etschmann 1 1
Division of Early Life Origins of Chronic Lung Diseases, Research Center Borstel, LeibnizCenter for Medicine and Biosciences, Member of the German Center for Lung Research (DZL), Borstel, Germany *Presenting author
Background: Prenatal exposure to nicotine due to maternal smoking increases the risk for the development of chronic obstructive lung diseases such as asthma later in life. But not only maternal nicotine exposure is a risk factor for the development of asthma. Even grandmaternal smoking can affect the grandchildren’s generation and more recent reports show also correlation to paternal smoking, suggesting epigenetic effects. To elucidate the underlying mechanisms regarding transgenerational effects of nicotine exposure, model organisms such as mice are commonly used. However, transgenerational experiments can be time consuming and expensive. The fruit fly Drosophila melanogaster develops much faster, produces a lot of genetically identical offspring and is more cost effective. The fly shares several aspects of airway development with vertebrates and moreover has a high homology with human disease related genes, thus can help elucidating developmental mechanisms, genetic risk factors and transgenerational effects of nicotine. Objective: The aim of this project is the establishment of Drosophila melanogaster as a model system for elucidating transgenerational effects of nicotine exposure on potential airway related developmental defects and gene expression. Concept: Fruit flies are exposed to nicotine by vaporising the drug from a heated nichrome coil and are subsequently mated. The following generation (F1) will be observed in different developmental stages, regarding potential developmental defects in general and specifically in the respiratory system. If effects of nicotine exposure can be observed in the F1generation, the same parameters will be assessed in the next F2 generation, etc. Moreover, the potential epigenetic effects of nicotine will be investigated by determination of genes, whose expression level is affected by nicotine exposure. The expression level of these genes will be compared between generations. Expectations: The establishment of a transgenerational model in Drosophila melanogaster would facilitate investigations on developmental and epigenetic effects of nicotine exposure.
Submitted as: Presentation 297 words of 300 possible words
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Abstract No. 231 Drosophila melanogaster - A promising model to study transgenerational effects of parental smoking Karolina-Theresa Neumann 1,* , Karin Uliczka 1, Sebastian Reuter 1, Susanne KraussEtschmann 1, and Christina Wagner 1 1
Research Center Borstel, Germany *Presenting author
Development of chronic inflammatory lung diseases such as bronchial asthma is affected by several environmental factors, inter alia by cigarette smoke. Since epidemiological studies indicate that prenatal exposure to (grand) maternal cigarette smoking is associated with a higher risk for childhood asthma, mechanisms of transgenerational inheritance are likewise responsible for disease onset. So far mouse models of tobacco smoke-related airway diseases have been used for transgenerational studies. However, they are restricted to only a few generations due to long generation times, high breeding costs and shortage of breeding space. Therefore, it is indispensable to utilize a model organism that allows studies on subsequent generations in a short period of time. In this context, the fruit fly Drosophila melanogaster has emerged as a suitable model system because of its short generation time, high fertility, ease of culture and the availability of various genetic tools. Furthermore, Drosophila has already been successfully used as a model system in respiratory research in order to study innate immunity and remodeling processes of airway epithelial cells. We aim at establishing a Drosophila smoking model mimicking key features of an antioxidant response phenotype in order to study transgenerational effects of parental smoke exposure on airway epithelial cells. After that, we will use this model to identify molecules and pathways mediating cigarette smoke-induced transgenerational alterations in airway epithelial cells. Ideally these results should be extrapolated to mammalian systems to identify novel regulators of asthma or COPD that also have the potential to serve as therapeutic targets in humans. (Supported by the BMBF (DZL and joint Leibniz research project EXASENS)
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Abstract No. 238 Development of asthma phenotypes: Predictors and Mechanisms – Aims of WP1 in the Asthma and Allergy (AA) Disease Area in DZL2.0 Bianca Schaub 1,* , Anna-Maria Dittrich 2, and Holger Garn 3 1
Dr. von Haunersches Kinderspital, LMU München Klinik für Pädiatrische Pneumologie, Allergologie und Neonatologie, MH Hannover 3 Institut für Laboratoriumsmedizin, Philipps-Universität Marburg *Presenting author 2
Rationale: Asthma represents a disease comprised of several clinical phenotypes with different underlying pathomechanisms which require further characterization regarding cellular and molecular aberrations as basis for stratified/personalized therapeutic strategies. Aims: Two major aims are investigated in close cooperation between the AA WP1 ALLIANCE and basic research study groups 1) Identification of novel predictive biomarkers for development of asthma and asthma phenotypes 2) Elucidation of asthma phenotype-specific pathomechanisms as basis for development of stratified therapeutic concepts Two alternative hypotheses are tested: • the “quantity hypothesis”, assuming that differences in the strength of signals are decisive for disease/phenotype development, i.e. exceeding of thresholds determines disease or phenotype occurrence; • the “quality hypothesis”, supposing that specific signal patterns are key for disease/phenotype development. Methods: Cellular/molecular phenotypes are identified by comprehensive analyses of cohort biomaterials and correlated to identified clinical phenotypes accompanied by experimental systems comprising different complexity levels e.g. cell culture systems including cocultures, air liquid interface and complex tissue models, drosophila and endotyperepresenting murine models. Four major areas have been identified: The role of (modified) allergens is tested by use of HDM-allergen panels to query their efficacy in predicting clinical phenotypes and disease course. Analysis of epithelial cell function is focused on identification of phenotype-specific activation/transcription patterns including functional characterization of phenotype-associated mediators (e.g. E1/E2concept) and targets for epithelial cell regeneration. Investigations on phenotype-specific patterns of immune regulation are focused on the role of IL-10 and IL-1 family members, the inflammasome pathway and dysregulation of T-, B- and innate lymphocyte subpopulations analyzed in humans and endotype-representing murine and primate models. Phenotypespecific miRNA patterns will be characterized. Conclusion: Synergistic investigations of mechanisms in human cohorts and models will develop a comprehensive understanding upon the development of asthma phenotypes and prepare ground for the development of novel approaches for their prevention and treatment.
Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 242 T-cell dependency of HDM-induced neutrophil airway inflammation Stefanie Hagner 1, and Garn Holger 1,* 1
Laboratory Medicine and Pathobiochemistry *Presenting author
Background: Pathomechanisms leading to differential asthma phenotype development esp. for non-type-2 phenotypes are poorly understood. While an activation of innate immune responses including epithelial activation with involvement of TNF a nd IL -1 been described, the functional role of T cells, mainly of the dominating Th17 and Th1 phenotypes, are still unclear. Aim: To decipher which cells initially decide on (allergic) airway inflammation phenotype development – epithelial cells (innate immunity) or T cells (adaptive immunity). Methods: Balb/c wildtype (WT) and RAG-/- mice were sensitized and challenged intranasally with house dust mite (HDM) extract according to a previously developed protocol for the induction of type-2-independent airway inflammation. Innate and allergic airways inflammation was characterized by BAL, cytokines and histological analyses. Results: Exposure to HDM extract resulted in an increase in total leukocyte numbers in BAL in WT mice in comparison to sham-treated mice. Furthermore, differential gene expression signatures for cytokines/chemokines characteristic for non-Th2 asthma phenotype in humans have been validated by microarray analyses. In contrast, HDM-exposed RAG-/mice showed total leukocyte numbers nearly comparable to sham-treated mice. As expected, determination of differential cell counts revealed a predominant recruitment of neutrophils accompanied by an increase of lymphocytes in WT HDM-treated mice in comparison to sham-treated control mice. Neutrophil recruitment was significantly less pronounced in HDM-exposed RAG-/- mice. These observations were also reflected at the histological level which showed no airway inflammation and mucus production as well by cytokine analyses. Conclusion: Airway neutrophilia/lung inflammation is significantly reduced in Rag-deficient HDM-exposed mice in comparison to WT HDM-exposed mice suggesting that cells of the adaptive immune system, such as T cells are important players in the initiation of a non-type-2/neutrophil inflammation phenotype. This conclusion has to be further proven by T cell transfer experiments in the future.
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Abstract No. 246 IRF-1 SNPs influence the risk for childhood allergic asthma in the CLARA-study: a critical role for pro-inflammatory immune regulation Katja Landgraf-Rauf 1,* , Andreas Boeck 1, Elisabeth Klucker 1, Vanessa Vogelsang 1, Susanne Schmidt 1, 1, Sonja Kunze 2, Anke Graessel 3, Carsten Schmidt-Weber 3, Erika von Mutius 1, Michaela Schedel 4, and Bianca Schaub 1 1
Dr. von Haunersches Kinderspital Helmholtz Zentrum München 3 Technische Universität and Helmholtz Center Munich 4 National Jewish Health *Presenting author 2
Background: Allergic and non-allergic childhood asthma has been characterized by distinct immune mechanisms. While interferon regulating factor 1 (IRF-1) single nucleotide polymorphisms (SNPs) influence atopy risk, the effect of SNPs on asthma phenotypespecific immune mechanisms is unclear. We assessed whether IRF-1 SNPs modify distinct immune regulatory pathways in allergic (AA) and non-allergic (NA) childhood asthma. Methods: In a cross-sectional childhood asthma study, asthma was characterized by doctor´s diagnosis and AA vs NA by positive or negative specific IgE. Children were genotyped for four tagging SNPs within IRF-1 (n=172). Associations between AA, NA and IRF-1 single SNPs and haplotypes were calculated and correlated with gene expression (qRT-PCR) and cytokine levels (Luminex). Results: Carrying the polymorphic- allele of IRF-1 SNPs rs10035166, rs2706384 or rs2070721 was associated with significantly increased risk for AA (defined as risk-alleles), while the presence of the polymorphic allele of rs17622656 was associated with lower risk for AA but not NA (defined as non- risk allele). Only in AA children carrying the risk- alleles an increased expression of pro-inflammatory genes ICAM3, IRF-8, XBP-1, IFN-γ, RGS13, RORC, and TSC2 was observed. Innate NOD2 expression was downregulated in AA children in the presence of IRF-1 risk- alleles of rs2706384 and rs10035166 and with risk haplotype GCCG. Further, AA children carrying the risk haplotype showed increased secretion of IL-13 (p=0.014; unstimulated). NA children carrying the risk allele of rs2070721 compared to NA children carrying the non-risk allele of rs17622656 showed significantly upregulated calcium-, innate-, mTOR-, neutrophil- and inflammatory-associated genes. Conclusion: IRF-1 polymorphisms influence the risk for childhood allergic asthma being associated with increased pro-inflammatory gene regulation.
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Abstract No. 252 Nuclear localization of Suppressor of Cytokine Signaling (SOCS)-1 regulates local immunity in the lung Jana Zimmer 1, Michael Weitnauer 1, Sébastien Boutin 1, Bernd Arnold 2, Günter Küblbeck 2, Felix Lasitschka 3, Lars Lunding 4, Michael Wegmann 4, and Alexander Dalpke 1,* 1
Dept. of Infectious Diseases, Univ. Heidelberg German Cancer Research Center (DKFZ), Heidelberg 3 Institute of Pathology, Heidelberg 4 Division of Asthma Exacerbation & Regulation, Research Center Borstel *Presenting author 2
Immune responses are tightly regulated to ensure for effective defense of pathogens but also to avoid excessive immune responses. The negative feedback inhibitor SOCS1 is of special interest, as its reported main function - inhibition of JAK/STAT signaling - is in the cytoplasm, however, SOCS1 is localized mainly in the cell nucleus. To study the role of nuclear SOCS1, a BAC transgenic mouse model was established, expressing non-nuclear mutant Socs1 within a Socs1-/- background (Socs1-/-MGLtg mice). Socs1-/-MGLtg mice are rescued from early lethality as compared to Socs1-/- mice, which die due to excessive interferon signaling within three weeks after birth. Classical interferon signaling is not altered in Socs1-/-MGLtg mice as shown by tyrosine phosphorylation of STAT1, by regulation of classical interferon gamma target genes and by whole genome expression analysis. However, a subset of NFκB inducible genes is dysregulated. Although cytoplasmic SOCS1 is crucial for survival, Socs1-/-MGLtg mice spontaneously develop lowgrade inflammation in the lung. Mice lacking nuclear SOCS1 show increased percentage of Gata3+ CD4+ cells and Th2-type cytokines. Upon ovalbumin (OVA) sensitization and challenge, Socs1-/-MGLtg mice show increased airway eosinophilia. Decreased transepithelial electrical resistance (TER) in trachea epithelial cells from Socs1-/-MGLtg mice suggests disrupted epithelial cell barrier. In summary, Socs1 / MGLtg mice present a valuable tool to study the nuclear function of SOCS1 in vivo and will allow investigating control of local immunity in the lung by nuclear SOCS1.
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Abstract No. 264 Characterization of cellular sources for non-quantal ACh release in mouse airways Lucas Delventhal 1, Katrin Audrit 1,* , Silke Wiegand 1, Wolfgang Kummer 1, and Christina Nassenstein 1 1
Institute of Anatomy and Cell Biology *Presenting author
Acetylcholine (ACh) is the major transmitter to induce contraction of the airway smooth muscle (ASM). In addition to the quantal vesicular ACh release after stimulation of parasympathetic neurons, non-quantal ACh (nqACh) release is observed in tracheal preparations after eserine treatment, which is capable to induce ASM contraction. In order to identify which cell population in the airways contributes to nqACh synthesis, expression of the choline acetyltransferase (ChAT), high affinity choline transporter-1 (CHT1), and the lowaffinity choline transporter-like family (CTL1-5) was evaluated by RT-PCR in primary isolated vagal and dorsal root ganglia, parasympathetic neurons, tracheal epithelial cells, lymphocytes, granulocytes, and in a smooth muscle and fibroblast cell line. All evaluated cell populations expressed ChAT-, CHT1- or CTL-mRNA, respectively. ACh release was functionally investigated by using M3WT4 cells, a reporter cell line overexpressing muscarinic M3 receptors. An increase in [Ca2+]i was induced in M3WT4 cells by supernatants of eserine-treated tracheal preparations and inhibited by atropine coapplication. However, supernatants of eserine-treated isolated cell populations or cells cocultured cells with M3WT4 cells, respectively, had no effect on [Ca2+]i levels. To address the question if mast cells might contribute to nqACh release, organ bath experiments with tracheas of mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmGlliJ mice (kindly provided by Frank Petersen, Leibniz Forschungszentrum Borstel, ARCN) were performed. ASM contraction in response to eserine in mast cell-deficient mice was unaltered in comparison to wild-type mice. In conclusion, we were able to show that many different cell populations located in the trachea express transporters and/or enzymes associated with nqACh release. The fact that we were not able identify the major source of nqACh may be due to a low sensitivity of the M3WT4 cell assay. Alternatively, the possibility that the effect of nqAChdependent ASM contraction is the sum of low-level ACh released from different cell populations has to be discussed.
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Abstract No. 272 B-cell subsets are modulated during OVA-induced allergic asthma but are not required for the development of respiratory tolerance in a murine model Anika Habener 1,* , Ann-Kathrin Behrendt 2, Jelena Skuljec 1, Adan Chari Jirmo 1, Almut Meyer-Bahlburg 1, and Gesine Hansen 1 1
Hannover Medical School University Medicine Greifswald *Presenting author 2
Objective: Allergic asthma is a widespread chronic inflammatory disease in children and adults. Besides well-known contributions of T- and other immune cells, the role of different B-cell subsets is largely unknown. B-cells act as immunoglobulin producing and antigen presenting cells and secrete cytokines. Especially regulatory B-cells may be important in asthma regulation. To better understand the role of B-cells in asthma, we analyzed the Bcell compartment in allergic airway inflammation and respiratory tolerance in a murine model. Methods: In wildtype (WT) and B-cell deficient (µMT) mice, ovalbumin (OVA)-induced allergic airway inflammation and respiratory tolerance was assessed by measuring invasive lung function, bronchoalveolar cell influx, lung inflammation and mucus production, and Th2 cytokine production. B-cell subsets were analyzed by flow cytometry and OVA-specific antibody secreting cells (ASC) were determined by ELISpot. Furthermore, B-cell subset induced T-cell proliferation and regulatory T-cell (Treg) induction were assessed in vitro. Results: Allergic WT mice displayed increased airway hyperreactivity, lung eosinophilia, mucus hypersecretion, pulmonary inflammation and Th2 cytokines. This was associated with elevated expression of MHCII and CD23 on follicular mature B-cells in lungs, bronchial lymph nodes (bLN) and spleens, which induced in vitro allergen-specific T-cell proliferation. Moreover, an elevation in germinal center B-cells in allergic mice was associated with increased OVA-specific ASC especially in bLN. In contrast, in respiratory tolerance these Bcell alterations were clearly attenuated, and regulatory marginal zone precursor B-cells were increased, which displayed Treg inducing properties in vitro. In spite of these significant alterations in B-cell distribution and function during allergy and tolerance, µMT versus WT mice displayed no difference with regard to their asthma-like and tolerant phenotypes. Conclusions: Our data indicate that although distinct B-cell subsets are clearly affected by OVA-induced allergic airway inflammation and respiratory tolerance, B-cells are not essential for tolerance development in mice.
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Abstract No. 273 Extracellular microRNAs in pediatric asthma, the DZL All Age Asthma Cohort (ALLIANCE) Christine Happle 1,* , Ke Xiao 1, Franziska Wert 1, Janika Viereck 1, Jirmo Adan 1, Anika Habener 1, Christian Dopfer 1, Mareike Price 1, Malik Aydin 1, Anna-Maria Dittrich 1, Bianca Schaub 2, Oliver Fuchs 2, Markus Weckmann 3, Thomas Thum 1, Erika von Mutius 2, Matthias Kopp 3, Gesine Hansen 1, and ALLIANCE Study Group 4 1
Hannover Medical School University Children´s Hospital Munich 3 University Campus Schleswig Holstein, Lübeck 4 ALLIANCE Study Group *Presenting author 2
Introduction: Ubiquitously expressed microRNAs (miRNAs) are highly abundant gene regulators, which are differentially regulated in various pulmonary diseases, amongst them allergic asthma. To disclose novel diagnostic markers and therapeutic approaches in childhood asthma, we investigate specific miRNA alterations in pediatric allergic asthma and wheeze. Methods: Extracellular plasma miRNAs were extracted by standardized protocols from pediatric participants of the DZL All Age Asthma Cohort (ALLIANCE). In a first screening approach, n=80 matched case/control samples were selected for extracellular miRNA screenings. Cases were selected based on following criteria: age (6 months to 12 years), phenotype (wheezing (<6yrs) or asthma (>6yrs)) and atopy (spec. IgE >0.7 for at least one aero- or food allergen). Controls were matched for gender, recruitment center and age (tolerance 12 months). Screening for differentially regulated miRNAs was performed by array cards (n=754 miRNAs). Results: The mean age of n=80 participants included into the first screening was 5.7 yrs (3.3 yrs for wheezers and 8.1 yrs for asthmatics). 55% of these children were female (wheezers: 50%, asthmatics: 60%). Array card screening yielded high quality results which were normalized according to global miRNA expression and are currently being further analyzed. Conclusion: Within the pediatric arm of ALLIANCE, we collected >400 plasma miRNA samples. N=80 of these were screened for the regulation of n=754 extracellular miRNAs. Statistical analyses of data from this screening approach are ongoing. Target miRNAs differentially regulated in this first subset of patients will then be further validated by [1] qPCR in a larger set of probands, [2] in vitro and murine disease models and [3] analysis in harmonized cohorts such as the adult arm of ALLLIANCE or the in other Disease Areas (DA) such as the DA CF cohort.
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Abstract No. 282 THE CROSS-TALK BETWEEN AIRWAY EPITHELIUM AND IMMUNE CELLS IN ALLERGY RESEARCH Ulrich Zissler 1,* , Thomas Bahmer 2, Matthias Kopp 3, Gesine Hansen 4, Erika von Mutius 5, Klaus F. Rabe 2, Oliver Fuchs 5, Markus Weckmann 3, Henrik Watz 2, Anne-Marie Kirsten 2, Frauke Pedersen 2, Adam Chaker 6, and Carsten Schmidt-Weber 1 1
Center for Allergy & Environment (ZAUM), Technical University and Helmholtz Center Munich 2 Zentrum für Pneumologie und Thoraxchirurgie, LungenClinic Grosshansdorf GmbH 3 Pediatric Pneumology & Allergology, University Medical Center Schleswig-Holstein, Campus Centrum Lübeck 4 Zentrum für Kinderheilkunde und Jugendmedizin der Medizinischen Hochschule Hannover 5 Pediatric Allergology, Klinikum der Universität München 6 ENT-Department, Klinikum rechts der Isar of Technical University Munich / Center for Allergy & Environment (ZAUM), Technical University and Helmholtz Center Munich *Presenting author
BACKGROUND: Specific cytokine patterns (Th2-high vs. Th2-low) lead to immunological differentiation of airway epithelium into so-called E1/E2 cells. These cells are involved in mechanisms of sensitization, airway remodeling and resolution. Epithelial differentiation into E1/E2 cells is mirrored both in the upper and lower airways, and therefore nasal secretions could be useful non-invasive biosamples for investigation of distinct biomarker levels in different asthma phenotypes. METHODS: Biomarker levels in nasal secretions of 119 patients in the adult arm of the ALLIANCE cohort were measured by high-sensitive Multi-Array technology, a plate-based electrochemiluminescence detection technology. Mann–Whitney tests were used to determine significant differences between protein levels of patient groups. RESULTS: Levels of IL-1α, IL-8, IL-10, TNF-α, and CCL-26 were detectable in nasal secretions of all patients with asthma, while levels of IL-4, IL-22, IL-33, TSLP, G-CSF, and Periostin were beneath the lower limit of detection in about 5%-20% of patients. The amount of sputum eosinophils was significantly associated with increased levels of typical Th2 cytokines (i.e. IL-4, IL-5, and IL-13) in nasal secretions of asthmatic patients. Patients with predominantly allergic asthma (symptoms due to aeroallergens and allergic rhinoconjunctivitis) compared with non-allergic asthma had significantly different levels for IL-1α, TNF-α, IL-22, IL-4, IL-33, and TSLP. Patients with severe asthma compared to mild-to moderate asthma showed significantly different levels for CCL-26, IL-10, Il-1α, IL-8, TNF-a, IL-4, G-CSF, IL-33, Periostin, and TSLP. CONCLUSION: Cytokine levels in nasal secretions are reasonably detectable in adult patients with asthma, levels of typical Th2-cytokines in the upper airways correspond with markers of Th2-inflammation in the lower airways. Cytokine patterns measured in nasal secretions differ between clinical asthma phenotypes, supporting the methodological significance of these non-invasive biosamples, and warrant detailed analyses in the adult and pediatric cohort alike to better understand the cross-talk between airway epithelium and immune cells in asthma.
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Abstract No. 283 Towards Standardized Airway Hyperresponsiveness Measurements in Mice Winfried Möller 1, Andreas Stelzl 1, Oana V. Amerie 1, Oliver Eickelberg 1, Ali Ö. Yildirim 1, and Otmar Schmid 1,* 1
Comprehensive Pneumology Center Munich *Presenting author
Rationale: Airway hyperresponsiveness (AH) often represents a primary outcome in respiratory studies . The measurement protocol involves aerosolized delivery of a bronchoconstrictor (methacholine, MCH). It has been shown that the delivery protocol (respiratory and nebulizer parameters) affects the measured AH making it difficult to compare different studies on an absolute scale. Here we hypothesize that standardized AH measurements are possible, if the inhaled instead of the nominal MCH dose is used for AH measurement. Methods Aerosolized delivery of saline or MCH solutions were performed with a flexivent system (Legacy, EMKA/Scireq) for lung function measurements in mice. The inhaled aerosol dose was determined gravimetrically for a wide range of delivery protocols (different nebulizers, nebulizer settings, respiratory parameters; 60 different settings, n=3-5 each). Moreover, AH measurements in healthy wild-type C57BL/6 mice were performed. Results and Discussion The inhaled aerosol dose varied between 5 and 50% of the invested dose depending on various key parameters (nebulizer: output rate, droplet diameter, duty cycle; respiratory conditions). Here we introduce a method, which allows prediction of the inhaled aerosol dose directly from these readily known key parameters (R2 >0.85). In a proof-of-concept study we measured AH for identical mice (wild type C57BL/6) using two different MCH delivery protocols with 8.4% and 40.4% aerosol delivery efficiency. Presenting AH in terms of MCH concentration (standard method) suggested that the mice examined with the more efficient MCH delivery protocol were more hyperresponsive than the other ones. Obviously, this is a measurement artefact, since both AH measurements were performed with the same mouse strain. Conclusion: As these apparent differences in AH disappeared, if the data were presented in terms of inhaled MCH dose rather than MCH concentration, we conclude that standardized AH measurements on an absolute scale can be performed by switching from nominal MCH concentration to inhaled MCH dose.
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Abstract No. 288 Analysis of cytokine levels using the Bio-Plex multiplex system in the pediatric arm of the ALLIANCE cohort Markus Weckmann 1,* , Martin Pech 1, Christine Happle 2, Oliver Fuchs 3, Rösler Barbara 3, Dominik Thiele 4, Inke König 4, Gesine Hansen 2, Erika von Mutius 3, Matthias V. Kopp 1, and the ALLIANCE Study Group 5 1
University of Lübeck, Children´s Hospital, UKSH Campus Lübeck University Hospital of the Medizinische Hochschule Hannover (MHH) 3 Dr. von Hauner Children’s Hospital, Ludwig Maximilians University, Munich 4 University of Lübeck, Institute of Medical Biometry and Statistics 5 Member of the German Center for Lung Research *Presenting author 2
Introduction: Asthma is the most prevalent chronic respiratory disease in childhood, and population-based studies suggest that asthma is a syndrome rather than a single disease entity. Phenotypic differentiation is key to delineate underlying pathomechanisms and stratification of clinical assessment and therapy. Aims and objectives: In this study, we evaluated the use of a multiplex cytokine bead ELISA to determine phenotype specific cytokine profiles in unstimulated blood samples of children with new-onset of asthma/recurrent wheeze versus established disease in our multicenter cohort (pediatric arm of ALLIANCE). Methods: A cohort of children (n= 298) with either established diagnoses or new-onset asthmatic disease/recurrent preschool wheeze was investigated by standardized symptome questionnaire and for cytokine levels in peripheral blood samples. Aliquots of 15µL per patient from sera were analyzed using the human cytokine 27-plex (Bio-Rad) Standard curve fitting and cytokine level determination were performed with Bio-Plex Manager 6.0. Cytokine levels were analysed using principal component analysis (PCA) with factor ROC analysis and t-test (wheeze/asthma vs. healthy, whereby direction of change and p-value of least significant test is indicated). Results: Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin (IL)15 were not detected. Levels of IL-4 (down, p<0.0001), IL-8 (down, p<0.05), TNF-a (down, p<0.05), IL-12(p70) (up, p<0.05) and INF-g (down, p<0.01) were significantly different in both wheezers and asthmatics (vs. healthy). Factors (F) 1 to 7 together explained >60% of variation aggregating cytokines in F1 (G-CSF, IL-9, IL-7, IL-17A, VEGF, IL-12(p70), IL-13, FGF-basic), F2 (IL-8, Eotaxin, PDGF-b, TNF-a, IL-1b), F3 (IL-1ra, IL-10, IL-6), F4 (RANTES), F5 (MIP-1b, MIP-1a), F6 (IL-5), F7 (IP-10). Conclusion: Our study confirmed previous reports of differential cytokines expression in asthmatic children (1) and PCA aggregation of multiplex cytokines extends their deployment beyond single cytokine analyses for specific phenotype classification.
(1) Pukelsheim, K., Stoeger, T., Kutschke, D., Ganguly, K. & Wjst, M. Cytokine profiles in asthma families depend on age and phenotype. PLoS ONE 5, e14299 (2010).
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Abstract No. 290 Attenuated AHR and migration of dendritic cells to the lungs and mediastinal lymph nodes in absence of IL-17 in experimental asthma Adan Chari Jirmo 1,* , Mandy Busse 1, Christine Happle 1, Kathleen Deluege 1, Anika Habener 1, Jelena Skuljec 1, Immo Prinz 1, and Gesine Hannover 1 1
Hannover Medical School *Presenting author
Background: Several studies suggest an involvement of Th17 cells and related cytokines in the pathology of allergic asthma. However, cellular mechanisms involved in induction, maintenance and pathophysiology of asthma are multifaceted. IL-17 isoforms have been shown to be involved in the modulation of asthma both in humans and in murine models of allergic asthma, but the exact IL-17A and F dependent mechanisms in asthma still remain to be elucidated. Objective: Our aim was to decipher the cellular mechanisms involved in IL-17 dependent modulation of allergic asthma. Methods: WT, IL-17 AF-/- and IL-17F-/- mice were subjected to a murine experimental asthma model in which Ovalbumin was systemically and intranasally applied. Airway hyperresponsiveness, lung inflammation, antigen-specific IgG/IgE levels, cytokine levels, and dendritic cell (DC) trafficking was assessed. In Wt mice, anti-IL-17A specific monoclonal antibodies (mAbs) were used to neutralize IL-17A. Results: In IL-17AF-/- double knockout animals, allergic airway hyperresponsiveness determined through measuring invasive lung function, H&E assessed eosinophilic airway inflammation and mucus hypersecretion were significantly decreased. This was associated with a significant reduction of pulmonary and bronchial lymph node DC influx and decreased antigen specific Th2 responses in the draining lymph nodes of allergic IL-17AF-/- mice. Experiments employing IL-17F-/- single knockout and IL-17A neutralizing mAbs confirmed that IL-17A but not IL-17F is the decisive cytokine modulating allergic airway inflammation, whereas absence of IL-17F does not influence the asthma like phenotype in our model. Conclusions: Our data show that IL-17A plays an important role in DC migration under inflammatory conditions and is central in experimental asthma.
Submitted as: Presentation 253 words of 300 possible words
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Abstract No. 010 Neutrophil Extracellular Traps are Regulated by Chemokine Receptor CXCR2 in Chronic Obstructive Pulmonary Disease Frauke Pedersen 1,* , Benjamin Waschki 1, Sebastian Marwitz 2, Torsten Goldmann 2, Anne Kirsten 3, Klaus F. Rabe 1, Anna Malmgren 4, Kai Richter 5, Mohib Uddin 5, and Henrik Watz 3
1
LungClinic Grosshansdorf Research Center Borstel 3 Pulmonary Research Institute at LungClinic Grosshansdorf 4 Respiratory, Inflammation & Autoimmunity iMED & Global Medical Affairs, AstraZeneca, R&D Mölndal, Sweden 5 Respiratory, Inflammation & Autoimmunity iMED & Global Medical Affairs, AstraZeneca, R&D Mölndal, Swede *Presenting author 2
Neutrophil extracellular traps (NETs) contribute to airway inflammation in chronic obstructive pulmonary disease (COPD), yet the molecular mechanisms underlying their formation remain unclear. Therefore, we investigated NET formation in blood neutrophils and sputum derived from COPD patients using the selective CXCR2 receptor antagonist, AZD5069 ex vivo. Induced sputum and peripheral blood neutrophils were collected from clinically stable COPD patients. NET formation was stimulated in isolated blood neutrophils by incubation with sputum supernatant in absence or presence of 100 µM AZD5069. Spontaneous NET formation was assessed in sputum-derived neutrophils with or without100 µM AZD5069. NETs were visualized by immunofluorescence staining of histone H1, neutrophil elastase, DNA and quantified using ImageJ software (NIH USA) by selecting NETs as regions of interest (ROI). 171 blood and sputum samples of 12 COPD patients [mean age 67.8 years; FEV1 59.9% predicted] were analyzed. NET formations in each group, i.e. 1) unstimulated blood neutrophils, 2) stimulated blood neutrophils, 3) stimulated blood neutrophils with AZD5069, 4) sputum-derived neutrophils and 5) sputum-derived neutrophils with AZD5069, was measured three times in each patient and individual mean values were used for group comparison. Compared to unstimulated blood neutrophils (ROI median (interquartile range (IQR) of 49,680 (25,750-84,454) arbitrary units (AU)), NET formations were enhanced by sputum supernatant in blood neutrophils (ROI median (IQR) of 278,223 (187,732-498,593) AU; p = 0.002), while the increase in NET formations in stimulated blood neutrophils with AZD5069 was significantly lower compared to stimulated neutrophils without AZD5069 (ROI median (IQR) of 226,477 (110,872-260,184) AU; p = 0.010). Spontaneous NET formation in sputum with AZD5069 was significantly lower compared to sputum without AZD5069 (ROI median (IQR) of 867,410 (562147-1537469) AU versus 1,458,053 (903,763-1,611,397) AU; p = 0.034). These results provide evidence for a pathobiological role of CXCR2 in regulating induced and spontaneous NET formation in COPD.
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Abstract No. 012 Changes of cardiac function, morphology and wall stress in COPD during 1.5 years of COSYCONET follow up Peter Alter 1,* , Henrik Watz 2, Tobias Welte 3, Sven Gläser 4, Holger Schulz 5, Robert Bals 6, Sandra Söhler 1, Claus F. Vogelmeier 1, and Rudolf A. Jörres 7 1
University of Marburg Pulmonary Research Institute at LungClinic Grosshansdorf 3 Hannover Medical School 4 University of Greifswald 5 Helmholtz Center Munich, Institute of Epidemiology I, German Research Center for Environmental Health 6 Saarland University Hospital 7 Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig Maximilians University *Presenting author 2
Introduction. Based on a large data set from the German COPD cohort COSYCONET, it was previously shown by structural equation modeling (SEM) that the extent of lung function impairment and hyperinflation is associated with increased cardiac wall stress. Methods. For evaluation of longitudinal changes, follow up data obtained at visit 3 were compared with data from the baseline visit. Applying predefined criteria of completeness and quality, 773 patients were eligible for visit 1 and 3, with a mean (SD) follow-up duration of 575±48 days. Results. The study population comprised 92/85/320/229/47 patients of GOLD categories 0 to IV. The best fitting and most consistent SEM of visit 1 comprising two latent variables was also applicable to visit 3 (p≤0.005) and means values of visit 1 and 3 (p<0.001). Regarding the longitudinal changes from visit 1 to 3, a decline of FEV1%pred (p<0.001) and FEV1/FVC (p<0.001) and an increase of Reff (p<0.001) and ITGV%pred (p=0.0031) was observed. In parallel, a decrease of LV mass (p=0.012) and a trend towards an increase of enddiastolic volume (p=0.238) occurred, while the endsystolic LV volume remained virtually unchanged (p=0.635). Enddiastolic wall stress increased significantly (p<0.001), endsystolic wall stress by trend (p=0.228). The increase of enddiastolic LV wall stress during follow up tended to be greater in GOLD II to IV than in GOLD 0 to I (p=0.056). Conclusions. The decline of lung function parameters was in parallel with a decrease of LV mass and an increase of wall stress. The increase of enddiastolic wall stress appeared to be more pronounced in higher COPD stages, and LV mass decreased more than expected from epidemiological data. These findings suggest that the next two follow-up visits of COSYCONET will show changes of a magnitude that allows to analyze the associations between echocardiographic and lung function parameters longitudinally.
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Abstract No. 026 An epigenetic cell-of-origin approach to chronic obstructive pulmonary disease Reinhard Liebers 1,* , Michelle Paulsen 2, Marcus Mall 2, and Christoph Plass 1 1
German Cancer Research Center (DKFZ) Universitätsklinikum Heidelberg *Presenting author 2
Chronic obstructive pulmonary disease (COPD) is a progressive, incurable lung disease and a leading cause of mortality world-wide. Nevertheless, genetic analyses have only found genetic factors with low effect sizes. In order to pinpoint the molecular mechanism behind COPD we are using several genome-wide sequencing approaches on defined homogeneous cell populations from the βENaC-overexpressing mouse model. These mice show key characteristics of COPD, including mucous obstruction, inflammation, and emphysema. We address genome-wide methylation with tagmentation-based wholegenome bisulfite sequencing (T-WGBS), genome-wide chromatin accessibility and transcription factor footprinting by the Assay for Transposase-Accessible Chromatin sequencing (ATAC-Seq), and coinciding gene expression changes by ultra-low input RNASeq from sorted cells. Each analysis was carried out for populations that likely play a pivotal role in COPD development and progression, namely activated alveolar macrophages, type II alveolar cells, and ciliated cells. Determining the disease initiating cells will contribute to our understanding of COPD development and will allow improved diagnosis, prognosis, and treatment of this debilitating disease.
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Abstract No. 029 Identification of target genes in COPD-tissues: a molecular-clinical-histopathologic puzzle Lena Heinbockel 1, Ole Ammerpohl 2, Daniel Drömann 3, Henrik Watz 4, Klaus F Rabe 5, Sven Perner 1, Christian Kugler 5, Sebastian Marwitz 1, Karoline Gaede 1, and Torsten Goldmann 1 1
Research Center Borstel Christian-Albrechts-Universität zu Kiel 3 UKSH Universitätsklinikum Schleswig-Holstein 4 Pulmonary Research Institute 5 Lungenclinic Grosshansdorf 2
Chronic Obstructive Pulmonary Disease (COPD) is a continuous inflammatory manifestation in the lung, leading to an irreversible expiratory airflow limitation. Despite many efforts to identify the underlying mechanisms of COPD progression, real breakthroughs are still missing. Thus, an adequate therapy is still unavailable, although there have been some improvements in COPD management. The aim of this study is to determine candidate genes involved in the progression of the disease. Therefore, we performed transcriptome and methylome analyses of tumor-free lung tissues from patients suffering from COPD. On the basis of this analysis, we further characterised seven candidate genes by histology and quantitative real-time PCR. The corresponding proteins could be assigned to distinct cell types and some were found to be significantly regulated in COPD lungs. Aiming to find a target point to combat the disease, we will elucidate the mechanisms in which these proteins are involved.
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Abstract No. 046 microRNAs constitute a negative feedback loop in Streptococcus pneumoniae induced macrophage activation Kathrin Griss 1, Wilhelm Bertrams 1, Alexandra Sittka-Stark 1, Kerstin Seidel 1, Christina Stielow 1, Stefan Hippenstiel 2, Norbert Suttorp 2, Martin Eberhardt 3, Jochen Wilhelm 4, Julio Vera 3, and Bernd Schmeck 5,* 1
Philipps-University Marburg, Institute for Lung Research Charité – University Medicine Berlin, Department of Infectious Diseases and Respiratory Medicine 3 Erlangen University Hospital, Department of Dermatology, Laboratory of Systems Tumor Immunology 4 Justus-Liebig-University, Internal Medicine Clinic II 5 Philipps-University Marburg, Institute for Lung Research; University Medical Center Giessen and Marburg, Department of Medicine, Pulmonary and Critical Care Medicine *Presenting author 2
Streptococcus pneumoniae causes high mortality as a major COPD- and pneumoniaassociated pathogen. Control of innate immunity is necessary to prevent organ damage. We assessed the role of microRNAs (miRNAs) as regulators in pneumococcal infection of human macrophages. Exposure of primary blood-derived human macrophages with pneumococci resulted in transcriptional changes in several gene clusters and a significant deregulation of 10 microRNAs. Computational network analysis retrieved miRNA-146a as one putatively important regulator of pneumococci-induced host cell activation. Its induction depended on bacterial structural integrity and was completely inhibited by blocking Toll-like receptor 2 (TLR-2) or depleting its mediator MyD88. Furthermore, induction of miRNA-146a release did not require the autocrine feedback of interleukin 1β and tumor necrosis factor α released from infected macrophages, and it repressed the TLR-2 downstream mediators IRAK-1 and TRAF-6, as well as the inflammatory factors cyclooxygenase 2 and interleukin 1β. In summary, pneumococci recognition induces a negative feedback loop, preventing excessive inflammation via miR-146a and potentially other miRNAs.
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Abstract No. 047 Global profiling of bronchial epithelial cell response to Streptococcus pneumoniae infection André Wesener 1, Wilhelm Bertrams 1, Xin Lai 2, Kristin Surmann 3, Sascha Blankenburg 3, Uwe Völker 3, Julio Vera 2, Bernd Schmeck 4,* , and Evelyn Vollmeister 1 1
Philipps-University Marburg, Institute for Lung Research/iLung University Hospital Erlangen, Department of Dermatology, Laboratory of Systems Tumor Immunology 3 Ernst-Moritz-Arndt University Greifswald, Interfaculty Institute for Genetics and Functional Genomics, Department of Functional Genomics 4 Philipps-University Marburg, Institute for Lung Research; University Medical Center Giessen and Marburg, Department of Medicine, Pulmonary and Critical Care Medicine *Presenting author 2
Streptococcus pneumoniae is a gram-positive bacterium and an important pathogen causing COPD exacerbations and pneumonia. The process of streptococci infections is influenced by host and pathogen factors. Since the nowadays targeted factors possess limited therapeutic potential, we further investigated this host-pathogen-relationship. Therefore, we performed high-throughput profiling of proteins, mRNAs and miRNAs of a human bronchial epithelial cell line (BEAS-2B) infected with S. pneumoniae D39 for 9 h or 16 h compared to mock-infected and Lipoprotein-stimulated cells. Thereby, we focused on the interaction of mRNAs/proteins with microRNAs (miRNAs) that are crucial posttranscriptional regulators of gene expression and inflammatory host response in infectious diseases. The proteomic analysis was performed via SILAC method. In at least 2 of 3 biological replicates we detected and quantified 2315 proteins. Filtering for log fold change ±1.5 and significance (p<0.05) resulted in 18 proteins upregulated at 9 h and 16 h post treatment (hpt) (e.g. SOD2), and 12 proteins downregulated, respectively. Another 8 proteins changed their status of regulation between time points. Microarrays were used to examine mRNA expression; 11,500 were detected. Using the aforementioned filter criteria, we found 398 mRNAs significantly deregulation after treatment, 311 up- and 87 downregulated (e.g. IL-6). Furthermore, 8 genes, encoding for some of the proteins mentioned above, were also deregulation on mRNA level (e.g. SOD2). The miRNA sequencing and annotation revealed 175 significantly changed miRNAs. We observed several miRNAs deregulated in time and/or treatment dependent manner. In general, we detected more deregulated miRNAs in lipoprotein stimulation 9 hpt compared to 16 hpt. In contrast, we observed more miRNAs after S. pneumoniae infection 16 hpt than 9 hpt. The well-known miR-146a was one of few commonly deregulated. These data will now be combined to create a profound bioinformatics model of the human host response to S. pneumoniae.
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Abstract No. 052 Costs and health-related quality of life in COPD patients with alpha-1-antitrypsin deficiency: results of the COSYCONET COPD cohort Florian M. Karl 1, Rolf Holle 1, Robert Bals 2, Tim Greulich 3, Rudolf A. Jörres 4, Annika Karch , Stefan Karrasch 1, Armin Koch 5, Reiner Leidl 1, Holger Schulz 1, Claus Vogelmeier 3, and Margarethe E. Wacker 1,*
5
1
Helmholtz Zentrum München GmbH Saarland University Hospital 3 University of Marburg 4 Ludwig-Maximilians-Universität München 5 Medizinische Hochschule Hannover *Presenting author 2
Background: A deficiency of alpha-1-antitrypsin represents a rare genetic cause of COPD. Data from the COSYCONET COPD cohort offers the possibility of comparing costs and health-related quality of life (HRQL) between COPD patients with and without alpha-1-antritrypsin deficiency (AATD) as there is a lack of evidence on these aspects. Methods: Data from the baseline visit of the COSYCONET COPD cohort were used to compare 146 COPD patients with AATD (thereof 108 with augmentation therapy (AT)) and 2,034 COPD patients without AATD. Self-reported information on healthcare utilization, work absence and premature retirement were used to calculate societal costs in the year 2012. HRQL was assessed by the COPD Assessment Test (CAT), St.-George`s Respiratory Questionnaire (SGRQ) and the generic EQ-5D-3L questionnaire. The association of AATD (with or without AT) with costs and HRQL was analysed by generalized linear regression models while controlling for COPD GOLD grade, age, sex, education, smoking status, BMI and comorbidity as potential confounders. Results: Adjusted annual mean healthcare costs were €6.074 for AATD patients without AT, €6.626 for AATD patients with AT (excluding costs of AT) and €7.462 for COPD patients without AATD. Augmented AATD patients showed significantly higher outpatient, but lower inpatient costs compared to COPD patients without AATD. The group of AATD patients had significantly lower medication costs than the control group (when excluding costs of AT). Costs for work absence and premature retirement as well as generic and disease-specific HRQL scores did not differ between the groups. Discussion: Apart from the very high costs of AT, COPD patients with AATD showed slightly lower healthcare costs than COPD patients without AATD within the COSYCONET cohort. This cross-sectional analysis could not identify any advantages of AT regarding costs or HRQL. However, longitudinal analyses are necessary for a better contribution to the controversy about the cost-effectiveness of AT.
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Abstract No. 054 The real life data registry BeoNet - Leadoff results of the first use case: COPD Heidrun Lingner 1,* , Ines Aumann 2, Katrin Krüger 1, Anna Nickel 1, Margarethe Wacker 3, Reiner Leidl 3, Michael Kreuter 4, J.-Matthias von der Schulenburg 2, and Tobias Welte 1 1
Medizinische Hochschule Hannover Leibniz University of Hannover 3 Helmholtz Zentrum München GmbH 4 Thoraxklinik Heidelberg *Presenting author 2
Background Healthcare research needs real-life-data, but they are scarce. The BeoNet-Registry (“Beobachtungspraxen-Netz-Register”) compiles the complete information from electronic patient records (eR) of primary care practices (GPs and pneumologists) and links them with patient-reported outcomes. First use case: assessment and analysis of information about patients with COPD. Research question Are there any differences in demographics or absences from work between patients with COPD and without COPD or between the target-populations from the different sites or the literature reports? What impediments occur when working with data from eR? Methods Systems for real time pseudonymous routine data provision from GPs and pneumologists from Hanover, Munich and Heidelberg were set up and tested. eR data, including diagnoses, treatments, procedures and medication were transferred via a standardized secure interface and compiled for analysis. The results of paper based health related quality of life questionnaires were linked to eR information. First descriptive analyses assessed the incidence, age, sex and comorbidities of COPD+ and COPD- and their respective absences from work. Literature searches provided relevant publicly available data. Results Currently the weekly updated database (DB) contains 98,409 patient-IDs (female: 54 %). The BeoNet-incidence of COPD is 4,5% (4455 IDs). Our results demonstrate that COPD patients in primary care are older (COPD+: 69,1%; COPD-: 52,4%) and more often males (49,56 %) compared to patients without COPD (45,7%), are absent from work more frequently and most of them have more comorbidities as COPD-. Half of COPD+ have 3 comorbidities (median) (COPD-: 2); on average 5 additional illnesses (resp 4). More detailed results and the challenges encountered while working with real-life data will be presented at the conference. Conclusions The demographics fit well with the literature, supporting the reliability of the registry-data. Further research will analyze the additional information on quality of life of COPD+ and subgroup specific comorbidities correlations.
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Abstract No. 060 Transfer factor for carbon monoxide in patients with COPD and diabetes: Results from the German COSYCONET cohort Kathrin Kahnert 1,* , Tanja Lucke 1, Frank Biertz 2, Andreas Lechner 1, Henrik Watz 3, Peter Alter 4, Robert Bals 5, Jürgen Behr 1, Rolf Holle 6, Rudolf Maria Huber 1, Stefan Karrasch 1, Beate Stubbe 7, Margarethe Wacker 8, Sandra Söhler 4, Emiel Wouters 9, Claus Vogelmeier 4 , and Rudolf Jörres 1 1
University of Munich Hannover Medical School 3 Pulmonary Research Institute at LungenClinic Grosshansdorf 4 University of Marburg 5 Saarland University Hospital 6 Helmholtz Zentrum München (GmbH) 7 University Medicine Greifswald 8 Helmholtz Zentrum München (GmbH) - German Research Center for Environmental Health 9 Maastricht University Medical Center *Presenting author 2
Background An impairment of CO diffusing capacity has been shown in diabetic patients without lung disease. We analyzed how diffusing capacity in patients with COPD is affected by the concurrent diagnosis of diabetes. Methods Data from the initial visit of the German COPD cohort COSYCONET were used for analysis. 2575 patients with complete lung function data were included, among them 358 defined as diabetics with a reported physician diagnosis of diabetes and/or specific medication. Pairwise comparisons between groups and multivariate regression models were used to identify variables predicting the CO transfer factor (TLCO%pred) and the transfer coefficient (KCO%pred). Results COPD patients with diabetes differed from those without diabetes regarding lung function, anthropometric, clinical and laboratory parameters. Moreover, gender was an important covariate. After correction for lung function, gender and body mass index (BMI), TLCO%pred did not significantly differ between patients with and without diabetes. The results for the transfer coefficient KCO were similar, demonstrating an important role of the confounding factors RV%pred, TLC%pred, ITGV%pred, FEV1%pred, FEV1/FVC, age, packyears, creatinine and BMI. There was not even a tendency towards lower values in diabetes.
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Abstract No. 061 The effect of voluntary activity on obesity-associated pathological changes of the lung Julia Schipke 1,* , Elena Lopez-Rodriguez 1, Tobias Steingrüber 1, Nancy Ahrendt 1, and Christian Mühlfeld 1 1
Institute of Functional and Applied Anatomy *Presenting author
Obesity is a growing pandemic health problem and linked to a wide range of respiratory conditions including asthma and chronic obstructive pulmonary disease. Physical exercise has beneficial effects for patients suffering from a variety of diseases; however, the distinct mechanisms of action are diverse and not fully elucidated yet. We hypothesize that in obese mice voluntary activity reduces the body weight, improves the lung function and diminishes pulmonary structural changes. Male C57BL/6N mice were kept on control diet (CD) or high fat diet (HFD) and were housed in cages with or without running wheels resulting in the experimental groups CD, CD-Active, HFD, HFD-Active. Weight, food consumption and voluntary activity were weekly recorded. After 30 weeks, lung function testing was performed and left lungs were processed and analyzed according to design-based stereological standards. HFD resulted in significantly higher body weights in comparison to CD, which was not influenced by voluntary activity. Neither the feeding nor the presence of running wheels affected functional pulmonary parameters. However, in HFD animals the left lung volume was significantly increased what was due to both higher parenchyma and non-parenchyma volumes. Within the HFD parenchyma, septal volume, septal surface and septal thickness were enlarged in comparison to CD animals. In HFD-Active animals, left lung volume, parenchyma volume as well as septal volume and thickness were significantly reduced in comparison to HFD animals. We conclude that in alimentary obese mice, voluntary activity does not affect body weight or lung function, but has a normalizing effect on parenchyma volume as well as septal volume and thickness indicating an impact on obesity-related structural changes in the lung.
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Abstract No. 063 Breath volatile organic compounds (VOC) in COPD – first results from a large validation trial Olaf Holz 1, Arne Gaida 1, Bianca Lavae-Mokhtari 1, Lena Kruse 1, Sven Schuchardt 1, and Jens M Hohlfeld 1 1
Fraunhofer ITEM
Introduction In our recently completed dual center DZL study with a total of 190 patients (Gaida et al. 2016) we found evidence for COPD related breath VOCs. It was the aim of this study to assess the longitudinal variability of these VOCs and to study their relationship to neutrophilic airway inflammation assessed by sputum analysis. Methods Currently, 18 healthy non-smokers, 22 ex-smoking COPD patients (13 GOLDI/II, 9 GOLD III/IV), 5 healthy smokers, and 36 smokers with COPD (30 GOLDI/II, 6 GOLD III/IV) were included into this follow-up study. 26 of these subjects also participated in the previous study (median days between visits = 309) and from 74 subjects a sputum sample could be collected. Breath and room-air collected on Tenax tubes was analyzed using TD-GC-MS. Results Within each group of subjects, we found VOC mean levels that were comparable to the previous study. Larger differences were observed for breath VOCs considered to be roomair or environment related. Smoking related VOCs showed again higher levels in actively smoking subjects and the longitudinal analysis an unexpected good reproducibility for lifestyle related VOCs. For 3/7 COPD related VOCs we could confirm differences between COPD patients and controls. Significant relationships between VOC levels and sputum neutrophils were observed, but with low correlation coefficients (r<0.4). Discussion Our study provides data on the longitudinal variability of breath VOC levels in COPD and the influence of environmental factors. We show the limitations for breath “pre-conditioning“ by inhalation through charcoal filters and the importance to identify room air related VOCs in breath, which could substantially affect sensor based VOC analysis data. We demonstrate that it is important to validate VOC data from cross-sectional studies and show only a limited relationship between sputum neutrophils and the currently available breath VOC spectrum.
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Abstract No. 087 Chromatin modulator HMGN5 regulates cellular apoptosis of lung epithelial cells in pathogenesis of COPD Rim Sabrina Jahan Sarker 1,* , Thomas M Conlon 1, Julian Dorer 1, Gerald Burgstaller 1, Lisa Merthan 1, Valerie Gailus-Durner 2, Helmut Fuchs 2, Martin Hrabě de Angelis 2, Michael Bustin 3, Takashi Furusawa 1, Oliver Eickelberg 4, and Ali Önder Yildirim 1 1
Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany 2 German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, Munich, Germany 3 Protein Section, Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland, USA 4 Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver, CO, USA *Presenting author
Chronic obstructive pulmonary disease is characterized by chronic bronchitis, small airway remodeling and emphysema. Emphysema is the destruction of alveolar structures, leading to enlarged air spaces and reduced surface areas. Rising evidence suggest that epigenetic changes contribute to alter gene expressions involved in COPD pathogenesis. High mobility group nucleosome binding domain 5 (HMGN5), a chromatin remodeler competes with linker-histone H1 to bind the chromatin, thus affects cellular transcription, differentiation and cell repair. Here we investigated role of HMGN5 in development and progression of emphysema. The mRNA expression and protein localization of HMGN5 were performed on lung samples from COPD patients and healthy donors. Wild type (WT) C57BL/6) mice exposed to cigarette smoke (CS) for 6 months (500 mg/m3, 2X50 min/day) were analyzed for gene expression by quantitative real time PCR. WT mice and HMGN5 deficient mice oropharyngeally applied with porcine pancreatic elastase, PPE (40U/kg) were analyzed 28 days later lung function, gene expression and histology. RNA sequencing data from COPD patient samples demonstrated a reduced HMGN5 expression in lungs compared to healthy individuals (p<0.01). Immunohistochemistry revealed that HMGN5 is located in bronchial epithelial cells and alveolar epithelial type II (ATII) cells. A downregulation of HMGN5 was prominent in CS-exposed WT mice compared to filtered air-exposed control mice (p<0.05). PPE-treated WT mice showed reduced expression of HMGN5 after 28 days. HMGN5 deficient mice had greater impaired lung function compared to WT controls following PPE exposure (lung compliance 0.084±0.007 ml/cmH2O vs 0.070±0.006 ml/cmH2O respectively, p<0.001) with greater evidence of emphysema development upon histological analysis. Additionally, PPE-treated HMGN5 deficient mice showed significant number of Caspase-3 positive (ATII) cells compared to PPE-treated WT mice (p<0.01). The data demonstrated an association of epigenetic modulator HMGN5 with emphysema development through regulating cellular apoptosis and proliferation suggesting it as a promising therapeutic target to treat emphysema progression.
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Abstract No. 103 NON-CANONICAL NOTCH PATHWAY PARTIALLY REGULATES MACROPHAGES IMMUNE RESPONSE IN COPD Carolina Ballester López 1,* , Thomas M. Conlon 1, Zeynep Ertüz 1, Oliver Eickelberg 2, and Ali Önder Yildirim 1 1
Helmholtz Zentrum University of Colorado, Denver *Presenting author 2
Chronic Obstructive Pulmonary Disease (COPD) is mainly caused by exposure to exogenous particles, commonly cigarette smoke (CS). Chronic inflammation is a key factor in intensifying disease severity, but which signaling pathways regulate this response is not well understood. A genome-wide association study (GWAS) has reported that DNER (Delta/Notch-like Epidermal Growth Factor (EGF)-related Receptor), a novel non-canonical Notch ligand, positively correlates with GOLD stages (Hancock et al. PLoS Genet 2012). The aim of this study was to decipher the function of DNER in the immunopathology of COPD using a CS exposed mouse model and patient samples. DNER mRNA expression was quantified in lung tissue from healthy and COPD patients. Immunofluorescence co-staining of DNER with pro-SPC, acta-2, F4/80, Galectin-3, CD31, p63, Krt5, CD45 and CGRP were performed in COPD human and 4 months CS-exposed murine lung tissue. WT and Dner-/- bone marrow-derived macrophages (BMDM) and RAW264.7 cells were treated for 24h with LPS+IFNγ and Dner, Ifnγ, Tnfα, IL12a, IL6 levels were quantified by qPCR. Moreover, human bronchial epithelial (HBE) cells and murine isolated airways (MIA) were treated for 24h and 48h with CS-extract (CSE) or LPS to analyze Dner expression. We found that DNER mRNA expression is increased in COPD patients compared to healthy lungs and mainly localized to macrophages and airways. In addition, Dner was significantly upregulated in LPS+IFNγ-treated BMDM (3.93±1.86 fold change), RAW264.7 (12.26±8.46), 48h CSE-treated HBE (1.8±0.22) and 24h CSE-treated MIA (19.3±8.9) vs. non-treated controls. Interestingly, Dner-/- BMDM showed lower induction of Ifnγ (4.4±3.94 vs. 17.5±28.1) after LPS+IFNγ treatment compared to WT cells respectively. These results illustrated that DNER is increased in COPD patients as well as in CS-exposed mice and regulates IFNγ secretion in macrophages following LPS treatment. This study provides a novel pathway that could offer alternative therapeutic strategies for the treatment of COPD.
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Abstract No. 116 Insights into COPD pathogenesis by 2D cross-omics enrichment analysis of mice and human lung. Thomas Conlon 1,* , Martina Häckl 1, Jörg Bartel 2, Jan Krumsiek 2, Martin Irmler 3, Johannes Beckers 3, Fabian Theis 2, Oliver Eickelberg 4, and Ali Önder Yildirim 1 1
Institute of Lung Biology and Disease / Comprehensive Pneumology Center, Helmholtz Zentrum München 2 Institute of Computational Biology, Helmholtz Zentrum München 3 Institute of Experimental Genetics, Helmholtz Zentrum München 4 Institute of Lung Biology and Disease / Comprehensive Pneumology Center, Helmholtz Zentrum München and Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver *Presenting author
COPD, a leading cause of chronic morbidity and mortality worldwide, is driven by exposure to toxic gases and particles, especially cigarette smoke (CS), resulting in small airway remodeling, chronic bronchitis and emphysema. Our understanding of the pathological mechanisms are progressing, however there is still no curative therapy. We therefore undertook cross-omics integrated enrichment analysis on CS-exposed mice, compared against similar analyses of COPD patient cohorts to identify novel underlying pathological mechanisms for future drug targeting. C57BL/6 mice were exposed to CS or filtered air (FA) for 50min twice/day for 6 months. Lung tissue was used for both transcriptomics and label-free LC-MSMS-based proteomics. Differential expression analysis was undertaken using Limma. Lung tissue transcriptomics data from smokers and COPD patients was taken from NCBI GEO database GSE37768. Proteomics data from Ohlmeier et al. 2016. 2D cross-omics integrated GO-term enrichment analysis was undertaken using InCroMAP. Principle component analysis revealed clear separation in the gene expression profile of FA compared to CS exposed animals. 614 genes and 520 proteins were differentially regulated (adjP<0.05), 44 genes were regulated at both levels. Integrated enrichment analysis revealed an enrichment of 844 GO-terms (P<0.01). Cross-omics integrated enrichment analysis of human data revealed an enrichment of 384 and 361 GO-terms (P<0.01) from never smokers vs current smokers or COPD patients respectively. Analyzing pathways across mice and humans revealed an overlap of 11.1% increasing to 21.2% when comparing mice with current smokers or COPD patients respectively. This included the highly significant pathways: “inflammatory response” and “immune response” providing proof of principle. However, intriguing targets such as “lysosome” and “blood microparticle” along with potential candidates (NAPSA, GC and GSN) were identified. In conclusion, the identification of novel biological pathways common to both COPD patients and the CSexposed mouse, reveals exciting new targets that can be further investigated utilizing existing disease models.
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Abstract No. 123 A common coding variant in SERPINA1 increases the risk for large artery stroke Rainer Malik 1, Therese Dau 2, Evgeniia Edeleva 3, Jessica Götzfried 2, Patrick Wintrode 4, Dieter Braun 3, Dieter Jenne 5,* , and Martin Dichgans 1 1
Institute for Stroke and Dementia Research Institute of Lung Biology and Disease 3 Center for NanoScience 4 Department of Pharmaceutical Sciences 5 Institute of Lung Biology and Disease *Presenting author 2
Common single amino acid variations of proteins are traditionally regarded as functionally neutral polymorphisms as these substitutions are mostly located outside functionally relevant surfaces. In this study, we present an example of a functionally relevant coding sequence variation, which as we show here confers risk for large artery atherosclerotic stroke. The single residue variation M1(A213V) in SERPINA1 (encoding alpha-1-antitrypsin, AAT) is situated outside the protease-reactive inhibitory loop and is found in a ß-turn on the protein surface. We show that the Ala-to-Val exchange in the gate region of AAT alters its functional dynamics towards neutrophil elastase in the presence of complex lipid-containing plasma and also affects the overall structural flexibility of the protein.
Malik, Dau et al. Proc. Natl. Acad. Sci USA 2016, in revision. Dau et al., Quantitative analysis of protease recognition by inhibitors in plasma using microscale thermophoresis. Sci Rep. 2016 doi: 10.1038/srep35413 Submitted as: Presentation 113 words of 300 possible words
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Abstract No. 134 Targeting the mTOR signaling pathway to inhibit lung cell senescence in chronic obstructive pulmonary disease (COPD) Amal HOUSSAINI 1,* , Kanny Kebe 2, Marielle Breau 2, Elisabeth Marcos 2, Shariq Abid 2, Dominique Rideau 2, Christina Lukas 1, Jin Huang 2, Emilie Bizard 2, Pierre Validire 3, Bernard Maitre 2, Valerie Amsellem 2, Silke Meiners 1, and Serge Adnot 2 1
Helmholtz Zentrum München IMRB 3 Institute Mutualiste Montsouris *Presenting author 2
Chronic obstructive pulmonary disease (COPD) is a highly prevalent devastating disease with no curative treatment currently available. Exaggerated lung cell senescence may represent a key process in the pathogenesis of COPD. Here, we explored the role of the mTOR signaling pathway on cell senescence and lung alterations in COPD. Using lung specimens and derived cultured cells (pulmonary vascular endothelial cells (P-ECs) and smooth muscle cells) from patients with COPD and from age- and sex-matched control smokers, we show that cell senescence in COPD is linked to the activation of mTOR and that inhibiting mTOR by low dose of rapamycin prevents cell senescence and inhibits the pro-inflammatory senescence-associated secretory phenotype. To explore whether mTOR activation is causal in lung pathology, we generated mice with constitutive or conditional deletion of the tuberous sclerosis complex heterodimer TSC1 (a negative regulator of the mTOR complex 1), either in smooth muscle cells (SMC). We found that mTOR activation was sufficient to induce an early onset of replicative cell senescence in cultured pulmonary artery SMC from mice with TSC1 deletion. All these effects were prevented by rapamycin treatment. Our results provide evidence for a causal relationship between mTOR activation, cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a new therapeutic target for COPD.
Submitted as: Presentation 212 words of 300 possible words
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Abstract No. 137 Dependence of observed lung T1 relaxation time on echo time and oxygen concentration - a preparatory study Bertram Jobst 1,* , Mark Wielpütz 1, Peter Jakob 2, Hans-Ulrich Kauczor 1, and Simon Triphan 1
1
University Hospital Heidelberg University of Würzburg *Presenting author 2
Objective The T1 relaxation time represents a potential biomarker for the pulmonary health. It both depends on the concentration of dissolved molecular oxygen and differs significantly in value between diseased and healthy lungs. It was also discovered that apparent T1 depends on the measurement echo time (TE). The purpose of this work was to examine the interaction of the dependecies of T1 on both TE and oxygen concentration. Methods A 2D ultra short TE sequence described previously (Triphan et al, JMRI 2015) was used to acquire images at 5 TE along an inversion recovery curve at 21% and 100% oxygen in concurrent measurements. This experiment was performed in 7 healthy volunteers. From the images acquired at varying TE and inversion times, signal was averaged over a ROI and T1(TE) fitted for the entire lungs. Results At 21% O2, median 1/T1(TE1-5)=(950±22, 801±30, 746±22, 748±26, 721±39)μs-1 was found, increasing to 1/T1(TE1-5)=(1042±30, 879±36, 826±23, 813±30, 762±54)μs-1 at 100% O2, a significant change with p<0.001 at TE1-4 and p<0.05 at TE5. 1/T1 at 21% and 100% was both found to decrease with TE. The difference between those values, Δ1/T1(TE1-5)=(95±14, 95±17, 78±19, 65±16, 45±22)μs-1, was also found to decrease with TE. This change was not significant from TE1-3 (p<0.1-0.8), but significant from TE1 to TE4 (p<0.005) and TE5 (p<0.0005). Conclusions Since the observed T1 depends on the TE due to different contributions of extra- and intravascular protons, a specification of the TE used is recommended both for T1 measurements at 21% and 100% O2. Currently, the potential value of T1 as a biomarker for the pulmonary health is further evaluated within a subcohort of COPD patients from the multicentre COSYCONET study.
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Abstract No. 139 Repetitive exposure to cigarette smoke alters the airway structure of the fruit fly Drosophila melanogaster Marcus Thiedmann 1,* , Ruben Prange 1, Anita Bhandari 1, Kimberly Kallsen 2, Christine Fink 1 , and Thomas Roeder 1 1
Kiel University Research Center Borstel *Presenting author 2
Chronic obstructive pulmonary disease (COPD) comprises all diseases resulting in an airflow limitation due to the destruction of lung tissue (emphysema), fibrotic changes in the lung tissue (fibrosis) and mucus hypersecretion. It is estimated by the WHO to become the third leading cause of death in 2030 and already today over 64 million people suffer from it. The primary risk factor for COPD is cigarette smoke exposure, either directly or indirectly. In order to understand the responses of the airway epithelium to prolonged cigarette smoke exposure (CSE), we used the fruit fly Drosophila melanogaster as a model. For this, the tracheal epithelium was subjected to CSE with smoke of three cigarettes at an interval of three hours on two consecutive days each. After manual dissection of the tracheal system, RNA sequencing and qRT-PCR was performed. Additionally, the branching pattern of terminal tracheal cells was analyzed. Daily low dose CSE (1 cigarette per day) reduced the lifespan of adult Drosophila significantly in comparison to matching controls. Results of RNA sequencing suggest the involvement of the Keap1/Nrf2 and JAK/STAT pathways in the reaction to CS exposure. Among the regulated genes are those encoding for the cytochrome P450 family, Glutathione S-transferases and Mucin-like proteins. Additionally, the repetitive exposure to cigarette smoke shortens the length of the terminal tracheal cells and thus the respiratory surface. To better understand the molecular framework underlying COPD development, these results are the stepping stone to further expand the Drosophila emphysema model in particular and the Drosophila COPD model in general.
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Abstract No. 143 Natural history and therapy are associated with distinct phenotypes of alpha-1antitrypsin deficiency Sebastian Fähndrich 1,* , Frank Biertz 2, Annika Karch 2, Björn Kleibrink 3, Armin Koch 2, Helmut Teschler 3, Tobias Welte 2, Hans-Ulrich Kauczor 4, Sabina Janciauskiene 2, Rudolf Jörres 5, Claus Vogelmeier 6, and Robert Bals 1 1
Saarland University Hospital Hannover Medical School 3 University Hospital Essen, University Duisburg-Essen 4 University Hospital Heidelberg 5 Ludwig-Maximilians-University Munich 6 University Medical Center Giessen and Marburg *Presenting author 2
Rationale: Alpha-1-antitrypsin deficiency (AATD) is a rare inherited condition caused by mutations of the SERPINA1 gene and associated with liver and lung disease. Lung disease in AATD is considered as an early manifestation of COPD while the clinical phenotype is less well described. Objectives: The aim of this study was to analyze the clinical phenotype of AATD patients within The German COPD cohort study COSYCONET (“COPD and SYstemic consequences-Comorbidities NETwork”) cohort focusing on the pulmonary manifestation and cardiovascular comorbidities. Methods: The data from 2,645 COSYCONET patients with (n=139) or without AATD (n=2506) were analyzed by descriptive statistics and regression analyses. Measurements and Main Results: Patients with AATD were significantly younger as compared to non-AATD COPD patients, had smoked a lower number of cigarettes, had a lower CO diffusing capacity (TLCO), and a higher level of hyperinflation. In AATD patients, the reduction of TLCO was greater than expected from other parameters associated with emphysema in multivariate analysis. AATD patients under augmentation therapy presented a distinct phenotype characterized by more severe lung damage and significantly decreased prevalence of cardiovascular comorbidities. Bronchiectasis was more frequent in AATD individuals without augmentation therapy. Conclusions: Patients with AATD-based lung disease differ from non-AATD COPD patients in their lung disease phenotype and comorbidities. AATD is associated with an out-ofproportion loss of TLCO. AATD appears to be linked with significantly decreased prevalence of cardiovascular disease, the contribution of augmentation therapy to this association is not clear. These findings improve the understanding of COPD phenotypes and contribute to the development of individualized therapies.
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Abstract No. 158 The influence of cigarette smoke on the lung microbiota and its interaction with the bronchial epithelium Draginja Kovacevic 1,* , Sabine Bartel 1, Sebastian Reuter 1, Ulrich Schaible 1, and Susanne Krauss-Etschmann 1 1
Research Center Borstel, Leibniz-Center for Medicine and Biosciences *Presenting author
Background: Lung resident microbes form a layer between the respiratory epithelium and the external environment and play an important role in human health. Being directly exposed to the inhaled air, the lung microbiota is strongly influenced by a large number of environmental factors that could alter its composition, dynamics and functions and therewith cause respiratory diseases. It is known that cigarette smoking is a major risk factor for chronic lung diseases, but there is little known about its influence on the lung microbiota. Objective: Therefore, the aim of this study is to investigate how smoke exposure affects lung microbiota composition and function. Concept: We will analyse the longitudinal changes in composition and function of a fully developed murine lung microbiota (at age of 8 weeks) upon cigarette smoke (CS) exposure (SIREQ InExpose smoking robot, SIREQ, City, Canada) via 16S rDNA sequencing and metagenomics. Further, a possible regeneration of induced changes will be assessed. Obtained data, together with clinical samples from a human cohort of healthy smokers that undergo smoking cessation will help to identify CS-resilient or CS-vulnerable bacterial species. The interaction of identified bacteria, or their secreted metabolites with the host’s epithelial cells and their barrier function will be studied in detail in normal human bronchial epithelial cells (NHBE) (Lonza, Basel, Switzerland) exposed to cigarette smoke at the airliquid interface using the P.R.I.T. ExpoCube exposure system (Fraunhofer ITEM, Hannover, Germany). Finally, mice will be re-colonized with specific isolates (CS-vulnerable vs. CSresilient) to investigate the influence of a defined microbiota on CS-induced pulmonary changes. Expectations: This project will help to delineate consequences of cigarette smoke on the human lung microbiota and to extend understanding of the relationship between microbial communities and epithelial barrier functions. This knowledge is an important prerequisite for future exploitation of microbiome-based preventive strategies for chronic lung diseases.
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Abstract No. 164 YACTA as Tool for Big Data Analysis in Large Lung MDCT Data Sets – Preliminary Results of the COSYCONET study Oliver Weinheimer 1,* , Jeremy Stadter 1, Gregor Ojak 1, Hans-Ulrich Kauczor 1, Claus Peter Heussel 1, Mark Oliver Wielpütz 1, and Bertram Jobst 1 1
University Hospital of Heidelberg *Presenting author
Objectives: Within the multicenter COSYCONET study low-dose chest MDCT scans of 603 COPD patients were acquired in inspiration and expiration, using different scanners, reconstruction kernels and field of views. Since the total amount of CT data added up to approx. 1.5 TB it is a challenge to subtract useful information from this huge collection of CT data. Materials and Methods: Until now we analyzed 479 (79%) of the patients with the software YACTA fully automatically. The software generates information for the airways (e.g. wall percentage) and lung parenchyma (e.g. emphysema index, air trapping index) separately for the regions whole lung, left/right lung and the different lung lobes. The segmentation results of 327 patients were inspected visually afterwards to verify the correctness of the segmented regions. The software determined the tracheal Hounsfield value as a measure for CT image quality. All results were pushed in a database on the fly to allow further data analysis. Results: Lung segmentation was successful in 99.4%, lung separation in 98% and lobe segmentation in (71.2%). Tracheobronchial tree segmentation was successful in 99.6%, lobe bronchi were correctly identified in 93.9% of the cases. An average offset of +27 HU was measured for tracheal HU, which can be seen as drawback of the used low dose protocols influencing the quality of some of the derived numerical information. The emphysema index was: in average: 15.4%, min: 0%, max: 62.8%, air trapping index: 51.4%, 13.4%, 87.4% and airway wall percentage: 50.5%, 37.3%, 63.8%. Conclusion: Fully automatic software systems for medical image analysis such as YACTA offer a reliable way to subtract information from CT images efficiently and provide an essential component in the context of big data analysis. Importantly, they may be of value for monitoring the progression of COPD and the individual treatment of patients.
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Abstract No. 167 The phenotypic characterisation of patients with severe COPD Sandhya Matthes 1,* , Jürgen Barton 1, Jakob Stadler 1, Dieter Munker 1, Paola Arnold 1, Felix Ceelen 1, Gabriela Leuschner 1, Tobias Veit 1, Marion Frankenberger 1, Heidrun VillenaHermoza 1, Nikolaus Kneidinger 1, Jürgen Behr 1, and Claus Neurohr 1 1
University of Munich, Comprehensive Pneumology Center, Member of the German Center for Lung Research (DZL) *Presenting author
Background The aim of this study was to examine a group of patients with severe COPD and provide insight into the various phenotypes. Method 77 patients with severe COPD (ex-smokers of >6 months) on optimal therapy were examined for history of allergy and exacerbations, lung function tests, 6-minute walk test (6MWT), skin prick test, fractional exhaled nitric oxide (FENO), induced sputum, differential blood count and total serum IgE. Right-heart catheterisation (RHC) was used to assess for the presence of pulmonary hypertension (PH). Results 44 male and 33 female patients with a mean age of 59±6.9 and BMI of 23±4.3 kg/m2 were studied. Mean FEV1 was 0.73±0.4L (24.9±12.3% pred). 13/48 patients (27.1%) showed an increase in FEV1 of >12% following bronchodilation. Mean 6MWT was 252.5±116.9m. Mean FeNO Mittelwert was 23.9±18 ppb. Median IgE was 61 I.E/ml. We found blood esoniphilia of > 150 cells/μl in 32/67 (47.8%) of patients. Induced sputum was carreid out in 28 patients. The correlation coefficient between serum and sputum eosinophilia was r2 = 0,3. Bacteria was found in sputum cultures of 36.4% of patients. Skin prick testing was positve in 15/53 patients (28.3%). The presence of PH was 34.9 % in this cohort. 45/77 (58.4%) patients reported having ≥2 moderate to severe exacerbations of COPD in the past year. Patients with frequent exacerbations were significantly more likely to have bacterial colonisation in sputum (62% Vs 18.6%, p= 0.0005). There was no difference in lung function, blood eosinophilia or pulmonary arterial pressures. Conclusion Our analysis shows that blood eosinophilia, though a common finding in severe COPD, does not appear to influence the exacerbation rate or be a marker for airway reversibility. Patients with frequent exacerbations are significantly more likely to have bacteria in their sputum. Pulmonary hypertension is a common finding in patients with severe COPD.
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Abstract No. 181 The role of mitochondria in biological aging of the lung Claudia Fernanda Garcia Castro 1, Natascha Sommer 1, Martina Korfei 1, Mariola Bednorz 1, Bakyt Kojonazarov 1, Simone Kraut 1, Katharina Schäfer 1, Nasim Alebrahimdehkordi 1, Christiane Herden 2, Stefan Arnhold 3, Rainer Schulz 4, Norbert Weissmann 1, and Oleg Pak 1
1
ECCPS Institut für Veterinär-Pathologie 3 Institut für Veterinär Anatomie-Histologie und Embryologie 4 Physiologisches Institut 2
With advanced age, the lung may undergo structural and functional changes associated with decreased lung function. It has been suggested that mitochondrial dysfunction, release of mitochondrial reactive oxygen species (ROS) and apoptosis may play a crucial role during the aging pro-cesses. Different proteins are implicated in mitochondrial ROS metabolism and apoptosis, such as the adaptor protein p66shc and cyclophilin D (CypD), which is a regulatory component of the mitochondrial permeability transition pore. Thus, we investigated the role of p66shc and CypD in aging of the lung. Methods: Development of emphysema and pulmonary vascular remodeling were investigated in lungs of p66shc-/-, CypD-/- and wild type (WT) mice at different ages (2, 12 and 24 months) by histological determination of the mean linear intercept (MLI) and muscularization of pulmonary arteries, respectively. Histoimmunostaining against XBP-1 protein was used to detect endoplasmic reticulum (ER) stress. Results: Histological analysis did not reveal any significant changes in the mean linear intercept (MLI) and in the degree of muscularization of pulmonary arteries in WT mouse lungs at different ages. In contrast, the MLI in 24 months old and muscularization of pulmonary arteries in 12 and 24 months old p66shc-/- mice were increased compared to WT mice of the respective ages. 24 month old CypD-/- mice showed increased MLI, while the degree of muscularization of pulmonary arteries was similar compared to young CypD-/- or WT mice of the respective age. XBP1 expression was higher in p66shc-/- mice as well as in CypD-/- mice during aging and its expression was colocalized with alveolar epithelial type II cell and bronchial epithelial cells. Conclusion: The mitochondrial proteins p66shc and CypD may play an important role during aging of the lung and in development of pulmonary vascular remodeling and/or emphysema. Both proteins may promote development of emphysema via increasing ER stress.
1. Baines, C. P., Kaiser, R. A., Purcell, N. H., Blair, N. S., Osinska, H., Hambleton, M. A., & Robbins, J. (2005). Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature, 434(7033), 658-662. Brandenberger, Christina, and Christian Mühlfeld. "Mechanisms of lung aging." Cell and Tissue Research (2016): 1-12. 2. Balaban, R. S., Nemoto, S., & Finkel, T. (2005). Mitochondria, oxidants, and aging. Cell, 120(4), 483-495. 4. Trinei, M., Berniakovich, I., Beltrami, E., Migliaccio, E., Fassina, A., Pelicci, P., &
DZL Annual Meeting 2017 Giorgio, M. (2009). P66Shc signals to age. Aging (Albany NY), 1(6), 503-510. Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 183 Prevalence of Dyslipidemia in the COSYCONET cohort and its relationship to comorbidities and lung function Kathrin Kahnert 1,* , Tanja Lucke 1, Peter Alter 2, Jürgen Behr 1, Rudolf Maria Huber 1, Frank Biertz 3, Anja Vogt 1, Klaus Parhofer 1, Margarethe Wacker 4, Joachim Ficker 5, Rolf Holle 4, Beate Stubbe 6, Henrik Watz 7, Stefan Karrasch 1, Robert Bals 8, Claus Vogelmeier 2, and Rudolf Jörres 1 1
University of Munich University of Marburg 3 Hannover Medical School 4 Helmholtz Zentrum München (GmbH) 5 Klinikum Nürnberg 6 University Medicine Greifswald 7 Airway Research Center North 8 Saarland University Hospital *Presenting author 2
Background: Although dyslipidemia is a common comorbidity of COPD, its relationship to other comorbidities, risk factors and pulmonary function of COPD has not been studied in detail. Using data of the German COPD cohort COSYCONET we addressed this question.Methods: 1640 patients with complete data sets were selected (females 37.9%, age 64.8±8.4y, BMI 26.9±5.2 kg/m², GOLD categories 1-3 14.0/49.1/36.9 % – categories 0 and 4 were excluded due to systematic differences in comorbidities pattern). The analysis focused on comorbidities dyslipidemia, diabetes and cardiovascular complex (CVC, arterial hypertension and/or cardiac failure and/or ischemic heart disease) – which was established by doctor-diagnosis and specific medication – on risk factors BMI, packyears (PY) and lungfunction (FEV1, ITGV, sRaw, KCO). We used linear and logistic regression analyses as statistical methods. Results were implemented into path analysis (PA) models. Results: Prevalences of dyslipidemia, diabetes and CVC were 43.5/12.9/64.6%. Dyslipidemia was associated with diabetes and CVC; all comorbidities were linked to BMI and PY. Among lung function parameters, FEV1, ITGV and sRaw were associated with BMI, KCO and PY. FEV1, ITGV and KCO showed a link to dyslipidemia. Three PA models were analysed: the first comprised FEV1 and KCO. Dyslipidemia was linked to diabetes, CVC, BMI; FEV1 to dyslipidemia, CVC, BMI; KCO to PY, BMI, FEV1. The second additionally contained ITGV to account for lung volume. Dyslipidemia and BMI were related to ITGV; ITGV and FEV1 to KCO and PY. Adding sRaw, this structure was further modified. Instead of FEV1, sRaw was linked to CVC, ITGV and BMI. The relationship between dyslipidemia and ITGV remained stable. The consecutive models demonstrated the refinement of directional effects with increasing number of variables. Conclusions: The study provides prevalence estimates for comorbidities, particularly dyslipidemia. It suggests a potential role for dyslipidemia regarding lung volume beyond that mediated via BMI and diabetes.
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Abstract No. 192 Cigarette smoke-induced emphysema and pulmonary hypertension – The role of FGF10 Stefan Hadzic 1,* , Monika Malczyk 1, Mariola Bednorz 1, Michael Seimetz 1, Werner Seeger , Ralph Schermuly 1, Saverio Bellusci 1, and Norbert Weissmann 1
1
1
Excellence Cluster Cardio-Pulmonary System (ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany *Presenting author
Chronic obstructive pulmonary disease (COPD) is a collective term for chronic bronchitis and emphysema. It is a widespread and currently incurable disease, expected to be ranked as the third-greatest cause of death worldwide by 2030. The pathological concept is described by airway inflammation and remodeling. On the other hand, the main theory behind emphysema development is the destruction of the elastic architecture of the lung, leading to enlargement of distal airspaces. Influx of inflammatory cells, imbalance of proteolytic and anti-proteolytic activity, increased oxidative stress with the rise in number of apoptotic cells and decreased proliferation might be important upstream events. Besides the destruction of the lung parenchyma, COPD patients often also suffer from pulmonary hypertension (PH) due to significant structural remodeling of the vasculature. Fibroblast growth factor 10 (FGF10) is already known to be essential for lung development via controlling survival and proliferation of distal alveolar epithelial progenitor cells. Furthermore, FGF10 is involved in lung fibrosis. However, we showed that FGF10 expression levels are altered in different compartments of the lung from cigarette smokeexposed mice compared to non-smoke controls. Additionally, the treatment with L-NIL, a selective iNOS inhibitor which has been shown to ”cure“ smoke-induced emphysema and PH in mice, also upregulates FGF10 expression in different lung compartments. Furthermore, we observed that FGF10 promotes survival of pulmonary arterial smooth muscle cells and alveolar epithelial type II cells upon cigarette smoke extract treatment in vitro. Using different transgenic animals in our cigarette smoke model we aim to examine the role of FGF10 and FGF receptor 2b (FGFR2b) signaling pathway in pathology of COPD and its effect on the epithelial progenitor cells in the lung parenchyma during the repair process of the adult lung. Supported by the DFG, WE1978/8-1
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Abstract No. 194 Impact of different spirometric criteria on the prevalence of spirometrically defined COPD and its comorbidities in middle and advanced age Stefan Karrasch 1,* , Irene Brüske 2, Maia Smith 2, Barbara Thorand 3, Cornelia Huth 4, KarlHeinz Ladwig 3, Florian Kronenberg 5, Joachim Heinrich 2, Rolf Holle 6, Annette Peters 3, Holger Schulz 2, and KORA Study Group 7 1
Institute of Epidemiology I, Helmholtz Zentrum München; Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-Universität München 2 Institute of Epidemiology I, Helmholtz Zentrum München 3 Institute of Epidemiology II, Helmholtz Zentrum München 4 Institute of Epidemiology II, Helmholtz Zentrum München; German Center for Diabetes Research (DZD) 5 Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck 6 Institute of Health Economics and Health Care Management, Helmholtz Zentrum München 7 Helmholtz Zentrum München *Presenting author
A COPD diagnosis is based on spirometrically determined airflow obstruction. However, the appropriate spirometric criterion is subject to an ongoing debate. We examined the impact of different spirometric definitions on COPD prevalence and on the prevalence of common diseases and serum biomarker levels in COPD in a general population sample. Spirometry was performed in a population-based sample (KORA) from the Augsburg region (n=2256) covering an age range of 41-90y without bronchodilation. COPD was defined either by FEV1/FVC<0.7 (fixed ratio, FR) or by FEV1/FVC below the lower limit of normal (LLN) according to GLI reference values. The comprehensive examinations within KORA included the assessment of myocardial infarction, hypertension, stroke, diabetes, obesity, cancer, arthritis, anxiety/depression and gastrointestinal disease as well as the determination of high-sensitivity CRP (hsCRP) and IL-6 serum levels. Comorbidity prevalences and circulating biomarker levels were compared between subjects with or without COPD by the two criteria using logistic and multiple regression models. The prevalence of spirometrically defined COPD by FR increased with age from 10% in subjects aged <65y to 26% in subjects aged >75y, while for LLN-defined COPD it remained below 10% for all age groups. The severity of airflow limitation in COPD was predominantly mild to moderate. COPD diagnosis was not associated with specific comorbidities independent of the spirometric criterion, except for a lower prevalence of obesity in both FRand LLN-defined cases. IL-6 and hsCRP tended to be higher in cases by both criteria. Age-related COPD prevalence critically depended on the applied spirometric criterion, particularly for the elderly where differences between cut-offs become large. However, there was no conclusive association with comorbidity prevalences or inflammatory biomarkers in the predominantly mild COPD cases observed in the examined study population. Supported by the German Federal Ministry of Education and Research (BMBF FKZ 01ET0713, 01ET1003A and 01GI0882).
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Submitted as: Presentation 298 words of 300 possible words
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Abstract No. 195 Effects of electronic cigarette vapour extract exposure on primary isolated mouse lung cells Elsa T. Roxlau 1,* , Alexandra Pichl 1, Ralph T. Schermuly 1, Hossein A. Ghofrani 1, Werner Seeger 2, Jochen Wilhelm 1, Clemens Ruppert 1, Friedrich Grimminger 1, and Norbert Weissmann 1 1
Justus-Liebig-University Giessen. Excellence Cluster Cardio-Pulmonary System (ECCPS) Justus-Liebig-University Giessen. Excellence Cluster Cardio-Pulmonary System (ECCPS). Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany *Presenting author 2
E-cigarettes are gaining popularity as an alternative to cigarette smoking (CS) and the number of users are rapidly increasing. Although e-cigarettes are advertised as safer, little is known about health risks. This study aims to investigate the effects of e-cigarette vapour extract (ECVE) on cellular function and gene expression in lung cells. Primary isolated murine alveolar epithelial typeII cells (mAECII) and pulmonary arterial smooth muscle cells (mPASMC) as well as human pulmonary arterial smooth muscle cells (hPASMC) were used. ECVE was prepared by bubbling (15x4sec puff) the vapour of ecigarette liquids, containing 0 or 18mg/ml nicotine without flavours and CS extract (CSE) was prepared by continuously bubbling mainstream smoke of one research cigarette 3RF4 to 10ml culture medium. We determined the relative cellular proliferation, migration, glutathione level and performed mouse whole genome microarray. ECVE (with nicotine) and CSE inhibit cellular proliferation in both mPASMC and hPASMC. CSE significantly reduced migration of hPASMC and mPASMC. ECVE (without nicotine) represses migration only in mPASMC. Microarray data of mPASMC revealed that ECVE inhibits expression of genes involved in lysosomal, metabolic and glycolysis/gluconeogenesis pathways but induces expression of genes involved in cell cycle and spliceosomes. mAECII, ECVE induces expression of genes involved in xenobiotic stress and glutathione metabolism, and inhibits expression of genes involved in protein export and endocytosis pathways. Incubation of mAECII with ECVE or CSE diminishes glutathione level. These results indicate altered cellular functions of cells exposed to ECVE or CSE and differentially regulated pathways were involved in pathogenesis of neurodegenerative, immune and infectious diseases in humans. This study illustrate the importance of extensive short and long term animal experiments to validate the claim that e-cigarettes are safer.
Submitted as: Presentation 276 words of 300 possible words
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Abstract No. 196 Innate immune cells in a murine model of COPD-like inflammation Martin Wolff 1,* , Sebastian Reuter 1, Sabine Bartel 1, Heinz Fehrenbach 1, Holger Heine 1, and Susanne Krauss-Etschmann 1 1
Research Center Borstel *Presenting author
Introduction Chronic obstructive pulmonary disease (COPD) is a progressive and incurable disease affecting ~600 million patients worldwide. The main risk factor is chronic tobacco smoke exposure. Infection-driven exacerbations of COPD result in more rapid lung function decline with earlier lung failure. Early prediction of exacerbations could help to initiate treatment before symptoms reach a critical level. Here we investigated in a murine model of cigarette smoking how intranasal application of dsRNA as a mimic of viral infection affects innate immune cells. Methods Female C57Bl/6 mice were exposed to cigarette smoke (4 puffs/min; 1 hour daily for 24 days; TPM 824 mg, Casella MicroDust Pro; 2R4F research cigarettes, Kentucky Tobacco Research and Development Center; exposure via Inexpose® Scireq CA). Animals were challenged i.n. with 50 µl PBS without or with poly(I:C) (0.1, 1.0, 10 µg) per mouse one hour after the last smoke exposure. Innate immune cells were analyzed 24 hours later in bronchoalveolar lavage fluid (BALF) and lung homogenates. Results were analyzed by 2way ANOVA and Bonferroni post-hoc analyses (GraphPad Prism 5®). Results Smoking increased BALF neutrophils significantly (p<0.05) with a further significant increase induced by poly(I:C). Numbers and percentages of plasmocytoid (p)DCs (MHCII+, CD11c+, B220+), conventional (c)DCs (MHCII+, CD11c+, Ly6Clow, CD11b+) and inflammatory DCs (MHCII+, CD11c+, Ly6Chigh) showed a poly(I:C) dose-dependent increase in both exposure groups (air vs smoke; p>0.05). This increase was significantly higher in the smoke-exposed group for pDCs, cDCs and inflammatory DCs (1 µg poly(I:C); p<0.05)). Summary and Conclusion Poly(I:C) induces a significant increase of neutrophils and innate immune cells in murine lungs. In a next step, we will analyze the kinetic changes of these cells a well as of inflammatory cytokines.
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Abstract No. 199 Elastin microfibril interfacers in chronic obstructive pulmonary disease decrease lung repair via inhibition of WNT/β-catenin signaling Rita Costa 1,* , Darcy Wagner 1, Franziska Uhl 2, Daniel Weiss 2, and Melanie Königshoff 3 1
Helmholtz Zentrum München University of Vermont 3 University of Colorado, School of Medicine *Presenting author 2
The extracellular matrix is altered in Chronic Obstructive Pulmonary Disease (COPD) due to an imbalance between proteases and anti-proteases. It was previously thought that the lung loses its ability to repair tissue in COPD, however, it has become increasingly more evident that there may be aberrant attempts at repair. Elevated levels of a number of elastogenesis components were recently identified in larger COPD patient cohorts. Whether these components are beneficial or further drive disease pathogenesis remains unknown. Elastin microfibril interfacers (EMILINs) are a four member glycoprotein family critical for development of elastic fibers. Despite the fact that EMILIN1 knockout mice develop spontaneous airspace enlargement, nothing is known about EMILINs in COPD. EMILIN2 was recently shown to inhibit WNT/β-catenin signaling, a pathway known to be inhibited in COPD, through direct interaction with WNT ligands. We thus hypothesized that aberrant increases in EMILINs during lung emphysema pathogenesis might dampen pathways involved in lung repair. We analyzed the expression of EMILINs in lungs of cigarette smoke and elastase exposed mice and in COPD patients and found increased gene expression of Emilin1 and Emilin2 in lungs of cigarette smoke and elastase exposed mice, with trends towards increased EMILIN1 mRNA levels in COPD patients. In order to see if the extracellular matrix of emphysematous lungs can regulate WNT signaling, potentially through deranged EMILIN expression, we seeded a WNT luciferase reporter cell line in decellularized normal and emphysematous mouse lung scaffolds and found decreased WNT activity in decelled emphysematous mouse lungs scaffolds. Finally, we performed EMILIN1 overexpression and silencing in different human lung cell lines to see if EMILIN1 interferes with WNT signaling. EMILIN1 overexpression down-regulated, whereas silencing upregulated the WNT target gene AXIN2. Taken together, EMILINs have the potential to modulate WNT/β-catenin signaling, which might interfere with proper lung repair in the context of COPD.
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Abstract No. 202 Identification of novel compounds for WNT/β-catenin induced lung repair in Chronic Obstructive Pulmonary Disease Rita Costa 1, Darcy Wagner 1, Kenji Schorpp 1, Astrid Staschewski 1,* , Hoeke Baarsma 1, Ina Rothenaiger 1, Monica Campillos 1, Hadian Kamyar 1, and Melanie Königshoff 2 1
Helmholtz Zentrum München University of Colorado, School of Medicine *Presenting author 2
Chronic Obstructive Pulmonary Disease (COPD) is thought to be an incurable lung disease and is characterized by inflammation and progressive loss of tissue (emphysema). Among several pathways known to be deranged in COPD, the WNT/β-catenin has been increasingly recognized as being decreased in COPD. Previously, pharmaceutical activation of the pathway in an in vivo mouse model of emphysema and ex vivo in 3D human and mouse lung tissue slices decreased emphysematous markers such as aberrant overexpression of elastin and MMP12. These findings were obtained using glycogen synthase kinase 3 (GSK3) inhibition, using lithium chloride, however, concerns remain about the use of these agents in the clinic. We thus aimed to identify new pharmaceutical compounds which activate WNT/β-catenin signaling and could induce lung repair. We conducted a high-throughput drug screen, using an NIH3T3 cell line stably expressing luciferase under the control of WNT-responsive promoters (TCF/LEF). We screened 1.280 Food and Drug Administration (FDA) approved drugs (Prestwick Chemical library) and 30.000 novel compounds, belonging to small-molecule diversity libraries (ChemDiv, ChemBridge, Enamine and PPI). We identified 652 potential novel compounds which activated WNT/β-catenin signaling. 16 of these are FDA approved and of particular interest for potential quick translation into the clinic. In order to select compounds which might hold therapeutic potential for COPD, we predicted protein targets (>50% precision), using the target prediction tool HitPick, for 154 of the hits. We selected 15 compounds which were associated with the WNT pathway or components known to be deranged in COPD and with cell growth and repair. We selected 5 compounds from our FDA approved hits and were able to validate these compounds as WNT pathway activators. We are currently using complementary assays, closely related to disease pathomechanisms, to further characterize the biological effect of the candidate drugs.
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Abstract No. 211 Superior anti-inflammatory effects of Narrow spectrum kinase inhibitors in airway smooth muscle cells from subjects with COPD Juergen Knobloch 1, Kazuhiro Ito 2, David Jungck 1, Catherine Charron 2, Erich Stoelben 3, and Andrea Koch 4,* 1
Uniklinik Bochum-Bergmannsheil RespiVert Ltd, London, United Kingdom 3 Thoracic Surgery, Lungenklinik; Hospital of Cologne; Merheim 4 Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians-Universität, Munich, Germany *Presenting author 2
Corticosteroid-resistant airway inflammation is central for COPD progression. Narrow spectrum protein kinase inhibitors (NSKIs) are novel anti-inflammatory agents targeting p38MAPK and Src (RV568) or p38MAPK, Src and Syk (RV1088). As measured by ELISA, TNF was inhibited by FP in HASMCs of never-smokers but not of COPD, which, therefore, are cell culture models of corticosteroid-insensitivity. FP inhibited TNF - or LPS-induced GMCSF almost completely in HASMCs of both never-smokers and COPD , which, therefore, are cell culture models of corticosteroid-sensitivity. The BIRB796/Dasatinib/R343 triple was equally effective as the BIRB796/Dasatinib double. This suggests that p38MAPK and Src but not Syk have dominant roles in the regulation of TNF -induced GM-CSF. NSKIs RV568 and RV1088 resembled the effects of BIRB796/Dasatinib double or the BIRB796/Dasatinib/R343 triple and completely inhibited TNF -induced GM-CSF. We then investigated the effects of NSKIs in COPD HASMCs and compared with BIRB796 and FP. RV568 and RV1088 both almost completely suppressed TNF -induced GM-CSF without differences. Both NSKIs were superior to BIRB796. Both NSKIs were almost equally effective as FP. These data demonstrate that NSKIs block cytokine production in a COPD primary cell culture model of corticosteroid-sensitivity. The BIRB796/Dasatinib/R343 triple was superior to any single and double treatment. This suggests dominant roles of all three kinase families, p38MAPK, Src and Syk in the regulation of TNF -induced CXCL8. Both NSKIs were superior to any SKI in single treatments In COPD, RV568 partially inhibited TNF -induced CXCL8 and was more effective than BIRB796. RV1088 completely suppressed TNF -induced CXCL8 and was superior to RV568 and BIRB796. These data demonstrate that NSKIs block cytokine production in a COPD primary cell culture model of corticosteroid-insensitivity. Our data provide evidence for a utility of a new anti-inflammatory therapeutic strategy in COPD: NSKIs suppress corticosteroid-sensitive and -insensitive cytokine production in COPD.
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Abstract No. 212 Reduced Frizzled receptor 4 expression prevents Wnt/β-catenin-driven alveolar lung repair in COPD Wioletta Skronska-Wasek 1,* , Kathrin Mutze 1, Hoeke A. Baarsma 1, Ken R. Bracke 2, Darcy E. Wagner 1, Mariano Stornaiuolo 3, Ettore Novellino 3, Guy G. Brusselle 2, Ali Ö. Yildirim 4, and Melanie Königshoff 1 1
LRR Ghent University Hospital 3 University of Naples 4 ILBD *Presenting author 2
Objective: Chronic obstructive pulmonary disease (COPD) is characterized by small airway remodeling, emphysema and impaired tissue repair. It has been shown that Wnt/β-catenin signaling is reduced in experimental emphysema as well as in human COPD. Moreover, pharmacological Wnt/β-catenin activation attenuated experimental emphysema and led to epithelial cell activation in COPD patient-derived lung tissue cultures. The cause of Wnt/βcatenin signal reduction in COPD, however, remains unknown. Here, we aimed to investigate the potential involvement of Wnt receptors (Frizzled, FZD) on impaired lung repair in COPD. Methods: FZD receptor expression was analyzed in lung homogenates and primary alveolar epithelial type II (ATII) cells of never-smokers, smokers, and COPD patients, as well as two experimental emphysema models by qRT-PCR, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on WNT/β-catenin signaling and FZD4 function were investigated in primary ATII cells in vitro and 3D lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of increased/decreased FZD4 expression on alveolar epithelial cell wound healing and repair, and expression on elastognenic components. Results: FZD4 expression was reduced in human and experimental COPD lung tissues as well as primary human ATII cells from COPD patients. Cigarette smoke exposure downregulated FZD4 expression in vitro and in vivo, along with reduced WNT/β-catenin activity. Inhibition of FZD4 decreased WNT/β-catenin driven epithelial cell proliferation, migration, wound closure and interfered with ATII-to-ATI cell trans-differentiation and organoid formation, which was augmented by FZD4 overexpression. Moreover, FZD4 overexpression rescued the cigarette smoke-induced decrease in elastin. Conclusions: The Wnt receptor FZD4 is decreased in experimental and human COPD. Cigarette smoke and decreased canonical Wnt signal activity induced FZD4 downregulation in lung epithelial cells. Our results suggest that reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity, thus FZD4 represents a potential therapeutic target for COPD.
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Abstract No. 233 Does urinary peptide content differ between COPD patients with and without inherited alpha1-antitrypsin deficiency? Alfonso Carleo 1, Joanna Chorostowska-Wynimko 2, Sabine Wrenger 1, Harald Mischak 3, Tobias Welte 1, and Sabina Janciauskiene 1,* 1
Hannover Medical School National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland 3 Mosaiques Diagnostics and Therapeutics AG, Hannover, Germany *Presenting author 2
Differentiating between chronic obstructive pulmonary disease (COPD) patients with normal (PiMM) or deficient (PiZZ) genetic variants of alpha-1 antitrypsin (A1AT) is important not only for understanding the pathobiology of disease progression but also for improving personalized therapies. This pilot study aimed to investigate whether urinary peptides reflect the A1AT-related phenotypes of COPD. Urine samples from 19 clinically stable COPD cases (7 PiMM and 12 PiZZ A1AT) were analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS). We identified 66 peptides (corresponding to 36 unique proteins) that differed between PiZZ and PiMM COPD. Among these, peptides from the collagen family were the most abundant and divergent. A logistic regression model based on COL1A1 or COL5A3 peptides enabled differentiation between PiMM and PiZZ groups, with a sensitivity of 100% and specificity of 85.71% for COL1A1 and a sensitivity of 91.67% and specificity of 85.71% for COL5A3. Furthermore, patients with PiZZ presented low levels of urinary peptides involved in lipoproteins/lipids and retinoic acid metabolism, such as apolipoprotein A-I and C4, retinol-binding protein 4, and prostaglandin-H2 D-isomerase. On the other hand, peptides of MDS1 and EVII complex locus, gelsolin and haemoglobin alpha were found in the urine from COPD cases with PiZZ but not PiMM. These CE-MS-based results provide the first evidence that urinary peptide content differs between PiMM and PiZZ patients with COPD.
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Abstract No. 247 Plasminogen activator inhibitor-1 is elevated in patients with COPD independent of metabolic and cardiovascular function Benjamin Waschki 1,* , Henrik Watz 2, Olaf Holz 3, Helgo Magnussen 2, Olga Olejnicka 4, Tobias Welte 5, Klaus Rabe 1, and Sabina Janciauskiene 5 1
LungenClinic Grosshansdorf Pulmonary Research Institute at LungClinic Grosshansdorf 3 Fraunhofer Institute for Toxicology and Experimental Medicine 4 Trelleborg Hospital 5 Department of Respiratory Medicine, Hannover Medical School *Presenting author 2
Introduction Plasminogen activator inhibitor 1 (PAI-1), a major inhibitor of fibrinolysis, is associated with thrombosis, obesity, insulin resistance, dyslipidemia, and premature aging, which all are coexisting conditions of chronic obstructive pulmonary disease (COPD). The role of PAI-1 in COPD with respect to metabolic and cardiovascular function is unclear. Methods Within a prospective cohort study we measured serum levels of PAI-1 in 74 stable COPD patients (GOLD stage I to IV) and 18 controls without lung diseases. We also determined triglycerides, HDL cholesterol, fasting plasma glucose, waist circumference, blood pressure, smoking status, high-sensitive C-reactive protein (hs-CRP), adiponectin, ankle-brachial index, N-terminal pro-B-Type natriuretic peptide, and history of comorbidities. Results Serum levels of PAI-1 were significantly higher in COPD patients compared to controls, independent of a broad spectrum of possible confounders including metabolic and cardiovascular dysfunction. A multivariate regression analysis revealed triglyceride and hsCRP levels to be the best predictors of PAI-1 within COPD. GOLD stages II and III remained independently associated with higher PAI-1 levels in a final regression analysis. Conclusion Our data show that serum levels of PAI-1 are higher in patients with COPD independent of metabolic and cardiovascular disorders, and that hypertriglyceridemia and systemic inflammation play a role for a PAI-1 elevation. PAI-1 may be a potential biomarker candidate for COPD-specific and extra-pulmonary manifestations.
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Abstract No. 256 Cigarette smoke causes acute airways disease and exacerbates chronic obstructive lung disease in neonatal mice Thomas Conlon 1,* , Jie Jia 1, Carolina Ballester Lopez 1, Michael Seimetz 2, Mariola Bednorz 2 , Zhe Zhou-Suckow 3, Norbert Weissmann 2, Oliver Eickelberg 4, Marcus Mall 3, and Ali Önder Yildirim 1 1
Institute of Lung Biology and Disease / Comprehensive Pneumology Center, Helmholtz Zentrum München 2 Excellence Cluster Cardio-Pulmonary System (ECCPS), Justus-Liebig-University Giessen, Department of Internal Medicine, Universities of Giessen and Marburg Lung Center 3 Department of Translational Pulmonology, Translational Lung Research Center Heidelberg, University of Heidelberg 4 Institute of Lung Biology and Disease / Comprehensive Pneumology Center, Helmholtz Zentrum München and Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver *Presenting author
Epidemiological evidence demonstrates a strong link between postnatal cigarette smoke (CS) exposure and increased respiratory morbidity in young children. However, how CS induces early onset airway disease in young children, and how it interacts with endogenous risk factors, remains poorly understood. We, therefore exposed 10-day-old neonatal wild-type and β-epithelial sodium ion channel (β-ENaC)-transgenic mice with cystic fibrosis-like lung disease to CS for 4 days. Neonatal wild-type mice exposed to CS demonstrated increased numbers of macrophages and neutrophils in the bronchoalveolar lavage fluid (BALF), which was accompanied by increased levels of Mmp12 and Cxcl1. BALF from β-ENaC-transgenic mice contained greater numbers of macrophages, which did not increase following acute CS exposure; however, there was significant increase in airway neutrophilia compared with filtered air transgenic and CS-exposed wild-type controls. Interestingly, wild-type and β-ENaCtransgenic mice demonstrated epithelial airway and vascular remodeling following CS exposure. Morphometric analysis of lung sections revealed that CS exposure caused increased mucus accumulation in the airway lumen of neonatal β-ENaC-transgenic mice compared with wild-type controls, which was accompanied by an increase in the number of goblet cells and Muc5ac upregulation. We conclude that short-term CS exposure induces acute airway disease with airway epithelial and vascular remodeling in neonatal wild-type mice; and exacerbates airway inflammation, mucus hypersecretion, and mucus plugging in neonatal β-ENaC-transgenic mice with chronic lung disease. Our results in neonatal mice suggest that young children may be highly susceptible to develop airway disease in response to tobacco smoke exposure, and that adverse effects may be aggravated in children with underlying chronic lung diseases.
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Abstract No. 259 The minimal important difference for target lobe volume reduction following endoscopic valve therapy in patients with advanced emphysema Daniela Gompelmann 1, Ralf Eberhardt 1, Claus-Peter Heussel 1, Maren Schuhmann 1, Konstantina Kontogianni 1, and Felix JF Herth 1,* 1
Thoraxklinik at University of Heidelberg *Presenting author
Objective: Endoscopic valve therapy aims at target lobe volume reduction (TLVR) that is associated with improved lung function, exercise tolerance and quality of life in emphysema patients. In several trials, a TLVR greater than 350 ml was considered to be indicative of positive response to treatment. However, it is not really known what size of TLVR can be considered as being crucial following valve implantation. Methods: TLVR, FEV1 (forced expiratory volume in 1 second), RV (residual volume) and 6MWT (6-minute-walk test) were assessed prior and around 3 months after valve implantation in 119 patients treated from 2012-2013. TLVR was calculated based on MDCT scan analysis using quantitative imaging software (Apollo; VIDA Diagnostics, Coralville, IA). MID estimates were calculated by anchor-based methods (anchors: FEV1, 6-MWT, RV) and by distribution-based methods. Results: Patients treated by valves experienced a mean change of 0,11l in FEV1, of -0,51l in RV, of 46,6m in 6-MWT and a mean TLVR of 955ml. Using a linear regression based on each of the three anchors, and ROC analysis based on ΔFEV1 and ΔRV, the estimated MID for TLVR was between 890 ml and 1070 ml (between 41% and 54% of the baseline target lobe volume). Conclusion: These MID estimates for TLVR derived from the anchor-based method that identifies precisely the patients´ clinical outcome are much higher than assumed in previous trials and are useful for sample size determination in new studies.
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Abstract No. 260 Impact of fissure integrity on emphysema distribution in patients with severe chronic obstructive pulmonary disease Daniela Gompelmann 1, Maren Schuhmann 1, Konstantina Kontogianni 1, Claus-Peter Heussel 1, Juerg Tschirren 2, Ralf Eberhardt 1, and Felix JF Herth 1,* 1
Thoraxklinik at University of Heidelberg VIDA Diagnostics *Presenting author 2
Rationale: Incomplete interlobar fissures are common in healthy subjects as well as in patients with severe emphysema. As an incomplete fissure is a surrogate for collateral ventilation, it can be assumed that noxious agents can cross the parenchymal bridges thus resulting in a more homogeneous emphysema distribution. This analysis evaluates the impact of fissure integrity on emphysema distribution. Methods: Multidetector computed tomography scans (MDCT) of 433 patients who underwent endoscopic valve (n=355) or coil treatment (n=78) were assessed for fissure integrity and emphysema distribution by using a software (Apollo VIDA Diagnostics, Coralville, IA). A fissure was considered to be complete if the left oblique fissure and the right oblique fissure were measured as >90% complete, and considered incomplete otherwise. Using more restrictive criteria, a fissure was defined as complete if both major fissures were measured as >95% complete and as incomplete if both major fissures were measured <85% complete. Emphysema distribution was de fin percent emphysema measured at the -950 HU threshold for the upper lobe minus the percent emphysema measured at the -950 HU threshold for the lower lobe. Results: Fissure analysis using the 90% threshold revealed incomplete fissures in 222 and complete fissures in 221 patients. A signi -ficant complete and fissure-incomplete subjects was observed (p=0.02 [0.43; 5.04] for the left lung, p= 0.0005 [2.06; 7.22] for the right lung), whereby a more homogeneous emphysema distribution was seen if the fissure was incomplete. Using the more restrictive criteria for fissure completeness, there was a significant homogeneity difference only for the left lung between fissure-complete (n=134) and fissure-incomplete (n=39) patients (p=0.03 [-8.83; 0.50] for the left lung, p=0.41 [-7.65; 3.16] for the right lung). Conclusions: Interlobar fissure incompleteness is statistically associated with a more homogeneous emphysema distribution; the pathophysiological relevance however is unclear.
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Abstract No. 265 Lung volume reduction coil (LVR-coil) treatment of patients with advanced emphysema. Effectiveness and complications after 1 year Konstantina Kontogianni 1, Vasiliki Gerovasili 2, Daniela Gompelmann 1, Claus-Peter Heußel 1 , Felix Herth 1,* , and Ralf Eberhardt 1 1
Thoraxklinik Royal Brompton and Harefield, NHS *Presenting author 2
Background: LVR-coils treatment is established in daily endoscopic LVR routine. How safe and effective is it? Methods: Retrospective analysis of 86 patients (♂/♀:40/46, age: 64±7years) with bilateral incomplete fissures. A total of 10 coils were unilaterally implanted in a single lobe. 28 patients received additional treatment of a contralateral lobe. At 90, 180 and 365 days’ follow-up changes in pulmonary function tests (PFT), 6-Minute-Walk-Test (6MWT) and modified Medical Research Council (mMRC) dyspnea scale, as well as possible complications were recorded. Results: at 90 days the FEV1 did improve (p:0.001) but the improvement was not sustained at the 180- and 365-day follow up. VC improved at the 90- (p<0.001) and 180-day (p:0.13) follow up but improvement was also lost at 1 year. RV improved significantly at 90 days (p<0.0001) and at 180 days (p:0.008), but the improvement was not sustained further. Finally, 6MWT and mMRC also followed the same pattern. The severe adverse event rate was 60.5% (n=69) in a total of 114 procedures. 4 out of 86 patients passed away due to complications. Significant complications within the first 3 and then until the 12th post procedure(s) month included: severe hemoptysis 2.6% and 3.5%, pneumonia requiring hospitalization 28% and 7.9% and drained pneumothoraxes 6.1% and 1.7% respectively. 111 mild adverse events, 65 within the initial 3 and 46 within the following 9 months included light hemoptysis and orally treated pneumonias and COPD exacerbations. Conclusions: LVR-coil improved PFT, 6MWT and mMRC initially but improvement was lost after 365 days. Furthermore we observed 4 deaths and significant severe complications which need to be further elucidated.
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Abstract No. 266 Quantitative CT (QCT) analysis of emphysema and air trapping in Coil-based Lung Volume Reduction (LVRC) treatment Konstantina Kontogianni 1, Youbing Yin 2, Maren Schuhmann 1, Ralf Eberhardt 1, Daniela Gompelmann 1, Susan Wood 2, and Felix Herth 1,* 1
Thoraxklinik Vida Diagnostics *Presenting author 2
Background: LVRC treatment is promising for patients with severe emphysema and collateral ventilation Objective: To identify QCT parameters associated with positive treatment outcome Methods: The CT scans from 72 patients were acquired at full inspiration before LVRC treatment. FEV1, RV, TLC and 6-minute walk test (6MWT) data was collected prior to and at 3 months after treatment. Various QCT parameters like emphysema percentage, heterogeneity score, vascular volumetry and low attenuation cluster (LAC) distributions were derived using Apollo imaging software (VIDA Diagnostics, Iowa, US). Independent predictors of outcome were identified through stepwise linear regression analysis. Results: The response rates satisfying Δ6MWT>=26m, ΔFEV1>=12% or ΔRV>=10% are 54.8%, 37.1% and 42% respectively. 70.8% (51/72) patients met at least one response criterion. Positive Outcome
Predictors:
for Δ6MWT>=26m - 6MWT at baseline, p-value 0.0004 - Standard deviation of low attenuation cluster sizes in the peripheral region of the treated lung, p-value 0.037 for ΔFEV1>=12% - FEV1 at baseline, p-value 0.02 - Median size of low attenuation clusters in the central region of the target lobe, p-value 0.002 for ΔRV>=10% - TLC at baseline, p-value 0.001 - Vascular volumetric percentage of small vessels of the target lobe, p-value 0.003 - Mean size of low attenuation clusters in the peripheral region of the target lobe, p-value 0.007 Conclusions: Sicker patients with lower 6MWT, FEV1, and larger TLC at baseline tend to respond better. LAC distributions and vascular volumetry also constitute possible predicting factors of the response to treatment. QCT analysis of emphysema pattern might help improve patient selection for LVRC.
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Abstract No. 267 Assessment of Airway Instability and Respiratory Dynamics with Low-Dose 4D-CT in Chronic Obstructive Pulmonary Disease – Preliminary Results Anja Dutschke 1,* , Oliver Weinheimer 1, Roman Rubtsov 1, Ralf Eberhardt 2, Hans Ulrich Kauczor 1, Claus Peter Heußel 2, and Mark Oliver Wielpütz 1 1
University Hospital Heidelberg Thoraxklinik Heidelberg *Presenting author 2
Objectives: To evaluate the applicability of low-dose dynamic respiratory-gated multidetector CT (4DCT) as a new method to assess respiratory dynamics and airway instability in chronic obstructive pulmonary disease (COPD). Materials and Methods: In 60 COPD patients with suspected airway instability 4D-CT of the whole chest was performed at minimal radiation exposure (1.6 ±0,25mSv) during tidal breathing registered by a belt system. Raw-data-based iterative reconstruction and respiratory gating resulted in 21 stacks of volumetric datasets per patient. The trachea and main-stem bronchi were systematically assessed visually and each lumen area (LA) quantified semi-automatically with well-evaluated image processing software. A collapse of more than 50% of LA was considered diagnostic for instability. So far, the results of 16 patients could be analyzed (mean age 68y) and correlated with lung function parameters incl. forced expiratory volume in 1s predicted (FEV1%). Results: 7/16 (43.8%) patients showed an excessive dynamic collapse of the trachea. An instability of the right main-stem bronchus was detected in 14/16 (87.5%) patients and in 9/16 (56.3%) cases the left main-stem bronchus was affected. 1/16(6.3%) patient showed neither a significant collapse (≥50%) nor stenosis. Normalized minimal LA of the trachea and main bronchi correlated with R²=0.63 (p<0.001) with FEV1%. Visual analyzes for dynamic tracheobronchial collapse and image processing software results matched in 85.4% cases. Conclusion: Low-Dose 4D-CT provides full z-axis coverage and time-resolved volumetric datasets of the whole chest, which can be subjected to quantitative post-processing software. The latter allows novel insight into the phenotype of airway instability in COPD, often missed by nondynamic CT imaging or bronchoscopy.
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Abstract No. 270 Reversal of established cigarette smoke (CS)-induced pulmonary hypertension and emphysema in mice by sGC stimulation Mariola Bednorz 1,* , Alexandra Pichl 1, Michael Seimetz 1, Stefan Hadzic 1, Bakytbek Kojonazarov 1, Hossein Ardeschir Ghofrani 1, Ralph Schermuly 1, Werner Seeger 2, and Norbert Weissmann 1 1
Excellencecluster Cardio-Pulmonary System (ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany 2 Excellencecluster Cardio-Pulmonary System (ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany, Max Planck Institute for Lung and Heart Research, Bad Nauheim, Germany *Presenting author
Cigarette smoke-induced pulmonary hypertension (PH) and emphysema are still not reversible in human patients suffering from chronic obstructive pulmonary disease (COPD). Based on our previous study demonstrating that stimulation of sGC prevents cigarette smoke-induced pulmonary hypertension and emphysema we now aimed to investigate the regenerative potential of a sGC stimulation after established disease. Wild-type (WT) mice were chronically exposed to cigarette smoke or room-air for 8 months followed with a three months treatment. Mice were separated in different experimental groups (Methocel smoke-exposed, Methocel non-exposed and BAY 63-2521 smokeexposed). BAY 63-2521 treated animals received the drug once per day by gavage (3mg/kg body weight or 10 mg/kg body weight). After 11 months, lung function, hemodynamics and echocardiography were measured followed by structural analyses of the parenchyma and the vasculature via alveolar and vascular morphometry. WT smoke-exposed mice treated with Methocel developed emphysema reflected by worsened lung function and increased alveolar airspace. In addition, right ventricular systolic pressure and vascular muscularization were impaired causing PH. Such alterations were significantly diminished by BAY63-2521 treatment (3mg/kg and 10mg/kg). Also nitrotyrosine amounts were diminished in BAY63-2521 treated mice as an indication of less ROS formation. In conclusion, we here demonstrated that an sGC stimulation by BAY63-2521 administration in diseased mice can improve lung emphysema and PH suggesting BAY63-2521 as a novel therapeutic option for COPD/PH in future.
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Abstract No. 009 Randomized, double-blind, controlled pilot study on safety of hypertonic saline as preventive inhalation therapy in newborn patients with cystic fibrosis – follow-up after LOP Mirjam Stahl 1,* , Simon Gräber 1, Christian Dopfer 2, Lutz Nährlich 3, Matthias Kopp 4, Cordula Koerner-Rettberg 5, and Marcus Mall 1 1
ZKJM Heidelberg MHH 3 Universitätskinderklinik Gießen 4 Universitätskinderklinik Lübeck 5 Kinderklinik Bochum *Presenting author 2
CF is the most common lethal genetic multisystem disease in Germany. Although life expectancy increased over the last decades, most of the CF patients die in young adulthood due to chronic CF lung disease with respiratory failure. Preliminary work of our study group showed that CF lung disease in a transgenic mouse model of CF could be successfully treated with a preventive inhalation of hypertonic saline (HS). In patients with established CF lung disease, inhalation with 6% HS already is a simple, safe and inexpensive mucolytic therapy. So far, nothing is known about the preventive potential of HS in CF patients in the first 4 months of life. Therefore, the aim of this IIT is to verify the findings in a multicentric, randomized, double-blind, controlled pilot study on safety and efficacy of a preventive inhalation with HS in newborns and infants with CF. For this reason, 42 patients were included into the study. Participating patients were randomized to 6% HS or 0.9% isotonic saline (IS). In both groups, patients inhaled their study solution twice daily over 52 weeks. At the beginning, during and after completing the study, different measurements were undertaken to determine the endpoints (e.g., safety, number of patients with radiologic (MRI) and/or functional (MBW) alterations of the lung, number of exacerbations, time to first detection of a CF pathogen, and health-related QoL). In November 2016, the last two patients finished the study protocol. Completion of the data base, final monitoring and data base lock are currently ongoing. Therefore, first results on endpoints will be available in January 2017 and can be presented at the annual DZL meeting. Supported by BMBF (82DZL00401)
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Abstract No. 011 PYOCYANIN FROM P. AERUGINOSA INDUCES DYSBIOSIS AND INFLUENCES ESTABLISHMENT OF CHRONIC INFECTION Sebastien Boutin 1,* , Lisa Hennemann 1, Mirjam Stahl 1, Simon Graeber 1, Susanne Dittrich 2 , Marcus Mall 1, and Alexander Dalpke 1 1
University Heidelberg University Hospital Heidelberg *Presenting author 2
One of the major causes of mortality and morbidity in Cystic Fibrosis is chronic infection by Pseudomonas aeruginosa. The aim of the present study was to analyze the impact of infection by P. aeruginosa on the commensal airway microbiota and to study the interaction of P. aeruginosa with strains belonging to commensals genera, Streptococcus and Neisseria. We analyzed the microbiota of sputum samples, using 16S amplicon sequencing, from CF patients (classified as negative, intermittent or chronic according to established criteria) to evaluate the effect of the infection. We observed that dysbiosis only occurs in chronically infected patients, due to dominance of P. aeruginosa and a global decrease of commensals, while intermittent infection does not disturb the microbiota. We therefore tested the effect of 70 strains of P. aeruginosa isolates on the growth of commensals and vice versa. We also quantified biofilm formation of the various strains of P. aeruginosa. Our in vitro interaction assays showed that only strains producing pyocyanin strongly inhibited the growth of Neisseria and Streptococcus. Addition of pyocyanin alone in the growth media of the commensals was inducing the same reduction as P. aeruginosa full supernatant. The proportion of pyocyanin and biofilms producers was higher in the population of patients suffering of intermittent infection than the chronically infected one indicating that the ability to produce pyocyanin and to form biofilm is only adaptive for the early step of infection. Our data show that only chronic infection with P. aeruginosa is associated with profound changes of the lower airways’ microbiota indicating that early eradication of Pseudomonas may prevent severe dysbiosis in patients with CF. We demonstrate that pyocyanin production by P. aeruginosa helps to establish dysbiosis. We also demonstrate that long term adapted strain of P. aeruginosa do not inhibit commensals growth and form less biofilm.
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Abstract No. 014 The cystic fibrosis upper and lower airways microbial metagenome of patients with cystic fibrosis or immune deficiency Lutz Wiehlmann 1,* , Katarzyna Pienkowska 1, Patricia Moran Losada 1, Ulrich Baumann 1, Marie Dorda 1, Rebecca Hyde 1, Sebastian Fischer 1, and Burkhard Tümmler 1 1
Clinic for Pediatric Pneumology *Presenting author
In cystic fibrosis the basic defect predisposes to chronic airway infections with opportunistic pathogens. We applied shotgun metagenome sequencing to resolve the complex polymicrobial communities in upper and lower airways by collecting nasal lavage, throat swabs and induced sputa from 41 exocrine pancreatic insufficient (PI) CF patients, 21 exocrine pancreatic sufficient (PS) CF patients and 10 patients with immune deficiency (ID, positive disease control). Results. The samples contained on the average 97% of human DNA. In the remaining 3% of DNA hundreds of bacterial taxa and less than a dozen fungi, molds or DNA viruses were detected. Most viruses were bacteriophages accompanied by adeno- and herpesviruses in a few samples. Least bacterial species diversity was seen in the lungs of PI CF patients. The microbial communities were dominated by the opportunistic pathogens known from culturedependent diagnostics, i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Stenotrophomonas maltophilia plus some Rothia, Veilonella and Prevotella spp. The metagenomes became more and more similar with the progression of lung disease severity. In contrast, most bacterial species diversity was seen in the most mildly affected group of PS CF patients who presented metagenomes like those of healthy controls dominated by Streptococcus, Veilonella and Rothia spp. Nevertheless the microbial communities in nose, throat and lung were more similar between PI and PS CF than with ID. Moraxella and Haemophilus spp. were abundant in ID patients, but only minor members of the CF airway communities. In CF sputa the presence of Streptococci and Neisseria and of Bifidobacteria, Veilonella, Prevotella, Atopobium, Fusobacterium and the emerging pathogen Streptococcus anginosus was positively correlated with each other. In summary, we observed both habitat- and disease-specific signatures in the three niches and the three disease cohorts.
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Abstract No. 019 Non-contrast Enhanced Ventilation and Perfusion Magnetic Resonance Imaging in Early Cystic Fibrosis Lung Disease Mark Wielpütz 1,* , Sonja Gehlen 1, Patricia Leutz 1, Michael Puderbach 1, Mirjam Stahl 1, Olaf Sommerburg 1, Simon Gräber 1, Monika Eichinger 2, Yoshiharu Ohno 3, Hans-Ulrich Kauczor 1, Gregor Bauman 4, and Marcus Mall 1 1
University of Heidelberg Thoraxklinik at University of Heidelberg 3 University of Kobe 4 University of Basel *Presenting author 2
Rationale: Previous studies demonstrated that morpho-functional magnetic resonance imaging (MRI) detects structural and perfusion abnormalities in lungs of cystic fibrosis (CF) patients. With the present study we evaluate Fourier decomposition (FD-)MRI to detect ventilation and perfusion defects in infants and preschool children with CF without the need for exogenous contrast materials. Methods: In total, 30 children with CF (age 1.8±2.0y range 0–6y at first exam) contributed 85 MRI studies. Balanced steady-state free-precession imaging was performed in free breathing in three coronary slices through the central lung for FD-MRI, in addition to a standardized T1- and T2-weighted protocol including contrast-enhanced perfusion imaging. For FD-MRI lungs were segmented manually, and percent of ventilation and perfusion defects calculated by custom software. The remaining MRI protocol was assessed using the MRI score as previously evaluated. Results: Ventilation-weighted images could be generated from 81 (95%) and perfusionweighted images from all 85 (100%) datasets. Ventilation correlated substantially with perfusion defects (r=0.67, p<0.001). Mean ventilation and perfusion defects tended to be higher in older patients with 20.8±9.7% and 26.8±3.3% at 6y compared to 10.9±6.9% and 21.6±4.4% in newborns, respectively. Ventilation defects moderately correlated with overall disease severity documented by the MRI score (r=0.56, p<0.001), but perfusion defects did not (r=0.21). Conclusions: FD-MRI is feasible and detects ventilation and perfusion defects already in newborns, infants and preschool children with CF. These results support the development of FD-MRI for non-invasive functional lung imaging in this age group, and may render application of exogenous contrast materials unnecessary.
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Abstract No. 020 Characterization of genetic modifiers that alter the CFTR-mediated chloride conductance in Cystic Fibrosis epithelia Ellen Raddatz 1,* , Chidiebere Awah 1, Stefanie Tamm 1, Silke Hedtfeld 1, Georgios Tsiavaliaris 1, Burkhard Tümmler 1, and Frauke Stanke 1 1
Hannover Medical School *Presenting author
Modifying genes have been identified for lung function in cystic fibrosis [5], disease severity [1] and comorbidities [6]. We characterize genes that modify the CF basic defect [1,2,3]. Firstly, the regulatory SNP rs7901656 in the FAS gene modifies FAS gene expression [7], CF disease phenotype [1] and alters binding affinity for the transcription factors NF-KBp50, NF-KBp65 and HIF1a [4] which govern the cellular response to inflammation and hypoxia. As a second example, the transcription factor EHF, derived as a positional candidate from a genome wide scan [5], affects the transcriptome of CF patients’ intestinal biopsies in favor of a better processing of F508del-CFTR [3] which is confirmed in epithelial cell lines as siRNA provided against EHF results in a downregulation of MGAT2 and MGAT4, both of which are key enzymes for the complex glycosylation of proteins such as CFTR. Finally, the genetic background of CFTR is associated with CF disease severity and the CF basic defect [1]. The SNP J3.11 near CFTR alters a palindromic STAT binding motif and STAT3 mRNA levels correlate with the manifestation of the CF basic defect [2]. Findings derived from EMSA and Co-IP experiments demonstrate that STAT3 preferentially binds on the T-allele of J3.11. In conclusion, several gene regulatory mechanisms influence the CF basic defect. Interference with these pathways may results in better F508del-CFTR maturation, leading to better CFTR function in patient’s tissue and thereby promoting health in cystic fibrosis. References: 1: Stanke et al Med Genet 2011; 2: Labenski et al Eur J Hum Genet 2011; 3: Stanke et al Eur J Hum Genet 2014; 4: Awah et al BBA-GRM 2016; 5: Wright et al Nat Genet 2011; 6: Collaco & Cutting, CML cystic Fibrosis 2012; 7: Kumar et al Genes Immun 2008 Funding by: Deutsches Zentrum für Lungenforschung DZL; Mukoviszidose Institut gGmbH
Stanke et al J Med Genet 2011 Labenski et al Eur J Hum Genet 2011 Stanke et al Eur J Hum Genet 2014 Awah et al BBA-GRM 2016 Wright et al Nat Genet 2011 Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 024 Absence of T cells reduces structural lung damage in mice with cystic fibrosis-like lung disease Matthias Hagner 1,* , Simone Schmidt 1, Sandra Christochowitz 1, and Marcus Mall 1 1
Translational Lung Research Center Heidelberg, University of Heidelberg *Presenting author
Little is known about T cells in the in vivo pathogenesis of cystic fibrosis (CF) lung disease and their impact on chronic airway inflammation, mucus plugging and structural lung damage. Mice with airway-specific overexpression of the beta subunit of the epithelial sodium channel (βENaC-Tg) exhibit characteristic features of CF lung disease including airway mucus plugging, chronic inflammation and structural lung damage. To investigate the role of T cells in the in vivo pathogenesis of CF-like lung disease, we crossed βENaC-Tg mice with Rag1 deficient mice (Rag1-/-). We compared inflammatory cell counts in bronchoalveolar lavage (BAL), airway mucus obstruction and structural lung damage in juvenile (3-week-old) and adult (6-week-old) βENaC-Tg mice with βENaC-Tg/Rag1-/- mice, and their wild-type (WT) and Rag1-/- littermates. BAL neutrophils and eosinophils were elevated in βENaC-Tg and βENaC-Tg/Rag1-/- mice compared to their WT and Rag1-/littermates whereas they did not differ between βENaC-Tg and βENaC-Tg/Rag1-/- mice. Further, there was no difference in airway mucus obstruction between βENaC-Tg and βENaC-Tg/Rag1-/- mice at juvenile and adult ages. Interestingly, mean linear intercept was significantly decreased in juvenile and adult βENaC-Tg/Rag1-/- mice whereas destructive index was only reduced in juvenile βENaC-Tg/Rag1-/- mice. Gene expression of interleukin17 alpha (Il-17a), a cytokine associated with emphysema development and structural lung damage, was significantly elevated in lungs from βENaC-Tg mice. Flow cytometry revealed that γδ T, CD4+ T cells and ILC3 as major sources of IL-17A in lungs of βENaC-Tg mice. Our results suggest that T cells may not contribute primarily to the pathogenesis of airway inflammation and mucus obstruction in CF-like lung disease. However, absence of T cells reduces structural lung damage which could be related to decreased IL-17A levels in the lungs. Further studies are required to elucidate mechanisms triggering IL-17A secretion in mucostatic and chronically inflamed airways.
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Abstract No. 027 Altered Airway Macrophage Phenotype and Function in Mice with Mucociliary Clearance Dysfunction Michelle Paulsen 1,* , Jolanthe Schatterny 1, Simone Schmidt 1, and Marcus A. Mall 1 1
Translational Lung Research Center Heidelberg *Presenting author
Airway macrophages (AM) contribute to lung homeostasis through the clearance of surfactants, apoptotic cells and cell debris. Positioned at the interface of host tissue and environment they mediate key responses to inhaled pathogens. AMs show a remarkable plasticity with phenotype and function being tightly regulated by the airway microenvironment. In a recent study, we demonstrated that impaired mucus clearance in Scnn1b-Tg mice is associated with morphological macrophage activation, expression of signatures of alternative macrophage activation, and macrophage elastase (MMP12)dependent structural damage. However, the functional role and phenotype of AMs in mucociliary clearance deficiency needs further investigation. In AMs isolated from Scnn1bTg mice we identified significant changes in baseline expression of genes associated with alternative activation, like Arginase 1, MMP12, CCL17 and CCL22. In addition the M1associated surface marker CD86 was downregulated in macrophages from Scnn1b-Tg mice. Whereas increasing expression of the receptor Dectin-1 and TREM2 further emphasizes M2 polarization in Scnn1b-Tg mice. Exposure of AMs to LPS upregulated key genes involved in proinflammatory and anti-bacterial responses in Scnn1b-Tg and wt macrophages. However, expression levels of LPS-induced proinflammatory mediators, like IL-1α, IL-6, IL-12p40, CCL2 and NOS2 were significantly enhanced in AMs isolated from Scnn1b-Tg mice. Impaired macrophage clearance of apoptotic cells is associated with inflammatory lung diseases. Here we show that the in vivo efferocytosis capacity of AMs was markedly decreased in Scnn1b-Tg mice compared to wt controls. These results indicate that the mucostatic airway environment polarizes AMs toward alternative activation at baseline and primes them for augmented cytokine responses when challenged with LPS. Moreover, defective efferocytosis may result in increased inflammation and pathogenesis. These results suggest that alternative AMs may play an important role in the in vivo pathogenesis of chronic airway inflammation, and that a better understanding of the mechanisms underlying macrophage activation may lead to novel anti-inflammatory therapies.
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Abstract No. 031 Pseudomonas aeruginosa microevolution during chronic infection of cystic fibrosis airways Nina Cramer 1,* , Jens Klockgether 1, Sebastian Fischer 1, Lutz Wiehlmann 1, and Burkhard Tümmler 1 1
Medical School Hannover *Presenting author
Chronic airway infections with Pseudomonas aeruginosa typically determine the clinical course of patients suffering from cystic fibrosis (CF). During chronic infection the bacteria undergo microevolution which supposedly reflects the adaptation to the CF lung habitat and to the antimicrobial treatments administered over the years. Microevolution of P. aeruginosa was investigated for sequential isolates from twelve CFpatients, six with a mild and six with a severe clinical course. Therefore genomes of sequential isolates of the initially colonizing clone were sequenced. Screens for single nucleotide polymorphisms (SNPs), small indels, larger deletions and accessory genome variations were done by de novo assembly of sequencing reads and alignments to a reference sequence. This information was also used for clade reconstruction. All isolates were characterized in mutation rates and different phenotypic traits. Additionally, the competitive fitness of the sequential isolates was observed in vitro and in vivo. During the twelve courses >4800 mutations occurred. Loss of greater DNA blocks occurred rarely. Typically about five mutations per year were found within a course, whereas hypermutator lineages displayed dozens or even hundreds of mutations. The majority of these CF lung microevolution hotpots were associated with antimicrobial resistance, chemotaxis or adaptation and protection. For some courses, the phylogeny indicated the presence of only one lineage permanently evolving over the years. For others, several clades developed and persisted in parallel, demonstrating diversifying evolution in different niches of the CF lung. Severe courses show an increased occurrence of drastic amino acid exchanges and a lower number of persisting clades in the further infection course compared to mild courses. The fitness experiments will demonstrate if the pathoadaptive mutations in the CF-lung resulted in fitness advantages, and whether there is an impact on the competitive viability and infectious potential of single isolates.
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Abstract No. 039 Role of free versus membrane-associated neutrophil elastase activity in cystic fibrosis lung disease A. Susanne Dittrich 1,* , Iris Kühbandner 1, Clifford C. Taggart 2, Felix Herth 3, Carsten Schultz 4, and Marcus A. Mall 1 1
University of Heidelberg Queen’s University Belfast, United Kingdom 3 Thoraxklinik at the University Hospital Heidelberg 4 European Molecular Biology Laboratory *Presenting author 2
Experimental studies indicated that neutrophil elastase (NE) is an important pathogenic factor in the development of cystic fibrosis (CF) lung disease. Recent clinical studies identified free NE activity in bronchoalveolar lavage fluid and sputum as a key risk factor of early bronchiectasis and decline of lung function in patients with CF. However, until now research focused on “free” NE measured in supernatants of airway secretions, while knowledge on NE compartmentalization in the airways remains still limited. Therefore, the aim of this study was the comparison of soluble and membrane-associated NE activities with anti-proteases and pulmonary function. Inflammatory cells and cell-free supernatants were isolated from spontaneous and induced sputum of patients with CF (n=46) and healthy non-smokers (control, n=8). For the quantification of membrane-associated NE activity, cells were incubated with the lipidated FRET (Förster resonance energy transfer) reporter NEmo-2. NE activity was calculated as the ratio of donor to acceptor fluorescence and normalized to samples treated with the NE inhibitor sivelestat. In CF sputum, Soluble and membrane-associated NE activities were significantly increased, but exhibited only a weak correlation (rho=0.33, p<0.05). Further, NE protein and NE complexed with alpha-1-antitrypsin (NEAAT) were elevated while secretory leukocyte protease inhibitor (SLPI) was decreased. Membrane-associated, but not soluble NE activity correlated directly with NE protein in sputum supernatants. Conversely, soluble but not membrane-associated NE activity showed a direct correlation with NEAAT. Free and membrane-associated NE activity exhibited a moderate correlation with airflow limitation (FEV1 predicted, rho=-0.51, p<0.001 and rho=-0.46, p<0.01), but only membraneassociated NE activity was associated with air trapping (FRCpleth predicted, rho=-0.01, n.s. and rho=0.48, p<0.001). Collectively, these findings indicate that membrane-associated NE activity reflects the amount of freshly secreted NE not counteracted by the antiprotease shield. Therefore, membrane-associated NE activity could serve as valuable biomarker for the activation status of sputum neutrophils in CF.
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Abstract No. 057 Specific IgE production against Staphylococcus aureus, Escherichia coli, Hemophilus influenzae and Pneumococci in cystic fibrosis patients Anna-Maria Dittrich 1,* , Sheron Dzoro 2, Melanie Albrecht 1, Christian Dopfer 1, Annette Sauer-Heilborn 3, Felix Ringshausen 3, Burkhard Tümmler 1, Gesine Hansen 1, Rudolf Valenta 2, Belinda Hales 4, and Irene Mittermann 2 1
Pediatric Pneumology, Allergology and Neonatology, BREATH Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria 3 Pneumology, BREATH 4 Telethon Institute for Child Health Research, Centre for Child Health Research, The University of Western Australia, Perth, Australia *Presenting author 2
Background: Patients with cystic fibrosis are oftentimes colonized chronically with facultative pathogenic bacteria such as Hemophilus influenzae, Pneumococci, Escherichia coli and, most common in young children, Staphylococcus aureus. Specific IgE directed against these pathogens has not been comprehensively studied in these patients but might contribute to pulmonary disease course. Methods: IgE against Staphylococcus aureus and Escherichia coli were detected by immunoblotting, IgE against Hemophilus influenzae and Pneumococci were detected by DELFIA. Sera from 21 patients (7-40 years old) with genetically confirmed cystic fibrosis with elevated total IgE were included in our analyses. Results: We could detect production of IgE against all pathogens. Analyses included consecutive measurements from selected patients which revealed changes of sensitization patterns with time, suggesting that presence of IgE to S.aureus is associated with course of disease. Production of IgE against Staphylococcus aureus was most frequent (29% of all patients), followed by sensitization against Escherichia coli and Pneumococci and Hemophilus influenzae. Immunoblots revealed distinct IgE patterns compared to patients with atopic dermatitis who also produce Staphylococcus aureus-specific IgE. Conclusions: Patients with cystic fibrosis produce specific IgE against several facultative pathogenic bacteria known to colonize their airways. IgE against Staphylococcus aureus, encountered most frequently, displays a unique IgE pattern compared to patients with a different disease, possibly attributable to different sensitization routes. In the future analyses of cohort samples and correlation with clinical data will provide insight into the clinical relevance of this immune response.
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Abstract No. 072 Visualization of channel-activating protease (CAP) activity by peptide-based FRET probes Verena Rickert-Zacharias 1,* , Marcus Mall 2, and Carsten Schultz 1 1
Cell Biology and Biophysics Unit; EMBL Heidelberg Department of Translational Pulmonology, Heidelberg University *Presenting author 2
The epithelial sodium channel (ENaC) and the proteolytic processing of its alpha- and γsubunits by channel-activating proteases (CAPs) play a crucial role in sodium transport across different epithelial membranes, including the airways, and in airway surface liquid (ASL) homeostasis. It was shown that CAP expression is elevated in primary bronchial epithelial cultures of Cystic Fibrosis (CF) patients compared to tissue from healthy subjects. However, the impact of CAPs on ENaC activity, ASL regulation and CF lung disease is unknown. In order to study the role of CAPs to CF lung disease and their regulation in the disease context, substrate-based Förster Resonance Energy Transfer (FRET) probes were developed for measuring protease activity. Proteolytic cleavage of the probe resulted in a loss of FRET, giving a ratiometric readout reflecting enzyme activity. The attachment of a lipid anchor enabled the detection of membrane-associated enzyme activity in a spatially resolved manner. In heterogeneous cell populations, it is possible to distinguish cell types with proteolytic activity on their surface from cells without significant CAP activity, representing a strong advantage of our probes over commercial substrates. Probes based on different substrate sequences have been evaluated with respect to selectivity and biophysical properties, namely solubility and maximal dynamic range upon cleavage. In vitro characterization using recombinant enzymes as well as cell-based characterization using broad-spectrum inhibitors identified the probe based on the CAP cleavage site in the human ENaC γ-subunit as the most selective one towards CAP activity. As proof of concept, the probes were successfully tested in human primary nasal epithelial cells and primary murine tracheal epithelial cells. Both experimental sets suggested that our FRET probes are potential biomarkers for human specimen as well as research tools for further investigation of CAP activity in chronic lung diseases, including CF, idiopathic pulmonary fibrosis and lung cancer.
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Abstract No. 079 Lack of Slc26a9 Cl- channel produced neonatal mortality due to airway mucus plugging Pamela Millar-Büchner 1,* , Johanna J Salomon 1, Stephan Spahn 1, and Marcus A Mall 1 1
Department of Translational Pulmonology, Translational Lung Research Center (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany *Presenting author
Introduction: Several genetic studies have associated variants of the SLC26A9 gene encoding an epithelial Cl- channel with a higher risk of developing asthma and bronchiectasis. Further, SLC26A9 has been identified as a modulator of lung function in patients with Cystic Fibrosis (CF), supporting the potential role as a modifier of lung disease. The aim of this project was to determine the in vivo role of SLC26A9 in the lung in the very early phase of life. Methods: To achieve this goal, we compared the pulmonary phenotype of wild-type and Slc26a9 knockout (Slc26a9 -/-) mice including survival, histology, wet/dry measurements and µCT morphometry analysis. Results: All genotypes were represented according to Mendelian genetics when mice were born. In the first 30 minutes of life, abnormal breathing patterns were observed in Slc26a9-/mice and an increased mortality of 50% was found on the first day of life (p<0.05). In order to determine the cause of death, histological analyses were performed and revealed mucus plugging in the trachea (p=0,009), the proximal (p= 0,002) and distal airways (p=0,001) in deceased Slc26a9-/- compared to wild-type (WT) mice. Histological analysis of the vital organs in prenatal animals (d17) did not show any differences. Further evidence was provided by µCT imaging studies, where preliminary results indicated that lungs of Slc26a9/- mice were not fully inflated. To further investigate whether this was due to a lung liquid clearance dysfunction, wet/dry measurements were performed. Here, no differences were found between the two genotypes (p=0.56). Conclusion: Altogether, deletion of Slc26a9 causes an early onset of mortality in mice, due to the accumulation of airway mucus plugging. Our data demonstrate an essential developmental role of the Cl- channel SLC26A9 in sustaining postnatal lung mucus clearance.
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Abstract No. 089 Altered epithelial ion transport across cultured nasal epithelia Johannna Salomon 1,* , Tobias Albrecht 2, Heike Scheuermann 1, Ingo Baumann 2, and Marcus Mall 1 1
Translational Pulmonology, University of Heidelberg Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Heidelberg *Presenting author
2
Rationale. Chronic rhinosinusitis (CRS) is a very common chronic disorder of the upper airways characterized by nasal congestion and discharge and highly impacts the quality of life of patients. It is a heterogeneous disease which may be linked to impaired mucociliary clearance, sinonasal inflammation or altered ion transport. However, the pathologic mechanisms remain unknown and here, we aimed to study whether the epithelial ion transport is affected in cultured nasal epithelia of patients with CRS. Methods. Human nasal epithelial primary cells (hNEpC) were freshly isolated from nasal tissue from the paranasal sinuses of patients with CRS undergoing polyps resection (CRS) and turbinate tissue from healthy controls. HNEpC were cultured for 14 days and transepithelial short-circuit current (Isc) was measured in Ussing chambers under a Clgradient. Results. Ussing chamber studies demonstrated substantial changes in ion transport of cultured hNEpC cultures from CRS compared to controls (CTL). The basal Isc (Isc-CRS = 19.9±1.4 µA/cm² vs. Isc-CTL = 42.4±6.6 µA/cm², p<0.01), as well as the amiloride-sensitive Isc (ΔIsc-CRS = 2.4±0.3 µA/cm² vs. ΔIsc-CTL = 3.3±0.4 µA/cm², p<0.05) and the amilorideinsensitive current reflecting constitutive Cl- secretion (ΔIsc-CRS = 17.2±1.5 µA/cm² vs. ΔIsc-CTL = 38.4±6.7 µA/cm², p<0.01) were significantly diminished in CRS compared to control cultures. Further, UTP-induced responses reflecting Ca2+-activated Cl- secretion were significantly lower in CRS (ΔIsc-CRS = 0.0±0.2 µA/cm²) vs. control cultures (ΔIsc-CTL = 0.7±0.2 µA/cm²). No differences were found in CFTR-mediated Cl- transport (cAMPstimulated Isc and CFTRinh-172-sensitive current, respectively). Conclusions. Taken together, we observed an abnormal epithelial ion transport in hNEpC cell monolayers from patients with CRS compared to controls. It will be important to further investigate the primary causative mechanism in the pathogenesis of CRS and whether the epithelial dysfunction is a cause or consequence of chronic inflammation in paranasal sinuses.
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Abstract No. 090 Assay development and high-throughput screen installation for identifying SLC26A9 chloride channel activators Anita Balázs 1, Aliaksandr Halavatyi 2, Johanna J. Salomon 3, Marko Lampe 4, Rainer Pepperkok 5, and Marcus A. Mall 3 1
Department of Translational Pulmonology, University of Heidelberg, Germany; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany; Advanced Light Microscopy Facility, EMBL, Heidelberg, Germany 2 Department of Translational Pulmonology, University of Heidelberg, Germany; Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany 3 Department of Translational Pulmonology, University of Heidelberg, Germany; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany 4 Advanced Light Microscopy Facility, EMBL, Heidelberg, Germany; Department of Translational Pulmonology, University of Heidelberg, Germany; Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany 5 Advanced Light Microscopy Facility, EMBL, Heidelberg, Germany; Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany;Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany
Introduction&Aims The SLC26A9 chloride channel represents a promising candidate to provide apical chloride transport in the absence of functional CFTR, thus circumvent the primary defect in cystic fibrosis. Recent evidence suggests that SLC26A9 Cl- channel function may be activated therapeutically by compounds that increase translocation of the protein into the apical plasma membrane. To systematically identify therapeutic target genes and lead compounds promoting trafficking of SLC26A9, we aim to perform high-throughput siRNA and chemical library screens. For that we are developing two complimentary, cell-based assays to measure:(i) apical membrane localization of SLC26A9 and (ii)SLC26A9 function using a membrane potential sensitive (FLIPR) dye. Methods We generated CFBE41o- cells with stable expression of HA-tagged SLC26A9. Association of SLC26A9 to the plasma membrane is estimated using 3D confocal fluorescence microscopy. The apical membrane is labelled with Concavalin-A and SLC26A9 is stained via its HA-tag. Co-localisation of SLC26A9 with Concavalin-A is quantified as a correlation between 3D voxel intensities. Changes in membrane potential are measured by live-cell FLIPR time-lapse imaging in 96well format. After baseline measurements inhibitor is added manually. Intensity time-traces for individual cells are quantified after segmentation and image quality control. Results We established an initial image analysis pipeline to measure fraction of cytoplasmic protein co-localizing with plasma membrane. Currently we are optimizing sample preparation
DZL Annual Meeting 2017 protocol in order to increase dynamic range of the traffic assay. Using the FLIPR assay we quantified the baseline and the response upon adding inhibitor. We were able to detect significant difference between SLC26A9 over-expressing and control cells. Conclusions/Perspectives We are developing robust assays to monitor SLC26A9 function and membrane localisation. Currently we are adapting these assays for high-throughput experiments (high-throughput data processing and automated reagent addition synchronised with time lapse imaging). This platform will enable us in the future to identify therapeutic strategies to activate SLC26A9.
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Abstract No. 095 Characterization of myeloid cells after transplantation in a mouse model of cystic fibrosis Kerstin Brinkert 1,* , Stephanie Tamm 1, Theresa Buchegger 1, Silke Hedtfeld 1, Sabine Schild , Hegermann Jan 1, Rudolf Bauerfeind 1, Frauke Stanke 1, Mania Ackermann 1, and Antje Munder 1
2
1
Medizinische Hochschule Hannover Frauenhofer ITEM *Presenting author 2
Cystic fibrosis (CF) is a severe autosomal dominant monogenetic disease with an incidence of one out of 2,500 newborns in Europe. It is the result of around 2,000 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by mucus accumulation in all exocrine glands generating the ground for life threatening respiratory infections in CF lungs. For a long time CF research focused exclusively on the epithelial component of the disease, but in more recent literature the role of a CFTR defect in professional phagocytes such as macrophages and neutrophils was increasingly discussed (1). Own experiments in CF mice showed that transplantation of unaffected hematopoietic stem and precursor cells (HSPCs) reduced lung bacterial numbers and increased survival rates in these mice when infected with Pseudomonas aeruginosa in a standardized infection model. The profile of inflammatory cytokines was significantly altered as well. Based on these results, the actual project focuses on the role of myeloid cells in CF and aims to prove that transplanted HSPCs engraft in murine lungs, where they either differentiate into alveolar macrophages and hereby support the innate immune system or potentially differentiate into cells expressing epithelial characteristics and in this way reduce the lung damage induced by the bacteria (2). Investigating phagocytosis of bone marrow derived macrophages (BMDM) of CF and wild type mice, no clear differences in uptake and killing of bacteria could be detected between the two populations. Therefore, electron and confocal microscopy was started to get a more precise picture on the phagocytosis (3). Further experiments will try to detect Cftr in murine macrophages via Western Blot and epithelial features of transplanted cells will be checked by immune fluorescence.
(1) Haggie PM, Verkman AS. Defective organellar acidification as a cause of cystic fibrosis lung disease: Reexamination of a recurring hypothesis. Am J Physiol Lung Cell Mol Physiol. 2009;296(6):L859-67. (2) Wong AP, Keating A, Lu WY, et al. Identification of a bone marrow-derived epithelial-like population capable of repopulating injured mouse airway epithelium. J Clin Invest. 2009;119(2):336-348. (3) Zhang Y, Li X, Grassme H, Doring G, Gulbins E. Alterations in ceramide concentration and pH determine the release of reactive oxygen species by cftr-deficient macrophages on infection. J Immunol. 2010;184(9):5104-5111. Submitted as: Presentation 279 words of 300 possible words
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Abstract No. 110 The effect of the anti-inflammatory IL-1R antagonist anakinra in mice with CF-like lung disease and acute Pseudomonas infection André Schütte 1,* , Zhe Zhou-Suckow 1, Jolanthe Schatterny 1, Simone Schmidt 1, Selina Hassel 1, Michael Weitnauer 1, Alexander Dalpke 1, and Marcus A. Mall 1 1
Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg, Germany *Presenting author
Epithelial necrosis is a consequence of hypoxia in mucus-obstructed airways. This triggers the release of IL-1α and leads to a neutrophilic inflammation. The inhibition of the IL-1RMyD88-pathway by the treatment with the IL-1R antagonist anakinra reduces airway neutrophilia and structural lung damage, as recently demonstrated in Scnn1b-Tg mice with CF-like lung disease (Fritzsching B. et al., 2015). However, the effects of anakinra in the context of bacterial infection remain unknown. Thus, the aim of this study is to investigate whether anakinra impairs the antibacterial host defense within an acute Pseudomonas infection in mice with CF-like lung disease. Scnn1b-Tg and wild-type mice were treated with anakinra (5mg/10g body weight) or NaCl subcutaneously b.i.d. and subsequently instilled with the P. aeruginosa strain PAO1 (2.5x10^7 cfu/mouse) or vehicle (PBS) intratracheally. 24 hours post infection, bronchoalveolar lavage (BAL) was performed and analyzed using differential cell counts, cytometric bead assay to measure pro-inflammatory cytokines and quantitative microbiology by culture and MALDI. Furthermore, mucus volume density and structural lung damage was assessed by histological parameters. The instillation of PAO1 induces a robust neutrophilic inflammation in both wild-type and Scnn1b-Tg mice. However, treatment with anakinra significantly reduced the number of neutrophils in PAO1-infected Scnn1b-Tg mice (1,3x10^6 neutrophils/ml (untreated) vs. 4,4x10^5 neutrophils/ml (treated), n=13-15, P<0.001) but did neither aggravate the infection by Pseudomonas in wild-type nor in Scnn1b-Tg mice (1.9x10^3 cfu/ml (untreated) vs. 5.6x10^3 cfu/ml (anakinra-treated), n=13-14, P>0.4). Similarly, spontaneous infections by different bacterial species, as observed in Scnn1b-Tg mice (~1x10^2 cfu, n=6-10), were not increased by treatment with anakinra. In conclusion, anakinra may be used as a novel anti-inflammatory approach to control overwhelming neutrophilic airway inflammation without aggravating bacterial infection in CF. Certainly, clinical studies are necessary to test the safety and efficacy in patients with CF. (Supported by an EC 7th Framework Program No. 603038 CFMatters)
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Abstract No. 111 Role of the IL-1 signaling pathway in the development of type 2 airway inflammation in juvenile Scnn1b-Tg mice Ryan Brown 1,* , Simone Schmidt 1, Jolanthe Schatterny 1, Stephanie Hirtz 1, and Marcus Mall 1 1
Translational Lung Research Centre (TLRC), German Centre for Lung Research (DZL), University Hospital Heidelberg, Germany *Presenting author
The Scnn1b-Tg mice are a model of CF-like lung disease that is characterized by airway specific overexpression of the β subunit of the epithelial sodium channel ENaC under the control of the club cell secretory protein (CCSP) promoter, producing many of the features of the CF airway including airway mucus plugging, chronic inflammation, and progressive lung tissue destruction. Interestingly, during the first weeks of life a primarily type 2 inflammation predominates with upregulation of type 2 cytokines such as IL-13, influx of eosinophils to the airways, goblet cell metaplasia and mucus hypersecretion. Previous studies have demonstrated a role for IL-1α in the development of type 2 inflammation in allergen models. Additionally, in Scnn1b-Tg mice hypoxic epithelial necrosis in mucus obstructed airways results in elevated IL-1α. Thus, we hypothesize that IL-1α/IL-1R signaling may be implicated in type 2 inflammation in the Scnn1b-Tg mouse. To test this hypothesis we determined effects of IL-1R knockout on the Scnn1b-Tg phenotype. Scnn1b-Tg mice and WT littermates with or without IL-1R deletion were sacrificed at 2 weeks of age and BAL inflammatory markers, goblet cell metaplasia and mucus hypersecretion were analyzed. Scnn1b-Tg mice developed significant airway inflammation and mucus plugging when compared to WT mice. The knockout of IL-1R in the Scnn1b-Tg mouse significantly reduced airway eosinophilic inflammation (97x10^4 [Scnn1b-Tg] to 36x10^4 [Scnn1b-Tg/IL1R-/-] eosinophils/ml of BAL, p<0.001) and airway mucus plugging by ~65% (p<0.001) without affecting mucin, IL-13 or IL-5 expression. No effect of IL-1R knockout was observed in WT mice. Results so far suggest that IL-1α signaling through the IL-1R may contribute to eosinophilia and airway mucus plugging in the Scnn1b-Tg mouse. Further experiments are needed to elucidate the mechanisms behind the effects of IL-1R knockout on eosinophilic airway inflammation in juvenile Scnn1b-Tg mouse.
Submitted as: Presentation 289 words of 300 possible words
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Abstract No. 113 Genetic deletion of MMP-9 does not reduce chronic neutrophilic inflammation and structural lung damage in mice with cystic fibrosis-like lung disease Claudius Wagner 1,* , Jolanthe Schatterny 1, Zhe Zhou-Suckow 1, Carsten Schultz 2, and Marcus A. Mall 1 1
Department of Translational Pulmonology, University of Heidelberg, Heidelberg, Germany Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany *Presenting author 2
Cystic fibrosis (CF) lung disease is characterized by chronic neutrophilic airway inflammation and with an imbalance of leukocyte derived proteases and their respective antiprotease. This imbalance is thought to play a key role in structural lung damage leading to progressive decline in lung function. Elevated levels of matrix metalloproteases-9 (MMP9) in sputum have been shown to correlate with lung function decline in CF patients. The lung specific overexpression of the beta subunit of the epithelial sodium channel (βENaCTg) in mice reproduces key characteristics of CF lung disease with chronic inflammation and the development of emphysema. This mouse model of CF-like lung disease was used to study the impact of MMP-9 deletion on neutrophilic inflammation and structural lung damage. Therefore MMP-9+/- were crossed with MMP-9+/- βENaC-Tg mice. Total inflammatory cell counts and differentials were quantified in bronchoalveolar lavage (BAL) fluid and mean linear intercepts as an indicator of structural lung damage were evaluated in 6-8 week old mice. βENaC-Tg and MMP-9-/- βENaC-Tg mice showed a significant but similar increase in total leukocyte counts in BAL fluid by about 30% compared to WT mice (n= 12-26; p< 0.01). Neutrophil counts were equal in βENaC-Tg and MMP-9-/- βENaC-Tg mice (n= 16, 12; p >0.9) and both significantly elevated compared to WT mice (n=26, p< 0.001). Mean linear intercepts showed no reduction by MMP-9 deletion in βENaC-Tg mice while both were significantly increased compared to WT mice. The deletion of MMP-9 has no effect on inflammation and structural lung damage in βENaC-Tg mice arguing against a critical role in the in vivo pathogenesis. Inter-species differences, in particular a more predominant neutrophilia in humans and higher levels of antiproteases in the mouse lung, might account for the contrasting observations. Further studies are needed to address these apparent differences in MMP-9 effects between mice and patients.
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Abstract No. 201 Impact of age at diagnosis on development of lung disease in children with cystic fibrosis Eva Steinke 1,* , Mirjam Stahl 1, Marc O. Wielpütz 2, Olaf Sommerburg 1, Hans-Ulrich Kauczor 2, Michael Puderbach 3, Monika Eichinger 4, Simon Y. Gräber 1, and Marcus A. Mall 1
1
University Children’s Hospital Heidelberg Department of Diagnostic and Interventional Radiology, University of Heidelberg 3 Department of Radiology, German Cancer Research Center, Heidelberg, Germany 4 Department of Radiology, German Cancer Research Center, Heidelberg *Presenting author 2
Background Recent observational studies in children with cystic fibrosis(CF) demonstrated early onset and progression of lung disease, even without respiratory symptoms. However, the impact of early diagnosis and specialized care on the onset and development of lung disease remains poorly understood. We recently established imaging by MRI as a sensitive noninvasive outcome measure of abnormal lung structure. Aims/Methods The aim of this study was to evaluate the impact of the age and mode of diagnosis on the onset and progression of early CF lung disease. We performed annual longitudinal MRI studies in 96 children with CF over a period of 4 years from the time of diagnosis by i)NBS (pre-symptomatic,n=28), ii)clinical symptoms in the first 4 months of life (early clinical diagnosis, ECD,n=35), or by iii)clinical symptoms >4months of life (late clinical diagnosis, LCD,n=33) and compared morphological and functional abnormalities of the lungs using the CF-MRI score. Results 96 children with CF (48 males) met the eligibility criteria with a mean age at diagnosis of 0.1±0.1 years in the NBS group, 0.15±0.1 years in the ECL group and 1.7±1.3 years in the LCD group respectively with 89.6% being pancreatic insufficient. MRI scan was performed in 85 patients as part of their annual surveillance (NBS:n=27; ECD:n=29; LCD:n=29). Preliminary results from longitudinal analysis of structural abnormalities detected by MRI showed an increase over time for the whole cohort, but the 3 subgroups show distinct patterns. A detailed analysis is ongoing and results will be available in January 2017. Conclusions For the first time, we present longitudinal MRI data from infants and children with CF. Preliminary analysis shows different patterns of progression of early lung disease depending on the age and kind of diagnosis. The data of this preliminary study support early diagnosis and treatment to delay progression of structural lung damage.
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Abstract No. 218 Bicarbonate (HCO3-) improves mucus properties in β-ENaC-overexpressing mice with CF-like lung disease Mario Pieper 1,* , Hinnerk Schulz-Hildebrandt 2, Marcus Mall 3, Gereon Hüttmann 2, and Peter König 1 1
Institute of Anatomy, University of Lübeck, Germany Institute of Biomedical Optics, University of Lübeck, Germany 3 Department of Translational Pulmonology, University of Heidelberg, Germany *Presenting author 2
Viscous mucus is a hallmark of cystic fibrosis (CF). Recent data from experiments supplementing HCO3- in the CF pig model indicate that a lack of CFTR-mediated HCO3secretion during mucin/mucus secretion impairs normal mucus formation and suggested that this defect may be more important for the pathogenesis of viscous mucus in CF than airway surface dehydration. An alternative explanation for the effectiveness of HCO3- on mucus transport in CF models could be that it improves the properties of dehydrated mucus. Using intravital optical coherence microscopy (OCM) we tested if HCO3- can change mucus properties in mice that suffer from dehydrated mucus and develop CF-like lung disease. Images of mucus transport through the intact trachea were recorded with a custom-built OCM setup in anesthetized spontaneously breathing wild type (WT) and β-ENaCoverexpressing mice. Intranasal application of 30 µl 0.9% NaCl solution was compared to 30 µl 0.9 % NaCl solution supplemented with 115 mM NaHCO3. At baseline, WT and β-ENaC-overexpressing mice had only a very thin mucus layer. After intranasal application of HCO3- or saline control, a fast cough-like fluid transport was observed which cleared a significant amount of liquid. In WT mice, both tested substances only shortly increased airway surface liquid. In β-ENaC-overexpressing mice HCO3- and 0.9 % NaCl alone induced a substantial mucus mobilization. We did not detect differences in mucus amount, cilia-driven mucus transport, and epithelial height. However, mucus mobilized by HCO3- treatment led to a more even distribution of mucus over the epithelial surface. An effect we previously observed after application of 7 % NaCl. Our results demonstrate that treatment with HCO3- increases the transportability of dehydrated mucus in mice with CF-like lung disease to similar level obtained with hypertonic saline. Our data suggest that HCO3- can improve mucus transport, even after abnormal mucus has formed in vivo.
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Abstract No. 226 Characterization of Cystic Fibrosis-like Lung Disease in Mice: Preliminary Results of an intermodal ex vivo micro CT study Willi Wagner 1,* , Julia Dürr 1, Philip Konietzke 1, Wolfram Stiller 1, Maximilian Ackermann 2, Heinz Horstmann 1, Hans-Ulrich Kauczor 1, Marcus Mall 1, and Mark Wielpütz 1 1
Heidelberg University Johannes Gutenberg University Mainz *Presenting author 2
In previous studies, we monitored CF-like lung disease progression longitudinally in mice over-expressing the β-epithelial Na+ channel (βENaC-Tg). An intermodal approach was now used to characterize the development and progression of morphologic abnormalities of main conducting airways, distal airways and terminal bronchioles in βENaC-Tg mice in greater detail. To assess airway morphology, postnatal day 0.5, 14 and adult βENaC-Tg and wild-type littermate control lungs were perfusion fixed in situ to preserve airway lumen, luminal mucus, and airway mucus plaques in their orthotopic region. Global volumetric ex vivo measurements were performed by high resolution µCT (~9 µm voxel size). Selected regions of interest were subjected to ultra high resolution synchrotron radiation based tomography (~325 nm voxel size). Results were cross-validated by histology and block-face-SEM of same specimens. Gross examination of µCT scans revealed characteristic CF lung disease abnormalities such as airway mucus obstruction and atelectasis. Airway wall thickening was observed in the proximal bronchial tree, particularly in the bronchoalveolar duct junctions. Mucus plaques were localized in main conducting airways, distal airways and terminal bronchioles, as distal as the bronchoalveolar duct junction in the Tg group. Partially versus fully obstructed distal airways were defined. Atelectatic areas distal to fully obstructed bronchioles were identified, as well as hyperinflated parenchyma, as a microscopic correlate to air trapping as reported previously. Finally, mucus plaque volume and plaque-to-airway wall adhesions were visualized in three dimensions. Our study demonstrates the spatial arrangement of luminal airway mucus plaques throughout the bronchial tree, including terminal bronchioles and bronchoalveolar duct junctions in a mouse model of CF-like lung disease. 3D morphology of individual plaques, as well as abnormalities of distal lung parenchyma were characterized on a sub-micron level. The results suggest high and ultra high resolution micro-tomography to be a suitable tool to study small airways disease in mouse models.
Wielpütz MO et al. In vivo monitoring of cystic fibrosis-like lung disease in mice byvolumetric computed tomography. Eur Respir J 2011; 38: 1060–1070. Mall MA et al. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004; 10: 487–493. Mall MA et al. Development of chronic bronchitis and emphysema in b-epithelial Na+channel-overexpressing mice. Am J Respir Crit Care Med 2008; 177: 730–742. W. Denk, H. Horstmann et al. Serial block-face scanning electron microscopy to reconstruct
DZL Annual Meeting 2017 three-dimensional tissue nanostructure. PLoS Biol., 2 (2004), p. e329 Submitted as: Presentation 300 words of 300 possible words
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Abstract No. 229 Development of enzyme-cleavable FRET reporters to quantify MMP-9 activity in lung diseases. Victoria Halls 1 1
EMBL
There is a great clinical need for rapid, accurate testing of patient samples in lung diseases. One method that has potential in this area is the use of enzyme-cleavable FRET reporters, in which two fluorescent dye molecules are covalently linked via an amino acid sequence that is specifically cleaved by the enzyme of interest. The challenges in the synthesis are achieving a suitable solubility of the reporter so that it localises to the cellular region where the enzyme is active, and the specificity of the cleavage sequence (Hu et al, 2014). We are developing FRET reporters to investigate the involvement and activity of the enzyme matrix metalloprotease 9 (MMP-9) in various lung diseases such as cystic fibrosis and lung cancer. The goal is to quantify enzyme activity on the surface of MMP-secreting cells including those from lung epithelia. We based our design on a reported peptidic substrate (Tranchant et al, 2014) which is selectively cleaved by MMP-9 between a glycine residue and the unnatural amino acid iodophenylalanine responsible for its selectivity. To enable FRET measurements we attached Coumarin 343 as a donor to the N-terminus and TAMRA as acceptor to the C-terminus of the peptidic substrate. Additionally, we added palmitic acid to the N-terminus of the reporter to provide the necessary lipophilicity so that the reporter inserts itself in to the outer cell membrane, proximal to the known location of secreted MMP-9. We rapidly encountered a problem of rapid internalisation of the reporter into cells. Thus, we are currently modifying the N-terminus of our reporter by using negatively charged natural or unnatural amino acids to increase hydrophilicity and therefore reduce internalisation.
Hu et al, Biotechnol. J. 2014, 9, 266-281. Tranchant et al, Chem Biol 2014, 21, 1-6. Submitted as: Presentation 271 words of 300 possible words
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Abstract No. 251 Pseudomonas aeruginosa modulates the antiviral response of airway epithelial cells Julia Kantorek 1, Lauren Byrnes 1, Selina Tümkaya 1, Mirjam Stahl 2, Simon Gräber 2, Marcus Mall 2, Sébastien Boutin 1, Dao Nguyen 3, Alexander Dalpke 1,* , and Michael Weitnauer 1 1
Dept. of Infectious Diseases, University Heidelberg Dept. of Translational Pulmonology, Univ. Heidelberg 3 Dept. of Medicine, McGill University, CA *Presenting author 2
Pseudomonas aeruginosa (P. aeruginosa) often infects patients suffering from cystic fibrosis (CF). Furthermore, intermittently emerging pulmonary exacerbations, the main drivers of lung function worsening, are linked to P. aeruginosa infection. Since exacerbations in other respiratory diseases like asthma and COPD have been associated with infections with respiratory viruses, we investigated the influence of P. aeruginosa on the antiviral host immune response in an in vitro system. P. aeruginosa strains were grown and supernatant of bacterial cultures was used as conditioned medium (CM). Airway epithelial cells were pretreated with CM of different P. aeruginosa strains and subsequently infected with Respiratory Syncytial Virus (RSV). CM of P. aeruginosa could significantly repress the secretion and induction of both interferons (IFN) and antiviral proteins (MX1, OAS1) compared to the control, thereby facilitating viral replication and spreading. Of note, P. aeruginosa CM did not affect initial type III IFN induction, accordingly primary recognition of RSV did not seem to be altered. Further analysis revealed the direct degradation of type III IFN by CM of P. aeruginosa suggesting the involvement of secreted bacterial proteases. Additionally, experiments using P. aeruginosa isolates of patients suffering from CF at different infection stages revealed a dependency on the Quorum-sensing system LasR. Previous studies indicated that LasR, which is known to be a transcriptional activator of three proteases (LasA, LasB, AprA), is subject to a high mutation rate causing loss of function in chronic P. aeruginosa infection stages. A pilot study revealed a higher prevalence of rhinovirus in CF sputum sampled from CF patients being affected by an intermittent P. aeruginosa infection in comparison with those with a chronic course of disease. Abating the antiviral response through degradation of type III IFN via secreted proteases by P. aeruginosa might promote virus induced exacerbations in cystic fibrosis patients.
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Abstract No. 263 Magnetic Resonance Imaging Detects Mosaic Perfusion in Early Cystic Fibrosis Lung Disease Patricia Leutz 1,* , Michael Puderbach 1, Mirjam Stahl 2, Olaf Sommerburg 2, Hans-Ulrich Kauczor 1, Monika Eichinger 1, Marcus A. Mall 2, and Mark O. Wielpütz 1 1
Diagnostic and Interventional Radiology Pediatric Pulmonology *Presenting author 2
Objectives: To determine if MRI is sensitive to detect mosaic perfusion in lungs from infrants and preschool children with CF in stable clinical condition. Material and methods: 51 MRIs from CF- infants in clinical stable condition from 0,25-6 years were independently scored blinded for clinical and demographic data by two readers (Reader 1=R1, Reader 2= R2) of different reader experience with a previous established 4D perfusion MRI score. The extent of abnormalities for each morphology subscore/perfusion was rated in for whole lung and for each lobe from 0 (no abnormality) to 2 (abnormality>50% of lobe).Inspiratoray T2weighted MRI sequences and first pass perfusion imaging were used to detect signs of mosaic perfusion in lungs. Results: Mosaic perfusion can be detected on inspiratory MRI and Mosaic perfusion can be assessed by inexperienced readers. All MRI scans could be regularly completed in all 51 patients. Intrareader agreement between mosaic score and perfusion score was for R1 k=0.59 and 95% CI=0.45-0.72 and for R2 k=0.60and 95%CI=0,47-0.73. Interreader agreement for Mosaic scores were k=0.71 and 95%CI 0.63-0.79. Conclusions: This study demonstrates for the first time that MRI reliably detects mosaic perfusion early in CF. Inspiratory T2-weighted sequences as a routine protocol reflect pulmonary blood volume distribution and may be used for the assessment of perfusion if contrast material or alternative techniques are not available.
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Abstract No. 280 Early Development of Paranasal Sinusitis in Cystic Fibrosis Olaf Sommerburg 1,* , Mark O. Wielpütz 2, Monika Eichinger 3, Elzbieta Opdazaite 3, Mirjam Stahl 1, Michael U. Puderbach 3, Annette Kopp-Schneider 4, Eva Fritzsching 1, Ingo Baumann 5, Hans-Ulrich Kauczor 2, and Marcus A. Mall 1 1
Div. of Pediatric Pulmonology 3. Department of Diagnostic and Interventional Radiology 3 4. Department of Diagnostic and Interventional Radiology with Nuclear Medicine 4 5.Department of Biostatistics 5 6. Department of Otorhinolaryngology, Head and Neck Surgery *Presenting author 2
Background: Chronic sinonasal disease contributes to morbidity in cystic fibrosis (CF) and may constitute a repository for recurrent lower airway tract infection. Since little is known about sinus development in young CF patients this study was conducted to describe sinus pneumatization and onset of sinus disease in CF patients aging from 0 to 6 years. Methods: 28 infant and pre-school CF patients (3.72 ± 1.98 years) as well as 30 non-CF controls (3.47 ± 2.03 years) underwent magnetic resonance imaging (MRI). Axial dimensions and morphological changes including mucosal swelling, effusions, polyps, and mucopyoceles of the maxillary, frontal, sphenoid and ethmoid sinus were assessed by a newly developed standardized score. Results: Development and dimensions of paranasal sinuses were not significantly different between controls and CF. Mucosal swelling was the most prevalent pathology in 20% of control sinuses, but found in 90% of CF sinuses (P<0.001). Polyps were found in 2% of controls and 14% of CF (P<0.001). Mucopyoceles and maxillary sinus deformation occurred almost exclusively in CF with 61% and 75% (P<0.001), respectively, starting at the age of 1 year. The mean MRI sum score was significantly higher in CF (25.4±10.7) than in controls (4.5±7.6) (P<0.001). Conclusions: Sinonasal disease in CF starts in early childhood. The presence of mucopyoceles and maxillary sinus deformation are pathognomonic for CF and may be visible from infancy. Sinonasal disease may routinely be assessed at CF centers together with the lung by comprehensive MRI of the upper and lower airway tract.
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Abstract No. 286 Infection of fresh human lung tissue with Pseudomonas aeruginosa Laura Boge 1,* , Meike Müller 1, Danny Jonigk 2, Peter Braubach 2, Hans-Gerd Fieguth 3, Gregor Warnecke 2, Marcus Krüger 2, Armin Braun 1, Katherina Sewald 1, and Sabine Wronski 1 1
Fraunhofer ITEM Hannover Medical School 3 KRH clinics *Presenting author 2
Background: Pseudomonas aeruginosa is a major cause of morbidity for patients suffering from cystic fibrosis and thereby has a main impact on their life expectancy. Impaired mucus secretion leads to inefficient lung clearance resulting in infection with P. aeruginosa. The sensitivity of clinical isolates towards antibiotic treatment is usually determined via the MICAssay (determination of antibiotic minimal inhibitory concentrations). However, this standard assay does not consider the host-pathogen interactions in lung tissue. To mimic the in-vivo situation more closely, we infected fresh human lung tissue with Pseudomonas aeruginosa. Methods: PCLS from rat or human lungs were prepared and infected with P. aeruginosa strain PAO1 (105 /PCLS). Infected lung tissue was treated with different concentrations of tobramycin. Tissue viability was measured 6h and 24h post infection via LIVE/DEAD® staining and confocal microscopy or photometric quantification. Bacterial load was determined by dilution plating and counting of colony-forming units. Results: The ex-vivo infection of human lung tissue with P. aeruginosa was established successfully. The lung tissue remained viable after 6h infection. However, extended infection period led to cell death probably due to tissue necrosis. Addition of low doses of tobramycin allowed a prolonged cultivation period with a largely sustained tissue viability which is a prerequisite for the development of persistent pneumonia. Conclusion: Fresh lung tissue ex vivo can be infected with P. aeruginosa. This system can serve as a valuable tool for efficacy and toxicity testing of novel anti-infectious compounds in the microenvironment of lung tissue.
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Abstract No. 292 Epigenetic modulation of NRF2 and BACH1 in Cystic Fibrosis Bronchial Epithelial Cells by Curcumin and Histone Deactylase Inhibitors Rescues Heme Oxygenase-1 expression. Virajith Garapati 1, Martina Korfei 1, Poornima Mahavadi 1, Lutz Nahrlich 1, Claus Vogelmeier 2 , Andreas Guenther 1, and Shashi Pavan Chillappagari 1 1
Justus Liebig University, Giessen Philipps University Marburg
2
Cystic fibrosis (CF) is an inherited lung disorder caused due to defective CF transmembrane chloride channel regulator gene (CFTR) and is associated with recurrent pulmonary infections and increased oxidative stress. Hemeoxygenase-1 (HMOX1), an anti-oxidative stress response enzyme involved in heme degradation is severely curtailed in CF bronchial epithelial cells. Chromatin structural changes and alterations in the histone acetylation/deacetylation balance have been suggested to be involved in altered gene expression in several lung pathologies. In this study we aim to understand the role of HDACs in curtailed HMOX1 expression under CF conditions. In comparison to 16HBE14ocells we observed that Nrf2, a positive regulator of HMOX1 was 2- to 3-fold downregulated in CFBE41o- cells and BTB and CNC homology protein-1(BACH1), a negative regulator of HMOX1 was 3-5 fold increased in CFBE41o- cells. Altered expression of these transcription factors collectively influenced HMOX1 expression in CFBE41o- cells. Over expression of NRF2 using NRF2-GFP plasmids restored HMOX1 levels in CFBE41o- cells. Also therapeutic inhibition of HDACs using curcumin (diferuloylmethane), a dietary polyphenol, was shown to reduce BACH1 protein levels. In parallel pharmacological inhibition of HDACs using Tricostatin A and Panobinostat in NRF2 overexpressing CFBE41o- cells significantly rescued HMOX1 expression. To conclude, aberrant expression of transcription factors NRF2, BACH1 and HDACs in CFBE41o- cells may contribute towards curtailed expression of HMOX1 and increased oxidative stress. Therefore, inhibition of HDACs using natural dietary polyphenols such as curcumin may alleviate the HMOX1 mediated oxidative stress in CF.
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Abstract No. 293 Regulation of Ferroportin in Cystic Fibrosis Bronchial Epithelial Cells: Possible Role of TGF-β1, ENaC and Hypoxia. Gaurav Sarode 1,* , Virajith Garapati 2, Poornima Mahavadi 2, Andreas Guenther 2, Lutz Nahrlich 2, Markus Henke 3, and Shashi Pavan Chillappagari 2 1
Helmholtz Zentrum Munich Justus Liebig University Giessen 3 Asklepios Fachkliniken München-Gauting *Presenting author 2
Altered expression of hypoxia and their inducible pathways exert severe damaging effects on airway epithelial cells. There exists a long standing relation between hypoxia and iron homeostasis. We reported that impaired stabilization of hypoxia inducible factor-1α in cystic fibrosis (CF) bronchial epithelial cell lines (CFBE41o-) may contribute towards increased cellular levels of iron by involving HMOX1. However the underlying molecular mechanisms are still elusive. Thus, in this study we quantified the expression of two hypoxia inducible genes transforming growth factor beta (TGF-β) and ferroportin (FPN). Altered expression of TGF-β and FPN might potentially be involved in altered iron homeostasis in CFBE41o- cell lines. We identified a significantly decreased expression of TGF-β and FPN mRNA and protein levels in CFBE41o- cells. By rescuing the stability of HIF-1α using iron chelators we could restore TGF-β and FPN expression in CFBE41o- cells. On the other hand we observed that expression of TGF-β positively correlated with the expression of FPN1 in CF cell lines. We also identified a possible involvement of ENaC in ferroportin expression. Moreover inhibition of ENaC restored altered TLR4 and HO-1 expression in CF cell lines. Together, these data demonstrate that regulation of intracellular iron in CF airway epithelial cells may involve multiple signaling events to maintain cellular iron homeostasis.
Submitted as: Presentation 209 words of 300 possible words
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Abstract No. 003 Origin and fate of the lipofibroblasts during lung development and disease Saverio Bellusci 1,* , and Elie El Agha 1 1
Justus Liebig University Giessen *Presenting author
Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development and in disease conditions. We have pioneered the field through lineage tracing approaches during developmnent (1-3) and disease (4). In our follow up projects, we plan to manipulate different signaling pathways (FGF, PPARg, TGFb) in LIFs or their progeny and evaluate the impact of such changes during repair after injury.
El Agha et al. (2014) Fgf10-positive cells represent a multipotent progenitor cell population during lung development and postnatally. Development 141(2):296-306 Al Alam et al. (2015) Evidence for the involvement of Fibroblast Growth Factor 10 in lipofibroblast formation during embryonic lung development. Development, 142(23):4139-50 Li et al (2016) Mesodermal Alk5 Controls Lung Myofibroblast versus Lipofibroblast Cell Fate. BMC Biology 16(14):19 El Agha et al. (2017) Phenotypic plasticity of lipogenic and myogenic fibroblasts in the fibrotic lung: A novel mechanism for activated myofibroblast formation and potential resolution in idiopathic pulmonary fibrosis. Cell Stem Cell 20, 1-14 Submitted as: Presentation 132 words of 300 possible words
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Abstract No. 006 Prognosis and Longitudinal Changes of Physical Activity in Idiopathic Pulmonary Fibrosis Thomas Bahmer 1,* , Anne-Marie Kirsten 2, Benjamin Waschki 1, Klaus F. Rabe 1, Helgo Magnussen 2, Detlef Kirsten 1, Marco Gramm 2, Simone Hummler 3, Eva Brunnemer 3, Michael Kreuter 3, and Henrik Watz 2 1
LungenClinic Grosshansdorf Pulmonary Research Institute at LungenClinic Grosshansdorf 3 Thoraxklinik, University of Heidelberg *Presenting author 2
RATIONALE: Physical activity (PA) is associated with disease severity in idiopathic pulmonary fibrosis (IPF), but longitudinal studies evaluating its prognostic value and changes over time are lacking. METHODS: We measured PA (steps per day, SPD) in a cohort of 46 patients with IPF (mean age, 67 years; mean FVC, 76.1%pred.; SPD, 5,017±3,360) by accelerometry at baseline, recorded survival status during 3-years follow-up and repeated measurements in survivors. We compared the prognostic value of PA to established mortality predictors including lung function (FVC, DLCO) and 6-minute walking-distance (6MWD). RESULTS: During follow-up (median 34 months) 20 patients (43%) died. SPD and FVC best identified non-survivors (AUROC-curve 0.79, p<0.01). After adjustment for confounders (age, sex), a standardized increase (i.e. one SD) in SPD, FVC%pred. or DLCO%pred. was associated with a more than halved risk of death (HR<0.50; p<0.01). Compared to baseline, SPD, FVC, and 6MWD annually declined in survivors by 973 SPD, 130ml and 9m, resulting in relative declines of 48.3% (p<0.001), 13.3% (p<0.001) and 7.8% (p=0.055), respectively. SPD in survivors at follow-up (3482±2350) was comparable to SPD in non-survivors at baseline (3422±2655). CONCLUSION: While PA predicts mortality of IPF patients similar to established functional measures, longitudinal decline of PA seems to be disproportionally large. Our data suggest that the clinical impact of disease progression could be underestimated by established functional measures.
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Abstract No. 015 Analysis of Serum Metabolome in Idiopathic Pulmonary Fibrosis Alfonso Carleo 1,* , Sven Schuchardt 2, Benedikt Jäger 2, Thomas Illig 1, David DeLuca 1, and Antje Prasse 1 1
Hannover Medical School Fraunhofer ITEM, Hannover *Presenting author 2
Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a fatal Interstitial Lung Disease characterized by progressive loss of the alveolar integrity and excessive collagen deposition. Pirfenidone (5-Methyl-1-phenyl pyridine-2-one) is an antifibrotic synthetic drug approved by European Medicines Agency (EMA) and widely used in IPF treatment. Pirfenidone treatment attenuates mean FVC decline and improves progression-free survival. However, it is currently unclear how to predict which patient will benefit from pirfenidone treatment. The molecular mechanisms of this drug are not completely understood although inhibition of several fibrotic cytokines (as TGF-β, PDGF, and TNF-α) is reported in several in vivo studies. Aims: Methods: This work is studies the changes in the metabolome of the sera from 27 IPF patients in which we started treatment with pirfenidone. Using quantitative mass spectrometry (MS), 188 metabolites were measured at baseline before start of treatment and after 6 weeks during treatment. Results: The comparative analysis of MS data identified 78 metabolites in that the expression was changed with treatment. Serum concentrations of 22 molecules (in particular sphingolipids) decreased with pirfenidone treatment while 56 (mainly glycerophospholipids and aminoacids) increased. The analysis whether the serum metabolome is capable of treatment response prediction is ongoing. Conclusions: Treatment with pirfenidone leads to substantial changes in the serum metabolome, which may be useful to predict treatment response.
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Abstract No. 017 Improved alveolar dynamics and structure after alveolar epithelial type II cell transplantation in bleomycin induced lung fibrosis Elena Lopez-Rodriguez 1,* , Gemma Gay-Jordi 2, Matthias Ochs 1, and Anna Serrano-Mollar 2
1
Hannover Medical School Institute of Biomedical Sciences-CSIC *Presenting author
2
Idiopathic pulmonary fibrosis (IPF) is a progressively and ultimately fatal lung disease. Previously it has been shown that intratracheal administration of alveolar type II cell (AE2C) in the animal model of bleomycin-induced pulmonary fibrosis is able to reverse fibrosis and restores surfactant protein levels. However to date it has not been evaluated whether these changes involves any improvement in lung functionality. Consequently, the aim of the present work was study lung physiology after AE2C transplantation at different time points during the development fibrosis. Lung fibrosis was induced by intratracheal instillation of bleomycin (4U/kg) in rat lungs. The animals were transplanted with AE2C (2.5x106 cells/animal) 3 and 7 days after bleomycin instillation. Assessments were done at day 7 and 14 after the induction of fibrosis to plot time depending changes in lung physiology and functionality. To assess the pressures and rates at which closed alveoli reopens invasive pulmonary tests (Flexivent) including de-recruitability tests at different PEEP ventilations as well as quasi-static pressure volume loops were performed. Afterwards lungs were fixed by vascular perfusion and subjected to design-based stereological evaluation at light and electron microscopy level. AE2C delivered during the inflammatory phase (3 days) of the disease are only able to slightly prevent the increment of alveolar septal wall thickness however did not show either positive effects regarding ventilated alveolar surface nor any increase of lung compliance. Otherwise when AE2C are delivered at the beginning of the fibrotic phase (7 days after bleomycin instillation), an increase ventilated alveolar surface to control levels and reduce septal wall thickness can be observed. Moreover transplanted animals showed better lung performance, with increased inspiratory capacity and compliance. In conclusion, AE2C transplantation during fibrogenic phases of the disease improves lung performance in bleomycin-induced lung fibrosis.
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Abstract No. 021 Using Electron Microscopes to look into the Lung Jan Hegermann 1,* , Christoph Wrede 1, Lars Knudsen 1, Roman Grothausmann 1, Christian Mühlfeld 1, and Matthias Ochs 1 1
Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany *Presenting author
In the nineteenth century, there was a dispute about the existence of a lung alveolar epithelium which remained unsolved until the invention of electron microscopy (EM) and its application to the lung. From the early 1960s, Ewald Weibel became the master of lung EM. He showed that the alveolar epithelium is covered with a lining layer containing surfactant. Weibel also explained the phenomenon of "non-nucleated plates" observed already in 1881 by Albert Kölliker. Weibel's most significant contribution was to the development of stereological methods. Therefore, quantitative characterization of lung structure revealing structure-function relationships became possible. Today, the spectrum of EM methods to study the fine structure of the lung has been extended significantly. Cryo-preparation techniques are available which are necessary for immunogold labeling of molecules. Energy-filtering techniques can be used for the detection of elements. There have also been major improvements in stereology, thus providing a very versatile toolbox for quantitative lung phenotype analyses. A new dimension was added by 3D EM techniques. Depending on the desired sample size and resolution, the spectrum ranges from array tomography via serial block face scanning EM and focused ion beam scanning EM to electron tomography. These 3D datasets provide new insights into lung ultrastructure. Biomedical EM is an everdeveloping field. Its high resolution remains unparalleled. Moreover, EM has the unique advantage of providing an "open view" into cells and tissues within their full architectural context. Therefore, EM will remain an indispensable tool for a better understanding of the lung's functional design.
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Abstract No. 022 Functional proteomics of cellular mechanosensing mechanisms Anita A. Wasik 1,* , and Herbert B. Schiller 1 1
Comprehensive Pneumology Center *Presenting author
Cells actively sense their environment by translating mechanical properties of the extracellular matrix, such as stiffness, into biochemical signals. This cellular mechanosensing is crucial for development, tissue homeostasis and the outcome of many diseases. However, the precise molecular nature of this process is not well understood. Recent developments in phosphoproteomic workflows increase sensitivity of detection and sample throughput, which enabled us to follow the dynamic changes of the phosphorylation landscape of primary human lung fibroblasts during cell spreading, polarization and contraction at a depth of more than 9,000 quantified phosphosites. To uncover phosphosites that are involved in rigidity sensing we seeded the fibroblasts on fibronectin substrates with varying rigidities (0.5, 2, 8, 16, and 32 kPa) and measured phosphoproteomes either in suspension or 10, 20, 30, 60, 90, 120, and 240 minutes after seeding (n=5). ANOVA analysis of the various timepoints of cells seeded on stiff substrates resulted in 4548 significantly regulated sites (FDR=5%), with clusters of highly distinct temporal profiles. Using this data we grouped phosphorylation sites on proteins into initial events upon integrin engagement (0-30 minutes), signals that occur during cell polarization and establishment of focal adhesions and myosin-mediated contractility (30-120 minutes), and signals that may drive long term gene expression changes in response to the ECM substrate (120-240 minutes). The quantitative comparison of phosphopeptides between cells spread for 120 minutes on substrates with varying rigidities resulted in 1202 regulated (FDR=5%) phosphorylation sites, which were significantly enriched for RNA binding proteins, spliceosomal components, transcriptional regulators and endosomal proteins. Thus, we have generated the largest dataset of integrin adhesion dependent phosphorylation events in human lung fibroblasts and identified interesting subsets of sites that were controlled by extracellular matrix stiffness. We will analyze the functional implications of selected sites to uncover novel pathways that control fibroblast activity during tissue fibrosis.
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Abstract No. 025 Monocyte immunophenotyping reflects aberrant activation patterns in Interstitial Lung Disease patients Flavia Greiffo 1,* , Isis Fernandez 1, Marion Frankenberger 1, Almudena Ortega-Gomez 2, Jurgen Behr 3, Oliver Soehnlein 2, and Oliver Eickelberg 4 1
Helmholtz Zentrum München Institute for Cardiovascular Prevention, Ludwig-Maximilians-University, Munich, Germany 3 Asklepios Fachkliniken München-Gauting, Munich, Germany 4 Department of Medicine and University of Colorado, School of Medicine, Aurora, Colorado *Presenting author 2
BACKGROUND: Interstitial lung diseases (ILD), including idiopathic pulmonary fibrosis (IPF), are characterized by extracellular matrix deposition, disrupting the gas exchange process of the lung, affecting organ function. Monocytes are effector cells from the innate immune system that can migrate to and differentiate at the site of injury, acting as precursor cells of mature and potentially pro-fibrotic cell lineages. Monocytes are classified into classical, intermediate, and non-classical. Their activation status relates with induction of CX3CR1 expression. To date, the role of activated monocytes in the pathogenesis of ILD has not been elucidated. METHODS: Monocyte immunophenotype was performed in whole blood from control, IPF and non-IPF ILD patients, by flow cytometry. Monocytes subsets were detected in tissue by immunofluorescence. Monocytes mRNA levels were analyzed by qRT-PCR. RESULTS: Classical monocytes were significantly increased in non-IPF ILD when compared with control (p<0.001). Non-classical monocytes were significantly decreased in IPF (p<0.05) and non-IPF ILD (p<0.001) compared with control. The number of classical and non-classical monocytes significantly correlated with diffusing capacity of the lung for carbon monoxide (DLCO) (classical monocytes, non-IPF ILD r=-0.2909, p<0.05), (nonclassical monocytes IPF r=0.3181 p<0.05, non-IPF ILD r=0.3824 p<0.01). CD14lowCD16hi non-classical monocytes were detected in contact with blood vessels and abundant in ILD fibrotic areas, when compared with donor lungs. The mean fluorescence intensity (MFI) values of CX3CR1 were significantly decreased in non-IPF ILD patients in all three subsets of monocytes when compared with control. mRNA levels of cell-cell adhesion markers (CD31, CD34, CD151, and CTGF) were significantly downregulated in non-IPF ILD monocytes, compared with control. CONCLUSION: This study shows an altered expression of CX3CR1 in all monocytes subsets in ILD, and suggestive interplay with ECM deposition. Therefore, the depletion and/or activation of distinct phenotypes of monocytes, might contribute to ILD pathogenesis.
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Abstract No. 028 CDCP1/TGFβ1 cross-talk regulates (myo)fibroblast differentiation Nina Noskovicova 1,* , Katharina Heinzelmann 1, Gerald Burgstaller 1, and Oliver Eickelberg 1 1
Comprehensive Pneumology Centre *Presenting author
Rationale: Fibroblasts are the most important cell types producing extracellular matrix (ECM) in the lung. Transforming growth factor-beta 1 (TGFβ1) induces fibroblast(myo)fibroblast transdifferentiation, which leads to αSMA-expressing (myo)fibroblasts with increased ECM secretion. Nevertheless, little is known about specific receptors besides the TGF-beta receptors, which control this process. We have recently identified Cub domain containing protein 1 (CDCP1) in the cell-surface proteome analysis among the top downregulated proteins by TGFβ1. CDCP1 is a transmembrane glycoprotein, the expression and function of which in human lung fibroblasts remains to be elucidated. Methods: We used immunofluorescence staining to examine the localization of CDCP1 in primary human lung fibroblasts (phLFs). To study the mechanisms of regulation and function of CDCP1 in the presence/absence of TGFβ1 in phLFs, we performed Western blot, FACS analysis, and cell adhesion assays, together with siRNA-mediated knockdown of CDCP1. Results: Our data showed surface localization of CDCP1 in human lung fibroblasts. TGFβ1 decreases the percentage of surface CDCP1-positive cells (85.7% ± 10.0 to 73.5% ± 14.8, p=0.003), as well as in total protein level (p=0.0045). This effect occurs in a time-dependent manner. TGFβ1 induces CDCP1 down regulation via increased proteasomal degradation of CDCP1, but not MAPK or SMAD signaling. In addition, knockdown of CDCP1 increased the phosphorylation of Smad3 in the presence of TGFβ1 (p=0.033). Functionally, loss of CDCP1 expression resulted in increased cell adhesion of phLFs, independently from TGFβ1 (fold change siCDCP1/siScr= 1.78, p=0.01). Moreover, CDCP1-depleted cells displayed increased expression of collagen V, fibronectin, and αSMA independently from TGFβ1. Conclusion: CDCP1 is a novel negative regulator of TGF-beta signaling in fibroblast to (myo)fibroblast differentiation via potential CDCP1/TGFβ1 cross-talk.
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Abstract No. 032 Proteasome activator 200 (PA200) is dysregulated in fibrotic tissue remodeling Vanessa Welk 1,* , Thomas Meul 1, Juliane Merl-Pham 2, Martina Korfei 3, Nora Semren 1, Shrikant R. Mulay 4, Andreas Günther 3, Oliver Eickelberg 1, and Silke Meiners 1 1
Comprehensive Pneumology Center, Helmholtz Zentrum München Research Unit Protein Science, Helmholtz Zentrum München 3 Department of Internal Medicine, Universities of Giessen and Marburg Lung Center 4 Nephrologic Center, Medical Clinic and Polyclinic IV, Ludwig-Maximilians-University Munich *Presenting author 2
The proteasome is one of the main proteolytic systems of the cell and thus central for maintenance of cellular protein homeostasis. Fibrotic remodeling of the lung requires increased activity of the 26S proteasome (Semren et al., 2015). However, the regulation of other activators of the proteasome besides the 19S regulator has not been analyzed in this context so far. Here, we investigate the role of the proteasome activator 200 (PA200) during myofibroblast differentiation and fibrotic tissue remodeling. Protein levels of PA200 were significantly upregulated in fibrotic lungs of idiopathic pulmonary fibrosis (IPF) patients and in a mouse model of lung fibrosis. Its induction in fibrotic tissue of murine experimental kidney fibrosis suggests that this is a general feature of fibrotic tissue remodeling and not restricted to the lung. Immunohistochemistry analysis of IPF lungs identified myofibroblasts and abnormal hyperplastic basal cells as the main cell types overexpressing PA200 in the fibrotic lung. In isolated primary human lung fibroblasts (phLF), PA200 was upregulated by the profibrotic cytokine TGFβ both on mRNA and protein level. Native gel analysis revealed strong recruitment of PA200 to the 20S proteasome during TGFβ-induced myofibroblast differentiation. Remarkably, transient silencing of PA200 in phLF did not prevent myofibroblast differentiation but rather enhanced myodifferentiation and proliferation. To further elucidate the function of PA200 we performed coimmunoprecipitation experiments and identified so far unrecognized interacting proteins using mass spectrometry. Concluding, our findings show that PA200 is upregulated in fibrotic tissue remodeling and identify it as novel regulator of myofibroblast differentiation. Ongoing experiments aim to characterize the regulation of newly identified interacting proteins by PA200 to better understand its function.
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Abstract No. 036 Interim Analysis of the EXCITING-ILD registry (Registry for Exploring Clinical and Epidemiological Characteristics of Interstitial Lung Diseases) Michael Kreuter 1,* , Margarethe Wacker 2, Peter Hammerl 3, Conrad Wiederhold 4, Hans Joachim Kabitz 5, Lars Hagmeyer 6, Dirk Skowasch 7, Reiner Leidl 8, Andreas Hellman 9, Michael Pfeifer 10, Jürgen Behr 11, Dagmar Kauschka 12, Marcus Mall 13, Andreas Günther 14, Felix Herth 1, and Philipp Markart 15 1
Thoraxklinik Helmholtz Centre 3 Chest Clinic Immenhausen 4 Outpatient center for pulmonology, Fulda 5 Klinikum Konstanz 6 Clinic Solingen 7 University of Bonn 8 Helmholtz Institute 9 Bund Deutscher Pneumologen, Augsburg 10 University of Regensburg and Clinic Donaustauf 11 University Clinic Munich 12 Patient Support Group Lungenfibrose e.V 13 Translational Pulmonology, University of Heidelberg 14 University Hospital Giessen 15 Universtiy Hospital Giessen and Klinikum Fulda *Presenting author 2
Background: The epidemiologic knowledge on interstitial lung diseases (ILD) is limited. The multi-centre registry “EXCITING” which is lead by centers of the DZL collects data on characteristics, management and outcomes of all ILDs. Method: Since 10/2014 ILD patients are recruited prospectively. Results: Until 03/2016, 276 patients were included: 64% male, median age 64 years, 58% current/ex-smokers, symptoms >6 months before diagnosis: 51%. Medians: FVC 74%, DLCO 51%, GAP-ILD-Index 0-1 27%, 2-3 28%, 4-5 27%, 6-8 18%. Diagnostic procedures: 93% CT (47% HRCT), 73% BAL, 21% surgical-lung-biopsies; 57% multidisciplinary discussion. Patients had following diseases: IIP 45% (IPF 33%, NSIP 4%, DIP 2%, COP 3%, LIP 1%), hypersensitivity pneumonitis 14%, CTD-ILD 6%, pneumoconiosis 1%, sarcoidosis 21%, unclassifiable 6%, LAM 2%, drug-induced-ILD 3%, PAP 0.5%, eosinophilic pneumonia 0.5%, 2.9% familial forms. Relevant comorbidities: 24% GERD, 8% PH, 12% emphysema. Drug-therapies at baseline: Azathioprine 9%, Prednisolone 65%, NAC 12%, Pirfenidone 16%, Nintedanib 16%, Cyclophosphamide 4%, MTX 6%, MMF 2%, Rituximab 1%, ICS 14%, Sirolimus 1%, TNF-alpha-inhibitors 1%, clinical trials 5%. Further therapies: physiotherapy 3%, non-invasive ventilation 7%, long-term-oxygen 23%, inpatient pulmonary rehabilitation 5%; 0.4% listed for lung transplant. Hospitalisations within 6 months before inclusion: 52% (74% for ILD: pneumonia 12%, AE-ILD 19%, pneumothorax 2%). After 6 months follow-up: 25% hospitalisations (53% for ILD: 38% pneumonia), median relapsefree-survival (no decrease FVC ≥10%, DLCO ≥15%, death) is currently 24 months. Conclusions: Medical need and health care impact of ILDs are significant also as many patients present with severe disease (GAP-ILD index), and ILD associated hospitalizations are frequent.
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Submitted as: Presentation 251 words of 300 possible words
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Abstract No. 038 Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo Mareike Lehmann 1,* , Kathrin Mutze 1, Martina Korfei 2, Darcy Wagner 1, Stephan Klee 1, Rita Costa 1, Herbert Schiller 1, Michael Lindner 3, Andreas Günther 4, and Melanie Königshoff 5 1
Comprehensive Pneumology Center (CPC), Helmholtz Zentrum München and University Hospital of the Ludwig Maximilians Universität, Munich, Germany 2 Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Justus-Liebig-Universität Giessen, Germany 3 Center for Thoracic Surgery, Asklepios Biobank for Lung Diseases, Asklepios Clinic Munich-Gauting, Germany. 4 Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Justus-Liebig-Universität Giessen, Germany, Agaplesion Lung Clinic Waldhof Elgershausen, D-35753 Greifenstein, Germany, European IPF Network and European IPF Registry. 5 Comprehensive Pneumology Center (CPC), Helmholtz Zentrum München and University Hospital of the Ludwig Maximilians Universität, Munich, Germany. Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado, Denver. *Presenting author
Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating, age-related lung disease with limited therapeutic options. Aging-related mechanisms such as cellular senescence have been proposed as a pathogenic driver. The lung epithelium that contains progenitor cells, such as alveolar epithelial type II (ATII) cells that represent a major site of tissue injury in IPF. Senescence of ATII cells is likely detrimental to lung repair and regeneration. ATII cell senescence and its potential effects in IPF, however, remain poorly understood. Methods and results: Here we aimed to analyze cellular senescence and its potential pathomechanism in IPF. Gene set enrichment analysis of microarrays of different lung cells (GSE16846, GSE42564) derived from experimental lung fibrosis revealed significant enrichment of senescence associated genes in ATII cells. Expression analysis of whole mouse lungs after bleomycin challenge showed an increase in senescence associated cyclin-dependent kinase inhibitor 1a (CdKn1a) and 2a (Cdkn2a) gene expression. Fibrotic pmATII cells exhibited increased senescence-associated β-Galactosidase staining as analyzed by fluorescence-activated cell sorting(FACS) analysis and light microscopy. This was accompanied by enhanced gene expression of senescence associated Cdkn2a, CdKn1a and matrix metalloproteinase (MMP)2. Moreover, proteomic analysis of supernatants of cultured pmATII cells showed secretion of a number of factors associated with the SASP. Pharmacological depletion of senescent cells from fibrotic ATII cells or 3D lung tissue cultures reduced fibrotic marker gene expression and increased the ATII cell marker surfactant protein C (Sftpc). Importantly, human IPF samples showed increased senescence associated CDKN2A gene expression and enhanced staining as compared to donor samples, an effect which was also observed in phATII cells. Conclusion: In summary, fibrotic ATII cells exhibit increased senescence, which is accompanied by the secretion of SASP factors. Pharmacological depletion of senescent cells increased ATII cell markers and decreased fibrotic gene expression, suggesting that senescence contributes to impaired lung repair in IPF.
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Abstract No. 042 CURRENT PRACTICE OF DRUG TREATMENT IN CHILDREN WITH ILD: FIRST INSIGHTS FROM THE CHILD-EU REGISTRY Boglárka Szentes 1, Sabine Witt 1, Andrew Bush 2, Steve Cunningham 3, Nagehan Emiraliouglu 4, Lutz Goldbeck 5, Matthias Griese 6, Meike Hengst 6, Nural Kiper 7, Katarzyna Krenke 8, Joanna Lange 8, Reiner Leidl 1, Nicolaus Schwerk 9, and Larissa Schwarzkopf 1 1
Helmholtz Zentrum München Imperial College and Royal Brompton Hospital 3 Edinburgh Royal Hospital 4 Hacettepe Ihsan Dogramaca Children's Hospital 5 University Hospital Ulm 6 Ludwig Maximilian University Hauner Children´s Hospital 7 Hacettepe Ihsan Dogramaci Children's Hospital 8 Medical University of Warsaw 9 Hannover Medical School 2
OBJECTIVES: Childhood Interstitial lung disease (chILD) is an umbrella term for more than 200 rare disease entities with high mortality for which evidence on medical treatment regimens, healthcare utilization and clinical outcomes are broadly lacking. We therefore aimed to evaluate current drug utilization to benchmark European practice. METHODS: The chILD-EU registry was initiated in 2012 and includes children with ILD from 7 European countries. Demographics, medication and patient-reported utilization and outcomes (hospitalization, visits to their general practitioner/ specialist and rehabilitation 3 months prior to the survey) are recorded at baseline and at routine time intervals. Next to baseline characteristics, healthcare utilization is analyzed via logistic regression models adjusting for sex, age, country, education of the parents, subjective socioeconomic status, disease severity, disease status (incident/prevalent) and steroid intake. RESULTS: Among the current 202 patients (54.5% male) median age was 4.2 years (interquartile range (IQR) 1, 10.4), median disease duration 1.67 years (IQR 0, 4.6) with a median disease severity of 3 on the FAN 5 point severity scale. 46 % out of the 202 patients were on medication at baseline, predominantly on corticosteroids (26.8%), followed by macrolides (19.1%) and hydroxychloroquine (17.4%). However there were altogether 116 (57.4%) patients who already received corticosteroids until baseline or receive corticosteroids at baseline. Regression models show that patients taking corticosteroids are more often hospitalized (OR: 6.6 p=0.0116), but there is no association with other above mentioned utilization. Incident cases (newly diagnosed at inclusion in the database) have a higher probability to receive corticosteroids at baseline (OR: 3.7 p=0.0063). CONCLUSIONS:
DZL Annual Meeting 2017 Corticosteroids are the most frequently used treatments curerntly. To demonstrate whether corticosteroids or other medicaments can be linked to mortality or quality of life outcomes, longitudinal analysis of the chILD-EU registry will follow. Grant FP7-305653-chILD-EU.
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Abstract No. 049 High-throughput single cell mRNA sequencing reveals cell identity programs in the murine lung Maximilian Strunz 1,* , Lukas Simon 2, Christoph Mayr 1, Ilias Angelidis 1, Fabian Theis 2, and Herbert Schiller 1 1
Comprehensive Pneumology Center HMGU *Presenting author 2
Over 40 different potentially unique cell types with specific functions have been described in the lung. Single cell mRNA sequencing (scRNA-seq) of cells isolated freshly from their tissue context will revolutionize systems biology studies of dynamic multicellular processes in vivo. We employ the recently developed Drop-seq method, which uses microfluidics to capture single cells along with sets of uniquely barcoded primer beads into nanoliter-sized aqueous droplets. The smart barcoding approach in Drop-seq allows the massively parallel, and thus cost-effective, analysis of mRNA transcripts from thousands of individual cells simultaneously, while remembering the transcripts’ cell of origin. Using a 30 minute digestion protocol with a Dispase/Collagenase/Elastase enzyme mixture, we generated single cell suspensions from whole mouse lungs before and 14 days after bleomycin injury. Of note, to avoid any bias, single cell suspensions were used for the Dropseq procedure without prior presorting or depletion of selected cell populations. The scRNAseq libraries were sequenced on the Illumina High-seq 4000 platform. We quantified transcripts that were identified in at least 5 single cells using unique molecular identifier (UMI) counting, producing an expression matrix of 13641 genes that enabled us to stratify 396 cells from healthy lung and 252 cells from lungs after bleomycin injury into distinct groups of differential gene expression. Unsupervised statistical methods truthfully classified at least 10 distinct cell types, including most major epithelial, leukocyte and mesenchymal cell lineages of the lung parenchyma. Bleomycin injury resulted in the occurrence of distinct subsets of cells with altered gene expression compared to healthy control, mainly representing activated mesenchymal and leukocyte lineages. Applying this workflow to a complete 4-weeks time course of the bleomycin injury model at a depth of >20.000 cells sequenced, we anticipate obtaining a first comprehensive time and cell type-resolved model of tissue remodeling in lung regeneration.
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Abstract No. 050 Deep proteome profiling reveals common and distinct features of human lung and skin fibrosis Herbert Schiller 1, Maximilian Strunz 1, Christoph Mayr 1,* , Claudia Staab-Weijnitz 1, Beate Eckes 2, Pia Moinzadeh 2, Thomas Krieg 2, Jürgen Behr 3, Matthias Mann 4, and Oliver Eickelberg 5 1
Helmholtz Zentrum München University of Cologne 3 LMU University Hospital 4 Max-Planck-Institute of Biochemistry 5 University of Colorado *Presenting author 2
Rationale: Analyzing the molecular heterogeneity of organ fibrosis in human patients may reveal common and distinct factors, representing potential future targets for therapy. Objectives: We sought to use proteome-wide profiling of human tissue fibrosis to (1) identify common signatures across end-stage interstitial lung disease (ILD) cases, (2) characterize ILD subgroups in an unbiased fashion, and (3) identify common and specific features of lung and skin fibrosis. Methods: We collected samples of fibrotic lung tissue from biopsies of eleven ILD cases of different diagnostic classes and compared them with three healthy donor controls. We also collected fibrotic skin lesions of three patients with localized scleroderma and unaffected skin from the same identical patients. All samples were profiled by quantitative label-free mass spectrometry, exploratory statistics, and confocal imaging. Measurements and Main Results: We determined the abundance of >7900 proteins and stratified them by their detergent solubility profile. We observed common protein regulation across all ILD cases, as well as specific ILD subset signatures. Proteome comparison of lung and skin fibrosis identified a common upregulation of MZB1. We further characterized MZB1 in fibrotic lung and skin tissue sections and identified consistent accumulation of MZB1+/CD38+/CD20-/CD45- cells, representing tissue-resident plasma B-cells. Conclusions: Despite the presumably high molecular and cellular heterogeneity of lung fibrosis, common protein regulation is observed, even across organ boundaries. The surprising finding of abundant MZB1+/CD38+ plasma cells in tissue fibrosis warrants future investigations regarding the causative role of antibody-mediated autoimmunity in idiopathic cases of organ fibrosis, such as idiopathic pulmonary fibrosis (IPF).
Submitted as: Presentation 253 words of 300 possible words
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Abstract No. 058 FK506-BINDING PROTEIN 11, A PLASMA CELL-SPECIFIC PROTEIN FOLDING CATALYST, IS INCREASED IN PULMONARY FIBROSIS Stefan Preisendörfer 1,* , Larissa Knüppel 1, Leonhard Binzenhöfer 1, Isis Fernandez 1, Brenda Juan-Guardela 2, Rudolf Hatz 3, Jürgen Behr 4, Naftali Kaminski 2, Aloys Schepers 5, Oliver Eickelberg 6, and Claudia Staab-Weijnitz 1 1
Comprehensive Pneumology Center, Helmholtz-Zentrum München, Munich, Germany Pulmonary, Critical Care and Sleep Medicine, Yale School of Medicine, New Haven, Connecticut, United States of America 3 Thoraxchirurgisches Zentrum, Klinik für Allgemeine-, Viszeral-, Transplantations-, Gefäßund Thoraxchirurgie, Klinikum Großhadern, Ludwig-Maximilians-Universität, Munich, Germany; Asklepios Fachkliniken München-Gauting, Munich, Germany 4 Asklepios Fachkliniken München-Gauting, Munich, Germany; Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians-Universität, Munich, Germany, Member of the German Center of Lung Research (DZL) 5 Monoclonal Antibody Core Unit, Monoclonal Antibody Research Group, Helmholtz-Zentrum München, Munich, Germany 6 Pulmonary and Critical Care Medicine University, Colorado Anschutz Medical Campus, Denver, Colorado, United States of America *Presenting author 2
RATIONALE: We have recently identified the chaperone and peptidyl-prolyl isomerase FK506-binding protein 10 (FKBP10) as a profibrotic mediator in idiopathic pulmonary fibrosis (IPF). In this study, we sought to assess expression, localization, regulation and function of a related protein, FKBP11, in lung fibrosis. METHODS: Expression of FKBP11 in lung tissue was analysed during the onset and resolution of bleomycin-induced lung fibrosis (up to day 56) in C57BL/6N mice, as well as in lungs from IPF patients using Western Blot analysis. FKBP11 expression was also quantified in DNA-microarray data derived from an independent IPF cohort. Immunofluorescence of IPF-sections was used to determine the cell type expressing FKBP11 and to quantify FKBP11-positive cells in IPF tissue sections relative to donor and chronic obstructive pulmonary disease (COPD) tissue. Finally, FKBP11 expression was assessed in antibody-producing cell lines, including under conditions of endoplasmic reticulum (ER) stress. RESULTS: FKBP11 expression was increased in the fibrotic phase of bleomycin-induced lung fibrosis as well as in two independent IPF cohorts. Immunofluorescent stainings in IPF tissue sections confirmed these observations showing elevated numbers of FKBP11positive cells in contrast to healthy donor and COPD. Costainings with various markers for the hematopoietic lineage demonstrated specific expression of FKBP11 in CD45-/CD20/CD38+/CD27+/CD138+ plasma cells. Antibody-producing cell lines displayed strong expression of FKBP11 which was further increased by the ER stress inducer tunicamycin. CONCLUSIONS: The protein folding catalyst FKBP11 is increased in IPF, regulated by ER stress, and specifically localizes to plasma cells in the lung. Taken together, these results suggest a function of FKBP11 in plasma cell biology and B-cell differentiation and support the autoimmune hypothesis in IPF. Expression of FKBP11 in antibody-producing cell lines underlines their suitability as in vitro models for future genetic manipulation of FKBP11 expression and functional analysis.
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Submitted as: Presentation 292 words of 300 possible words
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Abstract No. 059 ABERRANT COLLAGEN SYNTHESIS AND PROLYL-3-HYDROXYLATION VIA INDUCTION OF PROLYL-3-HYDROXYLASE 1 MAY LEAD TO DYSREGULATED HOMEOSTASIS OF THE BRONCHIAL EPITHELIUM IN IDIOPATHIC PULMONARY FIBROSIS Claudia Staab-Weijnitz 1,* , Leonhard Binzenhöfer 1, Juliane Merl-Pham 1, Andrea Schamberger 1, Larissa Knüppel 1, Elisabeth Hennen 1, Rudolf Hatz 2, Jürgen Behr 2, Naftali Kaminski 3, Stefanie Hauck 1, and Oliver Eickelberg 4 1
Helmholtz Zentrum München Klinikum der Ludwig-Maximilians-Universität und Asklepios Fachkliniken München-Gauting 3 Yale School of Medicine, New Haven, Connecticut, USA 4 Colorado Anschutz Medical Campus, Denver, Colorado, USA *Presenting author 2
RATIONALE: In most types of organ fibrosis, epithelial injury leads to transforming growth factor-β (TGF-β)-driven activation of fibroblasts, which produce excessive amounts of collagen. Prolyl-3-hydroxylase 1 (P3H1) catalyses collagen prolyl-3-hydroxylation, a rare and poorly understood post-translational modification of collagen. Recent evidence from genome-wide association studies highlights the potential importance of the bronchial epithelium in the aetiology and progression of idiopathic pulmonary fibrosis (IPF). METHODS: In two independent cohorts, expression of collagen prolyl-3-hydroxylase 1 (P3H1, also LEPRE1) was assessed in lung tissue from IPF patients and healthy donor controls using Western Blot and immunofluorescent analysis. P3H1 protein levels were quantified by Western Blot analysis during in vitro differentiation of primary human bronchial epithelial cells (phBECs). Regulation by central fibrotic regulators was assessed in basal-like primary human bronchial epithelial cells by qPCR and Western Blot analysis. The effect of siRNA-mediated knockdown of P3H1 in basal-like phBECs was assessed by morphological assessment, proliferation and viability assays, as well as quantification of collagen synthesis, secretion, and post-translational modifications by liquid-chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: P3H1 protein was upregulated in both IPF cohorts relative to normal histology control. In IPF lung, P3H1 was specifically expressed by p63+/KRT14+ progenitor cells in aberrant hyperplastic-looking bronchial epithelium. P3H1 levels gradually decreased during bronchial epithelial differentiation in vitro. TGF-β1 and microRNA mir-29, but not endoplasmic reticulum stress, regulated P3H1 expression in basal-like phBECs. Loss-offunction experiments demonstrated attenuation of cell proliferation and gain of a squamous cell appearance as well as increased collagen I, but decreased collagen XVII synthesis in the absence of P3H1. Finally, prolyl-3-hydroxylations were identified in collagen I and XVII. CONCLUSIONS: P3H1 is upregulated in the IPF lung and localizes to a progenitor cell type in aberrant hyperproliferative bronchial epithelium. The results suggest a role of collagen prolyl-3-hydroxylation in aberrant bronchial epithelium homeostasis in IPF.
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Abstract No. 066 WISP1 mediates IL6-dependent proliferation in healthy and IPF-derived primary human lung fibroblasts Stephan Klee 1,* , Mareike Lehmann 1, Darcy E. Wagner 1, Hoeke A. Baarsma 1, and Melanie Königshoff 1 1
CPC *Presenting author
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. IPF is characterized by epithelial cell injury and reprogramming, increase in (myo)fibroblasts, and altered deposition of extracellular matrix. The Wnt1-inducible signaling protein 1 (WISP1) is involved in impaired epithelial-mesenchymal crosstalk in pulmonary fibrosis. Here, we aimed to further investigate WISP1 regulation and function in primary human lung fibroblasts (phLFs). We show that WISP1 is a common downstream target of the pro-fibrotic cytokines Transforming growth factor β1 (TGFβ1) and Tumor necrosis factor α (TNFα) in phLF using RT-qPCR and ELISA. TGFβ1 and TNFα significantly increased WISP1 mRNA expression and secretion. WISP1 was required for TGFβ1- and TNFα-dependent induction of IL6 expression and secretion in phLFs as determined by siRNA-mediated knockdown of WISP1 in phLFs. We found that IL6 production in phLFs by LPS is also dependent on the presence of WISP1. Importantly, we observed similar effects in phLFs derived from IPF patients, suggesting a conserved mechanism of WISP1-dependent IL6 expression in phLFs. Loss of WISP1 function by siRNA or neutralizing antibodies led to a significant reduction in phLF proliferation as analysed by WST-1 assay, cyclin D1 and PCNA expression by Western Blot and immunoflourescence staining, respectively. The reduced proliferation of phLFs was in part mediated by the reduction in IL6. Taken together, these results indicate that WISP1induced IL-6 expression contributes to the pro-proliferative effect on fibroblasts, which is likely orchestrated by a variety of profibrotic mediators, including Wnts, TGFβ and TNFα.
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Abstract No. 076 Pirfenidone exerts anti-fibrotic effects through inhibition of GLI transcription factors Miroslava Didiasova 1,* , Rajeev Singh 2, Jochen Wilhelm 1, Grazyna Kwapiszewska 3, Lukasz Wujak 1, Dariusz Zakrzewicz 1, Liliana Schaefer 4, Philipp Markart 1, Werner Seeger 1 , Matthias Lauth 2, and Malgorzata Wygrecka 1 1
Universities of Giessen and Marburg Lung Center Philipps University, Center for Tumor Biology and Immunology 3 Ludwig Boltzmann Institute for Lung Vascular Research 4 Institute of Pharmacology and Toxicology, Goethe University School of Medicine *Presenting author 2
Pirfenidone is an anti-fibrotic drug, recently approved for the treatment of patients suffering from idiopathic pulmonary fibrosis (IPF). Although pirfenidone exhibits anti-inflammatory, anti-oxidant and anti-fibrotic properties, the molecular mechanism underlying its protective effects still remains largely unknown. Here, we link pirfenidone action with the regulation of the pro-fibrotic Hedgehog (Hh) signaling pathway. Specifically, we demonstrate that pirfenidone selectively destabilizes the glioma-associated oncogene homolog (GLI) 2 protein, the primary activator of Hh-mediated gene transcription. Consequently, pirfenidone decreases overall Hh pathway activity in IPF patients and in patient-derived primary lung fibroblasts and leads to diminished levels of Hh target genes such as GLI1, Hh receptor Patched-1, α-smooth muscle actin, and fibronectin and to reduced cell migration and proliferation. Interestingly, Hh-triggered transforming growth factor (TGF)β1 expression potentiated Hh responsiveness of primary lung fibroblasts by elevating the available pool of GLI1/GLI2, thus creating a vicious cycle of amplifying fibrotic processes. As GLI transcription factors are not only crucial for Hh-mediated changes but are also required as mediators of TGF-β signaling, our findings suggest that pirfenidone exerts its clinically beneficial effects through dual Hh/TGF-β inhibition by targeting the GLI2 protein.
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Abstract No. 084 Nuclear miRNA/exosome-mediated transcriptional silencing within the context of TGFB1 signaling and Idiopathic Pulmonary Fibrosis. Indrabahadur Singh 1,* , Karla Rubio 1, Adriana Contreras 1, Julio Cordero 1, Stephanie Dobersch 1, Aditi Mehta 1, Stefan Günther 1, Pouya Sarvari 1, Soni Pullamsetti 1, Andreas Günther 2, Marcus Krüger 1, Klaus T Preissner 2, Werner Seeger 1, Thomas Braun 1, and Guillermo Barreto 1 1
Max-Planck-Institute for Heart and Lung Research, Parkstraße 1, 61231 Bad Nauheim, Germany 2 Justus-Liebig-University, 35932 Giessen, Germany *Presenting author
Non-coding RNAs (ncRNAs) are important regulators of different biological processes in the nucleus of the cell as part of the machinery controlling the chromatin structure at specific loci. However, the full extent of the function of ncRNAs in the nucleus has remained elusive, since the involvement of nuclear microRNAs (miRNAs) has not been investigated. Here we deciphered the function of a specific miRNA during transcriptional regulation. We show that the mature miRNA directly binds to nuclear ncRNAs and the RNA exosome complex to induce heterochromatin through histone methyl transferases, SUV39H1 and EZH2, thereby silencing transcription. The mechanism of miRNA/exosome-mediated transcriptional silencing is biologically relevant within the context of transforming growth factor (TGFB1) signaling. Furthermore, we confirmed the mechanism of transcriptional regulation presented here using primary cells from control donors and patients with idiopathic pulmonary fibrosis (IPF), opening new possibilities for the development of novel therapeutic approaches against IPF.
Singh I, et al., (2015) Cell Research; Jul;25(7):837-50 Singh I, et al., (2014) BMC Biol; Mar 24;12:21 Ozturk N, Singh I, et al., (2014) Front Cell Dev Biol.; Mar 6;2:5 Submitted as: Presentation 148 words of 300 possible words
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Abstract No. 086 Development of Pulmonary Fibrosis in Adult Nedd4-2 Deficient Mice Dominik Leitz 1,* , Simon Fraumann 1, Julia Duerr 1, Jolanthe Schatterny 1, Simone Schmidt 1, and Marcus Mall 1 1
Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany *Presenting author
Diffuse Parenchymal Lung Diseases (DPLD) constitute a heterogeneous group of pulmonary diseases characterized by inflammation, fibrotic remodeling and impaired gas exchange. Several mutations in different proteins important for epithelial cell function have been reported to be associated with DPLD, but the pathogenesis is still poorly understood. In this studies, we used a mouse model enabling conditional lung-specific deletion of Nedd4-2, a E3 ubiquitin ligase that was shown to be critical for regulation of the epithelial sodium channel (ENaC), surfactant protein-C biosynthesis and TGF- s igna ling. We found that conditional deletion of Nedd4-2 in the pulmonary epithelium of adult mice resulted in a chronic interstitial lung disease with fibrotic remodeling, restrictive lung physiology and poor blood oxygenation. To characterize disease progression we studied pulmonary morphology, inflammation, and lung mechanics after different periods of induction of the Nedd4-2 deletion. Our results demonstrate that mice induced for 2 weeks and 2 months did not show changes in inflammatory cell counts in the bronchoalveolar lavage and lung physiology compared to control mice. But after 3, 4 and 5 months of induction we observed a progressive decrease of the compliance accompanied by an elevation of inflammatory parameters that exhibit a peak after 4 months of induction. Further, we detected microscopic honeycomb cysts and distal airway remodeling characterized by a higher abundance of ciliated cells, goblet cells and Muc5b expressing cells in mice with an advanced stage of disease. We conclude that DPLD phenotype in Nedd4-2 deficient mice is chronic progressive and shares key features of lung disease in patients with IPF. This model may be a powerful tool to study new biological pathways and define novel biomarkers in early pathogenesis and develop novel therapeutic strategies for patients with IPF and potentially other DPLD.
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Abstract No. 093 LRP1 controls the transcriptional activity of SMAD3 in idiopathic pulmonary fibrosis Jennifer Schnieder 1,* , Lukasz Wujak 1, and Malgorzata Wygrecka 1 1
Biochemisches Institut *Presenting author
Accumulation of myofibroblasts and excessive deposition of extracellular matrix are hallmarks of idiopathic pulmonary fibrosis (IPF). These pathogenic processes are driven by the overactivated TGF-β signaling. Low density lipoprotein receptor-related protein (LRP) 1 is a scavenger/signaling receptor which was described to modulate TGF-β signaling and to contribute to kidney fibrosis. Based on these findings, we hypothesized that LRP1 may regulate the TGF-β signaling in IPF. LRP1 expression was downregulated in lung fibroblasts isolated from IPF patients and bleomycin-treated mice. Knock-down of LRP1 enhanced expression of α-SMA in IPF-derived lung fibroblasts and in mouse embryonic fibroblasts (MEF) on the protein and mRNA level. Dual-luciferase reporter assay showed that loss of LRP1 increased the transcriptional activity of the TGF-β/SMAD3 axis in MEF indicating that LRP1 may function as a negative regulator of SMAD3. Accordingly, knock-down of SMAD3 reduced -SMA upregulation upon LRP1 silencing in IPF-derived lung fibroblasts. Interestingly, LRP1 knock-down enhanced the activation status of extracellular signalregulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 which are known to be involved in SMAD3 regulation. Inhibition of ERK showed no impact on -SMA expression after LRP1 silencing, however, blockage of JNK1/2 abrogated induction of -SMA expression after LRP1 knock-down in IPF-derived lung fibroblasts. In summary, our studies point out that loss of LRP1 in IPF induces transcriptional activity of SMAD3 in a JNKdependent manner which results in upregulation of -SMA expression.
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Abstract No. 097 The European IPF Registry: Clinical Data from a European Registry for Patients with Idiopathic Pulmonary Fibrosis and other Interstitial Lung Diseases Jasmin Wagner 1,* , Fotios Drakopanagiotakis 1, Bettina Paul 1, Athol Wells 2, Philippe Bonniaud 3, Carlo Vancheri 4, Bruno Crestani 5, and Andreas Günther 1 1
Universities of Giessen and Marburg Lung Center, Giessen, Germany Interstitial Lung Disease Unit, Royal Brompton Hospital, London, United Kingdom 3 Service de Pneumologie, Centre Hospitalier Universitaire Dijon, Dijon, France 4 Dept. of Clinical and Molecular Biomedicine, Università degli Studi di Catania, Catania, Italy 5 Competence Center for Rare Pulmonary Diseases, Hopital Bichat, Paris, France *Presenting author 2
The European IPF registry (eurIPFreg) is a pan-European registry and biobank for patients with Idiopathic Pulmonary Fibrosis (IPF) and other Diffuse Parenchymal Lung Diseases (DPLD). Patients suffering from other lung diseases are included as Controls. Here, we describe the baseline data of 525 IPF patients recruited for the eurIPFreg in the time-period November 2009 until October 2016. The analyzed data comprises demographics, symptoms and signs, comorbidities, lung function tests, bronchoscopy findings, diagnosis practices and medical treatment. The mean age of included IPF patients was 68.1±11.1 years, 73.7% of these were males. Two third of the patients were ex-smokers. The most common symptoms reported by the patients were dyspnea (90.1%), weakness and latitude (69.2%), dry cough (53.2%), and symptoms of an unresolved upper airway infection (32.6%). Crackles were found in 95.5% of the patients. Digital clubbing was reported by 30.8% of the patients. The mean FVC at the time of inclusion into the eurIPFreg was 2.39 ±0.87 liters (68.3% ± 22.6 % of predicted), TLC was 4.28 ± 1.23 liters, and DLCO was 42.1± 17.8 % of predicted. Diagnosis was based on clinical and radiological findings alone in 69.7% of the cases. Bronchoscopy was performed in 67.9% of the patients. The percentage of patients diagnosed with a surgical lung biopsy was 50% in 2009 and 2.3% in 2016. This is in accordance with the changes in practice regarding the diagnosis of patients with IPF due to the publication of the ATS/ERS/ALAT/JRS Guidelines in 2011 (Raghu et al, AJRCCM 2011; 183: 788–824). The percentage of patients treated with prednisolone, azathioprine and acetylcysteine was progressively reducing from 2009 to 2016 due to the commercial release of pirfenidone and nintedanib for patients with IPF.
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Abstract No. 099 Exploring Efficacy and Safety of Oral Pirfenidone for Progressive, non-IPF Lung Fibrosis (RELIEF-Study) Jürgen Behr 1, Petra Neuser 2, Antje Prasse 3, Michael Kreuter 4, Klaus Rabe 5, Carmen Schade-Brittinger 2, Jasmin Wagner 6, and Andreas Günther 6 1
Department of Internal Medicine V, Comprehensive Pneumology Center, University of Munich and Asklepios Fachkliniken München-Gauting, Munich, Germany 2 Coordinating Center for Clinical Trials, Philipps University of Marburg, Germany 3 Department of Respiratory Medicine, Hannover Medical School, Hannover, Germany 4 Department of Pneumology and Respiratory Critical Care Medicine, Thoraxklinik, University of Heidelberg, Heidelberg, Germany 5 Lungenclinic Grosshansdorf and University of Kiel, Germany 6 Universities of Giessen and Marburg Lung Center, Giessen, Germany
The anti-fibrotic drug pirfenidone is approved in the EU for the treatment of mild to moderate Idiopathic Pulmonary Fibrosis (IPF), but not for other forms of progressive fibrotic lung diseases, in which conventional anti-inflammatory therapy is not sufficiently effective. As pirfenidone exhibits a favorable benefit-risk profile in IPF, it appears likely to be an effective treatment option for patients with other fibrotic lung diseases. The RELIEF study, a randomized, double-blind, placebo-controlled, parallel-group, multicenter, phase II trial, aims to explore the efficacy and safety of oral pirfenidone for progressive, non-IPF lung fibrosis. The study population comprises patients with collagen/vascular disease-associated lung fibrosis (CVD-LF), fibrotic non-specific interstitial pneumonia (fNSIP), chronic hypersensitivity pneumonitis (cHP), and asbestos-induced lung fibrosis (ALF). Disease progression has to be proven by slope calculation of at least three Forced Vital Capacity (FVC) measurements obtained within 6 to 24 months prior to inclusion, documenting an annualized decline in percent predicted FVC of 5 % (absolute). The study medication (pirfenidone or placebo) is administered on top of existing antiinflammatory medication, which has to be stable for at least three month. As primary endpoint the efficacy will be assessed by the absolute change in percent predicted FVC from baseline to week 48 in the Pirfenidone and placebo group. Data will be analyzed using a rank analysis of covariance (ANCOVA) model. A number of secondary endpoints will be analyzed, e.g. time to disease worsening, progression-free survival, categorical relative change of FVC from baseline to week 48. Patient recruitment started in April 2016 with currently 15 study centers including all five DZL sites. 48 (11 CVD-LF, 17 fNSIP, 18 cHP, 2 ALF) of 374 planned patients have been randomized so far (December 2nd 2016).
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Abstract No. 105 Antifibrotic effects of Nintedanib and Pirfenidone on alveolar epithelial cells in 2D and 3D culture Lara Buhl 1,* , Mareike Lehmann 1, Stephan Klee 1, Darcy Wagner 1, Jürgen Behr 2, Michael Lindner 3, and Melanie Könighoff 1 1
CPC, Helmholtz Zentrum München Medizinische Fakultät, LMU München 3 Asklepios Fachkliniken München-Gauting *Presenting author 2
Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive tissue scarring and impaired lung function. To date, Pirfenidone and Nintedanib are the only approved drugs known to slow disease progression. As dysfunctional epithelium is central to the pathogenesis of IPF, we aimed to investiagte how Pirfenidone and Nintedanib exert their anti-fibrotic effects via epithelial cells. Effects of Nintedanib and Pirfenidone were investigated using murine and human 3D ex vivo lung tissue cultures (3D-LTCs) as well as primary murine alveolar epithelial cells (pmATII) obtained from mouse lungs subjected to bleomycin or PBS. The pmATII and 3D-LTCs were cultured with different concentrations of Nintedanib (100nM-10µM) and Pirfenidone (100µM2.5mM) in the presence or absence of TGF-β. Changes on mesenchymal and epithelial markers were assessed by qPCR, Western blotting, ELISA and immunofluorescence. Both Nintedanib and Pirfenidone inhibited mesenchymal gene expression in fibrotic 3DLTCs (Collagen1a1, Fibronectin, Tenascin C) as well as TGF-β-induced upregulation of mesenchymal gene expression in 3D-LTCs and pmATII already at low µM concentrations. Further, both drugs increase alveolar epithelial type I and II cell marker (Surfactant Protein C, T1α) gene and protein expression in murine and human 3D-LTCs and pmATII cells, while the gene expression and secretion of profibrotic epithelial mediator WISP1 was inhibited. In summary, our data suggest that nintedanib and pirfenidone lead to lung fibrosis reduction by targeting alveolar epithelial cell gene and protein expression.
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Abstract No. 107 Bleomycin Induced Pulmonary Fibrosis in Rats and Mice Show Similar Progression in Lung Function, Biochemical and Histological Analyses Dorothee Walter 1,* , Dirk Schaudien 1, Katherina Sewald 1, Armin Braun 1, and Heinz-Gerd Hoymann 1 1
Fraunhofer ITEM *Presenting author
Bleomycin-induced pulmonary fibrosis in rodents is widely used for efficacy testing of antifibrotic drugs. Here, similarities and differences in bleomycin-induced fibrosis between mice and rats are shown. Female C57BL/6 mice or male Wistar rats were treated oropharyngeally with 1.2 U/kg (mice) or intratracheally in two doses with a MicroSprayer aerosolizer with 2.9 U/kg bleomycin (rats). On Day 21, lung function was measured in spontaneously breathing animals. Elevated fibrosis scores were seen in both models (mice: 1.44 vs. 0.0, p<0.01; rats: 2.38 vs 0.0, p<0.01). Analysis of Fulton index revealed an elevation, which might indicate right heart hypertrophy in bleomycin-treated mice and rats. Histomorphometric analysis showed higher levels of collagen-positive areas (mice: 0.16 vs. 0.09 x 105, p<0.01; rats: 0.18 vs. 0.13 x 105, p<0.01). Higher amounts of hydroxyproline were found in lungs of bleomycin-treated rodents (mice: 298.6 vs. 206.12 µg/right lung, p<0.01; rats: 2.07 vs. 0.78 mg/lung, p<0.01). Lung function measurements showed decreased compliance (mice: 0.015 vs. 0.018 ml/cmH2O, p=0.069; rats: 0.191 vs. 0.391 ml/cmH2O, p<0.01). Treatment with bleomycin led to an increase in lung resistance (mice: 0.95 vs. 0.59, p<0.05; rats: 0.9 vs. 0.56 cmH2O/ml, p<0.01). Moreover, breathing frequency was increased in rats (76 min-1 vs. 50 min-1, p<0.01) but not in mice (189 min-1 vs. 184 min-1, p = 0.065). Cell differentiation revealed accumulation of macrophages in bleomycin-treated animals (mice: 8.09 vs. 4.39 x 105, p<0.01; rats: 6.22 vs. 3.59 x 105, p<0.01). Also, lymphocytes were elevated (mice: 0.27 vs. 0.08 x 105, p<0.01; rats: 0.71 vs. 0.03 x 105, p<0.01). We showed similar progression of fibrosis in both rodent species. Hereby, the extent of lung injury and fibrosis induction was confirmed on a lung functional, a biochemical and a histological level. Based on these data, both, mice and rats, seem suitable for preclinical testing of antifibrotic drugs.
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Abstract No. 109 Regulatory role of dendritic cells in mice with pulmonary fibrosis Meritxell Tort Tarrés 1,* , Franziska Aschenbrenner 1, Regina Maus 1, Jennifer Stolper 1, Lars Knudsen 1, Danny Jonigk 1, Tobias Welte 1, Jack Gauldie 2, Martin Kolb 2, and Ulrich A. Maus 1 1
Hannover Medical School McMaster University, Hamilton, Canada *Presenting author 2
Dendritic cells (DC) accumulate in lungs of patients with idiopathic pulmonary fibrosis. However, their pathogenetic relevance in pulmonary fibrosis is poorly defined. Here we examined the role of lung dendritic cells (lung DC) in a mouse model of lung fibrosis triggered by adenoviral gene transfer of biologically active TGF-ß1 (AdTGF-ß1). We found that WT mice exposed to AdTGF-ß1 demonstrated significantly increased accumulation of both CD11b DC and CD103 DC subsets in their lungs along with significantly increased lung collagen contents, relative to control vector-treated mice. Lung DC were found to accumulate within thickened alveolar septae of fibrotic lung lesions, as judged by CD11c/MHCII-specific immunohistochemistry. Opposed to WT mice, FMS-like tyrosine kinase-3 ligand (Flt3L) KO mice characterized by congenital lack of lung DC subsets responded with progressive lung fibrosis and significantly impaired lung function to AdTGFß1 exposure, relative to WT mice, which could be reverted by reconstitution of Flt3L KO mice with recombinant Flt3L protein. Gene expression profiling of sorted lung DC from AdTGF-ß1 versus control vector exposed mice revealed that CD11b DC expressed profibrotic markers and markers of extracellular matrix (ECM) turnover, suggesting their particular contribution to regulation of lung tissue remodeling. Consistent with this notion, CD103-deficient mice mounted a similar lung fibrotic response to AdTGF-ß1 as WT mice. Together, pulmonary fibrosis is characterized by increased accumulation of DC subsets in the lungs of mice, and lack of DC mobilization exacerbates lung fibrosis, which can be attenuated by administration of Flt3L. These data point to regulatory functions of dendritic cells in diffuse parenchymal lung diseases, and suggest that lung DC may be a therapeutic target to limit interstitial fibrosis in DPLD.
Submitted as: Presentation 271 words of 300 possible words
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Abstract No. 114 PDGF dependant alveolar pathology following postnatal lung injury Prajakta Oak 1,* , Nona Kamgari 2, Tina Pritzke 1, Markus Kosclig 3, Daphne S Mous 4, Tobias Reicherzer 5, Harald Ehrhardt 6, Gerrit John-Schuster 7, Jie Jia 1, Ali Önder Yildirim 1, Robbert J. Rottier 4, Tushar J. Desai 8, and Anne Hilgendorff 1 1
University Hospital of the University of Munich and Helmholtz Zentrum Muenchen, Munich, Germany Member of the German Lung Research Center (DZL) 2 Helmholtz ZentruUniversity Hospital of the University of Munich and Helmholtz Zentrum Muenchen, Munich, Germany Member of the German Lung Research Center (DZL) 3 HelmhUniversity Hospital of the University of Munich and Helmholtz Zentrum Muenchen, Munich, Germany Member of the German Lung Research Center (DZL) 4 Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam 5 Perinatal Center Grosshadern, Ludwig-Mximilians University, Munich 6 Justus-Liebig-University and Universities of Giessen and Marburg Lung Center (UGMLC), Giessen 7 Helmholtz ZUniversity Hospital of the University of Munich and Helmholtz Zentrum Muenchen, Munich, Germany Member of the German Lung Research Center (DZL) 8 Stanford University School of Medicine, Stanford, CA, USA *Presenting author
Introduction Neonatal chronic lung disease (nCLD) is a complication in preterm and term infants with significant long-term consequences. The characteristic extensive interstitial remodelling and consequently impaired development of the gas exchange area is induced by pre- and postnatal stressors. Critical for pathophysiologic concepts and effective treatment strategies is a deeper understanding of the intertwined regulation of multiple disease relevant processes by central growth factors. Several unique and clinically relevant mouse models of the disease using both pre- and postnatal injury mechanisms allowed us to study important signalling pathways and their critical role in disease development. Methods and Results Prenatal lung injury in mice was induced by cigarette smoke (CS, 500mg/m3, 50 min/2Xday, gestational day 7-18), whereas postnatal injury using moderate hyperoxia and/or mechanical ventilation (MV-O2) in newborn mice. We showed that prenatal smoke significantly affects alveolar septation in the developing lung with apoptosis of PDGF receptor (PDGF-R)-α positive myofibroblasts present in the gas exchange area. This phenotype aggravated upon postnatal injury, i.e. MV-O2 and in transgenic mice with impaired PDGF-Rα signaling associated with this phenotype. The affection of vascular lung compartment with reduction in microvessel number observed after prenatal smoke was addressed in studies showing a causal relationship between impaired PDGF-Rα signalling and endothelial survival through production of VEGF-A as shown by in-vivo rescue experiments in neonatal mice undergoing MV-O2 and in-vitro studies. Translation of these findings into the human system using lung tissue and isolated cells from patients confirmed the central role of PDGF signalling in provoking alveolar and vascular pathology in nCLD. Conclusion Our data provide causal evidence for the central role of PDGF signalling in the translation of both pre- and postnatal stressors into disease relevant pulmonary pathophysiology. The
DZL Annual Meeting 2017 translational data as well as the treatment experiments indicate valuable therapeutic potential for the findings obtained.
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Abstract No. 124 Monocyte-centred inflammatory response and consecutive TGF-β signalling as central drivers of neonatal interstitial lung disease Prajakta Oak 1,* , Tina Pritzke 1, Harald Ehrhardt 2, Markus Koschlig 1, Melina Kossert 1, Kai Foerster 3, Marion Frankenberger 1, Luisa Biebach 1, Daphne S Mous 4, Robert J Rottier 4, Christina M Alvira 5, Tushar J Desai 6, and Anne Hilgendorff 1 1
Comprehensive Pneumology Center, Helmholtz Zentrum Muenchen, Munich, Germany, Member of the German Lung Research Center (DZL) 2 Department of General Pediatrics and Neonatology, University Hospital of Giessen and Marburg, Giessen (UGMLC), Germany, Member of the German Lung Research Center (DZL) 3 Department of Neonatology, Dr. von Haunersches Children`s Hospital, Ludwig-Maximilians University of Munich, Munich, Germany 4 Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, The Netherlands 5 Dept. of Pediatrics, Stanford University, Stanford, California, USA 6 Department of Internal Medicine, Stanford University School of Medicine, Stanford, CA, USA *Presenting author
Introduction Neonatal chronic lung disease (nCLD) is common complication of preterm infants undergoing prolonged mechanical ventilation (MV-O2) and forms basis to study injury and repair in developing organ with potential relevance for other chronic lung diseases in infants and adults. The characteristic inflammatory cells influx into the lung in nCLD leads to imbalanced expression and activation of chemokines and growth factors. To identify key players and central regulatory mechanisms, we characterized early inflammatory response in nCLD using pre-clinical mouse model and samples obtained from nCLD patients. Methods and Results FACS analysis showed MV-O2 dependent elevation of proinflammatory monocytes in blood from preterm infants. Consequently, analysis of cytokines and growth factor panel using Luminex® xMAP® technology indicated a monocytic-centered response, i.e. elevated MCP-1, Il-8, TNF-α and EGF in cord blood of infants later developing nCLD. Follow-up studies confirmed increased proliferation in fibroblasts from tracheal aspirate of infants with elevated EGF levels at birth. As a therapeutic approach, we mechanically ventilated (MV-O2) newborn TNF-α deficient mice. Conversly, TNF-α deficiency further increased TGF-β driven lung injury in these mice via altered NFκB activation reversible in in-vitro experiments with ligand TNF-α in primary myofibroblasts. The understanding of TGF-β dependent increased apoptosis and ECM remodeling was significantly extended, when PDGF signalling regulation was causally related to pulmonary TGF-β expression. We showed TGF-β dependant PDGF-Rα (critically important in septation) downregulation in cord blood from preterm infants, and newborn mice undergoing MV-O2. In-vitro and in-vivo analyses established defective PDGF-Rα signalling coregulating stretch induced defective primary human and mouse myofibroblast migration and driving increased endothelial apoptosis with reduced pulmonary microvessel number via diminished VEGF-A expression. Conclusion Our data establish the central role of monocyte related TGF-β activation in modulating MV-O2 associated lung injury, leading to impaired cellular function and both
DZL Annual Meeting 2017 alveolar and vascular pathology in neonatal lung.
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Abstract No. 125 Diagnosis of Bronchopulmonary Dysplasia using novel biomarkers and MR imaging analysis, The AIRR study (attention to infants with respiratory risk) Kai Förster 1,* , Steffen Sass 2, Birgit Ertl-Wagner 3, Harald Ehrhardt 4, Andreas W. Flemmer , Prajakta Oak 5, Judith Gronbach 6, Oliver Eickelberg 7, Robbert J. Rottier 8, Andreas Pomschar 3, Daphne S. Mous 8, Christoph Hübener 9, Andreas Schulze 1, Lutz Nährlich 6, Oliver Dietrich 6, Fabian J. Theis 2, and Anne Hilgendorff 1
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Dept. of Neonatology, Perinatal Center Grosshadern, Dr. von Hauner Children`s Hospital, Ludwig-Maximilians-University, Munich, Germany 2 Institute of Computational Biology, Helmholtz Zentrum München, Munich, Germany 3 Institute for Clinical Radiology, Ludwig-Maximilians-University Hospital Munich, Munich, Germany, Member of the German Lung Research Center (DZL) 4 Department of General Pediatrics and Neonatology, University Hospital of Giessen and Marburg, Giessen, Germany 5 Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany 6 Department of General Pediatrics and Neonatology, University Hospital of Giessen and Marburg, Giessen, Germany, Member of the German Lung Research Center (DZL) 7 Comprehensive Pneumology Center, Helmholtz Zentrum München, Munich, Germany, Member of the German Lung Research Center (DZL) 8 Department of Pediatric Surgery, Erasmus Medical Center – Sophia Children’s Hospital, Rotterdam, The Netherlands 9 Clinic for Gynaecology and Obstetrics, Perinatal Center Grosshadern, Ludwig-MaximiliansUniversity, Munich, Germany *Presenting author
Rationale: Bronchopulmonary Dysplasia (BPD) is a consequence of pre- and postnatal injury to the immature lung with significant long-term consequences. Diagnostic tools for early diagnosis and improved structural characterization are urgently needed to improve current clinical practice and thus patient care. Method: In a prospective study, forty preterm infants (27.7±2.09wks, 984±332g) were enrolled to obtain i) serial plasma samples for proteomic screening (SOMAscan™) from birth until discharge and ii) advanced MR imaging (3-Tesla) at the time of diagnosis, complemented by lung function testing. Key findings were validated in an independent study cohort (n=21 infants). Statistical analysis used penalized and Poisson regression analysis; confounder effects were subtracted by Lasso regression. Results: Proteomic screening revealed characteristic plasma levels for three proteins in the first week life with high sensitivity for BPD. The results were confirmed by ELISA in an independent cohort, pulmonary expression was proven by immunohistochemistry in human lung tissue. Analysis of MR imaging showed increased T2- and decreased T1-relaxation times to characterise the BPD lung, mirrored by functional data. Analysis using the count variables ‘days of oxygen or mechanical ventilation’ confirmed the results. Further development of a MR imaging-score (regional scoring on a 6-point semi-quantitative Likert scale in sagittal and transverse images) showed a higher score for interstitial thickening in all BPD grades, whereas severe BPD cases were indicated by increased scores for emphysematous changes. High intra- and inter-reader agreement for all scores confirmed the results. Conclusions: In a next step, the implementation of the identified biomarkers and the new protocol for MR imaging in clinical care for infants with BPD through an interventional study
DZL Annual Meeting 2017 will allow for the i) early detection and ii) sensitive and specific characterization of the disease, a prerequisite for the development of individualized treatment strategies.
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Abstract No. 127 A dual role of SFRP1 in lung fibrosis Arunima Seal 1, Bettina Oherle 1, Oliver Eickelberg 2, and Gerald Burgstaller 1 1
ILBD Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver, CO, USA 2
Migration of activated fibroblasts within the alveolar compartment, their transition to myofibroblasts, and extensive secretion of extracellular matrix constituents are indicative for active fibrogenesis. By using a three dimensional collagen-based invasion assay we generated a transcriptome-wide signature of invading fibroblasts by microarray analysis. Within this signature the secreted frizzled-related protein (SFRP1), a Wnt signaling pathway inhibitor, was identified as a negative regulator of fibroblast invasion. Treatment with the profibrotic master regulator TGFb1 augmented the fibroblasts’ invasive capacity but further suppressed the expression of SFRP1. However, in a proteomic profiling of the matrisome of fibrotic mouse lungs during a time course, SFRP1 expression was found to be highly upregulated in the total tissue homogenates of fibrotic lungs. This increased expression of SFRP1 in fibrotic lungs was further confirmed on transcript and protein levels, as well as by immunohistochemical stainings. In-depth immunohistochemical analyses of fibrotic mouse lungs revealed high levels of SFRP1 expression in E-Cadherin and partially in Desmin positive cells, but not in aSMA positive myofibroblasts. Strikingly, in human IPF sections a strong expression of SFRP1 was discovered in basal cells of the bronchial epithelium, which may indicate a role of SFRP1 in tissue remodeling, repair and regeneration. In conclusion, we argue for a dual role of SFRP1 in lung fibrosis showing low SFRP1 expression in invading fibroblasts and myofibroblasts, and a high SFRP1 expression in the remodeled and regenerated epithelial compartment.
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Abstract No. 130 Distinct niches in the extracellular matrices of decellularized 3D lung tissue cultures (3D-LTCs) instruct cellular behavior Gerald Burgstaller 1,* , Darcy Wagner 1, Sarah Vierkotten 1, Deniz Ali Bölükbas 1, Silke Meiners 1, Melanie Königshoff 1, and Oliver Eickelberg 2 1
ILBD Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver, CO, USA *Presenting author 2
In the last decades the study of cell behavior in mesenchymal cells was typically accomplished in flat, two-dimensional environments such as either uncoated or with extracellular matrix (ECM) coated plastic dishes. During recent years, there has been considerable interest in cell biology to use three-dimensional (3D) scaffolds from synthetic and natural materials in order to mimic, in vitro, the natural micro-environment found in vivo. Here we report successful decellularization and subsequent recellularization of 3D ex-vivo lung tissue cultures (3D-LTCs) derived from healthy and diseased mouse models for fibrosis (bleomycin), emphysema (elastase) and cancer (K-Ras). Confirmation of the efficiency of decellularization of 3D-LTCs was confirmed by Western blotting, qPCR and immunohistological stainings. Reseeded murine and human fibroblasts effectively attached to, spread and proliferated on the decellularized 3D-LTCs over a time period of 72 hours. Engraftment of the fibroblasts in various niches of the 3D-LTCs (alveolar, fibrotic, emphysematous, airway, vessel, and mesothelium) was found to be significantly different. When compared to fibroblasts seeded on conventional 2D plastic dishes, fibroblasts reseeded on 3D-LTCs exhibited differential expression of cellular tension markers, contraction regulators, cell surface receptors, cytoskeletal regulators and phosphorylated signaling proteins. Notably, adhesion of fibroblasts to 3D-LTCs was accomplished by focal adhesions, thus advocating a substantive functional relevance of adhesive multi-protein complexes in vivo. Furthermore, reseeded fibroblasts displayed a tremendous morphological and migratory plasticity which was dependent on the extracellular microenvironment found in the pleural, alveolar and bronchial compartments, as well as in fibrotic and tumor areas. In conclusion, fibroblasts reseeded on 3D-LTCs were found to follow intrinsic cues within the ECM leading to a great plasticity of cellular behavior, which is substantially distinctive from 2D cell culture systems.
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Abstract No. 133 The role of Notch signaling pathway in the etiology and pathogenesis of idiopathic lung fibrosis Roxana Wasnick 1,* , Martina Korfei 1, Katarzyna Piskulak 1, Jochen Whilhelm 1, Irina Shalashova 1, Daniel von der Beck 1, Poornima Mahavadi 1, Lutger Fink 1, Oleksiy Klymenko 1 , Heiko Witt 1, Saverio Bellusci 1, Walter Klepetko 1, Oliver Eickelberg 2, Thomas Braun 1, Werner Seeger 1, Ruppert Clemens 1, and Andreas Guenther 1 1
UGMLC CPC-M *Presenting author 2
Notch signaling is involved in proliferation, differentiation and cell fate decision in the epithelial compartment of the developing lung. Here we hypothesize that Notch1 plays a crucial role in IPF. We demonstrate that Notch1 is the preferential Notch receptor expressed by Alveolar Type II cells (ATII cells) and that plays an important role in ATII cell proliferation and differentiation in human and experimental lung fibrosis. We demonstrate that Notch1 activation leads to transcriptional activation of the downstream target gene Hes1 in ATII cells of IPF patients. To further characterize the role that Notch1 signaling plays in ATII cells proliferation and differentiation, we generated transgenic mice overexpressing activated Notch1 (NICD1) specifically in ATII cells. This alone was sufficient to induce a fibrotic phenotype, characterized by increased collagen deposition, thickening of the alveolar septae, and increase expression of profibrotic markers. Additionally an increase in cell proliferation in NICD1 overexpressing cells was noticed, which was paralleled by a decrease in differentiated function of ATII cells as evident by impaired processing of surfactant protein B (SP-B) and C (SP-C). To study the role of defective surfactant processing in the generation and maintenance of fibrotic lesions, mice were treated with Pepstatin A, a potent aspartyl protease inhibitor, thus inhibiting surfactant processing. Pepstatin A treated mice showed extensive SP abnormalities, chronic ER stress and developed fibrotic lesions similar to those described in Notch1 overexpressing mice, suggesting that Notch1 inhibition of SPB processing is sufficient to induce lung fibrosis. We further demonstrate that human IPF bronchoalveolar lavage fluid (BALF) mirrors the defective SPB processing observed in ATII cells and substitution of human IPF BALF with mature SP-B restored normal surface tension, suggesting that SP-B processing is essential to surface tension maintenance. Taken together our data suggests that Notch1 is an early event in the pathogenesis of IPF.
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Abstract No. 136 Dissecting the involvement of the autophagy marker protein, MAP1LC3B in the development of lung fibrosis Vidya sagar Kesireddy 1,* , Saket Ahuja 1, Shashi Chillappagari 1, Lars Knudsen 2, Ingrid Henneke 1, Matthias Ochs 2, Clemens Ruppert 1, Martina Korfei 1, Andreas Guenther 1, and Poornima Mahavadi 1 1
JLU-Giessen Hannover Medical School *Presenting author 2
Lysosome associated disturbances have been reported in the alveolar epithelial cells type II (AECII) of patients with idiopathic pulmonary fibrosis (IPF) and in animal models of lung fibrosis. Autophagy is a lysosome related quality control pathway that primarily aims at maintaining cellular homeostasis. Several autophagy-related (Atg) proteins have been identified which interact with each other to facilitate the initiation and progression of autophagy. Yeast Atg8 or mammalian microtubule-associated protein 1 light chain 3 beta is an important autophagy related protein and its lipidated form, LC3BII is a reliable marker of the autophagosomes. In this study, we aim to decipher the involvement of LC3BII in the development of IPF. We identified that AECII express LC3B by western blotting and immunofluorescence. Electron microscopic analysis revealed that LC3B is present on the limiting membrane of the lamellar bodies in healthy AECII and that the lamellar bodies remain in close association with the autophagosomes in AECII of fibrotic mice. In order to further understand the functions of LC3B in alveolar epithelial cells, we overexpressed and co-immunoprecipitated LC3B in mouse lung epithelial cells. Analysis of the immunoprecpitates by liquid chromatography-mass spectrometry revealed 249 interacting partners for LC3B. Parallely lung tissue from LC3B-/- mice revealed alterations in surfactant forms in addition to reduced airspaces and apoptosis in aged mice. We propose that LC3B, by interacting with proteins of the secretory pathway, plays essential roles in AECII and might contribute to the cellular stress events during the progression of lung fibrosis.
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Abstract No. 155 Comparative Analysis of the Effects of Nintedanib and Pirfenidone on Collagen Synthesis and Maturation in Donor and IPF Fibroblasts Larissa Knüppel 1,* , Katharina Heinzelmann 1, Rudolf Hatz 2, Jürgen Behr 3, Oliver Eickelberg 1, and Claudia Staab-Weijnitz 1 1
Helmholtz-Zentrum München Ludwig-Maximilians-Universität, Munich 3 Asklepios Fachkliniken München-Gauting *Presenting author 2
Rationale: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung disease characterized by excessive deposition of collagen. The two IPF therapeutics nintedanib and pirfenidone decelerate disease progression, but the underlying mechanisms are poorly understood. This study comparatively analyzes the effects of both drugs on collagen synthesis and maturation between donor and IPF fibroblasts. Methods: Primary human fibroblasts isolated from healthy donors and IPF patients were treated with nintedanib (0.01-1.0µM) or pirfenidone (0.1-1.0mM) with or without TGF-ß1. Effects on the expression of collagens, the collagen chaperones FKBP10 and HSP47, and other fibrotic markers were monitored by Western Blot analysis and qPCR. Moreover, the influence of both therapeutics on collagen secretion was assessed by Western Blot analysis of collagens precipitated from cell culture supernatant. Results: Nintedanib affected expression of collagen I, collagen V, fibronectin and the collagen chaperones FKBP10 and HSP47 similarly in donor and IPF fibroblasts. In contrast, collagen I and III secretion was reduced by nintedanib in IPF, but not in donor fibroblasts. Pirfenidone tended to upregulate collagen I and FKBP10 expression in donor fibroblasts and decreased secreted collagen I in IPF fibroblasts. Additionally, pirfenidone showed a trend to upregulate secretion of collagen I and III in donor fibroblasts. Conclusions: Both drugs act on different regulatory levels in collagen synthesis and processing. In general, nintedanib was more effective than pirfenidone and had comparable effects in IPF and donor fibroblasts. Pirfenidone, in contrast, may exert different effects on collagen I and FKBP10 expression and collagen secretion on donor and IPF fibroblasts.
Submitted as: Presentation 249 words of 300 possible words
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Abstract No. 163 Regulation and role of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) in Idiopathic Pulmonary Fibrosis. Oleksiy Klymenko 1, Martin Huehn 1, Jochen Wilhelm 1, Roxana Wasnick 1, Clemens Ruppert 1, Ingrid Henneke 1, Stefanie Hezel 1, Irina Shalashova 1, Poornima Mahavadi 1, Christos Samakovlis 2, Werner Seeger 3, Andreas Guenther 4, and Martina Korfei 1,* 1
Biomedical Research Center Seltersberg (BFS), Justus-Liebig-University Giessen, German Center for Lung Research (DZL) 2 Excellence Cluster Cardiopulmonary System (ECCPS), Justus-Liebig-University Giessen, German Center for Lung Research (DZL) 3 Excellence Cluster Cardiopulmonary System (ECCPS), Justus-Liebig-University Giessen, Max Planck Institute for Heart and Lung Research in Bad Nauheim, German Center for Lung Research (DZL) 4 Biomedical Research Center Seltersberg (BFS), Justus-Liebig-University Giessen, Excellence Cluster Cardiopulmonary System (ECCPS), Agaplesion Lung Clinic WaldhofElgershausen in Greifenstein, German Center for Lung Research (DZL), European IPF Network and European IPF Registry *Presenting author
We have recently identified severe and pro-apoptotic Endoplasmic Reticulum (ER) stress as major pathomechanism for alveolar epithelial cell (AEC) injury in Idiopathic Pulmonary Fibrosis (IPF). In line with this, the transcription factor CHOP, which has been suggested to cause ER stress-induced apoptosis, was strongly induced in AECII from patients with IPF. We therefore aimed to elucidate the regulation and role of CHOP overexpression in induction of epithelial apoptosis and lung fibrosis in vitro and in vivo. The promoter of the human CHOP gene was extensively analyzed in epithelial cells employing Luciferase reporter gene assays and ChIP. We identified a new mechanism for the regulation of CHOP expression during ER stress. According to our data, AP-1 and cETS-1 transcription factors are up-regulated in the lung epithelium under ER stressconditions, interact with each other and jointly bind to the CHOP promotor, thereby inducing the CHOP gene expression. Chop-overexpression in vitro was performed using an inducible "Tetracycline-On"-vectorsystem in stably-transfected epithelial MLE12 cells or by an adenoviral gene-transfer in isolated, primary murine AECII. Both in vitro-models revealed induction of cleaved caspase3 and thus apoptosis in response to Chop-overexpression. In addition, incubation of murine lung fibroblasts with supernatants of Chop-overexpressing cells resulted in increased fibroblast-proliferation and collagen-I-biosynthesis. Moreover, in vivo-data supported the results of in vitro-studies. Conditional Chopoverexpression in AECII in vivo was achieved by crossing SP-C rtTA to tetO7-Chop mice. In response to doxycycline-feeding (4/8 weeks), Chop was induced in AECII and led to caspase-3-activation and thus apoptosis of these cells. Employing qPCR, induction and upregulation of Chop-target genes was detected in lungs of double-transgenic mice. However, Chop-overexpressing mice did not show development of lung fibrosis after 4/8 weeks doxycycline-feeding, although significant up-regulation of pro-fibrotic markers could be detected. Thus, involvement of Chop in the development of lung fibrosis requires further investigation.
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Abstract No. 174 Species comparison of pro-fibrotic biomarkers in ex vivo lung tissue slices Christina Hesse 1,* , Samuel Mang 2, Heiz-Gerd Hoymann 1, Monika Niehof 1, Peter Braubach 3, Danny Jonigk 3, Mark Kühnel 3, Gregor Warnecke 3, Olaf Pfennig 4, Hans-Gerd Fieguth 4, Armin Braun 1, and Katherina Sewald 1 1
Fraunhofer Institute for Toxicology and Experimental Medicine Boehringer Ingelheim Pharma GmbH & Co. KG 3 Hannover Medical School 4 KRH Clinics Hannover *Presenting author 2
Pulmonary fibrosis is characterized by an abnormal repair process resulting in uncontrolled deposition of extracellular matrix, and destruction of cellular architecture of the lung. Yet, medical treatments are limited, and the development of new therapies is hampered by the lack of experimental models that entirely reflect all features of the disease. With the use of fresh lung tissue, the aim was to identify specific biomarkers of pulmonary fibrosis in order to improve the investigation of novel pharmacological approaches. Human Precision-cut lung slices (PCLS) were prepared from tumor-free lung tissue and cultured in the presence of TGF-β and TNF-α to induce a pro-fibrotic profile. Rat PCLS were prepared from lung tissue of bleomycin-treated rats or NaCl-treated controls and were cultivated in medium only. The mRNA profile in lungs from bleomycin-treated rats displayed an upregulation of important pro-fibrotic genes as compared to the controls. Importantly, PCLS prepared from these animals retain this pattern in culture for 2 to 5 days. Amongst others, pro-angiogenic genes, e.g. chymase1 was found >30-fold elevated. Furthermore, extracellular matrix genes, e.g. collagen1a1 or fibronectin were found significantly up-regulated as compared to the controls. A comparable pattern of up-regulated pro-fibrotic genes was found in human PCLS stimulated with TGF-β and TNF-α. Comparison of the mRNA profiles, by using INGENUITY pathway analysis software, revealed identical canonical pathways and upstream regulators induced in both species. In addition, pro-fibrotic mediators, e.g. IL-β, involved in the initial wound repair mechanism and early fibrotic response, were significantly elevated in both experimental ex vivo systems. Overall, we describe here the induction of identical pro-fibrotic pathways in bleomycintreated rats and TGF-β- and TNF-α-stimulated human lung tissue slices. This characteristic pattern of pro-fibrotic biomarkers enables the investigation of pulmonary fibrosis with high translational relevance in both experimental systems.
Submitted as: Presentation 291 words of 300 possible words
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Abstract No. 182 An ex vivo human Model of Idiopathic Pulmonary Fibrosis using Precision Cut Lung Slices Hani Alsafadi 1,* , Claudia Staab-Weijnitz 1, Mareike Lehmann 1, Michael Lindner 2, Britta Peschel 1, Melanie Königshoff 1, and Darcy Wagner 1 1
Comprehensive Pneumology Center Asklepios Fachkliniken München-Gauting *Presenting author 2
Idiopathic Pulmonary Fibrosis (IPF) is an interstitial chronic lung disease characterized by lung tissue scarring, high morbidity and unknown etiology. Lung epithelial injury, (myo)fibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression. Therapies to reverse the disease are non-existent. Part of the difficulty in identifying effective therapies may reflect the inability of existing mouse and human models to fully recapitulate the complexity of human disease. The use of precision cut lung slices (PCLS) could aid in overcoming the limitations of current models by more closely mimicking the native human lung environment. We hypothesized that the use of a mixture of known profibrotic growth factors, pro-inflammatory cytokines, and signaling molecules, could induce IPF-like characteristics in non-IPF human PCLS. PCLS were treated with a fibrosis cocktail (FC) over a period of five days. Metabolic activity was quantitatively assessed using WST-1. Fibrotic changes were quantitatively assessed using RT-qPCR, WB, and ELISA and qualitatively assessed using histological and full tissue immunofluorescence. Our data demonstrate an increase of well-known fibrotic markers such as FN1, SERPINE1, COL1A1, CTGF, ACTA2, and the recently identified biomarker MMP7 as early as 24 hours after FC treatment, with persistent or further increases at 120 hours. Alveolar epithelial type II cell (ATII) markers, such as SFTPC, were decreased following FC treatment with evidence of attempted alveolar repair as assessed by increases in the ATI marker PDPN. Protein levels of FN1, TNC, and VIM were increased after 48 hours of FC treatment and immunofluorescence revealed increasing FN1 deposition. Furthermore, collagen secretion and deposition increased after 48h of FC treatment. This novel fibrotic assay induces characteristics of IPF in non-IPF human PCLS and could potentially be used to evaluate novel therapeutic targets, and may contribute to better understanding of pathomechanisms driving IPF onset and progression.
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Abstract No. 188 Cultivating bronchial epithelial cells collected through bronchoscopic microsampling in patients with interstitial lung disease Nicolas Kahn 1,* , Michael Meister 1, Ralf Eberhardt 1, Thomas Muley 1, Marc Schneider 1, Felix JF Herth 1, and Michael Kreuter 1 1
Thoraxklinik, University Hospital Heidelberg *Presenting author
Molecular biomarkers in ILDs may improve diagnosis and risk assessment. In contrast to bronchoalveolar lavage, endobronchial epithelial lining fluid (EELF) can be collected localization-specific in the airway of areas with active disease. Recently, we demonstrated that sampling of EELF for transcriptomic analysis is feasible and reliable by bronchoscopic microsampling (BMS). The cellular source of the analyzed markers has not been investigated yet. The aim of this study was to investigate the ability to collect cell culture suitable material from airways of patients with ILDs via BMS. EELF was collected from 10 ILD patients during bronchoscopy by BMS from areas with specific radiological patterns. For cell culture preparation samples were diluted in Airway Epithelial Cell Growth Medium (PromoCell) with antibiotics. Cells were expanded for three weeks and transferred onto filters to generate an air liquid interface (ALI) culture setup. Resulting air liquid culture composition was analyzed using bronchial epithelium specific antibodies. No complication occurred with the BMS procedures. Cytospin analysis of BMS probes showed a clear majority of epithelial cells (80-90). Generating cell culture out of the sampled material was possible in 60% of cases; in 40% this was prohibited due to low cell count and/or contamination with bacteria/fungi. Antibody staining confirmed BEC in the ALI. Here we demonstrate for the first time the ability of BMS to acquire primary BEC cultures from EELF. Ongoing studies are designed to investigate the molecular biomarkers in freshly collected EELF in comparison to cell cultures of the same material.
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Abstract No. 191 Autophagy in lung fibrosis: Exploring the mitophagy pathways Jennifer Arndt 1,* , Shashi Chillappagari 2, Moritz Schäfer 1, Clemens Ruppert 1, Martina Korfei 1, Andreas Günther 1, and Poornima Mahavadi 1 1
Justus-Liebig University Giessen, Germany Member of the German Centre for Lung Research (DZL), Giessen *Presenting author 2
Autophagy is an important cellular homeostasis mechanism and primarily aims to maintain cell survival. However, severe autophagic stress has also been indicated to lead to apoptosis. Autophagy has been implicated in the pathology of idiopathic pulmonary fibrosis (IPF) and in this study we aim to dissect the contribution of the yet unexplored autophagy proteins in general and novel mitochondrial autophagy (mitophagy) proteins in particular towards autophagic stress in IPF as well as in amiodarone (AD) model of lung fibrosis. Our preliminary results indicate an increase in the master regulator of autophagy, Transcription Factor E Box (TFEB) in the lungs of both IPF as well as AD-treated mice and cells. In addition, nuclear localization of TFEB was observed in IPF lungs. Mitophagy markers namely PTEN-induced putative kinase (PINK1) and Parkin were altered in IPF lungs, while the levels of these proteins did not change in AD-treated mice lungs. On the contrary, the selective autophagy marker as well as the autophagy substrate protein, sequestosome 1 (SQSTM1/ p62) was significantly increased under both conditions. In AD-treated mouse lung epithelial cells, mitophagy was activated via binding of p62 to dysfunctional mitochondria in addition to increased interaction of p62 with Keap1 and localization of Keap1 to p62 positive vacuolar structures, indicating the activation of its competitive interacting partner, Nrf2. We conclude that in response to AD treatment, mitochondrial autophagy pathway is activated involving the p62-Keap1-Nrf2 axis. In addition, preliminary evidence suggests context-specific role of autophagy in the development of lung fibrosis. The analysis of other specific mitophagy proteins and their contribution to the disease process is currently underway.
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Abstract No. 198 Stereological characterization of progressive pulmonary remodeling in conditional Nedd4-2 deficient mice Theresa Engelmann 1,* , Dominik Leitz 2, Julia Duerr 2, Marcus Mall 2, Lars Knudsen 1, and Matthias Ochs 1 1
Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL) 2 Department of Translational Pulmonology, Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany *Presenting author
The ubiquitin-ligase Nedd4-2 is involved in post-translational processing of a diversity of proteins including pro-surfactant protein C (proSP-C). Conditional Nedd4-2 knockout in pulmonary epithelial cells in mice leads to mistrafficking of proSP-C and subsequent fibrotic remodeling in the lung resulting in end-stage lung disease after 3 to 4 months. However, the time course of disease progression at structural level and above all the initial structural alterations have not yet been investigated in detail. The purpose of this study was to monitor the progress of structural changes after knockout. To that end wildtype and Nedd4-2 knockout lungs were instillation fixed after 2 weeks, 2 months and 3 months of induction and samples were investigated quantitatively by design-based stereology at light and electron microscopic level. After 2 weeks there were no differences between the knockout and control group in total volume of septal wall tissue per lung, mean septal wall thickness, and volume density of septal wall tissue. After 2 months the values of these parameters increased significantly while the surface area of the alveolar epithelium as well as the total volume of alveolar airspaces remained roughly stable. After 3 months the volume of septal wall tissue and septal wall thickness further increased and nearly doubled compared to the control group. In conclusion Nedd4-2 knockout and proSP-C mistrafficking lead to progressive remodeling in lung tissue within a period of 3 months. The stable surface area of alveolar epithelium and volume of alveolar airspaces in concert with progressive thickening of septal wall tissue suggests a predominantly interstitial remodeling process without intraalveolar fibrosis or collapse induration.
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Abstract No. 204 Modulated deposition of extracellular matrix proteins in a high content imaging assay Michael Gerckens 1,* , Katharina Heinzelmann 1, Oliver Eickelberg 2, and Gerald Burgstaller 1 1
Comprehensive Pneumology Center, Helmholtz Zentrum München, München Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver, CO, USA *Presenting author 2
RATIONALE: Idiopathic lung fibrosis is a severe pulmonary disease with limited pharmacotherapy options. A central mechanism of the disease progression is the activation of fibroblasts, e.g. by TGFβ1, and their consequent excessive deposition of extracellular matrix (ECM). We hypothesized that an appropriate screening assay can identify new small molecule entities (NMEs) inhibiting excessive deposition of ECM by primary human lung fibroblasts (pHLFs). METHODS: We established an automated 96-well plate assay using immunofluorescence confocal imaging to measure the cells’ deposited volume of distinct ECM molecules, such as Collagen Type 1, Type 5 and Fibronectin. As proof of concept, we assessed effects of cytokine stimulation and pharmacological inhibition of ECM deposition. Additionally, we isolated single cell clones (SCCs) of pHLFs using serial dilutions and expanded these under hypoxic culture conditions. Subsequently, we analyzed the SCCs in respect of differential mesenchymal protein/gene expression, immunocytochemistry and ECM deposition behavior. RESULTS: The established high-content assay detects quantifiable changes in ECM deposition by primary human lung fibroblast as effects of TGFβ1 stimulation. In addition, SCC-derived fibroblast populations reveal large differences in expression levels of αSMA (up to >10-fold), CD90 (up to >5-fold) and in ECM deposition behavior (up to >2.5-fold). CONCLUSION: Our assay is able to detect significant changes in ECM deposition of pHLFs. Regarding the expressional and functional differences between the fibroblastic SCCs, we conclude that subpopulations among cells with fibroblastic phenotype exist that might be differently involved in lung fibrosis. OUTLOOK: Our assay might be beneficial for a non-destructive measurement of ECM deposition by pHLFs and further high-throughput screenings to identify NMEs preventing excessive ECM deposition in lung fibroblasts. Additionally, further characterization of clonal lung fibroblast heterogeneity patterns may contribute to the understanding of the role of different fibroblast subtypes in the pathogenesis of fibrotic lung diseases.
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Abstract No. 213 CD82 is a novel mediator of fibroblast proliferation Katharina Heinzelmann 1,* , Nina Noskovicovà 1, Mareike Lehmann 1, Michael Gerckens 1, Michael Lindner 2, Rudolf Hatz 3, Jürgen Behr 4, Melanie Königshoff 5, and Oliver Eickelberg 5
1
Comprehensive Pneumology Center, Munich Asklepios Fachkliniken München-Gauting 3 Asklepios Fachkliniken München-Gauting/Thoraxchirurgisches Zentrum, Klinikum Großhadern, LMU, Munich 4 Asklepios Fachkliniken München-Gauting/Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians-Universität, Munich 5 Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver, CO, USA *Presenting author 2
Fibroblasts are the main ECM producing cells in the lung and play an important role in wound healing. In pulmonary fibrosis, fibroblasts are characterized by a highly proliferating and migrating phenotype, strong expression of aSMA and enhanced secretion of ECM components. Little information exists about their surface marker expression, that is why their definitive isolation and characterization remains challenging. In a previous study, we analyzed the surface proteome of primary human lung fibroblasts and defined a panel of highly expressed cluster of differentiation markers. We characterized these markers over time in culture for expression changes and functional effects. Fibroblasts were isolated from human lung biopsies, taken in culture and analyzed between passage 2 and 10. We determined marker expression and percentage of positive cells by using WB, immunofluorescent stainings, and FACS. Cell status of replicative senescence was analysed by b-Galactosidase activity assay and p16/p21 expression. Changes in proliferation in the absence/presence of CD82 were assessed by gene-specific knock down. A heterogenous expression pattern was observed for desmin, aSMA, and the surface markers CD36 and CD97. The majority of markers showed a stable expression over 6 passages, but a significant increase of positive cells was observed for CD36, CD54, CD82, CD106, and CD140a. Fibroblasts turned senescent from passage 10 on, which correlated with the highest number of cells being positive for CD82. Knockdown of CD82 did not change replicative senescence, but led to reduced proliferation of cells. We identified a panel of stable surface markers expressed in early passages, which can be used for future isolation from tissue. The heterogenous expression of single markers and their changes over time in culture, as observed for e.g. CD36 and CD82, highlights the existence of distinct fibroblast subtypes, which might have different functions, since CD82 could be defined as a positive regulator of proliferation.
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Abstract No. 221 The role of p50ATF6 and sXBP1 in the pathogenesis of lung fibrosis Irina Shalashova 1, Martin Hühn 1, Clemens Ruppert 2, Roxana Wasnick 2, and Andreas Günther 3 1
Universities of Giessen and Marburg Lung Center (UGMLC), Department of Internal Medicine and Biomedical Research Center Seltersberg, Justus-Liebig-University Giessen 2 Universities of Giessen and Marburg Lung Center (UGMLC), Department of Internal Medicine and Biomedical Research Center Seltersberg, Justus-Liebig-University Giessen, German Center for Lung Research (DZL) 3 Universities of Giessen and Marburg Lung Center (UGMLC), Department of Internal Medicine and Biomedical Research Center Seltersberg, Justus-Liebig-University Giessen, German Center for Lung Research (DZL),Agaplesion Lung Clinic Waldhof Elgershausen
Idiopathic pulmonary fibrosis is a lethal disease affecting older adults. Although the pathogenesis of IPF is not clearly understood, chronic injury of type II alveolar epithelial cells (AECIIs) is believed to play an important role in IPF development. Previously our group showed increased level of ER stress and proapoptotic markers in AECIIs in the lungs of IPF patients. While ER stress mediators can act as pro-survival signals, when ER-stress is overwhelming they drive cells into apoptosis. To investigate the role of ER stress in AECII injury, we generated conditional AECII specific p50ATF6 and spliced form of XBP1 overexpressing transgenic mice. Thus the lungs of transgenic mice were harvested following 6 months of transgene induction and the phenotype was characterised by histology, FACS, IF staining, Real-Time PCR and Western Blot. While ER stress markers were successfully induced, we could not observe the induction of full fledge lung fibrosis. However we noted induction of some pro-fibrotic markers like Collagen I and AECII hyperplasia. Taken together our data indicates that overexpression p50ATF6 or sXBP1 during 6 months is not sufficient to induce lung fibrosis in mice but could contribute to it. Further experiments will assess the effect of overexpressing XBP1 and ATF6 during bleomycin induced lung fibrosis.
Submitted as: Presentation 204 words of 300 possible words
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Abstract No. 230 Surfactant replacement therapy reduces acute lung injury and collapse induration related lung remodeling in the bleomycin model Lilian Steffen 1,* , Clemens Ruppert 2, Heinz-Gerd Hoymann 3, Manuela Funke 4, Simone Ebener 4, Christina Kloth 1, Christian Mühlfeld 1, Matthias Ochs 1, Lars Knudsen 1, and Elena Lopez-Rodriguez 1 1
Institute of Functional and Applied Anatomy, Hannover Medical School, Hanover, Germany Department of Internal Medicine, Justus-Liebig-University Giessen, Giessen, Germany 3 Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM), Hanover, Germany 4 Departments of Pulmonary Medicine, Inselspital Berne, and Clinical Research, University of Berne, Berne, Switzerland *Presenting author 2
Bleomycin-induced lung injury leads to surfactant dysfunction and permanent loss of alveoli due to a remodeling process called collapse induration. Collapse induration also occurs in acute interstitial lung disease and idiopathic pulmonary fibrosis in humans. We hypothesized that surfactant dysfunction aggravates lung injury and remodeling resulting in collapse induration within 7 days after lung injury. Rats received bleomycin to induce lung injury and either repetitive surfactant replacement therapy (SRT: 100mg Curosurf®/kg BW=surf group)) or saline (0.9% NaCl=saline group). After three (D3) or seven (D7) days, invasive pulmonary function tests were performed to determine tissue elastance (H) and static compliance (Cst). BAL was taken for surfactant function and inflammatory markers; lung tissue was harvested for design-based stereology and hydroxyproline measurement. SRT significantly improved minimum surface tension of alveolar surfactant as well as H and Cst at D3 and D7. At D3 decreased inflammatory markers such as neutrophilic granulocytes, IL1β, IL-6 and MCP-1 correlated with reduced intraalveolar edema formation after SRT. Numbers of open alveoli were significantly increased at D3 and D7 in SRT groups while at D7 there was also a significant reduction in septal wall thickness and tissue volume without difference in hydroxyproline. Numbers of open alveoli and septal wall thickness highly correlated with improved lung mechanics after SRT. In conclusion, reduction in surface tension was effective to stabilize alveoli linked with an attenuation of parameters of acute lung injury at D3 and collapse induration at D7. Hence, surfactant dysfunction contributes to lung injury and later on collapse induration.
Submitted as: Presentation 249 words of 300 possible words
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Abstract No. 236 Quantitative Proteomics Reveals Novel Fibrotic Networks of Myeloid-Derived Suppressor Cell and Monocytes in IPF Isis E. Fernandez 1,* , Riti Roy 2, Flavia Greiffo 1, Marion Frankenberger 1, Jürgen Behr 3, Alistair Forrest 2, and Oliver Eickelberg 1 1
Helmholtz Zentrum Muenchen Harry Perkins Institute of Medical Research 3 Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians- Universität *Presenting author 2
Idiopathic pulmonary fibrosis is a fibroproliferative disease with irreversible loss of lung function. Myeloid-derived suppressor cells (MDSC) are activated immature myeloid cells, which suppress immune responses in cancer, and other inflammatory conditions. We reported, that MDSC are increased, functionally active, and reflect disease status in IPF, in cross-sectional and longitudinal analysis. Monocytic MDSC are the predominant subtype in IPF, and yet, differences between mature monocytes and monocytic MDSC, and their interaction in IPF have not been explored. Using label-free quantitative MS-analysis, monocytes and MDSC isolated from 10 IPF patients were analyzed. In total, we identified and quantified more than 7000 proteins. PCA unequivocally discriminated both cell types. 4345 proteins were detected in at least 2/3 of all samples, and subject of further analysis. Comparing the proteins identified in both cells, we found 502 MDSC enriched and 1224 monocyte enriched proteins (2 to >30 log10 intensity ratios). Next, based on published datasets we examined cell-cell communication potential, by identification of the receptors-ligands expression and interaction. In combined dataset 200 ligands and 153 receptors were detected. From the cell-to-cell communication analysis we identified both autocrine signaling edges from monocyte to monocyte (339), MDSC to MDSC (290), and paracrine signaling edges from monocyte to MDSC (311) and MDSC to monocyte (316). Specific ligands predicted to signal from monocyte to MDSC included: ANXA1, CCL18, CXCL2, HSP90AA1, ICAM1, TGFB2, amongst others. MDSC to monocyte included: COL1A1, FN1, HLA-C, HSPG2, MMP1, S100A8-9, TGFB1, amongst others. In summary, this study explores the MDSC proteome in fibrosis and identifies novel targets expressed in IPF MDSC, and monocytes. Using network analysis, we found autocrine and paracrine signals from and between monocytes and MDSC. Furthermore, confirmation by flow cytometry of exclusively expressed surface receptors, might lead to identification of novel proteins useful for cell targeting in IPF.
Peripheral blood myeloid-derived suppressor cells reflect disease status in idiopathic pulmonary fibrosis. Fernandez IE, et al. Eur Respir J. 2016 Oct Submitted as: Presentation 297 words of 300 possible words Page 161 of 286
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Abstract No. 237 Pro-fibrotic role of Histone Deacetylase 9 isoforms in pulmonary fibrosis Pouya Sarvari 1,* , Prakash Chelladurai 1, Martina Korfei 2, Eric Olson 3, Andreas Guenther 2, Werner Seeger 1, and Soni Savai Pullamsetti 1 1
Max-Planck-Institut für Herz- und Lungenforschung University of Giessen 3 University of Texas Southwestern Medical Center *Presenting author 2
Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal interstitial pulmonary disease with no proven drug therapy. The disease is characterized by the presence of phenotypically altered fibroblasts that exhibit hyper-proliferation, apoptotic resistance, increased migration and trans-differentiation. Recently, the role of epigenetic modifiers such as histone deacetylases (HDACs, Class IIa) has been identified in the pathophysiology of many fatal diseases such as cancer and cardiovascular diseases. However, the role of class IIa HDACs in the progression of IPF and specifically in driving IPF-fibroblast phenotype is yet to be investigated. We examined fibrotic lungs and lung fibroblasts isolated from patients with IPF and comparative controls and assessed the expression of class-II HDAC family members (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and its alternatively spliced isoform HDRP and HDAC10). We found that HDRP expression is up- regulated exclusively in fibroblasts isolated from IPF patients compared to fibroblasts isolated from healthy controls/donors. Assessing the pro-fibrotic mediators that can regulate the expression of HDACs, we observed that the TGF-β, leads to up regulation of both HDAC9 and HDRP expression, However the expression of remaining class IIa HDACs (HDAC4, HDAC5 and HDAC7) remain unaltered. Our functional data shows that the overexpression of HDRP and HDAC9 leads to fibroblast-to myofibroblast trans-differentiation and apoptosis-resistance in donor Fibroblast. In addition, down-regulation of HDRP using siRNA not only reverses the myofibroblast phenotype of the TGF-β stimulated donor fibroblasts as well as of the IPF fibroblasts but also increases the apoptosis in IPF fibroblasts. The mass spectrometry analysis highlights diverse functions for HDAC9 and HDRP including the regulation of Focal adhesion signalling. We additionally confirmed the interactions with the Coimmunoprecipitation. Our primary data suggests that TGF-β mediated regulation of HDRP and HDAC9 modulates lung fibroblast phenotypes and hence contributing to the fibrotic pathology in the pathogenesis of IPF.
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Abstract No. 239 WNT5A is secreted by extracellular vesicles in pulmonary fibrosis and increases fibroblasts proliferation Aina Martin Medina 1,* , Sarah Vierkotten 1, Mareike Lehmann 1, Darcy E. Wagner 1, Hoeke A. Baarsma 1, Thomas Hofer 1, Marion Frankenberger 1, Jürgen Behr 2, Michaela Aichler 3, and Axel Walch 3 1
Comprehensive Pneumology Center Asklepios Fachkliniken München-Gauting 3 2Institut of Pathology Helmholtz Zentrum München *Presenting author 2
Objective: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of yet unknown etiology characterized by lung epithelial injury, myofibroblast activation, and increased extracellular matrix deposition. The non-canonical WNT5A ligand has been linked to fibroblast proliferation in IPF. Recently, it has been discovered that WNT ligands can be secreted on extracellular vesicles (EVs), which are released by cells to regulate the intercellular communication. Nevertheless, the contribution of EVs in the pathogenesis of IPF has not been explored yet. Here we hypothesize that WNT5A contributes via EVs to impaired cellular crosstalk in pulmonary fibrosis. Methods and results: First, we performed a comprehensive profiling of EVs in experimental and human pulmonary fibrosis. EVs were isolated by ultracentrifugation from bronchoalveolar lavage fluid (BALF) and characterized by transmission electron microscopy, nanoparticle tracking analysis and Western Blotting. Notably, we found increased EV secretion in BALF obtained from fibrotic mice (particles/ml: PBS 2.68x108±3 x107, Bleo 4.65x108±1.77 x108) which carried high amounts of WNT5A. In addition, we confirmed increased EVs carrying WNT5a in BALF from IPF patients compared to non-IPF. Interestingly, WNT5A was upregulated in EVs derived from TGFβtreated primary human lung fibroblasts (phLFs), suggesting that lung fibroblasts are a source of EV-WNT5A secretion in IPF. Functionally, phLFs-EVs significantly induced autocrine proliferation at 48h. Furthermore, this effect was significantly reduced after antibody-mediated neutralization or silencing of WNT5A (%metabolic activity to control: Ev+IgG 23.7±8.4, EV+W5A-AB 10±11.9; scr-EV 16.9±17, siRNA-EV 1.3±9). Conclusions: We report an increase of WNT5A-containing EVs in experimental and idiopathic pulmonary fibrosis. Furthermore, EVs derived from phLFs induced proliferation in a WNT5A-dependent manner in the same cell type. All in all our data suggest a new role of EV-derived WNT5A in the disease.
Submitted as: Presentation 279 words of 300 possible words
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Abstract No. 249 Nanoparticle exposure of persistently herpesvirus-infected cells reactivates latent virus and restores features of an acute virus infection Christine Sattler 1, Franco Moritz 2, Shanze Chen 1, Beatrix Steer 3, David Kutschke 1, Martin Irmler 4, Johannes Beckers 4, Oliver Eickelberg 1, Philippe Schmitt-Kopplin 2, Tobias Stoeger 1 , and Heiko Adler 3,* 1
Institute of Lung Biology and Disease Research Unit BioGeoChemistry 3 Research Unit Lung Repair and Regeneration 4 Institute of Experimental Genetics *Presenting author 2
Both inhalation of environmental (nano)particles (NP) and persistent herpesvirus-infection have been implicated to contribute to the development of chronic lung disease. We hypothesized that the combination of NP exposure and virus infection leads to a different outcome than exposure to each factor alone. Latent virus infection and additional exposure to NP might, for example, result in disruption of the immune control of viral latency and induce virus reactivation. To test this hypothesis, we conducted in vitro studies in latently infected human and murine cells. Additionally, we performed in vivo studies using the model system of murine gammaherpesvirus 68 (MHV-68). Latently infected murine or human cells were exposed to NP in vitro. The amount of virus in supernatant was determined by Plaque assay or qPCR, and expression of viral genes was analyzed by qRT-PCR. To investigate the effect of pulmonary NP exposure in vivo, latently infected mice were instilled with NP or left untreated. After 24 hours, whole lung tissue was harvested for detection of lytic viral proteins by immunohistochemistry (IHC), analysis of the transcriptome and the metabolome. NP exposure of latently infected cells induced lytic virus production and expression of the viral transactivators Rta and BZLF1, respectively. In the lungs of latently infected mice, IHC demonstrated an increase in lytic viral proteins after exposure to NP. Simultaneously, gene expression analysis revealed a signature with considerable parallels to the one seen during acute infection. Analysis of the metabolome demonstrated an enrichment of phospholipids in latently infected mice after exposure to NP, matching profoundly with the pattern observed during acute virus infection. Our results indicate that the combination of NP exposure and persistent herpesvirus infection restores a molecular signature found in acute virus infection, boosts production of lytic viral proteins, and induces an inflammatory response in the lung.
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Abstract No. 254 Disruption of the hepcidin/ferroportin regulatory circuitry causes pulmonary iron overload and restrictive lung disease Joana Neves 1,* , Dominik Leitz 1, Christina Brandenberger 2, Christian Mühlfeld 2, Raman Agrawal 1, Marcus A. Mall 1, Sandro Altamura 1, and Martina U. Muckenthaler 1 1
University of Heidelberg Hannover Medical School *Presenting author 2
Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in organ pathologies that are so far not considered classical iron-related disorders.
Submitted as: Presentation 121 words of 300 possible words
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Abstract No. 257 Rescue of mutant ABCA3 by small molecular correctors Susanna Kinting 1,* , Stefanie Höppner 1, Thomas Wittmann 1, Ulrike Schindlbeck 1, Jacqueline Harfst 1, Jan Hegermann 2, Ralf Zarbock 1, and Matthias Griese 1 1
Dr. von Haunersches Kinderspital - Klinikum der Universität München Funktionelle und Angeandte Anatomie - Medizinische Hochschule Hannover *Presenting author
2
Introduction: ABCA3 is a lipid transporter expressed in alveolar type II pneumocytes. It is involved in the transport of phospholipids into lamellar bodies (LBs) and is therefore essential for the assembly of pulmonary surfactant and LB biogenesis. Mutations in the ABCA3 gene display a common genetic cause for respiratory distress syndrome in newborns and interstitial lung disease in children. Aim: As ABCA3 shares several features with CFTR (ABCC7), small molecules that are able to correct mutated CFTR proteins were tested for ABCA3 mutations. Methods: A549 cells stably expressing HA-tagged wildtype ABCA3 or variants Q215K, M760R, A1046E, K1388N, or G1421R, were treated with a range of different correctors to identify molecules that are able to restore ABCA3 processing and function. Cells were analyzed in regard to ABCA3 protein pattern by Western blotting and immunofluorescence. Activity of corrected ABCA3 protein was quantified using a lipid transport assay with TopFluor-conjugated phosphatidylcholine. Results: C17 corrected misprocessing of all ABCA3 variants except for M760R and led to a WT-like appearance in Western Blotting and immunofluorescence . Lipid transport into ABCA3-HA-positive vesicles was increased by corrector treatment for WT ABCA3 and all variants except for M760R ABCA3. Conclusion: The identification of molecules with the ability to restore processing and function of mutated ABCA3 proteins are an important step towards the development of drugs for the treatment of children suffering from respiratory distress syndrome or interstitial lung disease. Supported by Deutsches Zentrum für Lungenforschung (DZL)
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Abstract No. 274 iPSC-derived Pulmonary Macrophage Transplantation Therapy in a murine model of hereditary PAP Christine Happle 1,* , Nico Lachmann 1, Mania Ackermann 1, Martin Wetzke 1, Adele Mucci 1, Anja Mirenska 1, Torsten Glomb 1, Silke Glage 1, Oliver Dittrich-Breiholz 1, Thomas Moritz 1, and Gesine Hansen 1 1
Hannover Medical School *Presenting author
Introduction: Transplantation of induced pluripotent stem cell (iPSC) derived cells harbors great potential for the treatment of hereditary and acquired diseases, but only limited data on the therapeutic benefit of human iPSC-derived cells exists. Here, we analyzed the potential of pulmonary transplantation of iPSC derived macrophages in the treatment of hereditary Pulmonary Alveolar Proteinosis (PAP), a disease caused by GM-CSF receptor mutations and a differentiation block of alveolar macrophages (AM). Methods: iPSC derived macrophage culture was upscaled in order to generate sufficient numbers of iPSC macrophages for consecutive pulmonary transplantation into humanized PAP mice. Mice were transplanted four times in four consecutive weeks, and treatment effects were analyzed two months after the first treatment. Results: Pulmonary transplantation of iPSC derived macrophages led to pulmonary engraftment and in situ differentiation of iPSC derived macrophages into alveolar macrophage (AM) like cells. These cells displayed the typical morphology, function, and transcription profile of AMs. Importantly, the transplantations significantly improved the PAP phenotype as demonstrated by markedly diminished alveolar protein content, reduced bronchoalveolar lavage fluid (BALF) proteinosis and reduced BALF turbidity. Conclusion: To our knowledge, this data represents the first proof of concept for a therapeutic effect of human iPSC derived macrophages in a pulmonary disease. This may have profound implications beyond the rare disease of PAP.
Submitted as: Presentation 213 words of 300 possible words
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Abstract No. 277 Classical Transient Receptor Potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis Susanne Fiedler 1,* , Katharina Hofmann 1, Sarah Vierkotten 2, Jonas Weber 1, Stephan Klee 2 , Jie Jia 2, Wolfgang Zwickenpflug 1, Veit Flockerzi 3, Ursula Storch 1, Yildirim Önder 2, Thomas Gudermann 1, Melanie Königshoff 2, and Alexander Dietrich 1 1
Walther-Straub-Institute of Pharmacology and Toxicology, Member of the German Center for Lung Research (DZL), Ludwig-Maximilians-Universität München 2 Comprehensive Pneumology Center, Member of the German Center for Lung Research (DZL) 3 Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg *Presenting author
Pulmonary fibrosis (PF) is a progressive lung disease ultimately leading to death. PF may be induced by chronic exposure to toxic drugs like the cytostatic bleomycin during tumor chemotherapy. One of the pathological features of PF is the formation of myofibroblast foci and the secretion of collagen. Many different cell types are suggested to transdifferentiate into these activated myofibroblasts e.g. alveolar epithelial cells and resident fibroblasts. Although the mechanism of PF is not fully understood, the profibrotic transforming growth factor β (TGFβ) is considered to play a crucial role. TRPC6 is an unselective cation channel playing an important role in myofibroblast transdifferentiation of cardiac and dermal fibroblasts (1). To study a potential role of TRPC6 in PF we analyzed lung function in wildtype (WT) and TRPC6-deficient (Trpc6-/-) lungs utilizing a bleomycin-induced PF-model. WT mice died more frequently than Trpc6-/- mice during the fibrotic phase from day 7 after application of bleomycin, most probably due to severe changes in lung function which was similar to control mice in Trpc6-/- mice. Moreover, collagen accumulation in bleomycintreated TRPC6-deficient lungs was indistinguishable to PBS-treated control animals, while WT mice treated with bleomycin showed increased collagen levels (2). To analyze TRPC6 function on a cellular level, we isolated primary murine lung fibroblasts (pmLFs) from mice of both genotypes and incubated them with TGFβ. Most interestingly, formation of actin stress fibers were significantly reduced in TRPC6-deficient pmLFs in clear contrast to WT cells, which also showed increased TRPC6-expression after exposure to TGFβ (2). Electrical cellular impedance sensing system (ECIS) measurements of TGF-β1 treated WT and TRPC6-/- pmLF showed significant higher increase in cellular resistance in WT pmLF reflecting an increased cellular barrier function triggered by increased myofibroblast differentiation. Therefore, defining TRPC6 function might help to identify pharmacological targets for new therapeutic options in bleomycin-induced PF.
1.) Davis et al. (2012). A TRPC6-dependent pathway for myofibroblast transdifferentiation and wound healing in vivo. Developmental cell, 23(4), 705-715. 2.) Hofmann et al. (2017) Classical transient receptor potential (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1863: 560-568. Submitted as: Presentation 299 words of 300 possible words Page 168 of 286
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Abstract No. 285 3T MRI in infants without medical sedation – a novel and gentle way to achive MRI imaging Andreas Pomschar 1,* , Sophia Stöcklein 1, Birgit Ertl-Wagner 1, Anne Hilgendorff 1, and Kai Förster 1 1
LMU - Munich University *Presenting author
Introduction MRI imaging in infants is of increasing relevance. Although the risk of this examination is considered very low, it is limited by the need for medical sedation with the accompanying risks and high costs. The AIRR Study examined infants in a 3T MRI and imaging of the lung and brain was achieved. To avoid sedation, the infants were imagined in a vacuum mattress, following a special protocol, avoiding medical sedation. We evaluated the quality of this medication-free examination and compared it to a group with medical sedation. Subjects&Methods 13 randomly selected datasets were retrospectively analyzed for motion artifacts. Informed consent was obtained from the parents. The infants were first fed and their diapers changed. A pulsoxymeter and hearing protection were applied. They were placed in a vacuum mattress and then in the MRI. Imaging began once the infant was sleeping. The procedure was paused when the infant became too excited. Lung images were rated by an experienced radiologist on a 5-point Likert scale (1=nomovement;5=non-diagnostic)for movement and diagnostic quality. A fMRI dataset with 176 repetitions was analyzed and frames with movement above 0.5 mm were excluded. We compared to a collective of 13 infants who had received medical sedation. Results All examinations were finished successfully and were of diagnostic quality. However the medication-free group showed more movement with a median score of 2,range 14vs1,range 1-2. Significantly more fMRI frames were excluded in the medication-free group with a mean of 29±31.7vs8±14.4, however only 3 examinations were considered unusable for further fMRI analyses. A reduction of atelectasis was noted. Discussion Movement is generally higher without medical sedation, however our novel method still allows to perform extensive clinical MRI examinations of good quality in most infants, avoiding medical sedation. Very sensitive examinations such as fMRI may be limited by the additional movement.
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Abstract No. 294 Temporal Subtraction Method for detection of ILD progression on successive thoracic CT Hoen-oh Shin 1, Tom Kracke 1, and Antje Prasse 1 1
Medical School Hannover
Purpose The aim of the study was to determine whether a novel computed tomography (CT) postprocessing technique (Temporal Subtraction) is superior to visual CT scoring as judged by functional correlations in idiopathic pulmonary fibrosis (IPF). Material and Methods Paired CT scans of 24 patients were non-linearly registered. The subtraction image was obtained by the density difference of two consecutive CT scans of each patient and compared to visual scoring and pulmonary function tests. Results Preliminary results showed that TS-derived estimates of ILD extent demonstrated stronger correlations than visual scores for most pulmonary function tests. Even subtle changes in parenchymal patterns in paired CT scans could be assessed with TS. Conclusion In longitudinal studies, early morphological changes in CT may be quantitatively assessed by TS. TS might be used as an imaging biomarker for monitoring disease in IPF.
Submitted as: Presentation 137 words of 300 possible words
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Abstract No. 008 Cytokine expression in humanized mice reflects development of primary graft dysfunction in lung transplant recipients Ann-Kathrin Knöfel 1,* , Thierry Siemeni 1, Nodir Madrahimov 1, Wiebke Sommer 1, Murat Avsar 1, Fabio Ius 1, Katharina Jansson 1, Linda Pauksch 1, Jawad Salman 1, Igor Tudorache 1 , Christian Kühn 1, Axell Haverich 1, Christine Falk 1, and Gregor Warnecke 1 1
MHH *Presenting author
Primary graft dysfunction (PGD) is the main cause of early mortality after lung transplantation. In humanized mice, we studied the impact of the baseline condition of the lung donor endothelium on the development of transplant arteriosclerosis. Segments of the pericardiophrenic arteries were procured from surplus donor lung tissue and implanted into the abdominal aorta of immunodeficient mice and development of transplant arteriosclerosis was determined at day 28. Experiments were assigned to two groups depending on the early post-transplant course of the respective lung transplant recipient with 11 patients developing PGD grade ≥ 2 (PGD+) and 11 patients developing PGD grade ≤ 1 (PGD-) within 24 hours after transplantation. Mice bearing aortic transplants were divided into six treatment groups with group A mice receiving aortic vessels from PGD+, group B mice from PGD- patients. Group C mice with aortic vessels from PGD+ patients were reconstituted with the respective allogeneic recipient human peripheral blood mononuclear cells (PBMC), group D mice additionally received CD4+CD25high Treg cells. Group E mice with aortic vessels from PGD- patients received PBMC and group F mice additionally CD4+CD25high Treg cells. Luminal occlusion of aortic vessels was significantly higher in PGD+ group A mice compared to those from PGD- group B mice in the absence of PBMC which was paralleled by IFN- s e rum conce ntra tions . Addition of P BMC in group C & E m ice furthe r incre a s e d occlusion and IFN- le ve ls . Tre g of P GD+ a nd P GD- recpients showed similar suppressive capacity. IFN--inducible chemokine CXCL10, TNF-α and the soluble high affinity IL-2 receptor showed similar Th1 patterns. We conclude that the donor endothelial system contributes to PGD development which is driven by systemic TH1 responses. This inflammatory response is suppressed by CD4+CD25high Treg cells indicating a potentially important target for future interventions in lung transplantation.
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Abstract No. 051 Breath VOC patterns of lung transplant recipients with and without chronic lung allograft dysfunction (CLAD) Lucas Küppers 1,* , Olaf Holz 1, Bianca Lavae-Mokhtari 1, Frank Günther 1, Linda Häsler 2, Konstantina Zang-Pappa 2, Jens Gottlieb 2, and Jens M Hohlfeld 1 1
Fraunhofer ITEM Hannover Medical School (MHH) *Presenting author 2
Introduction: Despite the recent development in post transplantation care and decrease of acute rejection, the chronic lung allograft dysfunction (CLAD) with its clinical correlative of bronchiolitis obliterans syndrome (BOS) is considered as one of the major limiting factors for long-term graft survival. BOS is difficult to detect in early stages and its diagnosis is particularly based on the deterioration of lung function. In our study we compared breath volatile organic compounds (VOC) between different stages of BOS to assess the potential of this non-invasive measurement as a novel tool for the early detection of BOS. Methods: In this preliminary analysis we included 73 subjects (24 BOS 0, 25 BOS 1/2, 24 BOS 3) with bilateral lung transplantation (6 with combined heart-lung transplantation). Key exclusion criteria were active smoking, oxygen therapy and acute infection. Patients inhaled room air through a VOC and sterile filter and exhaled into an aluminum reservoir tube. Breath was loaded onto Tenax adsorption tubes and analyzed by GC-MS. Results: The three study groups differed with respect to the duration since transplantation and the number of patients receiving azithromycin treatment. In the univariate analysis we found 10 VOCs that were related to BOS stage. Three of these were among the 9 VOCs that were different for patients treated with azithromycin. Antibiotic treatment affected the level of breath indole, which is known to originate from bacteria. Differences in VOC patterns were also observed within BOS 3 patients with respect to immunosuppressive treatment and for those with additional anti-inflammatory treatment. Conclusion: We showed that the collection of breath samples using our reservoir sampler in the clinical environment was feasible. The results of the univariate analysis suggests that breath VOCs related to the severity of BOS and to the different treatment regimens exist.
Submitted as: Presentation 292 words of 300 possible words
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Abstract No. 067 Cooperation between nicotinic acetylcholine receptor subunits α7, α9 and α10 is mandatory for cholinergic inhibition of IL-1β release in monocytes Katrin Richter 1,* , Anna Zakrzewicz 1, Bijan Fink 1, Robin Siebers 1, Gabriela Krasteva-Christ 2 , Winfried Padberg 1, J. Michael McIntosh 3, and Veronika Grau 1 1
Justus-Liebig University Giessen, Germany Julius-Maximilians-University Wuerzburg, Germany 3 University of Utah, USA *Presenting author 2
Introduction: Increased production of the proinflammatory cytokine interleukin-1β (IL-1β) plays a pathogenic role in inflammatory diseases, like acute respiratory distress syndrome. We previously demonstrated that the ATP-dependent release of IL-1β in human monocytes is inhibited by a cholinergic mechanism involving nicotinic acetylcholine receptors (nAChR). Here, we tested if lyso-phosphatidylcholine (LPC) and glycerophosphocholine (GPC) function as novel ligands of nAChR (α7/α9/α10) and investigated, which nAChR subunits are essential for the immune-modulatory function. Methods: In human monocytic U937 cells the P2X7 receptor was stimulated with BzATP in absence and presence of nAChR agonists. IL-1β was measured in cell culture supernatants by ELISA. To test a possible cooperation of nAChR subunits (α7/α9/α10), IL-1β release was investigated in U937 cells using specific nAChR antagonist and siRNA-treated cells. Furthermore, experiments were performed on peripheral blood mononuclear cells (PBMC) isolated from nAChR gene-deficient mice. Electrophysiological two-electrode voltage-clamp (TEVC) measurements were performed on Xenopus oocytes expressing human nAChR subunits (α7/α9/α10) to test for ionotropic functions of LPC and GPC. Results: LPC and GPC inhibit BzATP-induced IL-1β release from U937 cells. Using mice PBMC and siRNA-treated U937 cells, we demonstrated that inhibition of ATP-dependent release of IL-1β by nicotine, acetylcholine and phosphocholine depends on the cooperation of subunits α7, α9 and α10. Signalling of LPC and GPC requires the cooperation of the α9 nAChR subunit with one of the two subunits (α7, α10). In α7/α9/α10-expressing oocytes, acetylcholine induced ionotropic receptor functions, whereas phosphocholine, LPC and GPC did not. Conclusion: The nAChR subunits α7/α9/α10 cooperate in monocytes. Classical nAChR agonists (nicotine, acetylcholine, phosphocholine) inhibit P2X7 receptor function via cooperation of nAChR subunits α7/α9/α10. For metabotropic signaling of LPC and GPC the cooperation of two nAChR subunits is needed, among them α9 is mandatory. LPC and GPC seem to regulate immune functions without perturbing canonical ion channel functions of nAChR.
Submitted as: Presentation 300 words of 300 possible words
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Abstract No. 073 Lung volumes predict survival in patients with chronic lung allograft dysfunction Nikolaus Kneidinger 1,* , Katrin Milger 1, Silke Janitza 2, Felix Ceelen 1, Gabriela Leuschner 1, Julien Dinkel 1, Melanie Königshoff 3, Thomas Weig 1, Rene Schramm 1, Hauke Winter 1, Jürgen Behr 1, and Claus Neurohr 1 1
Comprehensive Pneumology Center (CPC-M), University of Munich University of Munich 3 Comprehensive Pneumology Center (CPC-M), Helmholtz Zentrum München *Presenting author 2
Background: Identification of disease phenotypes might improve understanding of patients with chronic lung allograft dysfunction (CLAD). The aim of the study was to assess the impact of pulmonary restriction and air trapping by lung volume measurements at the onset of CLAD. Methods: 396 bilateral lung transplant recipients were analysed. At onset, CLAD was further categorized based on plethysmography. A restrictive CLAD (R-CLAD) was defined as a loss of total lung capacity (TLC) of baseline. CLAD with air trapping (AT-CLAD) was defined as an increased ratio of residual volume (RV) to TLC. Outcome was survival after CLAD onset. Patients with insufficient clinical information were excluded (n=95). Results: Out of 301 lung transplant recipients 94 (31.2%) developed CLAD. Patients with RCLAD (n=20) and AT-CLAD (n=21), respectively had a significantly worse survival (p<0.001) than patients with non-R/AT-CLAD. Both R-CLAD and AT-CLAD were associated with increased mortality when controlling for multiple confounding variables (HR=3.57 [1.39; 9.18]; p=0.008 and HR=2.65 [1.05; 6.68]; p=0.039). Further, measurement of lung volumes was useful to identify patients with combined phenotypes. Conclusion: Measurement of lung volumes in the long-term follow-up of lung transplant recipients allows identification of patients who are at risk for worse outcome and warrant special consideration.
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Abstract No. 121 Assessment of Flow Dynamics in a Blood Oxygenator - Implications for Endothelialisation Strategies Daniele Dipresa 1,* , Panagiotis Kalozoumis 1, Bettina Wiegmann 1, Christian Kϋhn 1, Axel Haverich 1, and Sotiris Korossis 1 1
MHH, Hannover Medical School *Presenting author
Extracorporeal membrane oxygenators (ECMO) have been used clinically for more than 40 years as a bridge to transplantation. Owing to their high gas transfer and low flow resistance, hollow fiber oxygenators have gained in popularity compared to conventional systems. In spite of the technological advances in ECMOs, the inevitable contact of the circulating blood with the artificial hollow-fibre gas-exchange membrane, and the subsequent thrombus formation, limits their usages to short-term clinical use (2-4 weeks). In addition, non-uniform flow can further limit gas-exchange efficiency and influence susceptibility to thrombus formation. Endothelialisation of the hollow-fibre gas-exchange membrane offers a potential solution to the thrombogenicity of these devices. However, abnormal shear stresses and inhomogeneous blood flow in the oxygenator could affect the function and activation status of the seeded endothelial cells (ECs). In this study, the blood flow through a commercially available (iLA Novalung) and a miniaturized oxygenator was modelled using computational fluid dynamics (CFD), with a view to assessing the shear stress magnitude/distribution and flow fields in the oxygenators. The study developed pseudo-homogeneous models, which considered the hollow-fibers as porous medium, and heterogeneous models taking into account the full hollow-fiber architecture, immersed in a viscous incompressible fluid (blood). Clinically-relevant blood boundary conditions were prescribed at the upstream and downstream of the oxygenator. The computational simulations predicted high inhomogeneity in the blood flow velocity and wall shear stress distribution along and around the hollow fibers and throughout both devices, as well as blood recirculation areas, implying a variable physical stimulation on the seeded ECs. The study highlighted the importance of optimizing oxygenator design prior to EC seeding. Future work will focus on parametric studies to optimize the geometry and operating conditions of the oxygenators, with a view to homogenizing the flow and wall shear stress distributions and optimize gas-transport across the oxygenators.
Bartlett RH. Extracorporeal life support: history and new directions. Semin. Perinatol. 2005;29:2-7. Bezzant TB, Mortenses JB. Risks and hazards of mechanical ventilation: a collective review of published literature. Disease-a-Month.1994; 40:581–640. Zhang J, Nolan TDC , Zhang T, Griffith BP, and Wu ZJ. Characterization of membrane blood oxygenation devices using computational fluid dynamics. J. Membr. Sci. 288:268– 279, 2007. Wiegmann B, von Seggern H, Höffler K, Korossis S, Dipresa D, Pflaum M, Schmeckebier S, Seume J, Haverich A. Developing a biohybrid lung - sufficient endothelialization of poly-4methly-1-pentene gas exchange hollow-fiber membranes. JMBBM. 2016; 60:301-11. Pelosi A, Sheriff J, Stevanella M, Fiore GB, Bluestein D, Redaelli A. Computational
DZL Annual Meeting 2017 evaluation of the thrombogenic potential of a hollow-fiber oxygenator with integrated heat exchanger during extracorporeal circulation. Biomech M. M. 2014; 13(2): 349–361. Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 131 Alternatively activated naive human T cells exhibit strong suppressive capacity in a humanized transplant arteriosclerosis model Linda Pauksch 1,* , Wiebke Sommer 1, Katharina Jansson 1, Ann-Kathrin Knöfel 1, Tomoyuki Nakagiri 1, Axel Haverich 1, and Gregor Warnecke 1 1
Hannover Medical School *Presenting author
Objectives: Transplant arteriosclerosis (TA) is one major limitation to allograft survival. It is characterized by a fibroproliferative neointimal thickening, which is driven by alloantigenreactive processes. Regulatory T cells (Tregs) are highly anti-inflammatory cells, able to reduce TA. But since the number of circulating Tregs in peripheral blood is limited, other suppressive cell types should be considered for anti-inflammatory treatment. The aim of this study is to examine the immunosuppressive potential of alternatively activated naïve T cells regarding TA using an allogenic humanized mouse model. Methods: PBMC were isolated from leukapheresis products of healthy donors. Cells were separated into CD4+CD25+CD127lo Tregs and CD4+CD25- naïve T cells by FACS and expanded in vitro by addition of CD3/CD28 antibody-coated beads and high amounts of recombinant IL2. After verification of phenotype and in vitro suppressive capacity, cells were used for in vivo application. To assess in vivo regulatory function of the expanded cells in a TA model, PBMC -/+ expanded T cells were injected into immunodeficient NOD.rag(−/−)γc(−/−) mice and blood samples were drawn on day 28 for immunophenotyping. Mice were sacrificed on day 28 and allogeneic artery grafts were harvested for histopathological examination. Results: Strong stimulation of naïve T cells lead to a >600 fold increase in cell number after 21 days. These cells displayed a phenotype similar to those of in vitro expanded Tregs, with a high expression of CD25 and FoxP3. Both cell types revealed a strong suppression capacity in vitro. In vivo application of the stimulated naïve T cells revealed reduction of luminal occlusion, intimal hyperplasia and cell infiltration. Conclusion: In vitro expansion of induced naïve T cells allows for the production of sufficient numbers of highly immunosuppressive T cells for in vivo applications. These polyspecific suppressor cells are functional in vivo and could enable cell therapy studies in clinical transplantation.
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Abstract No. 145 STEROID PULSE THERAPY FOR LOSS OF LUNG FUNCTION IN LUNG TRANSPLANT RECIPIENTS AFTER EXCLUSION OF ACUTE CELLULAR REJECTION Dieter Munker 1,* , Paola Arnold 1, Nikolaus Kneidinger 1, Felix Ceelen 1, Gabriela Leuschner 1 , Tobias Veit 1, Sandhya Matthes 1, Marion Frankenberger 1, Julien Dinkel 1, Simone Reu 1, Jürgen Behr 1, and Claus Neurohr 1 1
Campus Großhadern *Presenting author
The gold standard for the treatment of the acute cellular rejection after lung transplantation (LTx) is high dose steroids. Our analysis evaluates the value of high dose steroids in lung recipients after progressive loss of forced expiratory volume in one second (FEV1) after exclusion of acute cellular rejection. Included were 25 consecutive patients [15 male (60%), age: 52.6 ± 12.4] after LTx (84% with DLTX) with progressive decrease of FEV1 before steroid pulse therapy (SDH 500 mg for 3 days and 100 mg for 2 days). Patients underwent HR-CT, bronchoscopy, BAL and TBB. Patients with acute cellular rejection (A≥1 according to ISHLT) and other reasons for loss of FEV1 were excluded. FEV1 was measured 5 days, one month and 3 months after steroid pulse therapy. The mean decrease of FEV1 was 840ml ± 630 ml (-30.2 % ± 15.6 %) from the Best-FEV1. In comparison to the FEV1 directly before steroid pulse therapy the value increased after 5 days for +110 ml ± 170ml (p=0.02), after 30 days for +60 ml ± 280 ml (p=0.92) and decreased after 3 months for -60 ml ± 320 ml (p=0.61). After 3 months 20% (n=5) of the patients had a higher FEV1 (positive response to steroid pulse therapy was defined by an FEV1 more than 10% compared to the FEV1 before therapy). Our data suggest that a minority of patients after lung transplantation with progressive loss of FEV1 and after exclusion of an acute cellular rejection by TBB, could benefit of a steroid pulse therapy.
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Abstract No. 170 Generation of disease-specific iPSCs and development of transgenic reporter cell lines for cystic fibrosis disease modelling and drug screening Madline Schubert 1, Sylvia Merkert 1, Alexandra Haase 1, Lena Engels 1, Saskia Ulrich 1, Nico Lachmann 2, Jeanne de la Roche 3, Burkhard Tümmler 4, Luis Galietta 5, Ruth Olmer 1, and Ulrich Martin 1 1
Leibniz Research Laboratories for Biotechnology and Artificial Organs, MHH Institute of Experimental Hematology, MHH 3 Institute for Neurophysiology, MHH 4 Clinic for Paediatric Pneumology, Allergology and Neonatology, MHH 5 Istituto Giannina Gaslini, Genova, Italy 2
The genetic disorder Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for a cAMP-activated chloridechannel. So far, immortalized cell lines overexpressing mutant CFTR-variants have been used to screen compound libraries. In fact, CFTR-modulators have been identified, but show modest effects at best. Obviously, the complexity of the mutant CFTR-maturation and turnover kinetics including the influence of genetic modifiers require the use of advanced personalized cellular models that closely recapitulate the properties of the clinically most affected organs. To address these unmet needs we focus on the generation of induced pluripotent stem cell (iPSC) lines from CF-patients homozygous for F508del mutation. CFiPSCs were generated via reprogramming of CD34+ cells isolated from small volumes of non-mobilized peripheral blood. The resulting CF-iPSCs were analysed regarding their karyotype, pluripotency status and potential to differentiate. Moreover, different transgenic iPSC and embryonic stem cell (ESC) lines were generated overexpressing a halide sensitive yellow fluorescent protein (YFP) monitoring CFTR-function, in combination with the overexpression of an artificial CFTR or an endogenous CFTR-tomato-fluorescence-reporter. Several CF-iPSC lines were established and characterized in detail. The generated YFPreporter cell lines showed stable transgene expression also during in vitro differentiation and general functionality of the YFP-reporter could be confirmed. Directed differentiation of YFPexpressing CFTR-tomato-reporter iPSCs towards intestinal/biliary epithelium revealed YFP+/tomato+ cells, displaying CFTR-channel specific response after Forskolin application, which was inhibited after CFTR(inh)-172 treatment. Furthermore, CF-iPSCs with heterozygous correction of F508del mutation are able to recover CFTR function comparable to wild type CFTR. Hence, the stable integration of the halide reporter into CF-iPSCs in combination with integration of the CFTR-tomato-reporter should enable disease modelling of F508del-based CF with regard to the individual genetic context and the implementation of high-throughput screening for novel correctors and potentiators of CFTR-trafficking mutations.
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Abstract No. 178 Lung transplant under current therapy with pirfenidon or nintedanib: a case series Gabriela Leuschner 1,* , Florian Stocker 1, Tobias Veit 1, Nikolaus Kneidinger 1, Felix Ceelen 1 , Paola Arnold 1, Dieter Munker 1, Sandhya Matthes 1, Marion Frankenberger 1, Jürgen Behr 1 , and Claus Neurohr 1 1
Ludwig Maximilians University, Klinikum Grosshadern, Comprehensive Pneumology Center (CPC-M), Munich, Germany *Presenting author
BACKGROUND: Pirfenidone and nintedanib have been shown to reduces the loss of forced vital capacity in patients with idiopathic pulmonary fibrosis (IPF). Still it remains unclear if an antifibrotic treatment has an impact on post-operative outcome after lung transplant (LuTX). PATIENTS AND METHODS The study included all patients with IPF who received pirfenidone or nintedanib until undergoing LuTX at the university of Munich from January 2012 until October 2016. Pulmonary functions test, comorbidities, and post-transplant outcome were reviewed. RESULTS Our study population consisted of 27 patients with antifibrotic therapies, 21 (77.8%) with pirfenidone and 6 (22.2%) with nintedanib. 21 patients (77.8%) were male. The mean age at LuTX was 59.6±3.6 years and the mean time of pirfenidon and nintedanib treatment before LuTx was 603.6±427.1 days and 113.3±112.8 days respectively. Before LuTX the mean vital capacity was 48.7±13.4% pred., the total lung capacity was 52.6±12.0% pred., the diffusing capacity was 24.3±6.1% pred. and the 6-minute walking distance was 271.3±115.8m. In 14 patients (51.2%) single-LuTX was performed. The most common post-OP complications were the need for surgical revision (n=13; 48.1%) and bleeding anaemia requiring blood transfusion (n=5; 18.5%). In 13 patients (48.1%) an intra-operative ECMO was used and 6 (22.2%) patients needed a post-operative ECMO-therapy. Patients were extubated after a median time of 4 days (range 1-45 days). Within the first 30 days no patient died. In the first year after LuTX 5 patients died (1-year mortality 18.5%). During the study period 32 IPF-patients without an antifibrotic therapy underwent LuTX. There was no significant difference in lung function before LuTX, post-operative complications and survival compared to patients with antifibrotic therapy. However patients without antifibrotic therapy were significantly younger when receiving LuTX (p=0.033). CONCLUSION: Our data suggest that there is no increased post-operative risk in LuTX in patients with an existing antifibrotic therapy.
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Abstract No. 180 Stepwise Generation of CFTR-expressing Airway Epithelial Cells from Human Pluripotent Stem Cells Saskia Ulrich 1,* , Sandra Baus 1, Sylvia Merkert 1, Lena Engels 1, Ruth Olmer 1, and Ulrich Martin 1 1
MHH *Presenting author
Pluripotent stem cells (PSCs), like embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer promising new options for the treatment of lung diseases like cystic fibrosis (CF) by cellular/tissue replacement therapies, disease modelling and drug screening. These approaches crucially rely on the efficient differentiation of PSCs into respective lung epithelial cells, which is the aim of our present study. We made use of the human ESC reporter line hES3_NKX2.1-eGFP (kindly provided by A. Elefanty) expressing eGFP under the endogenous NKX2.1 promoter. Furthermore, we took advantage of a lab-internal generated double transgenic cell line based on the hES3_NKX2.1-eGFP cells additionally expressing dTomato controlled by the endogenous CFTR promotor. Using the commercially available STEMdiff™ Definitive Endoderm (DE) Kit by STEMCELL Technologies resulted in the robust and efficient generation of a highly enriched DE population of ~ 98 % CXCR4pos/c-Kitpos cells and CXCR4pos/EpCAMpos cells demonstrated by flow cytometric analysis, additionally verified by co-expression of the transcription factors FOXA2 and SOX17 based on immunofluorescence staining. Further differentiation resulted in a distinct FOXA2pos/SOX2pos anterior foregut endoderm (AFE) population of ~ 76 %, which gave rise to ~ 55 % NKX2.1-eGFPpos cells. Subsequent maturation of purified NKX2.1-eGFPpos cells under air-liquid-interface conditions demonstrated the formation of ~ 30 % CFTR-dTomatopos airway epithelial cells partly coexpressing the club cell markers SP-B and CCSP. In summary, we were able to stepwise generate almost pure DE followed by the induction of a distinct AFE population resulting in a decent percentage of NKX2.1-eGFPpos cells. Further maturation demonstrated the generation of CFTR-dTomatopos airway epithelial cells, most likely representing (partly) club cells. Future work will focus on the adaption of the established differentiation strategy to in-house generated CF-iPSCs to provide a suitable cell source for high throughput screening approaches for the identification of new therapeutic drugs to treat CF.
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Abstract No. 186 Investigation of the Effect of Oxidative Stress on the behaviour of ECs Seeded onto ECMO Membranes Kanchan Chauhan 1, Michael Pflaum 1, Sabrina Schmeckebier 1, Daniele Dipresa 1, Christian Kϋhn 1, Bettina Wiegmann 1, Axel Haverich 1, and Sotirios Korossis 1,* 1
Hannover Medical School *Presenting author
Endothelialisation of the blood-contacting surfaces of extracorporeal membrane oxygenators (ECMO) is considered to be the most effective strategy to provide complete haemocompatibility. Although the successful generation of a confluent and functioning endothelial cell (EC) monolayer on gas exchange membranes under standard culture conditions has been reported previously, there have been no studies addressing the function of ECs under ECMO operating conditions (21-100% pO2). Prior studies have reported that mechanical-ventilation-induced hyperoxia, led to intracellular accumulation of reactive oxygen species (ROS) and subsequent detrimental effects on cell viability and function in the absence of effcient intracellular ROS scavenging mechanisms. Moreover, it has been reported that ECs from different vascular beds differ in oxidative stress tolerance. This study focused on investigating the behaviour of ECs under ECMO operating conditions and, in particular, under exposure to a hyperoxic environment. With a view to analysing intracellular ROS accumulation, ECs from different sources (cordblood-derived HCBECs from 3 donors, and hTERT-immortalized pulmonary capillary ECs ST1.6R) were seeded on gas exchange membranes (PMP films) and exposed to a hyperoxic environment (85% O2, 5% CO2). Flow cytometry analysis, carried out directly after 12h or 24h of hyperoxia showed that ROS levels were not significantly elevated in either cell type. However, expression analysis of a panel of genes, that have been reported to be regulated upon ROS-mediated stress response, revealed cell-source-dependent differences. Specifically, the ST1.6R ECs demonstrated no detectable change in the expression of GCLM, KLF2, SOD1, SOD2, NQO1 or TXN10 after 24 h in hyperoxic condition. On the other hand, HCBECs showed a 6-fold increase in the expression of the GCLM, which has been shown to be involved in the gluthatione-mediated ROS scavenging mechanism. The results indicated that HCBECs were more sensitive to hyperoxia than ST1.6R ECs, and up-regulated ROS-associated genes earlier to prevent oxidative damage.
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Abstract No. 187 ORTHOTOPIC LUNG TRANSPLANTATION IN A SINGLE-MISMATCH-BASED MOUSE MODEL SHOWS SIGNS OF CHRONIC LUNG ALLOGRAFT DYSFUNCTION ASSOCIATED TO iBALT FORMATION Natalia Smirnova 1,* , Thomas Conlon 1, Carmela Morrone 1, Önder Yildirim 1, and Oliver Eickelberg 2 1
Helmholtz Zentrum München University of Colorado Denver *Presenting author 2
While murine models of acute or progressive allograft rejection, based on full donor/recipient mismatch, have been described, their routine use is limited because of a lack of human-like features of bronchiolar obliteration and inducible Bronchus Associated Lymphoid Tissue (iBALT) formation. We here sought to establish a mouse model of chronic lung allograft rejection based on indirect allorecognition of donor tissue. Left lungs from C57BL/6J (syngeneic group) or mice on a C57BL/6J background carrying a single human HLA-A2.1 molecule (allogeneic group) were orthotopically transplanted into C57BL/6J recipients and analyzed two months after orthotopic single lung transplantation by morphometry, immunostaining and flow cytometry. Lung grafts obtained from the syngeneic group appeared normal, as assessed by dark-field imaging and histological analysis. In contrast, the allogeneic group presented significantly less scattering in dark-field imaging, strongly suggesting a partial loss of graft lung function. Histological analysis demonstrated injury and altered cellular composition of the bronchiolar epithelium. The subepithelial peribronchiolar compartment was infiltrated by inflammatory cells and subepithelial fibrosis was evident in allogeneic transplant recipients. Overall, two months after LTx, the allogeneic lung grafts exhibited signs of lymphocytic bronchiolitis. Immunofluorescence, in the allografts, but not the syngeneic grafts, showed peribronchial and perivascular joint T and B cells accumulation, strongly suggesting iBALT formation. Furthermore, these structures were GL7+, revealing intragraft germinal centers. The CD138+ plasma cells, surrounding the follicles suggested that these iBALT structures are functional and capable of generating specific antibodies inside the allografts. Thus, a single indirect allorecognition-triggering mismatch provokes a chronic rejection of the graft. Epithelial changes resemble those of CLAD patients and are likely to represent early processes inducing long-term bronchiolar obliteration. We describe for the first time iBALT formation in chronic lung allograft rejection in the mouse, therefore providing a suitable model to evaluate new treatments targeting iBALT formation of progression.
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Abstract No. 205 Efficient generation of expandable and genetically stable endothelial cells from human pluripotent stem cells in scalable suspension culture Ruth Olmer 1,* , Lena Engels 1, Mónika Szepes 2, Sandra Menke 1, Michael Pflaum 3, Sabrina Schmeckebier 3, Sotirios Korossis 3, Ina Gruh 2, and Ulrich Martin 1 1
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH) and Member of the German Center for Lung Research (DZL) 2 Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), REBIRTH - Center for Regenerative Medicine, Hannover Medical School, Hannover 3 NIFE - Lower Saxony Centre for Biomedical Engineering, Implant Research and Development Hannover Medical School, Hannover *Presenting author
Applications like full endothelialisation of gas exchange membranes in extracorporal membrane oxygenation (ECMO) devices for improved hematocompatibility, cell therapy or tissue engineering require large numbers of (patient-specific) endothelial cells (ECs). Isolation of ECs from peripheral blood or explanted vessels is well established however especially cells from older individuals show a limited proliferation capacity and a high probability for karyotypic abnormalities. ECs from pluripotent stem cells (hiPSCs) might be an alternative cell source. The opportunity to generate large amounts of undifferentiated hiPSC in defined media under scalable conditions allows for the generation of cell numbers in dimensions which are suitable for envisioned applications. By differentiation of these well monitored cell populations a virtually unlimited number of (autologous) ECs may become available for disease modelling, tissue engineering approaches and biofunctionalization of ECMO devices. The growth factors BMP4 and VEGFA as well as modulation of the WNT pathway were utilized for the differentiation of the scalable suspension cultures in agitated Erlenmeyer flasks to endothelial cell types. Resulting cell populations were analyzed by flow cytometry and quantitative RT PCR for expression of endothelial cell markers. Differentiation approaches resulted in up to 75% of CD31 positive cells. Expression of endothelial cell markers like, VEGFR2 (FLK1), von Willebrand Factor (vWF) and VEcadherin (CD144) could be shown by qRT-PCR. Sorting of CD31pos populations resulted in almost pure, expandable hiPS-EC. Phenotypic analysis showed network formation on matrigel as well as uptake of Dil-LDL. Furthermore no karyotypic abnormalities could be detected until passage 8. We were able to generate ECs from scalable cultures, which showed a stable phenotype and no karyotypic abnormalities in culture. In the future the resulting patient- specific iPSCderived ECs will represent a novel cell source for disease modelling or biofunctionalization of gas exchange membranes as well as for vascularization of tissue engineered constructs.
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Abstract No. 215 Hypothermic preservation of endothelialised surfaces Hayan Merhej 1, Michael Pflaum 1,* , Erik Beckmann 1, Sabrina Schmeckebier 1, Daniele Dipresa 1, Kanchan Chauhan 1, Bettina Wiegmann 1, Wim Wolkers 2, Sotirios Korossis 1, and Axel Haverich 1 1
Hannover Medical School Leibniz University Hannover *Presenting author 2
Endothelialization of blood contacting surfaces has been proposed as a strategy to increase the haemocompatibility of cardiovascular devices, such as biohybrid lung or vascular prostheses. However, the isolation and the expansion of autologous endothelial cells (EC) usually require multiple processing steps and time for cell proliferation in order to gain sufficient cell numbers, which excludes this strategy from application in acute situations. For an instant “off-the-shelf” availability, cardiovascular devices, pre-seeded with nonimmunogenic allogeneic ECs, potentially could be stored under hypothermic conditions until required. In this study, the metabolic activity, of human cord blood ECs was measured by calcein fluorescence intensity, and the integrity of the derived EC monolayers was compared after storage for up to two weeks in different solutions, which have been commonly used for the transportation of cells, whole organs or tissue grafts. While storage at 4°C in normal growth medium (EGM-2™) for 1 hour resulted in a massive disruption of the EC monolayer and a significant decline in metabolic activity 3 hours after re-warming to 37°C, EC monolayers could be stored for up to 14 days in Chillprotec™ or Chillprotec+™, where the integrity of the monolayer was much less affected. Furthermore, the metabolic activity of EC monolayers that were hypothermically stored in these media and rewarmed for 3 hours to 37°C was increased at day 14 compared to the control (confluent monolayer in EGM-2 before hypothermic storage). The re-warmed EC monolayers that were stored in these media were still viable after additional 24 hours under standard culture conditions, recovering full confluency. EC monolayers preserved in Celsior™ or Tiprotec™ medium showed a significant decrease in metabolic activity at day 12. The study demonstrated the feasibility to store EC monolayers at 4°C for up to two weeks, indicating the possibility to apply pre- endothelialized cardiovascular devices on demand.
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Abstract No. 216 Generation of a NKX2.1/p63 knockin human induced pluripotent stem cell reporter line for monitoring the generation of respiratory cells Sandra Baus 1,* , Saskia Ulrich 1, Sylvia Merkert 1, Ruth Olmer 1, and Ulrich Martin 1 1
Medical School Hannover *Presenting author
One promising option to cure hereditary pulmonary diseases like cystic fibrosis might be a cell replacement therapy comprising the generation of patient specific autologous induced pluripotent stem cells (iPSCs), followed by the correction of the underlying genetic mutation, in vitro differentiation into the needed airway epithelial cell type and replacement of the endogenous diseased cells. For long term restoration, most likely lung stem cells like basal cells will be required. A requirement of this strategy is the development of an efficient and robust protocol for the generation of basal cells from human iPSCs (hiPSCs). The transcription factor NK2 homeobox1 (NKX2.1) expressed by lung epithelial progenitor cells represents an appropriate marker for optimizing differentiation protocols towards lung epithelial progenitor cells. Combination with the tumor protein 63 (p63) should allow for monitoring of basal cell generation in sequential differentiation protocols. The aim of the present study was the generation of a hiPSC double transgenic reporter line targeting the NKX2.1 and p63 locus. Therefore we designed one targeting vector for a non-disruptive integration of an eGFP coding sequence into the NKX2.1 locus and one targeting vector for the disruptive integration of nuclear localized Venus coding sequence into the p63 locus. Furthermore, the p63 targeting vector introduces a Neomycin selection cassette under control of the endogenous p63 promoter by the use of a 2A-site located behind the Venus coding sequence. This established hiPSC-NKX2.1/p63 reporter line represents an optimal tool for the improvement of protocols for the differentiation of hiPSCs into basal cells and enables their selection which is indispensable for further in vitro and in vivo analysis.
Submitted as: Presentation 263 words of 300 possible words
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Abstract No. 228 Reduced Preoperative Treg levels Remain Suppressed Following Lung Transplantation and Represent a Risk factor for Increased Acute Rejection Rates Nicole Strobl 1,* , Hauke Winter 1, Gregor Warnecke 2, Elfriede Nößner 3, Ann-Kathrin Knöffel 2 , Rene Schramm 1, Rudolf Hatz 1, Axel Haverich 2, and Gerhard Preissler 1 1
Ludwig-Maximilians-Universität München Medizinische Hochschule Hannover 3 Helmholtz-Zentrum München *Presenting author 2
Introduction: Regulatory T cells (Treg) are key modulators of the immune response in transplantation. Nevertheless, it has not been evaluated so far, if their pre-transplant levels correlate with the incidence of rejection episodes following lung transplantation (LTX). Therefore, Treg immunomonitoring was performed in lung transplant recipients and correlated with the clinical course. Methods: In total, 76 (m/f: 39/37, age: 52 ± 2y, LAS: 51 ± 3) patients undergoing single (n=24) or bilateral (n=52) LTX were included in the study. Blood samples were taken prior to LTX (day 0) and 7, 14, 21, 90, 180 and 365 days thereafter. Following FACS-analysis of Tregs (CD3+/CD4+/CD25+/FoxP3+) at day 0, 19 patients (age: 45 ± 3y, LAS: 59 ± 5) could be assigned to a low-Treg group (≤ 1.75% of CD4+) and 19 recipients (age: 55 ± 2y, LAS: 48 ± 5) to a high-Treg group (≥ 5.15% of CD4+). Patients treated with steroids due to a biopsy proven (A≥1/ B≥1) and/or clinical rejection episode (FEV1-loss), were defined positive for acute rejection (AR). Data are given as mean ± SEM. Results: Leukocyte count, CRP and trough-levels of FK506 were not significantly different between the groups. Treg levels of the two groups differed significantly from day 0 (1.2 ± 0.1 vs. 7.4 ± 0.5) until day 180 (2.6 ± 0.5 vs. 5.6 ± 0.5) and maintained the same trend up to 365 days after LTX. Interestingly, patients with a constantly low Treg frequency also showed continuously reduced CD39+ mTreg-levels (by 24 - 44%). Furthermore, in the low-Treg group an increased amount of AR (26.32% vs. 5.26%) was noted. Conclusion: Reduced Tregs prior to LTX maintain suppressed at least as long as one year after LTX. This is associated with a significantly increased risk for the incidence of AR episodes.
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Abstract No. 289 Reliability and validity of the Transplant Evaluation Rating Scale (TERS) in lung transplant candidates Sarah Weusthoff 1, Johannes Beneke 1, Igor Tudorache 1, Jens Gottlieb 1, and Martina de Zwaan 1,* 1
MHH *Presenting author
Background: Lung transplantations are the third largest group in solid organ transplantation in Germany. Evaluation of psychosocial functioning prior to transplantation can help to anticipate behavioral difficulties and to screen for psychological distress that might be harmful for post-transplantation outcomes and adjustment. Objective: We used the Transplant Evaluation Rating Scale (TERS) which is a widely used structured expert interview for the psychosocial evaluation prior to transplantation. The TERS has not been differentially investigated with respect to its psychometric properties in candidates for lung transplantation. Method: In a cross-sectional single-center study, the level of psychosocial functioning of N = 75 patients before lung transplantation was rated during a routine clinical interview using the TERS. Internal consistency and factor structure of the scale were investigated. In addition, concurrent validity was as-sessed by investigating the relationship between the TERS score and self-report questionnaires on psychological distress, disease severity, and health behaviors. Results: Results support the factorial structure, reliability and concurrent construct validity of the TERS in lung transplantation candidates. Lower levels of psychosocial functioning were significantly associated with higher levels of depressive and anxiety symptoms, lower quality of life, and more dysfunctional health behaviors. No associations were found with lung disease and symptom severity. Patients with severe impairments of psychosocial functioning were less likely to get listed for transplantation. Conclusion: The TERS appears to be a reliable and valid measure also in lung transplant candidates. However, associations between TERS ratings prior to lung transplantation and relevant longitudinal outcomes post-transplantation (e.g., survival, number and duration of hospitalization episodes) need to be investigated in future studies to increase the understanding of psychosocial functioning for patients’ well-being in more detail.
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Abstract No. 001 Mutation analysis of circulating DNA to monitor non-small cell lung cancer Steffen Dietz 1, Anja Lisa Riediger 1, Uwe Schirmer 1, Clémentine Mercé 1, Nikolas von Bubnoff 2, Edgar Dahl 3, Arne Warth 4, Michael Meister 5, Thomas Muley 5, Michael Thomas 5 , and Holger Sültmann 1,* 1
German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT) University Medical Center Freiburg 3 RWTH Aachen University 4 Heidelberg University Hospital 5 Thoraxklinik at University Hospital Heidelberg *Presenting author 2
Monitoring of tyrosine kinase inhibitor (TKI) therapy course in NSCLC patients is crucial since the tumors acquire TKI resistance. Here, we show the results of a continuous monitoring of tumors over time based on the level of mutant circulating free (cf) DNA. To this end, serial plasma samples (n=107) from 16 NSCLC patients, treated with either gefitinib or erlotinib, were used to quantify EGFR and KRAS mutations in cfDNA by digital PCR. Variations in mutation levels over time were found in almost all patients, with the earliest sign for response 26 hours after therapy onset. Low levels of mutant DNA were concurring with stable disease. Progressive disease and occurrence of resistance were detected up to several months prior to radiological progression. Vast numbers of mutations in cfDNA were associated with poor prognosis and short overall survival. Our data indicate that tumor progression can be monitored by digital PCR and might inform the demand for a therapy switch earlier than currently possible. Furthermore, to detect mutations besides the prominent hotspots and to investigate the molecular representation of the tumor exome in cfDNA, we performed whole-exome sequencing (WES) of serum and matched tumor tissue samples from six NSCLC patients and two control sera. We identified cancer-associated mutations and variants at COSMIC annotated sites in all six patients. Our results provide evidence for cfDNA to inform about the molecular constitution of the disease in the six advanced cancer patients with up to 57% of the tumor variants represented in the matched serum samples. Moreover, we detected additional mutations of clinical relevance in cfDNA, which were not found in the primary tumors. In summary, we show that WES of cfDNA informs about the primary tumors’ molecular alterations and can provide complementary information about mutational patterns eventually deriving from in distant tumor clones.
Submitted as: Presentation 298 words of 300 possible words
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Abstract No. 002 p38 MAPK builds a hyaluronan niche to drive lung tumorigenesis Anna Brichkina 1,* , Thomas Bertero 2, Hui Mun Loh 3, Thi Minh Nguyet Nguyen 3, Alexander Emelyanov 4, Marius Ilie 4, Paul Hofman 4, Cedric Gaggioli 2, and Dmitry Bulavin 4 1
Institute of Molecular Oncology, Center for Tumor- and Immunobiology, Philipps University, Marburg, Germany; Institute of Molecular and Cell Biology, A*STAR, Singapore 2 Institute for Research on Cancer and Aging of Nice (IRCAN); Nice, France 3 Institute of Molecular and Cell Biology, A*STAR, Singapore 4 Institute for Research on Cancer and Aging of Nice (IRCAN); Centre Antoine Lacassagne; Nice, France *Presenting author
Lung cancer has been associated with the highest rate of cancer-related deaths worldwide. Although major advances have been made in understanding this disease, including the identification of novel potential therapeutic targets, overall survival has not significantly changed over the past several decades. It is becoming clear that the most effective approach for treating lung cancer requires therapies that target both tumor cells and the tumor microenvironment. In this respect, stromal fibroblasts could be attractive targets, because they are known to provide additional signals to support tumor growth, survival, and drug resistance. Unfortunately, no treatment option exists that successfully targets this component of the tumor microenvironment. We identify p38 MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. Systemic downregulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in stromal fibroblasts has a significant tumor suppressive effect on K-ras lung tumorigenesis. We identify fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to Carcinoma-Associated Fibroblasts (CAF), and cancer cell proliferation. Furthermore, both K-ras-driven mouse lung tumors and orthotopically-grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-thecounter inhibitor of hyaluronan synthesis. We propose that the hyaluronan cancer niche plays a major role in driving lung tumorigenesis and that chemical inhibition of p38 or hyaluronan synthesis could provide an important improvement for lung cancer treatment.
Submitted as: Presentation 254 words of 300 possible words
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Abstract No. 004 Prevalence of Somatic Mitochondrial Mutations and Spatial Distribution of Mitochondria in Non-Small Cell Lung Cancer Daniel Kazdal 1,* , Alexander Harms 1, Volker Endris 1, Roland Penzel 1, Mark Kriegsmann 1, Hendrik Dienemann 2, Thomas Muley 3, Albrecht Stenzinger 1, Nicole Pfarr 4, Wilko Weichert 4 , and Arne Warrth 1 1
Institute of Pathology, Heidelberg University Thoraxklinik at Heidelberg University, Department of Thoracic Surgery 3 Thoraxklinik at Heidelberg University, Translational Research Unit 4 Institute of Pathology, Technical University Munich *Presenting author 2
Introduction Targeted therapies of non-small cell lung cancer (NSCLC) based on tissue-derived biomarkers propelled the concept of lung cancer specific therapies and undoubtedly improved the patients´ survival. However, a better understanding of the complex interplay between tumor cells, stroma cells, immune cells, and components of the extracellular matrix seems to be of utmost importance for further improvements. In this regard mitochondria are considered as relevant players in many tumor entities and first data point towards beneficial effects of mitochondria-targeted antioxidants in both cancer prevention and anticancer therapies. Methods In order to further dissect the potential roles of mitochondria in NSCLC we comprehensively analyzed the following aspects and correlated the findings with clinicopathological characteristics: • Detection and evaluation of somatic mtDNA mutations in 26 ADC (whole mitochondrial genome panel sequencing) extended with a literature review (combined 352 lung tumors) • Systematic quantification of the spatial distribution of mtDNA in central sections of 16 ADC (5 mm stepwise segmentation of the central sections, qPCR). • Determination of the mitochondrial load on protein level in 171 ADC & 145 SQCC discriminating between tumor and tumor stroma (IHC, digital pathology) Results & Discussion We demonstrate that somatic mitochondrial mutations in NSCLC seem to have the character of passenger mutations rather than to be malignant. Large inter- and intra-tumoral heterogeneity was seen for mtDNA copy numbers in conjunction with a correlation to the predominant histological growth pattern, indicating a correlation to the tumor progression. However this correlation was not seen on protein level. Here the mitochondrial load in tumor cells was significantly higher compared to adjacent tumor stroma. In conclusion, the accumulation of mtDNA mutations seems to be negligible in NSCLC carcinogenesis, but the amount of mitochondria could play an essential role for the challenging approach of a mitochondria targeted treatment in this entity.
DZL Annual Meeting 2017 Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 005 Widespread epigenetic activation of ΔNp73 in small cell lung cancer causes vulnerability to Tip60-p400 inhibition Andrea Nist 1,* , Anna-Maria Krampitz 1, Katharina Schlereth 1, Marco Mernberger 1, MarieChristin Moßner 1, Thomas Muley 2, Reinhard Dammann 3, and Thorsten Stiewe 1 1
Philipps-University Marburg University Hospital Heidelberg 3 Justus-Liebig University Giessen *Presenting author 2
Lung cancer is the leading cause of cancer-associated mortality world-wide with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) being the most common subtypes. Although newly diagnosed SCLC is exquisitely sensitive to first-line chemotherapy, the disease eventually progresses rapidly in virtually all patients resulting in a 5-year survival of less than 10%. There is therefore an urgent need to develop novel therapeutic strategies. Unfortunately, SCLC are genetically driven by inactivating mutations in the tumor suppressor genes p53 and RB1 and not by druggable oncogenic drivers. Using targeted genome sequencing of SCLC patients we identified a few cases with structural rearrangements in the p73 transcription factor gene that delete the exons encoding the N-terminal p73 transactivation domain. More frequently, in approximately half of all SCLC cell lines and primary tumor samples, we observed loss of DNA methylation in an intronic CpG island of the p73 gene, which thereby gains promoter activity and drives expression of N-terminally truncated ΔNp73 proteins missing the transactivation domain. In both cases the resulting transactivation-deficient ΔNp73 proteins sequester and inactivate any tumor suppressive full-length p73 proteins. Given that ΔNp73, in contrast to transactivating full-length p73, represses transcription we analyzed the underlying mechanism by RNAi screening. We identified multiple components of the chromatin-modifying Tip60-p400 complex as required for transcriptional repression by ΔNp73. In a parallel RNAi-based viability screen, we found the Tip60-p400 complex to be also essential for survival of SCLC cells - in particular those with elevated ΔNp73 expression. Together these findings indicate that SCLC cells, due to either genetic or epigenetic alterations in the p73 gene, become dependent on ΔNp73-mediated gene repression via the Tip60-p400 complex which confers a vulnerability that could be exploited for cancer therapy.
Submitted as: Presentation 284 words of 300 possible words
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Abstract No. 016 Regulation of Glycodelin Expression – an Immunomodulatory and Pregnancy associated Protein in NSCLC Rebecca Weber 1,* , Marc A. Schneider 1, Thomas Muley 1, Michael Thomas 1, Holger Sültmann 2, and Michael Meister 1 1
Thoraxklinik at Heidelberg University Hospital German Cancer Research Center (DKFZ) *Presenting author
2
Background Glycodelin (gene name: progesterone-associated endometrial protein, PAEP) is a protein initially described as an immune system modulator during the establishment of pregnancy. Former studies determined an atypical expression of glycodelin in non-small cell lung cancer (NSCLC), the most common type of lung cancer. To date, there is not much known about the signaling pathway which regulates PAEP/glycodelin expression in cancer. Therefore, in this study we analyzed possible regulatory candidates to get insights into the signaling pathway of PAEP/glycodelin in NSCLC. Methods A lung adenocarcinoma (H1975) and a lung squamous cell carcinoma cell line (2106T) were transfected with siRNA targeting nuclear factor κB1 (NFκB1). Furthermore, they were treated with human chorionic gonadotropin (hCG), lysophosphatidic acid (LPA), phorbol 12myristate 13-acetate (PMA), epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), transforming growth factor-β (TGF-β), protein kinase C (PKC) activator bryostatin 1 and PKC inhibitor GF109203x, respectively. Combined treatments with GF109203x and EGF, HB-EGF or TGF-β were performed additionally. PAEP expression in the manipulated cells was determined by quantitative polymerase chain reaction, while glycodelin was detected by western blot analysis. Results NFκB1 siRNA transfection resulted in decreased PAEP and glycodelin amounts, whereas hCG, LPA (only in 2106T), PMA, EGF (only in 2106T), HB-EGF (only in 2106T) and TGF-β (only in 2106T) treatment led to higher levels. In bryostatin treated cells, PAEP/glycodelin expression was upregulated. The contradictory effect could be demonstrated for cells treated with the PKC inhibitor GF109203x alone and in combination with EGF, HB-EGF or TGF-β. Conclusion This study revealed that there are different regulatory mechanisms of PAEP/glycodelin induction in NSCLC. Especially, PKC seems to be involved as a key molecule. Elucidating the regulatory pathway of the immune system modulating protein glycodelin might reveal a potential strategy to weaken the immune system defense of lung tumors.
Schneider MA, Granzow M, Warth A, Schnabel PA, Thomas M, Herth FJ, Dienemann H, Muley T, Meister M. Glycodelin: A New Biomarker with Immunomodulatory Functions in Non-Small Cell Lung Cancer. Clin Cancer Res. 2015 Aug 1;21(15):3529-40
DZL Annual Meeting 2017 Schneider MA, Kahn, NC, Thomas M, Herth FJF, Muley T, Heussel, CP, et al. The Pregnancy Associated Protein Glycodelin as a follow-up Biomarker in a Male Non-Small Cell Lung Cancer Patient. Cancer Treatment Communications. 10.1016/j.ctrc.2015.09.005 Submitted as: Presentation 297 words of 300 possible words
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Abstract No. 023 The influence of EGF/HGF receptor stoichiometry on therapy resistance in NSCLC cell lines Florian Salopiata 1,* , Helge Hass 2, Sigfried Hänselmann 3, Yu Qiang 4, Marc Schneider 5, Michael Meister 5, Karl Rohr 4, Dirk-Peter Herten 3, Jens Timmer 2, and Ursula Klingmüller 1 1
Division of Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Centre for Systems Biology, University of Freiburg, Freiburg, Germany 3 Cellnetworks Cluster and Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany 4 Biomedical Computer Vision Group, Dept. Bioinformatics and Functional Genomics, University of Heidelberg, BIOQUANT, IPMB 5 Translational Research Unit, Thoraxklinik at University Hospital Heidelberg, Heidelberg, Germany. *Presenting author
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Unfortunately, the currently available targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment lead to only a few months increased survival due to the development of therapy resistance. It is suspected that this resistance can be mediated by the crosstalk of EGF and hepatocyte growth factor (HGF) induced signal transduction. Yet, the underlying mechanisms remain unknown. In this study time resolved quantitative data for EGF and HGF induced signal transduction was acquired in NSCLC cell lines. Three NSCLC cell lines were examined that harbor different mutations in the EGFR and differ in their expression levels of EGFR and HGF receptor (MET). The experiments showed a directed synergistic effect of EGF and HGF on MET phosphorylation leading to sustained signal transduction. This effect was shown to be cell line specific. Especially two cell lines showed a strong cooperativity in Met receptor phosphorylation upon co-stimulation with EGF and HGF that is absent in the other cell line. To gain insights into mechanisms regulating the interaction of the receptors, the data was used to develop a dynamic pathway model. The mathematical model predicted that the formation of EGFR and MET heterodimers is stoichiometry dependent and that heterodimers have a prolonged half-life leading to the observed cooperativity. Combining the data of the cell lines, our model suggested an important role of the receptor stoichiometry regarding the formation of signaling complexes and downstream signal activation. This was validated by artificially changing the receptor stoichiometry using retroviral transduction, siRNA and by screening nine other NSCLC cell lines with altered EGFR and MET expression ratios. With this strategy we propose a novel approach to develop strategies to avoid the emergence of therapy resistance depending on the relative receptor abundance.
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Abstract No. 033 Chemoresistant NSCLC cells are hypersensitive to metabolic drugs due to mTORmediated inhibition of autophagy Michael Wanzel 1,* , Niklas Gremke 1, Lea Schmoll 1, and Thorsten Stiewe 1 1
Center for Tumor Biology and Immunology (ZTI) *Presenting author
In non-small cell lung cancer (NSCLC) cisplatin is the first line chemotherapy and induc-es a strong apoptotic response mainly by inducing DNA interstrand crosslinks. However, the effectiveness of cytotoxic cancer therapy is often limited by the emergence of drug resistant tumor cells. To identify strategies for overcoming resistance to cisplatin, we treated the NSCLC cell line H460 continuously with DNA crosslinking agents resulting in resistant subclones that displayed cross-resistance to multiple DNA-damaging cancer drugs, which are commonly used in the clinic. Drug resistance was found to be mediated by mTORdependent upregulation of DNA repair via the Fanconi anemia pathway*. Strikingly, despite being multidrug-resistant, resistant cells were highly sensitive to treatment with the metabolic drugs 2-deoxyglucose (2-DG) and dichloroacetate (DCA), whereas parental H460 cells failed to respond. Moreover, xenograft studies in mice re-vealed, that DCA preferentially reduces growth of resistant tumor cells. Interestingly, mTOR inhibition by RNAi or pharmacological inhibitors not only reversed resistance to crosslinking drugs but in parallel mitigated the apoptotic response to metabolic drugs, thereby linking sensitivity to metabolic drugs with mTOR signaling in chemoresistant cells. Mechanistically, inhibition of autophagy by mTOR was found to be decisive. We observed elevated levels of the mTORinduced inhibitory S757 phosphorylation of the central autophagy-initiating kinase Ulk1 in chemoresistant tumor cells. Matching these observations, autophagosome formation was detected only in parental, but was abro-gated in resistant tumor cells upon DCA treatment. In line, direct inhibition of different steps of autophagy with pharmacological autophagy inhibitors or RNAi was sufficient to render parental cells sensitive to metabolic drugs. Failure to initiate autophagy resulted in lethal energy stress indicating that autophagy is absolutely required to survive meta-bolic drug treatment. Together these results demonstrate that mTOR-triggered re-sistance to DNA-damaging cancer drugs generates a therapeutic vulnerability to meta-bolic drugs due to mTOR-mediated inhibition of autophagy.
*Wanzel et al., Nat Chem Biol 2016, 12(1): 22-28. Submitted as: Presentation 298 words of 300 possible words
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Abstract No. 034 Contribution of CD4+ T cell subpopulations to lung carcinogenesis Ylia Salazar 1,* , Magdalena Huber 2, Anja Schmall 3, Werner Seeger 3, Friedrich Grimminger 4 , Soni Savai Pullamsetti 3, and Rajkumar Savai 3 1
Max-Planck-Institut für Herz- und Lungenforschung Philips Universität Marburg 3 Max-Planck-Institut für Herz- und Lungenforschung 4 Justus-Liebig-Universität Gießen *Presenting author 2
Tumor microenvironment and its immune components play a critical role in cancer development, progression, and control. In this study we aim to investigate the role of tumor infiltrating lymphocyte subpopulations in lung cancer progression. We demonstrate that conditioned media (CM) from co-cultures of lymphocytes with adenocarcinoma cells (A549) induces epithelial to mesenchymal transition (EMT) of cancer cells. CM from co-cultures of human-lymphocytes with adenocarcinoma cells induced the loss of the epithelial marker E-Cadherin and an increase of Vimentin and N-cadherin on mRNA and protein level. A549 cells also showed acquired spindled shape-like morphological changes and an increased migratory phenotype as revealed by woundhealing assay. Increased levels of cytokines such as, IL-8, IL-16, CCL2 and G-CSF were detected in the co-culture CM. Notably, lymphocyte-induced EMT was mediated via a TGFβ-independent pathway that involves phosphorylation of ERK1/2. To identify the specific T cell subpopulation responsible for the EMT effect observed in human data, Th subpopulations (Th0, Th1, Th9 and Th17) were generated. CM from each of these subpopulations was collected and its functional effects were assessed on tumor cells. We observed an increase in migration and mesenchymal marker expression in cancer cells stimulated only with Th9-CM and Th17-CM. Furthermore, the stimulation of A549 cells with IL9 itself resulted in EMT and increased migration. Interestingly, co-injecting tumor cells with Th subpopulations (Th9 and Th17) in mouse lung tumor xenograft/orthotropic models resulted in an increased lung tumor growth and metastasis. Additionally, tumor homogenates showed decreased mesenchymal markers and an increase in epithelial and angiogenesis markers. This study reveals that specific T lymphocyte subpopulations are able to induce EMT of lung cancer cells, accompanied with a more migratory phenotype.
Submitted as: Presentation 274 words of 300 possible words
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Abstract No. 035 Prognostic impact of CT-quantified muscle and fat distribution before and after firstline-chemotherapy in lung cancer patients. Johanna Nattenmueller 1, Raoul Wochner 1, Thomas Muley 2, Nadja Batora 1,* , Martin Steins , Simone Hummler 2, Birgit Teucher 2, Joachim Wiskemann 3, Hans-Ulrich Kauczor 1, Mark Wielpütz 1, and Claus Peter Heussel 4
2
1
Diagnostische und Interventionselle Radiologie Thoraxklinik 3 NCT 4 Diagnostische und Interventionselle Radiologie mit Nuklearmedizin *Presenting author 2
Introduction: Cachexia and sarcopenia are associated with poor outcome and increased chemotherapy-induced toxicity in lung cancer patients. However, the complex interplay of obesity, sarcopenia and cachexia, and its impact on survival in the context of firstlinechemotherapy is not yet understood. Methods: In 200 consecutively recruited lung cancer patients (70 female, mean age 62y; mean BMI 25 kg/m2; median follow-up 15.97 months) with routine staging-CT before and after chemotherapy (CTX, mean interval: 4.3 months), densitometric quantification of total (TFA), visceral (VFA), and subcutaneous-fat-area (SFA), inter-muscular-fat-area (IMFA), muscle-density (MD), muscle-area (MA) and skeletal-muscle-index (SMI) was performed retrospectively to evaluate changes under chemotherapy and the impact on survival. Results: We observed increases in TFA, VFA, SFA, VFA/SFA, and IMFA (p<0.05-0.001), while there were decreases in MA, MD and BMI (p<0.05-0.001) after chemotherapy. High pre-therapeutic VFA/SFA was a predictive factor for poor survival (HR=1.272; p=0.008), high pre-therapeutic MD for improved survival (HR=0.93; p<0.05). Decrease in BMI (HR=1.303; p<0.001), weight (HR=1.067; p<0.001) and SMI (HR=1.063; p<0.001) after chemotherapy were associated with poor survival. Patients with ≥4 CTX-cycles showed increased survival (17.6 vs. 9.1months), less muscle depletion (SMIdifference: p<0.05) and no BMI loss (BMIdifference: p<0.001). Conclusions: After chemotherapy, patients exhibited sarcopenia with decreased muscle and increased adipose tissue compartments, which was not adequately mirrored by BMI and weight loss but by imaging. Particularly sarcopenic patients received less CTXcycles and had poorer survival. As loss of BMI, weight and muscle were associated with poor survival, early detection (via imaging) and prevention (via physical exercise and nutrition) of sarcopenia may potentially improve outcome and reduce chemotherapy-induced toxicity.
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Abstract No. 037 Re-education of Tumor-Associated Macrophages by Modulating Histone Deacetylases in Lung Cancer Xiang Zheng 1,* , Poonam Sarode 1, Andreas Weigert 2, Stefan Guenther 1, Friedrich Grimminger 3, Soni Savai Pullamsetti 1, Werner Seeger 1, and Rajkumar Savai 1 1
Max Planck Institute for Heart and Lung Research Goethe-University Frankfurt, Institute of Biochemistry I, Frankfurt, Germany 3 Universities of Giessen Lung Center, Member of the German Center for Lung Research (DZL), Giessen, Germany *Presenting author 2
Within the established tumor microenvironment, tumor-associated macrophages (TAMs) are one of the most abundant stromal cell types. M1 macrophages possess anti-tumor capability and M2 macrophages manifest pro-tumor feature. This study investigated epigenetic mechanisms of macrophages polarization and re-education of TAMs by modulating histone deacetylases (HDACs). Human naive macrophages (M0) were generated from peripheral blood mononuclear cells and were polarized to M1 or M2 macrophages that were either used for cell culture or animal experiments. Additionally, M0 macrophages and lung cancer cells were co-cultured for 3-5 days to generate TAMs (M1-like TAMs and M2-like TAMs). Both, in vivo and in vitro, M2 macrophages led to increased tumor cell proliferation, migration and decreased apoptosis. Of note, next generation RNA-sequencing (RNA-Seq) analysis showed that HDAC2, belongs to class I HDAC family, as one of the significantly upregulated gene in M2 macrophages. Furthermore, upregulation of HDAC2 at protein level and significantly elevated HDAC activity was observed in M2 macrophages. Similar regulation of HDAC2 was found in M2 like TAMs. Interestingly, suppression of HDAC2 employing pharmacological (HDAC inhibitors; SAHA, VPA) or genetic (HDAC2-siRNA) approaches in human- and mouse- bone marrow-derived M2 macrophages and as well as in isolated TAMs from human- and mouse- lung tumors, led to upregulation of M1 markers (TNFα, IL8, CCR7, IL12) and downregulation of M2 markers (IL10, ALOX15, CD206). Notably, inhibiting M2 macrophages or M2-like TAMs by HDAC inhibitors or siRNA, followed by co-culture experiments reversed the tumor cells functions (proliferation, migration, and apoptosis). In addition, RNA-seq from HDAC2-siRNA M2 macrophages lead to identification of target genes that are involved in macrophage repolarization processes. Suppression of HDAC2 switches M2-like TAMs into M1-like phenotype and regulates tumor cell functions. Modulation of HDAC2 may provide a novel strategy for TAMs repolarization and cancer therapy.
Submitted as: Presentation 291 words of 300 possible words
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Abstract No. 040 Comparison of costs and care of lung cancer patients at the end-of-life in Germany depending on the time of survival after diagnosis Julia Walter 1,* , Amanda Tufman 2, Rolf Holle 1, and Larissa Schwarzkopf 1 1
Helmholtz Center Munich Hospital of the Ludwig-Maximilians-University (LMU) Munich *Presenting author 2
Because of its high mortality the end-of-life phase is of high importance in lung cancer. In this observational study of claims data we aimed to investigate whether the costs and care at the end-of-life differ depending on the length of survival after diagnosis. We analyzed 5,320 individuals with incident lung cancer in 2009 who: died until 2013, survived for 6 months after diagnosis and were treated with chemotherapy and radiotherapy. We defined two groups depending on the median survival time (363 days). Costs for the health insurance company for hospitalizations, doctor visits and medication in the last 30 days of life were modeled in generalized linear models with gamma distribution and reported as recycled predictions. Aspects of end-of-life care in the last month were modeled in logistic regression: death in hospital, intensive care treatment, unplanned hospitalization, more than one hospitalization, more than 14 days in the hospital, first palliative care treatment at least 30 days before death and chemotherapy in the last 14 days. All regression models were adjusted for age, sex, comorbidities, metastases at diagnosis and East vs. West Germany. We found significant differences in hospital costs (2,543€ vs. 1,828€, p-value=<0,0001) and inpatient medication (82€ vs. 35€, p- value =<0,0001) with lower costs in the group above Median survival. We also found significant differences between the two groups (reference above Median) in the site of death (OR=1,26 [1,12; 1,42]), chemotherapy (OR=1,30 [1,09; 1,54]), palliative treatment (OR=0,63 [0,54; 0,72]), intensive care treatment (OR=1,51 [1,33; 1,71]), hospitalizations (OR=3,30 [1,05; 10,43]) and hospital days (OR=1,47; KI:[1,29; 1,67]). Patients with shorter survival are more likely to be treated with a high intensity at the end-oflife. This leads to the question whether patients should be informed even earlier about possible palliative care to avoid possibly unnecessary intense treatment shortly before death.
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Abstract No. 043 Development of a high-throughput Drosophila model for lung cancer Judith Bossen 1, Karin Uliczka 2, Marcus Thiedmann 1, Mandy Mai 2, Christine Fink 1, Holger Heine 2, and Thomas Roeder 1 1
Kiel University Research Center Borstel
2
Lung cancer is accountable for 1.8 million new cases and 1.6 million cancer deaths annually and thereby one of the most aggressive types of cancer. The overall fatality rate of nonsmall cell lung cancer is extremely high. This is the consequence of late state detection and the lack of late state treatment options. Due to its simplicity Drosophila has already been successfully established as a model for studying chronic inflammatory diseases of the lung such as asthma and COPD. The Drosophila tracheal system shares comprehensive structural and functional similarities with the human lung, thus making it to an ideal model for studying the molecular framework underlying lung cancer development. Targeted overexpression of human oncogenes and their Drosophila homologs in the airway epithelium was chosen to induce cancer-like phenotypes in Drosophila. The animals were analyzed regarding hallmarks of cancer development, namely hyper- and metaplasia of the epithelium. Interventions showing a strong cancer-like phenotype were selected for treatment with potential anti-cancer drugs in a 96-well format. A number of oncogenes were able to induce epithelial meta- and hyperplasia indicating that they can induce tumor formation in the airway system. Ectopic overexpression of a constitutively active version of the EGFR gene in larval airway epithelia only was found to be lethal in early developmental stages. When treated with specific EGFR inhibitors these larvae can, in contrast to non-treated animals, successfully develop into adult flies. The results highlight the potential of Drosophila as a model in cancer research and its usefulness in high-throughput anti-cancer drug screenings. It is our goal to establish and use Drosophila lung cancer models to understand the molecular and genetic basis underlying tumor formation and progression and to identify lead compounds that can be used as therapeutic agents.
Submitted as: Presentation 288 words of 300 possible words
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Abstract No. 062 Non-invasive lung cancer diagnosis by detection of GATA6 and NKX2-1 isoforms in exhaled breath condensate. Aditi Mehta 1, Julio Cordero 1, Stephanie Dobersch 1, Addi J Romero-Olmedo 1, Rajkumar Savai 1, Johannes Bodner 2, Cho-Ming Chao 2, Ludger Fink 3, Ernesto Guzmán-Díaz 4, Indrabahadur Singh 1, Gergana Dobreva 1, Ulf R Rapp 1, Stefan Günther 1, Olga N Ilinskaya 5 , Saverio Bellusci 2, Reinhard H Dammann 2, Thomas Braun 1, Werner Seeger 1, Stefan Gattenlöhner 2, Achim Tresch 6, Andreas Günther 2, and Guillermo Barreto 1,* 1
Max-Planck-Institute for Heart und Lung Research Justus Liebig University, Giessen, Germany 3 Institute of Pathology and Cytology, UEGP, Wetzlar, Germany 4 Regional Hospital of High Specialties of Oaxaca (HRAEO), Oaxaca, Mexico 5 Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russian Federation. 6 Max Planck Institute for Plant Breeding Research, Cologne, Germany *Presenting author 2
Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Early LC diagnosis is crucial to reduce the high case fatality rate of this disease. In this case–control study, we developed an accurate LC diagnosis test using retrospectively collected formalinfixed paraffin-embedded (FFPE) human lung tissues and prospectively collected exhaled breath condensates (EBCs). Following international guidelines for diagnostic methods with clinical application, reproducible standard operating procedures (SOP) were established for every step comprising our LC diagnosis method. We analyzed the expression of distinct mRNAs expressed from GATA6 and NKX2-1, key regulators of lung development. The Em/Ad expression ratios of GATA6 and NKX2-1 detected in EBCs were combined using linear kernel support vector machines (SVM) into the LC score, which can be used for LC detection. LC scorebased diagnosis achieved a high performance in an independent validation cohort. We propose our method as a non-invasive, accurate, and low-price option to complement the success of computed tomography imaging (CT) and chest X-ray (CXR) for LC diagnosis.
Mehta A, Cordero J, et al., (2016) EMBO Mol Med; 8, 1380-1389 Mehta A, et al., (2015) Cancer Metast Rev; Jun;34(2):229-41 Mehta A, et al., (2015) Int. J. Mol. Sci; Feb 25;16(3):4492-511 Submitted as: Presentation 164 words of 300 possible words
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Abstract No. 065 Targeting the TGFβ pathway in lung cancer: Impact of Pirfenidone on non-small cell lung cancer Sebastian Marwitz 1,* , Kati Turkowski 2, Martin Reck 3, Werner Seeger 2, Rajkumar Savai 2, and Torsten Goldmann 1 1
Research Center Borstel Max Planck Institute for Heart and Lung Research 3 LungenClinic Großhansdorf *Presenting author 2
Despite recent developments in targeted therapies and immune-oncology, lung cancer remains a fatal disease. The majority of patients are diagnosed with an advanced disease and many patients have already distant metastases at the date of diagnosis. Among other pathways, the TGFβ pathway is known to be involved in activation of metastatic capabilities. Our recent study (Marwitz et al., Canc Res, 2016) could show that pathway activation is a hallmark of non-small cell lung cancer (NSCLC) and is joined by an elevated EMT phenotype. Furthermore, if the pathway activity was abolished by induced expression of a negative regulator, the invasive and metastatic capabilities were reduced. This study investigates the effect of TGFβ pathway inhibition by Pirfenidone in vitro (NSCLC cell lines) and in vivo (lung cancer xenograft models). Five different NSCLC cell lines were exposed to different dosages of Pirfenidone and cell viability, proliferation, migration as well as cell cycle phases were analyzed. Furthermore, protein array analyses were conducted to investigate the effect of Pirfenidone on intracellular signaling events. Using an in vivo animal model of tumor growth, the subcutaneous growth behavior of lung cancer cells was analyzed upon systemic treatment with Pirfenidone. Cell viability shows a reduction upon treatment of 5 NSCLC cell lines with Pirfenidone. Furthermore, Ki67 protein is significantly reduced and Pirfenidone induces cell cycle arrest in the G0/G1 phase. In addition, all five cell lines showed significantly reduced migration after treatment with Pirfenidone. The in vivo experiment suggests that oral application of Pirfenidone successfully reduces the tumor growth of murine lung cancer. Our data suggests, that Pirfenidone might have an impact on the treatment of NSCLC patients who did not qualify for targeted therapies or immune checkpoint inhibitors.
Submitted as: Presentation 281 words of 300 possible words
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Abstract No. 074 Study of angiogenesis-related biomarkers in patients with resected lung adenocarcinomas Helen Pasternack 1, Christiane Kümpers 1, Iris Watermann 2,* , Till Olchers 2, Mario Deng 1, Christian Kugler 2, Torsten Goldmann 3, Sven Perner 1, Ole Ammerpohl 4, and Martin Reck 2 1
Pathology of the University Medical Center Schleswig-Holstein, Campus Luebeck, Luebeck, Germany 2 LungenClinic Grosshansdorf, Großhansdorf, Germany, ARCN 3 Pathology of the University Hospital of Lübeck and the Leibniz Research Center Borstel, Borstel, Germany, ARCN 4 Institute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany, ARCN *Presenting author
Angiogenesis plays a major role in tumor growth, tumor maintenance and metastasis. Several antiangiogenic therapeutic strategies targeting VEGF-, FGF- and PDGF-signaling pathways have already shown promising results in the treatment of non-small cell lung cancer within clinical trials. Until now, there are no approved tumor biomarkers known predicting the successful application of anti-angiogenic therapies of patients with NSCLC. The aim of this study is to get more insight into the expression profile of biomarkers, gene expression-patterns, gene-alterations and epigenetic changes in the tumor / tumor stroma and its relation to early relapse. 100 patients with totally resected lung adenocarcinoma histology and cytologically or histologically confirmed stage IA-IIIA have been identified and separated into two equal groups: 1) Patients with relapse ≤ 2 years 2) Patients without relapse ≤ 2 years Formalin fixed paraffin embedded (FFPE) tumor blocks were selected for immunohistochemical-, molecular-, mRNA-expression- and epigenetic analyses. Patients are clinically characterized including age, gender, smoking status, therapies, tumor size, lymph node status, overall survival and progression free survival. FFPE-lung carcinoma tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34. Molecular analyses including KRAS- and EGFR-sequencing have been performed. FISHanalysis of ALK, ROS1, RET and PD-L1 are also enclosed as well as mRNA expression analyses with the PanCancer Immune Profiling Panel. First preliminary results of the epigenetic analyses are indicating an allocation of three different patients groups. Patients who relapsed within 2 years can be separated of patients without relapse within 2 years. In addition, an intermediate patient group is present. Analyses of DNA methylation profiling are indicating the classification of 3 groups. Next, clinical data have to be merged with data of immunohistochemical characterization, molecular analysis, mRNA-expression analyses and the epigenetic results.
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Abstract No. 077 Aberrant DNA methylation in the diagnostics of non-small cell lung cancer Swetlana Scheufele 1,* , Helene Kretzmer 2, Maren Kröger 1, Sebastian Marwitz 3, Iris Watermann 4, Niels Reinmuth 5, Christian Kugler 4, Peter Zabel 6, Ekkehard Vollmer 3, Sven Perner 3, Torsten Goldmann 3, Martin Reck 4, Reiner Siebert 7, and Ole Ammerpohl 1 1
Institute of Human Genetics, Christian-Albrechts-University Kiel, Schwanenweg 24, 24105 Kiel, Germany 2 Interdisciplinary Center for Bioinformatics, University of Leipzig, Haertelstraße 16-18, 04107 Leipzig, Germany 3 Pathology of the University Clinic Schleswig-Holstein, Campus Luebeck, and the Research Center Borstel, Clinical and Experimental Pathology, Parkallee 3, 23845 Borstel 4 LungenClinic Grosshansdorf, Wöhrendamm 80, 22927 Grosshansdorf, Germany 5 Asklepios Clinic Munich-Gauting, Robert-Koch-Allee 2, 82131 Gauting, Germany 6 Medical Clinic, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany 7 Institute of Human Genetics, University Hospital Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany *Presenting author
DNA methylation is a reversible covalent DNA modification, catalyzed by DNA methyltransferases. It plays an important role in gene regulation and can be influenced by environmental factors (e.g. tobacco smoke, asbestos or air pollution). Alterations of the DNA methylation pattern occur early during malignant cell transformation and are a typical hallmark of many cancers including non-small cell lung cancer (NSCLC). Therefore, this study aimed at identifying alterations in the DNA methylation pattern of various lung cancer entities and to subsequently validate their putative diagnostic value in both surgical samples, limited material collected during bronchoscopy as well as liquid biopsies. In the first approach we used Illumina Infinium HumanMethylation450k BeadChip to analyze 40 surgical lung cancer specimen and their corresponding controls. We identified 900 CpG loci aberrantly methylated in lung carcinomas as compared to controls (FDR < 1 x 10^-23, σ/σmax > 0.4). Moreover, we detected 1167 (FDR < 1 x 10^-4) loci differentiating between adeno- and squamous carcinoma of the lung. Additional 80 paired biopsy samples were collected during bronchoscopy and used for data validation. Further data validation has been performed using bisulfite pyrosequencing. In the second approach we performed Whole Genome Bisulfite Sequencing, to identify cancer and entity specific DNA methylation patterns of circulating tumor DNA isolated from plasma of patients suffering from adenocarcinoma (n=5, pooled material) or squamous carcinoma (n=4, pooled material). Plasma samples from individuals not suffering from a malignant disease (n=20, pooled material) were used as control. Our data analysis resulted in ~18 000 cancer and ~44 000 entity specific CpG loci aberrantly methylated in NSCLC. Finally, 800 best candidates from both described approaches were selected to generate an NGS based gene panel, which will be validated in future experiments.
DZL Annual Meeting 2017 Submitted as: Presentation 284 words of 300 possible words
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Abstract No. 080 Expression of TGFbeta-inducible Myosin-X predicts survival and chemotherapy resistance in squamous cell lung cancer Dmytro Dvornikov 1,* , Marc Schneider 2, Sebastian Ohse 3, Magdalena Szczygieł 1, Irina Titkova 1, Marcus Rosenblatt 4, Thomas Muley 2, Arne Warth 5, Felix Herth 2, Hendrik Dienemann 2, Michael Thomas 2, Jens Timmer 4, Hauke Busch 6, Melanie Börries 7, Michael Meister 2, and Ursula Klingmüller 1 1
German Cancer Research Center (DKFZ) Thoraxklinik at University Hospital Heidelberg 3 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg 4 Institute of Physics, University of Freiburg 5 Institute of Pathology, University of Heidelberg 6 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg; Institute of Experimental Dermatology, University of Lübeck 7 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg *Presenting author 2
Rationale: Squamous cell lung carcinoma (SCC) corresponds to about 25% of all lung cancers. Therapeutic approaches are very limited and platinum-based chemotherapy remains the main treatment option. Despite multiple studies, there are no generally accepted predictive biomarkers for SCC. Transforming growth factor-β (TGFβ) signaling was shown to be implicated in numerous pro-tumorigenic processes, including immune evasion, inflammation and cancer metastasis. Moreover, an epithelial-to-mesenchymal transition phenotype, which is commonly mediated by TGFβ, was widely observed in surgically resected specimens. However, the relation between TGFβ-induced changes and SCC progression remains unclear. Aim: Here we combined phenotypic and transcriptome-wide approaches to identify novel predictive biomarkers for SCC. Methods: Live-cell imaging, confocal microscopy and quantitative image analysis was used to examine migratory and invasive properties of the squamous cell carcinoma cell line SKMES1 upon TGFβ stimulation. Genome-wide changes in gene expression upon TGFβ treatment were examined using RNASeq. siRNA-mediated knockdown was used to validate the importance of candidate genes for TGFβ-induced 3D collagen gel invasion. Finally, a cohort of 362 NSCLC patients with defined clinical characteristics was used to examine the relevance of candidate genes in clinical settings. Results: We show that TGFβ treatment activated Smad-mediated signal transduction and resulted in the increase of migratory and invasive properties of SK-MES1 cells. Multiple cell motility-related proteins, including myosin motor proteins, such as Myosin-X were upregulated upon TGFβ stimulation. siRNA-mediated knockdown of Myosin-X completely abrogated TGFβ-induced collagen gel invasion. Finally, analysis of mRNA expression of Myosin-X in matched surgically resected tissues of SCC patients, showed that the expression ratio of Myosin-X in tumor and adjacent non-tumor tissue was predictive for overall survival and chemotherapy resistance.
DZL Annual Meeting 2017 Conclusion: TGFβ stimulation results in the up-regulation of numerous cell motility-related proteins. Myosin-X can be used as a biomarker to predict response to chemotherapy and overall lung SCC patient survival.
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Abstract No. 082 Immunoprofiling of patients with squamous NSCLC undergoing anti-PD-1 mAb maintenance treatment Jochen Behrends 1, Sebastian Marwitz 2, Iris Watermann 3,* , Torsten Goldmann 2, and Martin Reck 3 1
Core Facility Fluorescence Cytometry, Research Center Borstel, Borstel, Germany Pathology of the University Hospital of Luebeck and the Leibniz Research Center Borstel, Borstel, Germany, ARCN 3 LungenClinic Grosshansdorf, Großhansdorf, Germany, ARCN *Presenting author 2
Pembrolizumab is a humanized monoclonal antibody targeting programmed death 1 (PD-1). The antitumor activity in patients with advanced non–small-cell lung cancer (NSCLC) has already been shown within different clinical trials. In this phase II randomized, double-blind, placebo-controlled maintenance study of Pembrolizumab, patients with metastatic squamous NSCLC responding to first-line chemotherapy are enrolled. Within the translational part, blood samples of patients are analyzed by flow cytometry with the aim to generate immune profiles in the blood of these patients. 130 patients will be enrolled into this multicenter study. Patients fulfilling the inclusion/exclusion criteria will be randomized on a 1:1 basis into two groups: Patients in arm A receive Pembrolizumab 200 mg i.v. every 21 days until disease progression or nontolerable toxicity. Patients in arm B receive a placebo. Blood of patients is collected in TransFix®/EDTA vacuum blood collection tubes at three different time points for flow cytometric deep characterization of T-cell diversity on a BD LSRII (14 colors). Timepoints: I: within 14 days before the first cycle II. before the third cycle Pembrolizumab / placebo III: at time of progression Until now, 16 patients have been included in this study. 31 blood samples of 9 clinical centers have been analyzed by flow cytometry for the expression of several T-cell subset markers including regulatory, effector, memory and gamma/delta T cells. Measurement of fixed blood samples is working and differences in the expression levels of T cell subset markers, e.g. (PD-1) on leukocytes are detectable. Analysis of blood samples of the 130 patients shall be finished within the next two years for evaluating the results and to screen for differences between both patient groups and their dynamic changes. Next, functional T cell analysis including IFN gamma expression profiling will be performed in a small patients´collective treated at the LungenClinic Grosshansdorf.
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Abstract No. 091 Microsimulation model for an introduction of a population-based lung cancer screening program in Germany Ines Aumann 1,* , Marina Treskova 1, Heiko Golpon 2, Jens Vogel-Claussen 3, Tobias Welte 4 , and Alexander Kuhlmann 1 1
Center for Health Economics Research Hannover Clinic for Pneumology 3 Institut für Diagnostische und Interventionelle Radiologie 4 Clinic for Pneumology *Presenting author 2
Despite advances in treatment, long-term survival rate of lung cancer patients remains low. Detection of a tumor at the early stages can reduce lung cancer mortality. Two ongoing randomized control clinical trials examine efficacy of screening with low-dose (LDCT) in reduction of lung cancer mortality: NLST (USA), NELSON (Europe). The current results show reduction of lung cancer mortality. Aiming to reduce the harms without reduction of the benefits NELSON and NSLT apply eligibility criteria and protocols for the nodule management. This study aims to investigate the impact of the nodule management strategies used in the trials on the outcomes of lung cancer screening and to assess the benefits and harms of varying scenarios for implementation of a lung cancer LDCT screening program in Germany. The study is based on microsimulation model for lung cancer screening which is of modular design and comprises of the following structural modules: Population, Natural History (lung cancer biology), Clinical detection and Survival, and Screening. The screening cohort is characterized by the demographic structure and smoking behaviour of the German population. Five histological classes of lung cancer are simulated. Five years of six different screening scenarios are evaluated. Main health outcomes are life years saved and averted lung cancer specific deaths. Main comparator is no screening scenario. Sensitivity analysis is performed. Major driving factors of cost-effectiveness are eligibility criteria and settings of nodule management. For the German population the results show favourability for the screening program with the qualifying age range from 55 to 74, 30 pack-years and 15 years since quitting smoking and application of the NELSON approach to the nodule management. Main outcomes of this strategy include 7.55 detected cancer cases /1000 scans, 12.13 life years saved/ 1000 scans, 1.49 averted lung cancer /1000 scans and 11,741.96 Euro screening cost per life year saved.
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Abstract No. 092 microRNAs control of cancer cell – macrophage communication: role in tumor progression and metastasis Kati Turkowski 1,* , Sajo Kaduthanam 2, Holger Sültmann 2, Friedrich Grimminger 3, Werner Seeger 4, and Rajkumar Savai 1 1
Max Planck Institute for Heart and Lung Research German Cancer Research Center (DKFZ) 3 Universities of Giessen Lung Center 4 Max Planck Institute for Heart and Lung Research *Presenting author 2
In the lung cancer microenvironment, macrophages are a major component and one of the key effector cells regarding lung cancer development and progression. Cancer cells regulate several miRNAs in immune cells to limit their antitumor response and reprogram them to promote tumorigenesis. The molecular cues that control macrophage/ tumor crosstalk in the lung are only partially understood. Using a microRNA microarray- based approach we have found that miR-147 is highly induced in cancer cells co-cultured with macrophages. We could confirm the upregulation of miR-147 data in different human lung cancer cell lines co-cultured with macrophages as well as human lung cancer tissue. In vivo we could show that after macrophage depletion using the MAFIA-mouse model the expression of miR-147 is significantly downregulated, indicating a macrophage - dependent expression. Overexpression of miR-147 by A549 adenocarcinoma cells reveals a significant increase in proliferation, colony formation, and migration and resulted in epithelial to mesenchymal transition in vitro. To identify miR-147 target genes especially related to cancer pathways we used the NanoString approach. We found that most of the genes affected by overexpression of miR-147 are related to the MAPK, RAS, and PI3K pathways. Moreover, inhibition of miR-147 target gene PDE4D showed a dose-dependent reduction in tumor growth in vivo. We conclude that further investigations on miR-147 target genes and related pathways of these miRNAs will provide new opportunities for lung cancer targeted therapies.
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Abstract No. 100 Reversal of chemoresistance by targeting phosphodiesterase 5/10 mediated signaling in lung cancer Prema Subbarayal 1,* , Michael Wanzel 2, Thorsten Stiewe 3, Gary. A. Piazza 4, Friedrich Grimminger 5, Werner Seeger 1, Soni Savai Pullamsetti 1, and Rajkumar Savai 1 1
Max Planck Institute for Heart and Lung Research Center for Tumor Biology and Immunology 3 Center for Tumor Biology and Immunology 4 Drug Discovery Research Center, Mitchell Cancer Institute 5 Universities of Giessen Lung Center *Presenting author 2
Phosphodiesterases (PDEs) regulation has been described in various cancers including non-small cell lung cancer, which leads to the degradation of the intracellular secondary messengers such as cAMP and cGMP. The aim of this study was to identify whether the specific PDE inhibition has an anti-tumor effect by increasing the sensitivity towards chemotherapy. Among several PDE isoforms, we found the expression of PDE5/10 more in lung cancer tissues compared to the healthy tissues. Similarly, both were overexpressed in paclitaxel induced chemotherapy- resistant A549 tumor cells than the parental A549 cells. PDE5/10 mediated signaling pathway-induced paclitaxel drug resistance was evaluated by PDE5/10 dual inhibitor ADT-030 and by PDE5/10 gene silencing. ADT-030 with paclitaxel inhibited paclitaxel resistant tumor cell proliferation more effectively than other PDE5 or PDE10 inhibitors. Paclitaxel resistant cells upon PDE5/10 inhibition increased the intracellular secondary messenger levels, which further upregulated its endogenous protein kinases. β-Catenin has been involved in drug resistance of several cancers. PDE5/10 inhibition has downregulated β-Catenin as well as its downstream target genes. Hence challenging paclitaxel resistance cells upon PDE5/10 inhibition with paclitaxel regulates intracellular downstream signaling which sensitized the cells to chemotherapy treatment. Functional studies such as apoptosis, proliferation, migration, tumor colony formation and invasion assays revealed the possibility of reversing the paclitaxel resistance mechanism. Finally, we believe that the combination of PDE inhibitor and existing chemotherapeutic regimens targeting lung cancer merits consideration and offers a novel therapeutic window.
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Abstract No. 104 Erythropoietin Exhibit Angiogenic Potential And The Role of Erythropoietin Receptor In Lung Cancer Cells Xiaoqing Liu 1, Rosemarie Kief 1, and Rudolf M Huber 1,* 1
Medizinische Klinik V *Presenting author
Background Administration of EPO to cancer patients has been reported to be associated with decreased survival,and the mechanism remains controversial.In this study,we aimed to investigate the expression of EPO-R(EPO-receptor)in lung cancer cells,and whether EPO treatment affects growth and invasion of lung cancer cells.Moreover,the angiogenic effect of EPO was also explored. Material and methods The expression of EPO-R in lung cancer cell lines was measured by ELISA and cells proliferation was monitored by icelligence system.Vascular endothelial cells HUVEC tube formation assay and Transwell invasion assay in EPO or PBS treatment groups were also performed.Matrigel plug in was employed to observe the angiogenic ability of EPO in nude mice models.Microvessel density (MVD)was checked by using CD31 IHC staining. Results EPO-R can be detected in EGFR wild type cell line H838,while it was absent in cell lines H1650,H1975 and HCC827(EGFR gene mutation);Proliferation of cells H1650 was not affected by EPO treatment,interestingly,although EPO-R(+)in cell line H838,it also did not affect proliferation,demonstrating that EPO-R is not necessary for proliferation of lung cancer cells in vitro;Migration of lung cancer cell lines were not promoted by EPO;EPO significantly promoted HUVEC tube formation in vitro and induced new blood vessel in nude mice model. Conclusion The role of rHuEPO is beyond erythropoiesis,which also plays a strong role of angiogenesis and participates in the new blood vessel formation in lung cancer,implying that anti-EPO therapy may potentially act as one of the anti-angiogenic agents for cancer patients together with anti-VEGF treatment. Additionally,EPO-R may be co-expressed with EGFR.EPO-R mutation or chimeric receptor,and the role of EGFR and EPO-R in the mechanism of EGFRTKI (tyrosine kinase inhibitors) resistance on the possibility of cross-talk signaling pathway may be further investigated.
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Abstract No. 118 Investigating the dynamics of tumor–stroma interactions in lung cancer Magdalena Szczygiel 1,* , Martin Böhm 1, Bastian Kister 1, Michael Meister 2, and Ursula Klingmüller 1 1
DKFZ, Heidelberg Thoraxklinik, Heidelberg *Presenting author 2
Lung cancer is the leading cause of cancer-related deaths worldwide. Its fatal outcome is frequently related to late detection, early metastatic spread and frequent resistance to applied therapies. Recent reports indicate that metastatic spread and therapy resistance can be critically influenced by the tumor microenvironment. Cancer-associated fibroblasts modulate the extracellular matrix (ECM) and secrete a multitude of proteins including chemokines and growth factors. One of them is transforming growth factor beta (TGFβ), which regulates the epithelial-to-mesenchymal transition (EMT), a process that promotes tumor survival and spread. An important open question is to identify differences in communication between cancer cells and cancer-associated fibroblasts from different tumor stages and gain insights into how this unique interaction could drive tumor progression and resistance to therapies. We combine fibroblasts-derived matrix generation, tumor cell-fibroblast co-culture and novel cell-specific proteome labelling method to generate time-resolved proteomic data of tumor– stroma–ECM interaction. We show that collagen and fibronectin deposition by human lung fibroblast cell lines MRC5 and HFL1 was greatly facilitated by addition of Ficoll. Furthermore, TGFβ treatment resulted in differentiation of fibroblasts to myofibroblasts that was accompanied by time-dependent increase of alpha smooth muscle actin (αSMA) expression, a common marker of cancerassociated fibroblasts. ECM deposited by TGFβ-treated fibroblasts showed higher expression of components that were also differentially present in lung tumor-derived ECM of human patients. Finally, proteomic analysis of the co-culture secretomes of lung adenocarcinoma cell line H1975 with CCL-171 fibroblasts in comparison to the secretomes of mono-cultures alone showed differential up-regulation of several proteins related to tumor progression including TGFβ2 cytokine. These results suggest the existence of multiple feedback loops between cancer cells and fibroblasts. Such bidirectional interactions may further increase fibroblast differentiation and induce EMT in tumor cells that would facilitate tumor spread and therapy resistance.
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Abstract No. 120 Progastrin-releasing peptide (ProGRP) as a tool for response evaluation in patients with small-cell lung carcinoma (SCLC) Thomas Muley 1,* , Xiaotong Zhang 2, Stefan Holdenrieder 3, Catharina Korse 4, Xiu-yi Zhi 5, Rafael Molina 6, Zhongjuan Liu 2, Gunther Hartmann 3, Michael van den Heuvel 4, Kun Qian 5 , Ramon Marrades 6, Christine Engel 7, Ying He 7, Birgit Wehnl 7, Farshid Dayyani 7, and Felix Herth 1 1
Thoraxklinik Heidelberg Peking Union Medical College Hospital 3 University Hospital Bonn 4 The Netherlands Cancer Institute, Amsterdam 5 Xuanwu Hospital, Capital Medical University, Beijing 6 Hospital Clinic, University of Barcelona 7 Roche Diagnostics GmbH, Penzberg *Presenting author 2
Background: In SCLC response to chemotherapy is monitored by computed tomography (CT) scans. Baseline ProGRP levels were positively correlated with advanced SCLC, and a decline in ProGRP levels during treatment was associated with response (1). The objective of this study was to determine if progression could be ruled out by combining the changes in ProGRP levels over two chemotherapy cycles. Methods: Patients with SCLC were included from six centers in Europe and China. ProGRP levels were measured in serum or plasma samples using the Elecsys® ProGRP assay at baseline and after chemotherapy cycles 1 and 2. Only patients with blood samples taken at these time points and with elevated baseline ProGRP >100 pg/ml were eligible for this analysis. A logistic regression model was calculated to incorporate changes after the first cycle (i.e. from baseline to the end of cycle 1) and in between cycles (i.e. end of cycle 1 to the end of cycle 2). Response was evaluated by CT scan, according to RECIST v1.1 or WHO criteria. Results: 123 patients (n=108 non-progressors, n=15 progressors) satisfied the eligibility criteria. Median age was 62.0 years (range 36.0–83.0), 56% were male, and 78% were current or past smokers. A decline in ProGRP from both baseline to cycle 1 and from cycle 1 to cycle 2 was associated with non-progression (AUC 91.5%; sensitivity 100%; specificity 71%). All patients who experienced a >25% relative decline in ProGRP levels after the first chemotherapy cycle, followed by any further decrease (>0%) after the second cycle, were found to be non-progressors. Conclusions: By measuring the change in ProGRP levels at baseline and after each of the two subsequent chemotherapy cycles, we were able to identify patients with nonprogressive disease. This might reduce the need for interim CT scans by using ProGRP as a monitoring instrument.
1. Muley, et al. Journal of Thoracic Oncology 2015; 10(9) Supplement 2:MINI27.13 Submitted as: Presentation 298 words of 300 possible words Page 211 of 286
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Abstract No. 153 Inhalable nanoparticles for in vivo genome editing mediated by crispr-cas9 delivery for undruggable KRAS driven lung cancers Aditi Mehta 1,* , Georgios Stathopolous 2, and Olivia Merkel 1 1
Ludwig-Maximilians University of Munich Helmholtz Zentrum Munich *Presenting author 2
KRAS is the most frequently mutated gene in human cancers, and approximately 97% of the KRAS mutations involve codon 12/13. Despite its direct involvement in malignancy and decades of intensive effort, no effective pharmacological inhibitor of KRAS has been developed. Due to its important role in maintenance of cell signalling, picomolar affinity between KRAS and GTP, and the relatively smooth protein surface with very few grooves and/or pockets for small molecule binding, KRAS has proved as an impossible target for novel small molecule drugs, and KRAS tumours are often regarded untreatable. Compared to systemic chemotherapeutic anti-cancer drugs, direct localized administration of nucleic-acids via the pulmonary route allows higher retention in lung tissues and reduces systemic toxicity for better treatments of lung cancer. It was hypothesized that exploiting the efficient and precise CRISPR-Cas9 gene editing approach, KRAS mutations can be corrected leading to a sudden cessation of signaling and ultimate cell death. Inhibition of an initiating oncogene often leads to extensive tumour cell death, a phenomenon known as oncogene addiction. Utilizing highly specific receptor-ligand binding to target tumour cells, ligands were used as bait for cancer specific receptors for delivery of the plasmid DNA to lung cancer cells. Mouse cancer cells known to carry Kras mutations (AE-17, MC38 and LLC1) and WT Kras (B16F10, Pano2) were specifically transfected with the Cas9-sgRNArepair template complex leading to the 'repair' of the KRAS G12D mutation and hallmarks of cancer cells, such as cell survival, proliferation, migration and anchorage independent growth were evaluated. Ultimately, this project aims to address three major obstacles: 1. The 'undruggable' nature of KRAS driven cancers; 2. Efficient and safe pulmonary delivery of nucleic-acids to target cells; 3. The dosage and administration of nucleic-acids delivered to target cells, thereby modulating the duration and magnitude of nuclease expression controlling on-and off-target effects.
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Abstract No. 161 Ultra-early response capturing in the treatment of NSCLC using diffusion-weighted MRI: a prospective multicenter study (ERT) Nadja V. Batora 1,* , Gudula Heußel 2, Farastuk Bozorgmehr 2, Christiane Wiedemann 2, Davia-Elzbieta Optazaite 2, Paul Flechsig 1, Oliver Sedlaczek 1, Iven Fellhauer 1, Niels Reinmuth 3, Heiko Golpon 4, Jens Vogel-Claussen 4, Frank Wacker 4, Martin Reck 5, Susanne Stiebeler 5, Rudolf Maria Huber 6, Amanda Tufman 6, Julien Dinkel 6, Ullrich MüllerLisse 6, Andreas Günther 7, Gabriele A. Krombach 7, Hans-Ulrich Kauczor 1, Michael Thomas 2, and Claus Peter Heußel 2 1
University Hospital Heidelberg Thoraxklinik Heidelberg 3 Asklepios Fachkliniken München-Gauting 4 Medizinische Hochschule Hannover 5 Lungenclinic Grosshansdorf 6 Klinikum der Universität München 7 Universitätsklinikum Giessen *Presenting author 2
Purpose: There is an unmet need to evaluate response of NSCLC to systemic therapy as early as possible after treatment start. Ultra-early detection of lacking response offers the opportunity to adjust treatment early resulting in less adverse effects, possible better patient survival and lower costs. The goal of this prospective trial is to prove that DWI predicts response as early as 24h after the first dose of chemotherapy. The secondary goal is to demonstrate the feasibility of a standardized DWI protocol including data analysis in a multicenter setting. Methods and Materials: 150 patients suffering from adeno-ca of the lung will undergo DWI before and after chemotherapy within 3 years. MRI will be acquired 0-24h prior and either 24h after (platinum based) or 7+14d after (TKI based) first line chemotherapy. Histogram analysis of ADC maps will be generated by software-based analysis of user-defined ROIs (MINT). Ultra-early changes in DWI will be correlated with long-term RECIST 1.1. results. Results: Study protocol lasts 10min and is widely acceptable in clinical routine. Patient information was established at a single center and will be rolled out to another 4 national centers. So far, 17 pilot patients have been recruited including first staging results available for 10/17 patients. Clear signals of intraindividual diffusion change as caused by chemotherapy have already been observed in several of the pilot patients. The best strategy of ROI placement and data analysis are determined and protocol refinements are ongoing. Limitations: Heterogeneity of image quality from different centers is inevitable, while follow-up for individual patients is done using identical technology. Conclusion: This multicenter study will address the unmet clinical need of ultra-early detection of therapy response in NSCLC in order to translate this promising approach into broad clinical practice.
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Abstract No. 184 Interferon Regulatory Factor 9 mediated regulation of lung cancer progression and metastasis David Brunn 1,* , Friedrich Grimminger 2, Werner Seeger 1, and Rajkumar Savai 1 1
Max-Planck-Institute for Heart and Lung Research Justus-Liebig-Universität Gießen *Presenting author 2
Lung cancer is the leading cause of cancer-related death worldwide and accounts for about 300.000 deaths yearly in the European Union. The tumor microenvironment was shown to play a crucial role in tumor progression and metastasis. Beside numerous cytokines, chemokines and other factors secreted by the tumor stroma, Type-I-Interferons (IFN) are strong immune modulators, showing anti-proliferative and pro-apoptotic properties. We aim to study the essential role of the transcription factor IRF9 (Interferon Regulatory Factor 9) in the IFN pathway in lung cancer. Based on Kaplan-Meier estimators, high levels of IRF9 in lung cancer patients are associated with a significantly lower survival. Using tissue microarrays we could show that IRF9 is expressed in most of the lung cancer entities. In human lung cancer tissues, IRF9 is expressed in both, the solid tumor part and the tumor stroma, where we identified dendritic cells and tumor-associated macrophages. In vitro we used lentiviral particles to stably transduce the adenocarcinoma cell line A549 to overexpress (A549 LV IRF9) or to suppress IRF9 (A549 shIRF9). A549 LV IRF9 cells show an increase in proliferation and migration, whereas the knockdown of IRF9 leads to a reduction in proliferation and migration. In addition, these findings were confirmed in a subcutaneous in vivo xenograft model, where larger (A549 LV IRF9) and accordingly reduced (A549 shIRF9) tumor sizes were observed. These preliminary results suggest that IRF9 is one important transcription factor to target cancer and microenvironmental cells in lung cancer therapy.
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Abstract No. 190 Lipopolysaccharides Induce Radiotherapy Resistance in Non-Small Cell Lung Cancer Cell Lines - the Role of Protein Kinase-Activation Mira Yasemin Gökyildirim 1,* , Florentine Subtil 2, Ulrich Grandel 1, Werner Seeger 1, Rita Engenhart-Cabillic 2, Ulf Sibelius 1, Friedrich Grimminger 1, and Katja Hattar 1 1
Justus-Liebig-University Giessen Philipps-University Marburg *Presenting author 2
RATIONALE: Pulmonary infections are common complications in patients with lung cancer and worsen prognosis. These patients often show an acquired resistance to radiotherapy. Gram-negative bacteria are common pathogens in lung cancer. Their virulence is caused by cell wall components, especially by Lipopolysaccharides (LPS). LPS is known to activate multiple pathways in pulmonary epithelial cells. This could induce radiotherapy resistance in lung cancer cells. METHODS: Colony formation assays were performed to quantify the survival after ionizing irradiation. Human lung cancer cell lines were treated with different concentrations of LPS (0, 0.1, 1 and 10 µg/ml) and exposed to ionizing radiation (0, 1, 2, 4, 6 and 8 Gy). A defined number of treated cells were plated on dishes and after 10 days the colonies were counted. The plating efficiency and the survival rate were calculated. In parallel, proteins were isolated and proteome arrays were performed. Up-regulated target proteins where inhibited in LPS-treated cells before irradiation. RESULTS: Ionizing radiation induced a reduction in survival. However in LPS treated cells the effect of ionizing radiation was severely attenuated; thus LPS promotes radiotherapy resistance in the human adenocarcinoma cell line H1975. This effect was dose dependent and most pronounced when 10 µg/ml LPS were used. In H1975 cells the survival fraction increased significantly in the presence of LPS. The Proteome Array shows an upregulation of the cAMP response element-binding protein (CREB) and EGFR (Epidermal Growth Factor Receptor) after LPS treatment and radiation. After CREB binding protein (CBP) or EGFR inhibition the LPS-induced resistance to radiotherapy was decreased, meaning sensitivity to irradiation was restored. CONCLUSION: The LPS treatment of H1975 cells induces radiotherapy resistance. Inhibition of possible target proteins like CREB or EGFR may serve as a potential treatment to overcome resistance to radiotherapy.
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Abstract No. 203 Tumor infiltration by CD8+-cells correlates with better post-operative survival in stage IA-IIIA non-small cell lung cancer (NSCLC) Julia Stump 1,* , Simone Reu 2, Carmen Ballesteros-Merino 3, Amanda Tufman 1, Frank Berger 4, Michael Neuberger 5, Zipei Feng 3, Rudolf Hatz 6, Michael Lindner 7, Rachel E. Sanborn 3, John Handy 3, Bernard A. Fox 3, Carlo B. Bifulco 3, Rudolf M. Huber 8, and Hauke Winter 9 1
Ludwig Maximilian University of Munich and Thoracic Oncology Centre Munich, Munich, Germany; Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research, Munich, Germany 2 Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research, Munich, Germany; Institute of Pathology, University of Munich, Munich, Germany 3 Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Cancer Center 4 Department of Clinical Radiology, University of Munich, Munich, Germany 5 Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research, Munich, Germany; Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Cancer Center; Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital of the Ludwig Maximilian University, Munich, Germany 6 Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research (DZL, CPC-M), Munich, Germany; Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital of the Ludwig Maximilian University, Munich, Germany; Asklepios Clinic Munich-Gauting, Germany 7 Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research (DZL, CPC-M), Munich, Germany; Asklepios Clinic Munich-Gauting, Germany 8 Division of Respiratory Medicine and Thoracic Oncology, Department of Internal Medicine V, Ludwig Maximilian University of Munich and Thoracic Oncology Centre Munich, Germany; Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research (DZL, CPC-M), Munich, Germany 9 Comprehensive Pneumology Center (CPC) and Member of the German Center for Lung Research (DZL, CPC-M), Munich, Germany; Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital of the Ludwig Maximilian University, Munich, Germany *Presenting author
Non-small cell lung cancer (NSCLC) therapy is based on the histopathological evaluation, lymph node status and metastatic spread (TNM staging system). Nevertheless, stagespecific outcomes vary significantly: There is a need for additional prognosticators. Lymphocytic infiltrates were found in 6-11% of patients with NSCLC and were associated with increased disease-free and overall survival. We want to assess relationships between T-cells and FoxP3+ or PD-L1+-cells in tissue micro arrays (TMAs) of dense infiltrates at the invasive margin (IM) and center (CT) of NSCLC. TMAs were generated from formalin-fixed paraffin embedded tissue of 89 stage IA-IIIA NSCLC patients. TMAs included cores from the CT and cores from the IM, selected from areas with the most dense lymphocytic infiltrates. TMAs were immunolabeled with mIHC technique for PD-L1, CD8, CD3, FoxP3, CD163 and Cytokeratin. The CD8+-cells ratio in the CT compared to IM is significantly higher in tumors from patients
DZL Annual Meeting 2017 with stage I NSCLC. Based on the ratios of CD8+ and PD-L1+-cells at the CT compared to the IM we established an 'Invasive Score'. Score 0 patients (low CD8, low PD-L1) had a median overall survival of 45 months. Score 1 patients (high CD8 or PD-L1) had a median overall survival of 53 months. Score 2 patients (high CD8, high PD-L1) had a 62% survival at 72-months: Combining the rate of CD8-cell infiltrates with PD-L1 positivity in the tumor tissue is a stronger predictor than one based on the CD8 CT/IM ratio. Multispectral assessment of CD8 and PD-L1 does show a clear correlation with clinical outcome in stage I-III NSCLC: A tumor-controlling immune response appears to be associated with CD8-cell tumor permeability. This corresponds to reports that immune infiltrates are associated with improved outcome. Current studies are seeking to verify these findings in a larger cohort of patients. *Stump/Reu contributed equally to this study.
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Abstract No. 227 Modulation of tumor growth by bevacizumab and cisplatin within its dynamic human ex vivo microenvironment Sebastian Konzok 1,* , Susann Dehmel 1, Peter Braubach 2, Danny Jonigk 2, Gregor Warnecke 2, Marcus Krüger 2, Hans-Gerd Fieguth 3, Olaf Pfennig 3, Armin Braun 1, and Katherina Sewald 1 1
Fraunhofer Institute for Toxicology and Experimental Medicine Medical School Hannover 3 KRH clinics Hannover *Presenting author 2
Metastasis is being listed as the main cause of death in most cancers. However, the complexity of its formation remains challenging in basic research and drug development, as many studies fail to reflect the initial metastatic processes in a human organotypic microenvironment. In order to study early stages of tumor growth within its natural microenvironment, we add cancer cells to human Precision-Cut Lung Slices (PCLS) and modulate with established anti-cancer drugs. AdGFP-transduced human breast cancer cell line MDA-MB-231 was cultivated in co-culture with human PCLS over seven days. Timelapse of cancer cell growth, proliferation, morphology and dynamics were visualized using confocal microscopy and Imaris® analysis. Local release of soluble mediators VEGF and GM-CSF was determined with or without bevacizumab or cisplatin treatment in cancer cell-invaded PCLS. Efficacy of anti-cancer drugs was compared to tissue slices generated from tumor tissue and MDA-MB-231 2Dculture. Cancer cells integrate into the lung tissue and proliferate. Kinetics and morphology of MDAMB-231 cells within human ex vivo lung tissue follow a different pattern in comparison to 2D culture and decellularized tissue slices. Cisplatin treatment [50μM] reduced viability and cancer cell number in PCLS up to 37.5% and in tumor slices up to 48.7% after 72h. Cancer cell-invaded PCLS and tumor slices were less sensitive to their anti-cancer drug efficacy than the 2D culture. Extrinsic VEGF-levels were increased 3.5fold in cancer cell-invaded PCLS and 5.4fold in tumor slices after 48h. Bevacizumab treatment [200µg/mL] suppressed VEGF-release up to 24fold in cancer cell-invaded tissue and up to 25fold in tumor slices after 48h. Here we mimic cancer cell proliferation, growth and host cell interactions in human lung tissue. Pharmacological intervention with established drugs showed reduction of cancer cell growth and/or pro-tumor mediators. New drug targets may be discovered through studying allogeneous and autologous cancer growth in organotypic tissue.
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Abstract No. 232 Reprogramming of Tumor Associated Macrophages by modulating Wnt/β-catenin signalling in Lung Cancer Poonam Sarode 1,* , Xiang Zheng 1, Andreas Weigert 2, Alexsandra Tretyn 1, Soni Savai Pullamsetti 1, Friedrich Grimminger 3, Werner Seeger 1, and Rajkumar Savai 1 1
Max-Planck-Institute for Heart and Lung Research Goethe-University Frankfurt 3 Universities of Giessen Lung Center *Presenting author 2
Data from clinical and experimental studies suggest that Tumor Associated Macrophages (TAMs) contribute to cancer progression and metastasis. We aim to identify and manipulate TAMs specific signalling pathways, which are responsible for shift of macrophage from protumorigenic(M2 like-TAMs) to anti-tumorigenic(M1 like-TAMs).We established and characterized a novel in vitro model, training macrophages with tumor cell for 3 days (M1 like-TAMs) and 5 days (M1 like-TAMs). These M2 like TAMs showed reduced apoptosis in tumor cells, increased tumor cell migration and proliferation compared to M1 like-TAMs. RNA sequencing of M1 and M2 like-TAMs revealed differential activation of Wnt/β-catenin signalling. M2 like-TAMs showed significant upregulation in Wnt/β-catenin signalling. Immunostaining of β-catenin in TAMs of 70 human lung cancer sections revealed that βcatenin is activated in TAMs. Notably, interfering Wnt/β-catenin signalling in M2 like-TAMs by shRNAs ofβ-catenin, TNKS12 and β-catenin inhibitor resulted in cancer regression by phenotypic and functional switch of M2 like-TAMs to M1 like-TAMs. To study the role of Wnt/β-catenin signalling in cancer cells/TAMs interplay in vivo, we treated adenocarcinoma xenograft model with β-catenin inhibitor. Upon treatment, we observed significant reduction in tumor size and increased accumulation of M1 like-TAMs in tumor microenvironment. Interestingly, inhibition of Wnt/β-catenin signalling in TAMs from ex vivo isolated human and mouse lung tumors showed functional switch of M2 like-TAMs to M1 like-TAMs. Thus, intervention of Wnt/β-catenin signalling in TAMs will permit the development of new cancer therapeutics.
Submitted as: Presentation 231 words of 300 possible words
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Abstract No. 234 Circulating fibrocytes plays a key role in lung tumor progression by modulating macrophage phenotype, angiogenesis and endothelin system Alina Asafova 1, Xiang Zheng 1, Soni Savai Pullamsetti 1, Anja S chmall 1, Friedrich Grimminger 2, Werner Seeger 1, Andreas Weigert 1, and Rajkumar Savai 1,* 1
Max-Planck Institute for Heart and Lung Research Universities of Giessen Lung Center *Presenting author
2
Circulating fibrocytes (CFs) are bone marrow derived, mesenchymal progenitor cells that emerge as critical players in many diseases. So far, CFs role and regulation has not been delineated in lung cancer progression and metastasis. Here we show that the number of CFs (Col1+CD45+ or Col1+ CD68+) in human lung cancer and spontaneous mouse lung cancer (KRasLA2) models. To delineate their tumor promoting capabilities in vitro, we generated CFs and co-cultured with tumor cells, coculture conditioned medium (CM) led to increased proliferation and migration of tumor cells. Similarly, co-culture CM on endothelial cells and macrophages resulted in increased tube formation and differentiation of macrophages into M2 phenotype, linked to corresponding changes in pro-angiogenic cytokines and monocyte-chemo-attractant molecules. Further we employed three different approaches to evaluate the functional role of CFs in vivo: (i) Co-injection of tumor cells with CFs; (ii and iii) depletion of CFs in vivo by generating HSVTK (pCol1-TK/IRES-EGFP) mice, followed by LLC1 tumor cell injections and by crossbreeding KRasLA2 mice with HSVTK mice. All three-tumor models provide strong evidence that CFs promote primary and metastatic tumor growth by modulating macrophages and angiogenesis. Evaluating the molecular pathways regulated by CFs in tumor, we identified upregulation of endothelin system (EDNRA, EDNRB). Furthermore, CFs treatment with EDNRA and EDNRB antagonist - Bosentan in co-culture system, led to strong decrease in the tumor cells proliferation, migration and tube formation. Importantly, treatment of co-injected tumor model with Bosentan drastically reduced primary and metastatic tumors by modulating macrophages and angiogenesis. Thus, CFs plays an important role in lung cancer pathogenesis by regulating tumor cells and tumor microenvironment, and blocking of CFs recruitment and activity via endothelin receptor antagonism offers a potential therapeutic option for lung cancer.
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Abstract No. 235 Mechanism of resistance of pemetrexed and vinorelbine in adenocardinoma Fei Tian 1,* , Zhe Wang 1, Rosemarie kiefl 1, and Rudolf M Huber 1 1
LMU *Presenting author
Lung cancer is the leading cause of cancer deaths worldwide. Despite advances and progresses in surgery, chemotherapy, and radiotherapy over the last decades. The aim of this study was to investigate the mechanism of resistance of pemetrexed and vinorelbine in adenocardinoma.Vinorelbine and pemetrexed caused a strong dose-dependent cytotoxic effect in both HCC and cisplatin resistant HCC (HCC-res) cells. The IC50 values of vinorelbine against HCC and HCC-res cells were 10.34±1.12 nM and 9.98±2.12 nM, respectively. The IC50 values of Pemetrexed against these cells were 110.77±17.28 nM and 118.89±18.77 nM respectively. The application of different therapy schedules induced a significant time dependent cell growth inhibition on HCC naïve and cisplatin resistant cells. The therapy scheme of cisplatin→pemetrexed→vinorelbine showed the strongest inhibitory effect on both HCC and HCC-res cells. The application of different therapy schedules on HCC and HCC-res cells increased cytoplasma calcium concentration. Only the application of vinorelbine alone failed to increase calcium concentration in HCC cells. The most elevated calcium concentration was found in the cells treated with cisplatin→pemetrexed→vinorelbine in both HCC and HCC-res cells. The sequential application of cisplatin, vinorelbine and pemetrexed has a synergistic effect in cell growth inhibition, apoptosis induction, and calcium concentration elevation in HCC and HCC-res cells. The calcium overload could lead to apoptosis, which was related to the cell growth inhibitory effect of chemotherapeutics in lung cancer cells. It might cast a light to develop chemotherapy schedules for patients, and to overcome cisplatin resistance in lung cancer.Meanwhile the resistant cell lines have shown a significant different proliferation profile than the naive HCC cells.
Submitted as: Presentation 259 words of 300 possible words
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Abstract No. 243 Frequency and clinical relevance of EGFR-mutations and EML4-ALK-translocations in octagenarians with NSCLC Amanda Tufman 1,* , Simone Reu 1, Sandra Hasmann 1, Diego Kauffmann-Guerrero 1, Katrin Milger 1, Zulfiya Syunyaeva 1, Kathrin Kahnert 1, and Rudolf Maria Huber 1 1
LMU *Presenting author
Background: Novel therapies targeting genetic alterations have improved response rates and overall survival for some patients with NSCLC; however, only a minority of caucasian patients with lung cancer benefit from these treatments. Testing for EGFR mutation and ALK translocation is recommended for all patients with advanced adenocarcinoma, but the highest occurance of these driver mutations has been described in younger patients, females, and those with little or no smoking history. The frequency of driver mutations in elderly and very elderly patients has not been described. Methods: We reviewed the charts of all patients over age 70 treated at our centre in 2015. We report the frequency of EGFR and ALK alterations in patients aged 70-74 , 75-79 and >80 years. Results: Out of 179 patients 16 were 80 years or older at first diagnosis and 8 of 16 had non-squamous histology. Among these very elderly patients, 3 patients had EML4-ALK translocations and 3 patients had EGFR mutation (1 Del19, 1 L858R and 1 rare exon 19). This represents a 75% frequency of treatable driver mutations in octagenarians with nonsquamous NSCLC. Rates of genetic drivers were lower, but still clinically relevant, in nonsquamous NSCLC patients aged 70-74 (27.0%) and 75-79 (26.7%). Conclusion: Very elderly patients with non-squamous NSCLC were found to have high rates of EGFR mutation and ALK translocation. This is clinically relevant, as this often frail and comorbid population may not be suitable for treatment with chemotherapy and may benefit from first line treatment with a targeted tyrosine kinase inhibitor. Testing for these genetic alterations should not be restricted to younger patients. The biology of lung cancer in the very elderly may differ from that of moderately elderly patients, as the longevity of these patients may select for individuals more resistant to, or with little exposure to, environmental carcinogens.
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Abstract No. 244 Clinical and histological factors associated with SUV in PET-CT in patients with adenocarcinoma of the lung Amanda Tufman 1,* , Siokou Fiona 1, Ullrich Müller-Lisse 1, Frank Berger 1, Kathrin Kahnert 1, Thomas Pfluger 1, Simone Reu 1, Julia Stump 1, Hauke Winter 1, and Rudolf Maria Huber 1 1
University of Munich *Presenting author
Introduction: PET-CT is increasingly used for staging and treatment monitoring in NSCLC. The prognostic and possibly predictive value of SUV, as well as the clinical, molecular and pathological features contributing to a high or low SUV value, have not been well described. Methods: We retrospectively assessed patients staged with PET-CT and correlated SUV values before and during treatment with clinical and pathological features of the tumour including CRP, adenocarcinoma subtype, and Ki67. Results: Of 190 patients with adenocarcinoma of the lung, 110 had PET-CT staging and were included in this analysis. Tumour subtypes were: 50.0% solid, 16.4% acinar, 9.1% papillary, 7.3% lepidic, 1.8% micropapillary, 15.4% other. 70 patients received systemic treatment and 40 were treated surgically. The mean SUV for all patients was 11.1 (for patients treated medically 13.5 and for those treated surgically 8.6). Ki67 expression in the tumour was lowest in the group with SUV < 10 (38.6%) and highest in the group with SUV > 20 (56.0). The group with SUV 11-19 had a moderate Ki67 expression (47.9%). In patients with surgical tumour samples there was a trend towards higher SUV in patients with tumours showing 30% or more solid growth pattern (mean SUV 11.4) and lower SUV in patients with any lepidic growth (mean SUV 4.0) (p=0.002). Systemic markers of inflammation were significantly higher in patients whose tumours had SUV>10 (mean CRP 2.3 mg/dl; mean leukocytes 9.7 G/L) than in patients with low SUV tumours (<5) (mean CRP 0.4 mg/dl, p=0.0186; mean leukocytes 7.2 G/L, p=0.014). Discussion: Multiple factors correlate with SUV values including adenocarcinoma subtype, proliferation index and systemic inflammation. These factors should be taken into account when interpreting PET-CT SUV values in clinical practice.The correlation of PET-CT SUV values with inflamed tumour phenotypes and potential relevance for immune therapies should be further investigated.
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Abstract No. 248 IL-17C mediates the recruitment of tumor-associated neutrophils and lung tumor growth Christoph Beisswenger 1,* , Lars-Henning Schmidt 2, Christopher Jungnickel 1, Lisa Wolf 1, Rainer Wiewrodt 2, and Robert Bals 1 1
Saarland University University Hospital Münster *Presenting author 2
Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for lung cancer and an aberrant microbiota of the lung. Microbial colonization contributes to chronic neutrophilic inflammation in COPD. Nontypeable Haemophilus influenzae (NTHi) is frequently found in lungs of stable COPD patient and is the major pathogen triggering exacerbations. The epithelial cytokine IL-17C promotes the recruitment of neutrophils into inflamed tissues. The purpose of this study was to investigate the function of IL-17C in the pulmonary tumor microenvironment. We subjected mice deficient for IL-17C (IL-17C-/-) and mice double deficient for Toll-like receptor 2 and 4 (TLR-2/4-/-) to a metastatic lung cancer model. Tumor proliferation and growth as well as numbers of tumor-associated neutrophils were significantly decreased in IL-17C-/- and TLR-2/4-/- mice exposed to NTHi. The NTHiinduced pulmonary expression of IL-17C was dependent on TLR-2/4. In vitro, IL-17C increased the NTHi- and TNF-α-induced expression of the neutrophil chemokines keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2) in lung cancer cells but did not affect proliferation. Human lung cancer samples stained positive for IL-17C and in NSCLC patients with lymph node metastasis, IL-17C was identified as a negative prognostic factor. Our data indicate that epithelial IL-17C promotes neutrophilic inflammation in the tumor microenvironment and suggest that IL-17C links a pathologic microbiota, as present in COPD patients, with enhanced tumor growth.
Submitted as: Presentation 220 words of 300 possible words
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Abstract No. 250 Hypoxia and soluble mediators from H1975 Lung Adenocarcinoma Cells affect NaTransport in Human Alveolar Epithelium Ezgi Ermis-Kaya 1, Emel Baloglu 2, Michael Meister 3, Suna Cebesoy 4, Arne Warth 5, and Heimo Mairbäurl 1,* 1
Medical Clinic VII, Sports Medicine, University of Heidelberg Department of Pharmacology, Acibadem University Istanbul, Turkey 3 Thorax Clinic, Section Translational Research, University of Heidelberg 4 Science faculty, Department of Biology, University of Ankara, Ankara, Turkey 5 Institute of Pathology, University of Heidelberg *Presenting author 2
RATIONALE: Lung cancer cells generate a microenvironment by releasing signals that affect surrounding cells by causing cause abnormal function due to direct or indirect interaction. We ask the question whether cancer cells growing in close vicinity affect Natransport of healthy human alveolar epithelium. METHODS: Primary human alveolar epithelial cells (hATII) were isolated from tumor free regions of peripheral human lung tissue after lobotomy. hATII cells were grown on transwell filters in co-culture with H1975 adenocarcinoma cells using regular and oxygen-permeable culture plates. hATII cells were also treated with conditioned media from normoxic and hypoxic H1975 cells, normoxic media that were fractionated to exclude molecules >10.000 kD, and with exosomes released from H1975 cells. Ion transport was evaluated by measuring the short circuit current (ISC) in Ussing chambers to obtain the activity of epithelial Na channels (ENaC) and Na/K-ATPase. RESULTS: 24 h of co-culture causes a pronounced stimulation of ENaC and Na/K-ATPase. Exposure for more than 24 h causes inhibition which was prevented by culturing on gas permeable plates. H1975 conditioned media also stimulated ENaC and Na/K-ATPase whereas conditioned media from hypoxic H1975 cells decreased Na-transport. Neither the exosome fraction nor filtered conditioned media affected Na-transport. CONCLUSIONS: These results indicate that H1975 adenocarcinoma cells release soluble factors that stimulate hATII cell Na-reabsorption, and that these factors have a molecular weight > 10000 and are not contained in the exosome fraction. Co-culturing with H1975 cells and hypoxic H1975 conditioned media cause Na-reabsorption inhibition due to soluble factors released by hypoxic cells.
Submitted as: Presentation 249 words of 300 possible words
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Abstract No. 253 Heme and iron shape the phenotype of macrophages in the tumor microenvironment of non-small cell lung cancer Milene Costa da Silva 1,* , Carl Maximilian Thielmann 1, Margareta P. Correia 2, Michael O. Breckwoldt 1, Michael Meister 1, Thomas Muley 1, Adelheid Cerwenka 2, and Martina U. Muckenthaler 1 1
University of Heidelberg DKFZ *Presenting author 2
The tumor microenvironment, which impacts significantly on cancer progression and therapy response is characterized by high cellular complexity, including fibroblasts, stroma, blood vessels and infiltrates of immune cells. Tumor-associated macrophages (TAMs) are a critical component of the tumor microenvironment. They represent up to 50% of the mass of infiltrated cells and were shown to be of prognostic value. TAMs generally acquire an antiinflammatory M2 phenotype, contributing to immune suppression, angiogenesis, tumor growth and remodeling of the tumor microenvironment. Here we show that tumor samples from patients with non-small cell lung cancer and from experimental murine lung tumors accumulate iron in TAMs, whereas cancer cells are relatively iron spared. Iron loaded macrophages are located close to sites of red blood cell extravasation in hemorrhagic areas of the tumor microenvironment. Hemorrhagic areas not only show increased numbers of iron-loaded TAMs, but also enhanced infiltration of neutrophils and increased expression of cytokines and chemokines responsible for macrophage and neutrophil recruitment. Consistent with in vivo observations, treatment of macrophages with conditioned media from tumor cells polarizes macrophages towards an M2-like phenotype. Interestingly, incubation of these M2-like macrophages with hemolytic red blood cells triggers polarization towards an M1-like inflammatory phenotype and increases the expression of cytokines responsible for neutrophil recruitment. Functionally, macrophages treated with hemolytic red blood cells and co-cultured with T cells, show enhanced killing activity. In conclusion, our data suggest that iron and heme derived from leaking red blood cells of fragile vessels shape the phenotype of TAMs and trigger cytokine production. We propose that delivering iron to TAMs may be of value therapeutically to increase anti-cancer immune responses.
Submitted as: Presentation 266 words of 300 possible words
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Abstract No. 258 Progression patterns and prognostic factors of ALK-driven lung cancer treated with targeted therapies and conventional chemotherapy Petros Christopoulos 1,* , Albrecht Stenzinger 2, Mei Elsayed 1, Farastuk Bozorgmehr 1, Claus-Peter Heußel 1, Holger Sültmann 3, Felix Herth 1, Rocio Sotillo 3, Thomas Muley 1, Michael Meister 1, Helge Bischoff 1, and Michael Thomas 1 1
Thoraxklinik Heidelberg Institute of Pathology, Heidelberg 3 German Cancer Research Center, Heidelberg *Presenting author 2
Since the initial report of ALK rearrangements in ca. 5% of NSCLC patients in 2007, small molecule ALK tyrosine kinase inhibitors (TKI) have revolutionized treatment and transformed the course of the disease in this patient group. Crizotinib, an inhibitor of ALK, ROS1, and cMET, was the first targeted therapy developed and demonstrated superior response rates and progression-free survival intervals as well as better tolerability compared with first- and second-line chemotherapy. More recently, ceritinib and alectinib were approved for crizotinib-resistant disease. Several other compounds are currently in advanced clinical testing. Since virtually all patients on any TKI relapse, guidance of TKI-sequencing, understanding of resistance mechanisms and complementation with other therapeutic modalities become key. The impact of various treatment sequences on patient outcome and the different disease progression patterns under TKI and conventional chemotherapy will first be analyzed through a retrospective analysis of ALK+ NSCLC patients treated at the Thoraxklinik Heidelberg in the past 10 years (n=70). Furthermore, the prognostic role of different ALK fusion gene variants and immunologic parameters will be studied in cooperation with the Department of Molecular Pathology. Newly diagnosed patients will be added to the cohort and additionally monitored for tumor response and evolution by diffusion-weighted thoracic MRI, assays of circulating tumor DNA and sequential biopsies in the context of other translational DZL projects. Mouse models will be employed to define the role of chromosomal instability in the development of ALK-TKI resistance in vivo. The insights derived could pave the way for better therapeutic strategies, i.e. guide the decision to complement ongoing treatment with local therapies vs. early change of the systemic therapy in case of progression at selected sites, prompt to a more aggressive approach for patients with unfavorable ALK alterations or more pronounced chromosomal instability and suggest efficient ways of incorporating immunotherapy into current treatment concepts.
Submitted as: Presentation 300 words of 300 possible words
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Abstract No. 271 Immunohistochemical and molecular characterization of large cell lung cancer Alexander Harms 1,* , Daniel Kazdal 1, Mark Kriegsmann 1, and Arne Warth 1 1
Universitiy of Heidelberg *Presenting author
Large cell carcinoma is an undifferentiated non-small cell carcinoma that lacks the cytological, architectural and immunohistochemical features of small cell carcinoma, adenocarcinoma, or squamous cell carcinoma. Some pathologists therefore tend to call this sort of cancer a “waste-paper basket” because all other diagnoses have to be ruled out and the left overs should be classified as large cell cancer only in those cases when additional staining is negative, unclear or not available. The diagnosis can only be made among resected tumours and therefore cannot be made on non-resection or cytology specimens. Many tumours that have been classified as large cell carcinoma according to the WHO from 2004 are now reclassified as solid adenocarcinoma or non-keratinizing squamous cell carcinoma. Large cell carcinoma is rare, thus molecular data is sparse. For this study we searched our archive for all cases of large cell carcinoma of the lung, beginning in 2002 and, with the help of immunohistochemistry, reclassified them according to the new WHO and furthermore analyzed them via next generation sequencing to find possible targets. Due to its rarity little is known of its molecular background and possible targets for patient’s treatment are disguised. With this study we try to demystify large cell carcinoma of the lung and show additional molecular data.
Submitted as: Presentation 210 words of 300 possible words
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Abstract No. 291 Mathematical model for the optimal treatment of lung cancer-associated anemia. Agustin Rodriguez-Gonzalez 1,* , Max Schelker 2, Andreas Raue 3, Bernhard Steiert 4, Florian Salopiata 1, Lorenz Aldung 1, Martin Bohm 1, Markus Stepath 1, Sofia Depner 1, MarieChristine Wagner 1, Ruth Merkle 1, Bernhard Kramer 1, Susen Lattermann 1, Marvin Wäsch 1 , Andreas Franke 5, Edda Klipp 2, Patrick Wuchter 6, Anthony Ho 6, Wolf Lehmann 1, Michael Jarsch 5, Marcel Schilling 1, Jens Timmer 4, and Ursula Klingümller 1 1
DKFZ Humboldt-Universität zu Berlin 3 Merrimack Pharmaceuticals 4 University of Freiburg 5 Roche Diagnostics GmbH 6 University of Heidelberg *Presenting author 2
Background: With 1.8 million of new cases per year, lung carcinoma is the cancer with the highest incidence (1) and the associated anemia ranges from 50% to up to 90% in the advanced stages (2). Cancer-associated anemia reduces the response to chemotherapy and the quality of life. Erythropoiesis Stimulating Agents (ESAs) have been used to correct anemia in cancer, however it was reported that 30-50% of the patients do not respond and an increased risk of mortality has been observed (3-5). Meta analyses of several clinical trials failed to resolve the low efficacy and safety concerns regarding ESA treatment in cancer (5). As a consequence, despite of the possible advantages of ESAs over blood transfusions, their use in cancer-associated anemia was reduced. The high degree of heterogeneity among patients in the latest stage constitutes a complex question. A systems biology approach can identify patient-specific parameters that allow patient stratification and describe the patient heterogeneity at molecular level. Method: We combined a dynamic pathway model that describes the interaction of ESAs and the erythropoietin receptor (EpoR) with quantitative data from pharmacokinetic and pharmacodynamic studies of ESAs in human subjects. We utilized coupled ordinary differential equations that link the cellular scale with the body scale, and calibrated the model parameters based on quantitative experimental determinations at multiple experimental scales. Results: The mathematical model was able to describe the dynamic interaction of ESAs at molecular, cellular and systemic level in the human body. Further, the ODE model enabled to predict optimal dosing for ESAs to preferentially activate the EpoR in the hematopoietic context but not in the tumor context. Conclusion: This model can describe the dynamic interaction and the pharmacokineticpharmacodynamics of any EpoR ligand. Based on hemoglobin values of each patient, the model predicts a patient-specific safer minimal effective dose of ESA.
Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 295 Inflammation-related DNA methylation changes in blood lymphocytes as biomarkers for lung cancer risk Esther Schamschula 1,* , Yassen Assenov 2, Wolfgang Hagmann 2, Thomas Muley 3, Christoph Plass 4, Burkhard Tümmler 5, and Angela Risch 1 1
University of Salzburg DKFZ Heidelberg 3 Thoraxklinik Heidelberg 4 DZL Heidelberg 5 Medizinische Hochschule Hannover *Presenting author 2
Lung cancer is a leading cause of deaths worldwide. Inflammation and cancer are strongly associated and inflammatory processes constitute a risk factor for lung cancer development. Epigenetic changes play a major role in all types of cancer. Alterations in the DNA methylation pattern of peripheral blood mononuclear cells can serve as biomarkers for the risk of developing lung cancer and have been studied concerning smoking, the main risk factor for lung cancer. However, acquired differential methylation due to chronic inflammation as biomarker for lung cancer risk has not been investigated. Cystic fibrosis (CF) is a monogenetic disease caused by mutations in the CFTR gene that affects the respiratory and gastrointestinal tracts, leading to chronic inflammation. Therefore, in this study, CF is utilized as a model in order to explore acquired inflammation-related changes in DNA methylation. From the European CF Twin and Sibling Study, blood samples from 25 monozygotic twins with well-defined intra-pair discordance are available. The Illumina 450K methylation array was used to identify differentially methylated CpGs, that correlate with discordance of disease phenotype in monozygotic twins. A list of candidate loci was selected from the most differentially methylated probes based on the absolute pair-wise methylation differences, the intra-pair disease phenotype discordance and their genomic location. The candidate CpGs will be investigated in blood samples from a lung cancer case-control study. The intention is to investigate inflammation-related alterations in bloodDNA methylation as potential lung cancer risk markers.
Submitted as: Presentation 237 words of 300 possible words
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Abstract No. 094 Alveolar fibroelastosis and bronchiolitis obliterans after lung and stem cell transplantation: morphological and molecular motifs Danny Jonigk 1,* , Berenice Rath 1, Paul Borchert 1, Peter Braubach 1, Lavinia Mägel 1, Nicole Izykowski 1, Gregor Warnecke 2, Wiebke Sommer 2, Hans Kreipe 1, Robert Blach 1, Adrian Anklamm 1, Axel Haverich 2, Matthias Eder 3, Michael Stadler 3, Tobias Welte 4, Jens Gottlieb 4, Mark Kühnel 1, and Florian Länger 1 1
Institute for Pathology Division of Cardiac, Thoracic, Transplantation and Vascular Surgery 3 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation 4 Department of Respiratory Medicine *Presenting author 2
Chronic lung allograft dysfunction (CLAD) remains the major obstacle to long-term survival following lung transplantation (LuTx). Morphologically CLAD is defined by an obliterative remodelling of the small airways (bronchiolitis obliterans, BO) as well as collagenous obliteration of alveoli with elastosis summarized as alveolar fibroelastosis (AFE). Both patterns are not restricted to pulmonary allografts, but have also been reported following hematopoietic stem cell transplantation (HSCT) and radio chemotherapy (RC). We performed compartment-specific morphological and molecular analysis of BO and AFE lesions in 60 human lung explants from CLAD, HSCT and RC patients. Via conventional histopathology, laser-microdissection, PCR techniques and immunohistochemistry, fibrosisassociated gene and protein expression were assessed. We describe an evolutionary model for the AFE pattern: an unspecific fibrin-rich reaction to injury pattern triggers a misguided resolution attempt and eventual progression towards manifest AFE. Our data point towards an absence of classical fibrinolytic enzymes and an alternative fibrin degrading mechanism via macrophages, resulting in fibrous remodelling and restrictive functional changes. Four key results emerged from our analysis of fibrosis-associated genes: i) generally speaking, “BO is BO”. Despite the varying clinical backgrounds, the molecular characteristics of BO lesions were found to be alike in all groups. ii) “AFE is AFE”. In all groups of patients suffering from restrictive changes to lung physiology due to AFE there were largely – but not absolutely - identical gene expression patterns. iii) BO concomitant to AFE after LuTx is characterized by an AFE-like molecular microenvironment, representing the only exception to i). iv) Macrophages orchestrate the remodelling process. These data may serve as diagnostic adjuncts and help to predict the clinical course of respiratory dysfunction in LuTx and HSCT patients. Moreover, analysis of the mechanism of fibrinolysis and fibrogenesis may unveil potential therapeutic targets to alter the course of the eventually fatal lung remodelling.
Comparative Analysis of Morphological and Molecular Motifs in Bronchiolitis Obliterans and
DZL Annual Meeting 2017 Alveolar Fibroelastosis after Lung and Stem Cell Transplantation Danny Jonigk etal. 2016 The Journal of Pathology: Clinical Research DOI: 10.1002/cjp2.60 Submitted as: Presentation 297 words of 300 possible words
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Abstract No. 112 Implementation of a Data Management Workflow: Data Import, Cohort Management and Data Export for Statistical and Data Mining Analyses Daniel Firnkorn 1,* , Till Olchers 2, Michael Thomas 3, Petra Knaup 1, and Thomas Muley 3 1
Institute of Medical Biometry and Informatics LungenClinic Großhansdorf 3 Thoraxklinik at University Hospital Heidelberg *Presenting author 2
Introduction: Further advances in the pilot project based on data harmonization[1] and data integration tasks within the Disease Area Lung Cancer of the German Centre for Lung Research (DZL)[2] resulted in the establishment of a comprehensive data management workflow. This workflow covers three main steps: 1. Data Integration of a harmonized dataset into a research data warehouse (RDW), 2. cohort management inside the RDW based on the integrated parameters, 3. data export of a specified (sub-) cohort in a patient-oriented view for further analyses with statistical or data mining software. Methods: We extended the extract-transform-load (ETL) procedures implemented with Talend Open Studio to not only support i2b2 as RDW but also tranSMART. Within both RDW´s a user can manage patient cohorts by selecting parameters of interest. Each generated patient cohort is stored as a SQL representation inside the RDW. With a tool called Generic Case Extractor (GCE) these SQL statements can be used to either select a patient- or observation-oriented export of a cohort[3]. Results: We tested our three-step data management approach with a set of registry data of lung cancer patients. tranSMART provides advanced statistical analyses methods compared to i2b2 and is able to compare two sub-cohorts. The export generated with GCE is stored in a comma-separated-values (CSV) file. The user can specify export options like field delimiters or decimal delimiters. This file contains the harmonized parameters with defined characteristics and might be loaded into WEKA, a data mining program or into SPSS for statistical analyses. Discussion: In future, the integration of analytical data such as genotype data or expression data and their correlation with phenotype data might play a key role in all Disease Areas. As a first step in this direction, available microarray data will be integrated to gather experiences with these data formats.
[1] A Generic Data Harmonization Process for Cross-linked Research and Network Interaction: Construction and Application for the Lung Cancer Phenotype Database of the German Center for Lung Research. Methods Inf Med, 54(5), 455–460. [2] Data Harmonization and Data Integration Inside the Disease Area Lung Cancer of the German Center for Lung Research. Pneumologie, 69(07) [3] Unlocking Data for Statistical Analyses and Data Mining: Generic Case Extraction of
DZL Annual Meeting 2017 Clinical Items from i2b2 and tranSMART. In Studies in Health Technology and Informatics (Vol. 228, pp. 567–571). Submitted as: Presentation 297 words of 300 possible words
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Abstract No. 117 Using the DZL central data warehouse for queries and submitting data Raphael W. Majeed 1,* , Mark R. Stöhr 1, Clemens Ruppert 1, Jasmin Wagner 1, Daniel Firnkorn 2, Andreas Günther 1, and All members of the DZL plattform biobanking and data management 3 1
JLU Gießen University of Heidelberg 3 Platform biobanking and data management *Presenting author 2
In the DZL, more than 200 principal investigators conduct research, entering data into a manifold of study databases, cohorts, registries, research forms, bio banks and other software systems. The research data can only be accessed by the corresponding study personal at the research site. Calculating study populations or analyzing data across different un-connected registers/bio banks requires tremendous integration work on technical as well as semantic level. With the DZL central data warehouse, the DZL platform biobanking and data management provides a unified and simple interface for any researcher in the DZL to calculate simple statistics and patient counts across all studies, registers and biobanks: https://data.dzl.de/webclient/ With few mouse clicks, researchers can perform queries like the following: “How many male non-smoking patients aged between 70 and 80 are in the DZL with wholeblood-samples, no history of diabetes, FEV1 <80% and medication pirfenidone?” A technical or computer-science background is not required. Currently, the data warehouse contains only a small part of the total amount of patient related research data available in the DZL. In addition to the core dataset of all bio banks, a few selected registries are submitted to the data warehouse on a regular basis. Long-term goal is to make all relevant research data searchable through the data warehouse. To submit data to the data warehouse, the following steps are necessary: 1.Export the data from your primary system (e.g. Excel) 2.Download our data integration software 3.Configure parameters/field names (semantic annotation/harmonisation) 4.Run our software. Patient information is pseudonymized and securely transferred. Features and functionality of the central data warehouse are steadily extended over the next years. Future functionality includes partial data exports for statistical analysis, retrieving specimen sample counts as well as requesting bio samples for arbitrary patient collectives, integration of imaging data as well as genome sequencing/-omics data.
https://data.dzl.de/webclient/ Submitted as: Presentation 298 words of 300 possible words
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Abstract No. 132 Structured reporting of HR-CT examinations in post-lung transplantation patients Nina Hesse 1,* , Julien Dinkel 1, Nikolaus Kneidinger 2, Jessica Plum 1, Max Reiser 1, Wieland Sommer 1, and Felix Ceelen 2 1
Insitute for clinical radiology Department of Internal Medicine V *Presenting author
2
Purpose: To evaluate the effect of structured reporting of HR-CT studies of lungtransplanted patients on the content, clarity and clinical usefulness in clinical routine. Methods and Materials: Conventional and structured reports were generated for forty-nine lung transplanted patients who had undergone routine HR-CT. The template for the structured reports was designed to report on typical findings of acute and chronic allograft dysfunction. The conventional reports consisted of standard free-text. Two pulmonologist rated their degree of satisfaction on formative aspects, content, clarity and differentiation between CLAD-subtypes (BOS/RAS) from 1 (very dissatisfied or confusing) to 5 (very satisfied or very clear). Overall quality was rated from 1 (very good) to 6 (insufficient). Based on clinical and radiological information, clinicians had to hypothetically change therapy and decide for further diagnostic work-up. Wilcoxon and McNemar Test were used to test the differences between the report types. Results: Structured vs conventional reporting received in all categories significant higher ratings (p<0.0001): mean formative aspects 4.7 vs 2.9; mean content 4.4 vs 3.5; mean clarity 4.8 vs 3.5; differentiation between CLAD-subtypes 3.9 vs 2.9. Mean overall quality of structured reports was graded “good” and of conventional reports “satisfying” (1.5 vs 3.4; p<0.0001). There was no significant difference in patient therapy and diagnostic work-up. Conclusion: Referring clinicians perceive structured reports of HR-CT studies after lung transplantation as offering better content and greater clarity than conventional reports.
Verleden, S.E., et al., Linking clinical phenotypes of chronic lung allograft dysfunction to changes in lung structure. Eur Respir J, 2015. 46(5): p. 1430-9. Verleden, G.M., et al., Current views on chronic rejection after lung transplantation. Transpl Int, 2015. 28(10): p. 1131-9. 4. https://www.smart-radiology.com Submitted as: Presentation 228 words of 300 possible words
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Abstract No. 135 Case and sample management of the Lung Biobank Heidelberg – one year experience with STARLIMS Marc A. Schneider 1,* , Michael Meister 1, Christoph Doellinger 2, Mathias Wieland 2, Carmen Hoppstock 1, Christa Stolp 1, Andrea Bopp 1, Martin Fallenbuechel 1, Simone Krecik 1, Liz Meister 1, and Thomas Muley 1 1
Thoraxklinik at University Hospital Heidelberg Institute of Pathology *Presenting author 2
Introduction The lung research specimen biobank located at the Thoraxklinik Heidelberg (TK-HD) is an accredited subsidiary (tissue bank, DAP-IS-4153.04 according to DIN EN ISO / IEC 17020) of the National Center for Tumor Diseases Heidelberg (NCT), which is integrated into the BioMaterialBank Heidelberg (BMBH). Each year about 400-500 new tissue specimens and more than 2000 new blood samples are added to the biobank. Methods In 2015, we introduced 2D barcoded tubes and plates and STARLIMS as a case and sample management system. Results Using STARLIMS, samples are registered and stored traceable. Predefined filling schemes simplified the aliquoting steps. Pseudonymized clinical data can be linked to individual patient samples. An exit control as well as statistical overviews supports the biobank documentation and value. Discussion STARLIMS is an editable sample and case management system which simplifies the administration of sample data. Linking of samples with clinical data allows a fast overview on the availability of specific samples in phenotypically selected patient cohorts.
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Abstract No. 144 Visualizing perfusion and ventilation signal progression from non-contrast enhanced 2D-proton MRI measurements by use of phase maps David Bondesson 1,* , Olga Solyanik 1, and Julien Dinkel 1 1
University Hospital Munich, Ludwig-Maximilians-University Munich *Presenting author
INTRODUCTION Obtaining functional information in the human lung is of interest for characterizing lung pathologies. Fourier Decomposition (FD)[1] is a functional MRI method that extracts perfusion and ventilation information from contrast media-free untriggered fast gradient echo images. This work demonstrates the possibility of obtaining temporal highly resolved perfusion- and ventilation-weighted images, using phase information in the FD method to visualize signal progression. METHODS The technique utilizes a two-dimensional untriggered balanced steady-state free precession sequence (repetition time, 1.4 ms; echo time, 0.4 ms; acquisition time per image, 60 ms; flip angle, 50°; section thickness, 15 mm; matrix, 96 × 96). By applying Fourier transform along the temporal dimension, functional maps (perfusion and ventilation) were obtained. Phase information was then extracted from them. Ensuring that phase measurement started at zero phase shift and within an appropriate range (–π<ρ< π), all pixel were phase shifted with regards to a start pixel in the pulmonary trunk. By applying a running window over the full perfusion cycle, flow was visualized as a movie with 200 steps per cardiac or respiratory cycle. RESULTS Functional images of healthy volunteers and a patient diagnosed with COPD were successfully obtained. The generated images visualize the perfusion signal emanating from the pulmonary trunk, spreading through vessels and parenchyma towards the lung periphery in both the volunteers and COPD patient. Signal propagation of geometric changes of alveoli during breathing were about 120 ms. In areas of airway obstruction and emphysema, we found a distinct homogenous signal delay. DISCUSSION We have here presented a localized velocity measurement of perfusion signal flow within the lung and its ability to detect signal progression delay caused by COPD. The ventilation signal increases with alveoli density within pixels and thus visualize localized rates of inflation/deflation during inhalation/exhalation, and might be interesting for further clinical evaluation
1. Bauman G, Puderbach M, et al. Non-contrast-enhanced perfusion and ventilation assessment of the human lung by means of fourier decomposition in proton MRI. Magn Reson Med 2009;62:656–664. Submitted as: Presentation 299 words of 300 possible words
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Abstract No. 146 Biobanks of the DZL-Platform Biobanking Clemens Ruppert 1,* , Robert Bals 2, Karoline Gaede 3, Anne Hilgendorff 4, Thomas Illig 5, Ina Koch 6, Michael Lindner 6, Petra Ina Pfefferle 7, Thomas Muley 8, Andreas Günther 1, and all members of the Platform Biobanking and Data Management 9 1
UGMLC Giessen Biobank COSYCONET-Biobank Homburg 3 BioMateriaBank North @ Research Center Borstel 4 BioArchives @ Helmholtz Center Munich 5 Hannover Unified Biobank 6 Asklepios Biobank Gauting 7 Comprehensive Biomaterial Bank Marburg 8 LungenBioBank @ Thorax Clinic Heidelberg 9 DZL-Platform Biobanking & Data Management *Presenting author 2
Biomaterials are valuable tools in translational lung research. In order to provide research projects with biomaterials, currently 8 Biobanks from 6 DZL-sites are connected in the platform Biobanking and develop and harmonize standards and procedures regarding collection, processing and preservation of biomaterials. The samples itself are decentrally stored, and each local biobank has a different focus with regard to the collected phenotypes and specimen. The BioMaterialBank-North including Research+Center Borstel, LungClinicGrosshansdorf and UKSH-Lübeck, mainly collects blood, sputum and lavage samples from asthma, allergy, COPD, lung cancer, TB, sarcoidosis and other DPLDs. A specific feature is the use of the HOPE fixation technique and the isolation of primary cells. Hannover runs a central Biobank (HUB) for the Hannover medical School. Samples are stored in LN2-tanks and an automated -80°C repository (BiOS Hamilton). At UGMLC two biobanks exist. The Comprehensive Biomaterial Bank Marburg (CBBM) is the central biobank of the medical faculty and hosts lung tumor tissue and liquid samples from KIRA and ERA cohorts. The UGMLC-Giessen-Biobank has a strong focus on DPLD, PH, COPD and pneumonia. Next to liquid samples, it comprises a large collection of explanted lungs and isolated primary cells. The biobank is connected to large patient registries including the European-IPF-registry and the Giessen-PH-registry. The COSYCONET-biomaterialbank collects liquid biomaterials from recruited COPD patients in a long-term follow up. The LungenBioBank-Heidelberg (LBBH) is located at the Thoraxclinic and embedded in different network structures (NCTtissue-bank, BioMaterialBank-Heidelberg). The major expertise is on lung cancer (FFPE, native tissue, cells, liquids) and LC-data management. In Munic biobanking is established at two sites, the CPC-M Biomaterial Archive and the Asklepios Biobank for Lung Diseases at Gauting. Tissue, BALF, sputum, tracheal aspirates, blood derivatives are collected mainly from interstitial and chronic obstructive lung diseases, asthma. A special feature is primary cell extraction, immune monitoring and PCLS-culture.
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Abstract No. 147 Development and Implementation of a harmonized DZL-phenotype and specimen classification system Clemens Ruppert 1,* , Inga Bernemann 2, Karoline Gaede 3, Gudrun Helm 1, Anne Higendorff 4 , Thomas Illig 2, Ina Koch 5, Raphael Majeed 1, Thomas Muley 6, Britta Peschel 7, Marc Schneider 6, Jasmin Wagner 1, Bettina Weingard 8, and all members of the Platform Biobanking & Data Management 9 1
UGMLC Giessen Biobank Hannover Unified Biobank 3 BioMaterialBank North @ Reseacrh Center Borstel 4 BioArchives @ Helmholtz Center Munich 5 Asklepios Biobank Gauting 6 LungenBioBank @ Thorax Clinic Heidelberg 7 BioArcives @ Helmholtz Center Munich 8 COSYCONET Biobank Homburg 9 Platform Biobanking and Data Manegement *Presenting author 2
In frame of the harmonization procedure within the DZL Platform Biobanking and Data Management we developed a DZL-wide classification of lung disease phenotypes and biospecimen. The phenotype classification comprises lung diseases from all DZL Disease Areas divided into ten main categories. The clinical phenotypes are subcategorized in a modular system with a unique coding system. This code will allow an easy assignment of phenotypes defined at local sites within the central DZL data warehouse. If available ICD-10 codes are referenced with the DZL code and in the field of lung cancer the commonly used ICD-O codes are implemented. Biospecimen were categorized into blood derivates, swabs and brushings, lavages/aspirates/sputum, urine, tissue, isolated cells and others. A subcategorization was achieved by adding information for example with regard to type of sampling, origin, and fixation. A DZL-specific coding system has been implemented. Additionally, SPREC (Standard PREanalytical Codes) are provided if available. This uniform classification system will allow easy registration of biospecimen in the central DZL data warehouse and easy search for specific samples.
Submitted as: Presentation 170 words of 300 possible words
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Abstract No. 160 Regional gas-uptake measurements in the lung using hyperpolarized 129Xe magnetic resonance imaging and spectroscopy Agilo Luitger Kern 1,* , Filip Klimeš 1, Andreas Voskrebenzev 1, Marcel Gutberlet 1, Heike Biller 2, Kai Ruppert 3, Frank Wacker 1, Jens Hohlfeld 2, and Jens Vogel-Claussen 1 1
Hannover Medical School Fraunhofer Institute for Toxicology and Experimental Medicine 3 University of Pennsylvania *Presenting author 2
Lung diseases frequently manifest themselves by local pathologic alterations of lung tissue exhibiting impaired gas exchange. However, well-established methods for investigation of the lung’s diffusing capacity like DLCO are global measurements with limited sensitivity to local pathologies. We present hyperpolarized 129Xe dissolved-phase magnetic resonance imaging and chemical shift saturation recovery (CSSR) spectroscopy as means for local measurements of lung function. Upon inhalation, 129Xe diffuses through the alveolar walls and dissolves in lung parenchyma and blood, exhibiting a large chemical shift in Larmor frequency. A specially tailored RF pulse was used for frequency-selective excitation and imaging of the dissolvedphase xenon [1]. Relating the dissolved-phase xenon signal in one voxel to the corresponding gas-phase signal gives a quantitative measure for local gas-uptake in the lung. Using a Dixon-based method, the dissolved-phase signal can further be separated into a red-blood-cell and parenchyma/plasma phase, enhancing functional information. The dynamics of xenon uptake can be used for an estimation of various physiologic parameters related to lung function, such as the alveolar septal thickness, surface-to-volume ratio and capillary transit time of blood. A CSSR sequence was implemented and optimized for the available hardware (Avanto 1.5T, Siemens). After irradiation of a saturation pulse and subsequent spoiling of transverse magnetization, spectra of dissolved-phase xenon are acquired at different delay times. Physiologic parameters can then be obtained by fitting the Patz model function for gas uptake to the ratios of spectral peak areas [2]. The CSSR sequence does not feature any spatial encoding and hence provides a global measurement. Spectral localization has been achieved by use of a dedicated 16-channel phased-array coil, a subsequent acquisition of a ventilation image for coil sensitivity mapping and application of the Spectral Localization Achieved by Sensitivity Heterogeneity (SPLASH) method [3]. The described methods are now established and ready for use in clinical trials.
[1] Qing, K et al.: Regional Mapping of Gas Uptake by Blood and Tissue in the Human Lung Using Hyperpolarized Xenon-129 MRI. J Magn Reson Imag 2014 [2] Patz, S et al.: Diffusion of hyperpolarized 129Xe in the lung: a simplified model of 129Xe septal uptake and experimental results. New J Phys 2011 [3] An, L et al.: Spectral Localization by Imaging Using Multielement Receiver Coils. Magn Reson Med 2011
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Abstract No. 169 Diagnostic accuracy of MRI for the detection of pulmonary nodules using a chest phantom Olga Solyanik 1,* , Katharina Hellbach 1, Madeleine Bonert 1, David Bondersson 1, Thomas Gaaß 1, Natalie Thaens 1, Maximilian Reiser 1, and Julien Dinkel 1 1
Institute for clinical Radiology, LUM Campus Großhadern *Presenting author
Aim: To evaluate the sensitivity of modern MR-sequences for the detection of pulmonary nodules using a dedicated porcine chest phantom. Material and methods: Four porcine lungs containing a total of 131 variable sized nodules made of agar were inflated inside a phantom. Two three-dimensional (3D) gradient-echo (GRE) and one steady state free precession (SSFP) MR-sequences were acquired with optimized short TE (0.01 -0.6ms). Spiral computed tomography served as the standard of reference. Two blinded readers (with 2 (reader 1) and 4 (reader 2) years experience in chest radiology) analysed the MR-images independently and recorded the size and location of the nodules. Results: Pulmonary nodules were divided into four groups depending on the nodules´ size: A: 1.0-1.9 mm (n=57); B: 2.0-2.9 mm (n=50); C: 3.0-3.9 mm (n=14); D: 4.0-4.9 mm (n=10). Interobserver agreement was excellent (k=0.9) for D nodules with a sensitivity of 0.95 for SSFP-sequence and 0.75 for GRE-sequences. The interobserver agreement decreased (k=0.46) with smaller nodules. Reader1 showed a sensitivity of 0.60 (all nodules), 0.58 (A), 0.56 (B), 0.67 (C) for SSFP-sequence and - of 0.54 (all nodules), 0.50(A), 0.53(B), 0.60(C), 0.65(D) for 3D-GRE-sequences. Reader2 showed a sensitivity of 0.77 (all nodules), 0.70 (A), 0.83 (B), 0.71 (C) for SSFP-sequence and - 0.65 (all nodules), 0.58(A), 0.68(B), 0.71(C), 0.80(D) for 3D-GRE-sequences. Conclusions: MRI showed a high to good sensitivity for the detection of pulmonary nodules depending on the readers´ experience and nodule size. The SSFP-sequence showed higher sensitivity than 3D-GRE-sequences, reaching 0.95 sensitivity in nodules bigger than 4mm.
Submitted as: Presentation 249 words of 300 possible words
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Abstract No. 223 Computational imaging for assessing airway remodeling in Drososophila melanogaster Hinnerk Schulz-Hildebrandt 1,* , Mario Pieper 1, Thomas Roeder 2, Peter König 1, and Gereon Hüttmann 1 1
University of Lübeck University of Kiel *Presenting author 2
Intravital imaging provides important information on the morphology and function of biological tissues. Today, the workhorse of intravital imaging is 2-photon microscopy which provides cellular resolution down to a millimeter of depth. We recently introduced microscopic OCT (mOCT) as an alternative imaging modality. mOCT does not need special fluorescent markers and is not limited in imaging speed or imaging duration by photodamage. Tissue morphology and mucus transport were successfully imaged at micrometer resolution in trachea of spontaneous breathing mice using mOCT. Drosophila melanogaster was recently introduced as an animal models to study asthma, COPD or lung cancer. Aim of this study was to demonstrate, that mOCT can image remodeling of the fly's airways. Larvae of Drosophila melanogaster were imaged at about 1.5 micrometer resolution with a high resolution mOCT set-up, which consists of a supercontinuum light source (Super-K OCT, NKT Photonics) and a specially designed broad-band spectrometer (Thorlabs GmbH). Motion artifacts were considerably reduced by cooling of the animal. An optimization algorithm, which uses the phase of the OCT data, was applied to compensate aberrations as described previously [1]. Volumetric images were significantly degraded by defocus blur and sample induced aberrations. Using our computational aberration, correction image quality was improved and lateral resolution was restored to nearly diffraction limit. Changes of the airway morphology were successfully imaged in the drosophila larvae over time. The coherent detection of the scattered light enables computational correction of sample induced aberrations for a real volumetric microscopy (i.e. tomography at nanometer resolution). Our rather preliminary results demonstrate that mOCT combined with aberration correction is able to provide quantitative information on remodeling of airways in living larvae of Drosophila melanogaster.
1. D. Hillmann, H. Spahr, C. Hain, H. Sudkamp, G. Franke, C. Pfäffle, C. Winter, and G. Hüttmann, "Aberration-free volumetric high-speed imaging of in vivo retina," Scientific reports 6, 35209 (2016). 10.1038/srep35209. Submitted as: Presentation 274 words of 300 possible words Page 240 of 286
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Abstract No. 240 DZL Biobanking at BREATH Inga Bernemann 1,* , Markus Kersting 1, Jana Prokein 1, Kirstin Mittelstrass 1, Norman Klopp 1 , and Thomas Illig 1 1
Hannover Medical School (MHH) *Presenting author
With the Hannover Unified Biobank (HUB), the central biobank of Hannover Medical School (MHH), the DZL site BREATH is featured with a modern, harmonized and central biobank for all DZL biobank affairs at BREATH. As a member of the DZL platform biobanking HUB is involved in the development of harmonized DZL biobank solution covering quality and data management, as well as connecting biobanks and research databases to the DZL data ware house. HUB established the hosting of the DZL central ID-management of DZL in close cooperation with the MHH computing centre (ZIMt) and the BREATH data management group. Based on the DZL ID structure, HUB delivers data about quality and storage of the biomaterials for all DZL projects at BREATH to the central DZL data warehouse. To enable standardization in BREATH/DZL the modern infrastructure with hautomation in processing and storage can be used by all members of the DZL site BREATH. The HUB storage infrastructure comprises liquid nitrogen tanks and an automated -80°C repository with a -80°C cooled picking station for quality assured and temperature controlled composition of requested and issued samples. Local data management occurs in a professional Biobank Information Management System (BIMS). The BIMS stores and manages sample quality, processing, storage and retrieval data. Additionally it separately manages minimal clinical data information and complies thereby with the principles of data protection developed by TMF - Technology, Methods, and Infrastructure for Networked Medical Research. Thereby continuous sample tracking becomes possible and retention times as well as preparation steps are documented. Sample storage with continuous temperature monitoring and sample rearrangements is documented in the BIMS. The temperature monitoring system of HUB is validated. All freezing units are equipped with temperature loggers and connected to an alarm system. An emergency power supply and an emergency plan complete the intervene structure.
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Abstract No. 241 Biobanking at CPC-M Katharina Heinzelmann 1, Britta Peschel 1, Marion Frankenberger 1, Ina Koch 2, Michael Lindner 2, Jürgen Behr 3, Oliver Eickelberg 4, and Anne Hilgendorff 5,* 1
Comprehensive Pneumology Center/Helmholtz Zentrum München Asklepios Fachkliniken München-Gauting 3 Asklepios Fachkliniken München-Gauting/Medizinische Klinik und Poliklinik V, Klinikum der Ludwig-Maximilians-Universität, Munich 4 Comprehensive Pneumology Center/Helmholtz Zentrum München; Division of Respiratory Sciences and Critical Care Medicine, University of Colorado, Denver, USA 5 Comprehensive Pneumology Center/Helmholtz Zentrum München; University Hospital of the Ludwig-Maximilians University *Presenting author 2
Rationale. Access to human biomaterial is key for translational research to connect basic experimental research approaches with clinical studies to develop diagnostic and treatment procedures. The CPC-M is one of six collaborating sites within the biobank structure for the German Center for Lung Research (DZL). Methods. The CPC-M BioArchive comprises 9450 samples from 479 patients with acute and chronic lung diseases spanning studies in neonatal chronic lung disease, pediatric asthma, fibrosis/IPF, COPD/emphysema and cancer. With the University Hospital of the LudwigMaximilians University being one of the leading lung transplant sites in Germany, the CPCM BioArchive routinely collects samples from patients with end-stage lung disease undergoing lung transplantation including follow up samples and donor lung tissue. Sampling of biomaterial including tissue, BAL, blood, and sputum, among others, is done using standard operating procedures. The unique samples allow for routine isolation of primary cells from tissue and blood including fibroblasts, bronchial and alveolar epithelial cell, and monocytes. Further sample processing provides easy access to FFPE tissue sections, DNA, RNA and protein extracted from tissue and cells. Clinical phenotyping is matched by lung function and pulmonary imaging, including CT, MRI and PET scan. The implemented data warehouse structure integrates large data sets of clinical phenotyping including imaging data in context of exact tracking of obtained samples and analyses performed. Together with the establishment of a supervising board, the data warehouse allows for the complex design of new scientific ideas and the profound connection of different research areas. Summary and Conclusion. CPC-M provides numerous distinct sample types including serial sampling and primary cell isolation from different lung disease cohorts and spanning different diseases stages and age groups. The overall structure of sample collection allows easy access to high quality samples in context of clinical phenotyping, enabling translational research on a high international scientific standard.
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Abstract No. 268 Evaluation of biventricular systolic and diastolic cardiac function in experimental pulmonary arterial hypertension by micro computed tomography Baktybek Kojonazarov 1,* , Alexander Belenkov 2, Marian Kampschulte 1, Hossein Ardeschir Ghofrani 1, Friedrich Grimminger 1, Norbert Weissmann 1, Werner Seeger 1, and Ralph Schermuly 1 1
Universities of Giessen and Marburg Lung Center PerkinElmer *Presenting author 2
Introduction. Micro-computed tomography (µCT) is a powerful imaging modality for noninvasive and longitudinal investigation of animal models of human disease. Although assessment of the systolic heart function by µCT in small animals is highly appreciated, the feasibility of evaluating diastolic heart function has not been demonstrated yet. Methods. Severe angioproliferative PAH was induced in rats by combined exposure to the VEGFr antagonist SU5416 and hypoxia (SUHx). Echocardiography (Vevo2100, Visualsonics, Canada), contrast-enhanced cardiac µCT (Quantum GX, PerkinElmer, USA) and invasive hemodynamic measurements were assessed 35 days after SUHx or normoxic exposure. Multi-phase µCT reconstructions were performed with temporal resolution of 16.34 msec. Right and left ventricular (RV and LV) heart chambers were segmented in order to construct volume-time and dV/dt curves. RV and LV volumes and diastolic functional indices such as peak-filing rate (PFR) and time to peak-filing (TPFR) were derived. Results. SUHx rats developed severe pulmonary arterial hypertension, obstructive-like lesions of the small pulmonary arteries and RV hypertrophy. Noninvasive evaluation of the cardiac function demonstrated dilatation of the RV and decreased stroke volume (SV) in SUHx rats in comparison with normoxic healthy controls. The RV diastolic indices such as PFR and PFR/EDV were significantly lower in SUHx rats in comparison with healthy controls. In addition, TPFR and filling time prolonged significantly in SuHx rats in comparison with healthy animals. Moreover, echocardiographic derived e/e’ and invasively measured RV EDP correlated very well with µCT derived PFR and TPFR values. Conclusion. Here, we, for the first time, developed a method that extends the use of µCT for simultaneous interrogation of both systolic and diastolic heart function of the left and right ventricles in animal models of cardiovascular diseases. This method allows for rapid and accurate assessment of cardiac functional indices and paves the way for more extensive studies of cardiovascular research.
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Abstract No. 269 A systematic comparison of lung ventilation between Fourier Decomposition and Jacobian Determinant method Filip Klimeš 1,* , Andreas Voskrebenzev 1, Marcel Gutberlet 1, Frank Wacker 1, and Jens Vogel-Claussen 1 1
Institute of Diagnostic and Interventional Radiology *Presenting author
Introduction: Fourier Decomposition (FD) [1, 2] is able to assess lung ventilation and perfusion in free breathing without any contrast agent. After registration, the pulsatile blood influence needs to be separated from proton density changes to assess ventilation. The registration process itself contains information of voxel deformation, which by means of Jacobian Determinant (JD) can be used to quantify lung ventilation [3, 4]. According Patz et al. FD will be affected by blood volume changes during respiration and for this reason suggest the use of JD [5]. Considering the blood distribution, the main purpose of this study is the systematic comparison of FD and JD in ventral-dorsal direction. Material and Methods: Eleven healthy volunteers underwent an examination on a 1.5 T scanner. A 2D FLASH sequence was used to acquire images of coronal (middle, ventral, dorsal) slices during deep breathing. After registration towards the intermediate respiratory image, a fractional ventilation (FV) of FD and JD was calculated according to Zapke et al. [1] and Kjørstad et al. [4], respectively. Mean global fractional ventilation FVFD and FVJD were compared with ttests and Bland-Altman plots in ventral-dorsal direction. Additionally, the distribution of ventilation maps in apical-basal direction was evaluated using the moving average method. Result: Significant differences between FVFD and FVJD ventilation maps were found only in dorsal parts of lungs. Furthermore a strong apical-basal gradient of FVJD occurred for all slice positions. The most pronounced changes were present in middle (increase by 55%) and dorsal (increase by 157%) slice positions. Contrary, no significant gradients were found for FVFD in apical-basal direction. Conclusion: The results suggest further investigation regarding the influence of blood volume on FD.
Zapke, M.: Magnetic resonance lung function–a breakthrough for lung imaging and functional assessment? A phantom study and clinical trial. Respiratory Research. DOI: 10.1186/1465-9921-7-106. ISSN 14659921. Bauman, G.: Non-contrast-enhanced perfusion and ventilation assessment of the human lung by means of Fourier decomposition in proton MRI. Magnetic Resonance in Medicine, 2009, DOI: 10.1002/mrm.22031. ISSN 07403194. Chow, K.: Measurement of Regional and Global Lung Ventilation Using Non-Rigid Image Registration. Magnetic Resonance in Medicine, 2009. Kjørstad, Å.: Quantitative lung ventilation using Fourier decomposition MRI; comparison and
DZL Annual Meeting 2017 initial study. Magnetic Resonance Materials in Physics, Biology and Medicine, 2014. DOI: 10.1007/s10334-014-0432-9. ISSN 0968-5243. Patz, S.: Ventilation Dependent Blood Volume in Fourier Decomposition 1H Lung Imaging. Magnetic Resonance in Medicine. 2011. Submitted as: Presentation 274 words of 300 possible words
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Abstract No. 278 Depiction of pneumothoraces in a large animal model using x-ray dark-field radiography Katharina Hellbach 1,* , Andrea Bähr 2, Fabio De Marco 3, Konstanttin Willer 3, Lukas Gromann 3, Julia Herzen 3, Michaela Dmochewitz 2, Sigrid Auweter 1, Alexander A. Fingerle 3 , Peter B. Noël 3, Ernst J. Rummeny 3, Andre Yaroshenko 4, Ingo Maack 4, Thomas Pralow 4 , Hendrik van der Heijden 4, Nataly Wieberneit 4, Roland Proksa 5, Thomas Koehler 5, Karsten Rindt 4, Tobias Schroeter 6, Juergen Mohr 7, Fabian Bamberg 1, Birgit Ertl-Wagner 1, Maximilian F. Reiser 1, and Franz Pfeiffer 3 1
Ludwig-Maximilians-University Hospital Munich Ludwig-Maximilians-University Munich 3 Technische Universität München 4 Philips Medical Systems DMC GmbH 5 Philips GmbH Innovative Technologies 6 Karlsruher Institut für Technologie 7 Karlsruher Institut für Technologie *Presenting author 2
Purpose The aim of this study was to assess added clinical value of dark-field radiography in pneumothorax diagnosis using a pig model. Materials and Methods Experiments were performed using 2.5 months old, wild-type German landrace pigs (n=6). The animals were anesthetized, intubated and mechanically ventilated during the experiments. All pigs were imaged with an experimental grating-based large animal scanner to acquire x-ray transmission and dark-field radiographs before and after induction of a unilateral pneumothorax. All scans were performed in posterior-anterior (p.a.) direction under respiratory arrest. Image contrast ratios between lung tissue and the air filled pleural cavity were quantified for both, transmission and dark-field radiograms. Results Images revealed that all animals had developed a unilateral pneumothorax. Pneumothoraces displayed as areas with no dark-field signal next to the adjacent lung parenchyma, which generated a strong dark-field signal. The contrast ratio between the air filled pleural space of the pneumothoraces and lung tissue was significantly higher in the dark-field (2.95 ± 0.93) than in the transmission images (0.95±1.04; p < 0.05) when images were acquired in p.a. direction. Consequently, detection of pneumothoraces was easier when analyzing the dark-field images. Conclusion This study shows increased contrast between lung parenchyma and air in the pleural space in x-ray dark-field radiography as compared to conventional chest x-ray in a large animal model in p.a. images. This makes this technique a promising tool for facilitated diagnosis of pneumothoraces.
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Submitted as: Presentation 232 words of 300 possible words
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Abstract No. 279 X-Ray Dark-field Imaging to Monitor the Development of Acute Lung Injury in Mice Katharina Hellbach 1,* , Felix Meinel 1, Thomas Conlon 2, Konstantin Willer 3, Andre Yaroshenko 3, Astrid Velroyen 3, Sigrid Auweter 1, Maximilian Reiser 1, Oliver Eickelberg 4, Franz Pfeiffer 3, and Ali Önder Yildirim 4 1
Ludwig-Maximilians-University Hospital Munich Helmholtz Zentrum München 3 Technische Universität München 4 Helmholtz Zentrum München *Presenting author 2
Objective The aim of this study was to evaluate the feasibility of early stage imaging of acute lung injury in a murine model of pulmonary emphysema with grating-based X-ray dark-field imaging in vivo. Materials and Methods Acute lung injury was induced in female, 8-10 week old C57Bl/6N mice by orotracheal instillation of porcine pancreatic elastase (80 U/kg BW) (n=11). Control mice (n=10) received orotracheal instillation of PBS. Mice were imaged immediately before and 1 day after the application of elastase or PBS to assess acute changes in pulmonary structure due to lung injury. Subsequently, 6 mice from each group were sacrificed and their lungs were lavaged and explanted for histological analysis. A further 7, 14 and 21 days later the remaining mice were imaged to assess the development of acute lung injury as well as emphysema progression. All images were acquired with a prototype grating-based small-animal scanner to generate dark-field and transmission radiographs. At day 21, lungs were lavaged and obtained for histopathological analysis. Results Lavage confirmed that mice in the experimental group had developed acute lung injury one day after administration of elastase. Additionally, lungs showed signs of early pulmonary emphysema, which continuously worsened until the end of the experiment. Acute lung injury was visible as a striking decrease in signal intensity of the pulmonary parenchyma on darkfield images at day 1. Quantitative analysis confirmed that dark-field signal intensity at day 1 was significantly lower than signal intensities measured at the remaining timepoints. There was no significant alteration in median transmission signal intensity. Conclusion The development of acute lung injury in vivo can be monitored with dark-field radiography.
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Abstract No. 281 3D Lung Ventilation 1H Imaging using a Pseudo 3D Approach or Alternatively a SelfNavigated Sequence in Comparison with 2D Lung Fourier Decomposition Andreas Voskrebenzev 1,* , Marcel Gutberlet 1, Frank Wacker 1, and Jens Vogel-Claussen 1 1
Diagnostic and Interventional Radiology *Presenting author
Introduction: Fourier Decomposition (FD) is a proton 2D MRI technique to assess regional ventilation of the lung without the necessity for breath-hold and contrast agent [1]. Besides the advantages of FD it is not apparent how to combine the individual slice results to one consistent lung data set. Following the idea that an additional sagittal scan can be used as a navigator [2], the feasibility of a pseudo 3D fractional ventilation mapping (p3D-FV) is assessed and compared with a 3D self-navigation approach (3D-FV) and the contemporary method (FD-FV). Methods: Eight healthy volunteers were examined on a 1.5T scanner. Coronal FD scans covering the whole lung, a sagittal scan of the right lung and a 3D volume scan of the whole lung were acquired in free breathing. FD-FV was calculated as described previously [3]. For p3D-FV, the sagittal slice was used to create a dynamic pseudo 3D data set ranging from end-inspiration to end-expiration. For 3D-FV, a stack-of-stars gradient echo sequence with a golden angle increment [4] was used to reconstruct images in inspiration and expiration. For 3D-FV and p3D-FV, images in expiration were registered towards the inspiration images and quantified according to Zapke et al. [5]. Result: All three methods showed high vessel/parenchyma sharpness and a good qualitative agreement. The correlation of lung mean values resulted in high coefficient of determination for FD-FV and p3D-FV (R2=75%), FD-FV and 3D-FV (R2=85%) and p3D-FV and 3D-FV (R2=84%). Nevertheless, the quality of p3D-FV maps was not robust and the 3D-FV maps showed regions with artificially low FV values in some slices. As expected, 3DFV as a function of slice position showed the most consistent results. Conclusion: Besides minor stability and reconstruction problems, this study demonstrates the feasibility of p3D-FV and 3D-FV and a good concordance with FD-FV.
[1] G. Bauman et al.: Non-contrast-enhanced perfusion and ventilation assessment of the human lung by means of fourier decomposition in proton MRI. Magn Reson Med 62 (2009), S. 656-664 [2] Y. Masuda, H. Haneishi: 4D MR imaging of respiratory organ motion using an intersection profile method. Medical Imaging 7625 (2010), S. 76250Z [3] Voskrebenzev A, Gutberlet M, Kaireit TF, Wacker F, Vogel-Claussen J. Low-pass imaging of dynamic acquisitions (LIDA) with a group-oriented registration (GOREG) for proton MR imaging of lung ventilation. Magn Reson Med 2016;10.1002/mrm.26526 [4] R. Grimm et al.: Self-gating Reconstructions of Motion and Perfusion for Free-breathing T1-weighted DCE-MRI of the Thorax Using 3D Stack-of-stars GRE Imaging. Proc. Intl. Soc. Mag. Reson. Med. 20 (2012), S. 598 [5] M. Zapke et al.: Magnetic resonance lung function - a breakthrough for lung imaging and functional assessment? A phantom study and clinical trial. Respir Res 7 (2006), S. 106
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Abstract No. 284 Comparison of real-time dynamic fluorinated gas MRI in free breathing with clinical lung function test in patients with COPD Marcel Gutberlet 1,* , Andreas Voskrebenzev 1, Till Kaireit 1, Agilo Kern 1, Julia Freise 1, Tobias Welte 1, Jens Hohlfeld 2, Frank Wacker 1, and Jens Vogel-Claussen 1 1
Hannover Medical School Fraunhofer ITEM *Presenting author
2
Purpose: While lung function test used as clinical standard for diagnosis and monitoring of chronic obstructive pulmonary disease (COPD) assesses only global lung ventilation, realtime dynamic fluorinated (19F) gas magnetic resonance imaging (MRI) in free breathing may allow quantification of regional lung ventilation. Therefore, purpose of this work was to compare regional lung ventilation derived by dynamic 19F gas MRI averaged over the whole lung with spirometry as clinical test for COPD. Methods: 11 patients with COPD were enrolled in this study. Each patient inhaled 30l of a mixture of 79% fluorinated gas (C3F8) and 21% oxygen via closed face mask. For safety reasons, over the whole scan time patients were monitored for several physiological parameters1. Then, the subjects inhaled 100% O2 and the measurement of the 19F washout dynamics was started. After sorting data corresponding to the different respiratory phases, regional washout time was quantified with different parameters (washout time in seconds, number of breaths and fractional ventilation). Within the same day every patient underwent lung function test (forced expiratory volume in 1 second (FEV1) and ratio of FEV1/ forced vital capacity (FVC)). Results: After averaging over the whole lung, 19F gas washout parameters quantifying regional lung ventilation showed an excellent correlation compared to parameters of lung function test with highest correlation between number of breaths for 19F gas washout and FEV1 (%predicted) with Spearman’s correlation coefficient of 0.83 (p=0.007). Conclusion: Regional lung ventilation mapping derived by 19F gas washout MRI could add clinical value for monitoring of patients with COPD in the future.
1. Halaweish AF, Charles HC. Physiorack: an integrated MRI safe/conditional, gas delivery, respiratory gating, and subject monitoring solution for structural and functional assessments of pulmonary function. J Magn Reson Imaging. 2014;39(3):735-41. Submitted as: Presentation 255 words of 300 possible words
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Abstract No. 287 The Image eVAluation Service (IVA) – Software as a Service (SaaS) for the German Center for Lung Research (DZL) Iven Fellhauer 1,* , Simon Triphan 1, Oliver Weinheimer 1, Bertram Jobst 1, Mark Wielpütz 1, Kerstin Burmester 1, and Hans-Ulrich Kauczor 1 1
Heidelberg University Hospital *Presenting author
Obtained in 2016 and available in mid 2017, the Image eVAluation service (IVA) provides cloud-based computing power as a service for image quantification for the members of the German Center for Lung Research (DZL). The service is implemented as a computing grid to all DZL members. IVA provides a variety of software tools for CT and MRI lung imaging quantification, starting with a software solution for CT lung imaging data (YACTA). Depending on the requirements of the DZL members, IVA will be extended with additional software toolboxes for image quantification of other body parts e.g. brain volumetry. IVA is run in a fully virtualized environment to give access to toolboxes native to the Windows and Linux operating system. Both the results of the image analysis as well as the image data itself are transferred to the Image Bank and are available there for the generation of cohorts and as reference values for software benchmarks.
Submitted as: Presentation 154 words of 300 possible words
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Abstract No. 018 C-type lectin Mincle recognizes glucosyl-diacylglycerol of Streptococcus pneumoniae and plays a protective role in pneumococcal pneumonia Friederike Behler-Janbeck 1, Tomotsugu Takano 2, Regina Maus 1, Jennifer Stolper 1, Danny Jonigk 1, Meritxell Tort Tarres 1, Thomas Fuehner 1, Antje Prasse 1, Tobias Welte 1, Tomofumi Miyamoto 2, Sho Yamasaki 2, and Ulrich A. Maus 1,* 1
Medical School Hannover Kyushu University *Presenting author 2
Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We show that gene expression of Macrophage inducible C-type lectin Mincle was induced in lungs of mice infected with S. pneumoniae and in patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the pneumococcal ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.
Submitted as: Presentation 166 words of 300 possible words
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Abstract No. 044 Macrophages render alveolar epithelial cells hypo-responsive to Legionella pneumophila Christine Schulz 1, Xin Lai 2, Anna Lena Jung 1, Alexandra Sittka-Stark 1, Christina Herkt 1, Wilhelm Bertrams 1, Julio Vera 2, and Bernd Schmeck 3,* 1
Philipps-University Marburg, Institute for Lung Research Erlangen University Hospital, Laboratory of Systems Tumor Immunology, Department of Dermatology 3 Philipps-University Marburg, Institute for Lung Research; University Medical Center Giessen and Marburg, Department of Medicine, Pulmonary and Critical Care Medicine *Presenting author 2
Pneumonia is a leading cause of mortality worldwide. To secure organ function, pulmonary innate immune response has to be tightly regulated. Here, we analyze whether macrophages influence immune reactivity of type II alveolar epithelial cells during infection with an intracellular bacterial pathogen. For this purpose, human macrophage-like differentiated THP-1 cells were co-cultured with human alveolar epithelial A549 cells in a transwell-setting. Infection of THP-1 cells with the important respiratory pathogen Legionella pneumophila (L. pneumophila) resulted in the release of pro-inflammatory cytokines, e.g. IL-8, by co-cultured, non-infected epithelial cells. This effect was synergistically blocked by an IL-1 receptor antagonist (IL-1ra) and a TNF-α neutralizing antibody (anti-TNF-α). Furthermore, co-culture with infected THP-1 cells reduced cytokine expression by epithelial cells following direct encounter with L. pneumophila. This epithelial hypo-responsiveness could be mimicked by stimulation with IL1β. It was accompanied by an accelerated pro-inflammatory mRNA decay, long-lasting degradation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and reduced nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα)-degradation and less nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) translocation into the nucleus. miR 146a was induced, but did not mimic epithelial hypo-responsiveness in physiological concentrations. Likewise, histone modifying enzymes did not reproduce the effect. Corroborated by in silico simulations, our results demonstrate that macrophages can negatively regulate the responsiveness of lung epithelial cells to bacterial infection by the release of IL-1β resulting in a downregulation of IRAK-1. This intercellular communication may be critical for avoiding overwhelming inflammatory response in the lung.
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Abstract No. 045 Genome-wide chromatin profiling of Legionella pneumophila-infected human macrophages reveals activation of the pro-bacterial host factor TNFAIP2 Ilona Du Bois 1, Annalisa Marsico 2, Wilhelm Bertrams 1, Michal Schweiger 3, Brian E. Caffrey 4, Alexandra Sittka-Stark 1, Martin Eberhardt 5, Julio Vera 5, Martin Vingron 4, and Bernd Schmeck 6,* 1
Philipps-University Marburg, Institute for Lung Research/iLung Max Planck Institute for Molecular Genetics; Free University Berlin 3 University of Cologne, Functional Epigenomics 4 Max Planck Institute for Molecular Genetics, Berlin 5 University Hospital Erlangen, Department of Dermatology, Laboratory of Systems Tumor Immunology 6 Philipps-University Marburg, Institute for Lung Research; University Medical Center Giessen and Marburg, Department of Medicine, Pulmonary and Critical Care Medicine *Presenting author 2
Introduction Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. Infection leads to a broad host cell response, as evident e.g. on the transcriptional level. Chromatin modifications, which control gene expression, play a central role in the transcriptional response to L. pneumophila. Methods We infected human blood-derived macrophages with L. pneumophila and used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to screen for gene promoters with the activating histone 4 acetylation mark (pan-acH4). We transferred these findings to investigate the effect of L. pneumophila on blood-derived macrophages. Results We found the promoter of tumor necrosis factor, alpha-induced protein 2 (TNFAIP2) to be acetylated at histone H4. This factor has not been characterized in the pathology of L. pneumophila. TNFAIP2 mRNA and protein were upregulated in response to L. pneumophila infection of human blood-derived macrophages and human alveolar epithelial cells (A549). We show that L. pneumophila-induced TNFAIP2 expression is dependent on NF-κB. Importantly, a knockdown of TNFAIP2 led to reduced intracellular replication of L. pneumophila Corby in A549 cells. The TNFAIP2 promoter furthermore controls the expression of the long non-coding RNA linc00677 which might be accessory to TNFAIP2 expression. Discussion Taken together, genome-wide chromatin analysis of L. pneumophila-infected macrophages demonstrated induction of TNFAIP2, a NF-κB-dependent factor relevant for bacterial replication. Besides linc00677, we found numerous long non-coding RNAs that were promoter-acetylated upon L. pneumophila infection. These RNAs might also contribute to Legionella pathogenesis.
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Abstract No. 070 A Bacterial Signal Peptide Increases Mucociliary Clearance in Explanted Mouse Trachea Alexander Perniß 1,* , Bernd Bufe 2, Frank Zufall 2, Gabriela Krasteva-Christ 2, and Wolfgang Kummer 1 1
JLU Giessen University of Saarland *Presenting author 2
Objective: Bacterial signal peptides are known to trigger innate immunity responses by activation of formyl peptide receptors (FPRs) present in immune cells, e.g. leukocytes. Members of the FPR-family are also found in the murine vomeronasal organ where they are candidates for chemosensory recognition of bacterial pathogens. Here, we investigated the effects of bacterial signal peptides on mucociliary clearance in the murine trachea. Methods: The trachea of C57Bl6, TRPM5-deficient (transient receptor potential cation channel subfamily M member 5; a crucial component of the canonical bitter and umami taste transduction) and FVB/NCrl mice was explanted and particle transport speed (PTS) was visualized by tracking directed transport of dynabeads at the surface. The transcriptome of single tracheal ciliated and brush cells, a chemosensory epithelial cell type, was analyzed by single cell deep sequencing. Results: Deep sequencing showed FPR expression in both ciliated and brush cells. The Nformylated bacterial signal peptide FL185 increased PTS from 43.48±5.05 to 75.96±3.56 µm/s (N=8; p<0.0001) at 10 µM which addresses FPR1-3. Specific FPR1 and FPR2 inhibitors [cyclosporine H (1 µM) and t-BOC2 (10 µM)] did not reduce the effect. The effect was conserved in FVB/NCrl mice which are lacking a functional FPR3. In contrast, FL185 was ineffective in increasing PTS in TRPM5-deficient mice. More than ten other tested bacterial signal peptides did not increase PTS. Conclusion: A bacterial signal peptide stimulates cilia-driven mucociliary clearance, which could represent a novel defense mechanism against invasive bacteria in the trachea. This effect involves elements of the classical taste transduction cascade
Submitted as: Presentation 249 words of 300 possible words
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Abstract No. 075 Optogenetic mouse strains for studying airway innervation Amir Rafiq 1,* , Nodir Mirsaidov 2, Klaus Deckmann 1, Martin Bodenbenner-Türich 1, and Wolfgang Kummer 3 1
Institute for Anatomy and Cell Biology, JLU Giessen Department of Urology, Pediatric Urology and Andrology, JLU Giessen 3 Institute for Anatomy and Cell Biology, DZL, JLU Giessen *Presenting author 2
Airway function is profoundly controlled by parasympathetic, sympathetic and peptidergic sensory nerve fibers. Traditionally, the function of airway innervation is studied by electric field stimulation or by pharmacologic activation or inhibition. These methods allow only poor discrimination between fiber subtypes. Optogenetics is a new promising tool to solve these questions of specificity and selectivity. A blue light sensitive channelrhodopsin 2 (ChR2) from Chlamydomonas reinhardtii has been inserted in mice to create cell-specific expression of ChR2. Light stimulation induces depolarization and release of neurotransmitters from ChR2-expressing neurons. We here generated two mice strains expressing ChR2 in the major subtypes of neurons expected to induce bronchoconstriction, i.e. cholinergic (parasympathetic, expressing choline acetyltransferase = ChAT) and peptidergic sensory neurons, expressing transient receptor potential cation channel subfamily V member 1 (TRPV1), respectively, by crossbreeding ChAT and TRPV1 cre driver lines with Ai27D mice, expressing a ChR2-tdTomato fusion protein following exposure to Cre. In “cholinergic mice”, native tomato-fluorescence was observed in known cholinergic neurons in the central nervous system, in parasympathetic postganglionic fibers in the airways, heart, urinary bladder and in the enteric nervous system. Non-neuronal cholinergic epithelial cells of the trachea, gall bladder, urethra and thymic medulla lacked native tomatofluorescence. In organ-bath experiments, light stimulation activated cholinergic nerves of this strain leading to contraction of the urinary bladder and colon. In TRPV1-ChR2-tomato mice, native tomato-fluorescence was observed in sensory neurons of dorsal root ganglia and in nerve fibers in the trachea. Additionally, some cells in the gut wall and in very small arteries displayed tomato-fluorescence. These optogenetic mice offer a novel tool to functionally dissect the various neuronal airway constrictor pathways and to discriminate between non-neuronal and neuronal cholinergic mechanisms in particular. Funded by DZL and “Nachwuchsanschubfinanzierung”, FB11, JLU, to Amir Rafiq.
Submitted as: Presentation 288 words of 300 possible words
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Abstract No. 085 Modelling alveolar micromechanics in progressing bleomycin-induced lung injury Lars Knudsen 1,* , Elena Lopez-Rodriguez 1, Lennart Berndt 1, Caroline Boden 1, Lilian Steffen 1, Clemens Ruppet 2, Heinz-Gerd Hoymann 3, Jason HT Bates 4, Matthias Ochs 1, and Bradford Smith 5 1
Institute of Functional and Applied Anatomy Department of Internal Medicine 3 Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM) 4 University of Vermont College of Medicine 5 University of Colorado, Denver *Presenting author 2
Objectives: Surfactant-dysfunction related alterations of alveolar micromechanics such as intra-tidal alveolar recruitment and derecruitment (R/D) and dynamic heterogeneous ventilation contribute to lung injury by means of dynamic strain. The aim of this study was to model alveolar micromechanics in progressing bleomycin-induced lung injury. Methods: One (D1) and three (D3) days after bleomycin-instillation animals were subjected to invasive pulmonary function tests to determine tissue elastance (H) during PEEP ventilation between 1 and 10 cmH2O. Lungs were then perfusion fixed at end-expiratory airway opening pressures (Pao) of 1, 5, 10, and 20 cmH2O and assessed by design-based stereology. Another bleomycin-injured group received surfactant-replacement therapy (SRT) and was also analyzed at day 3. Lung mechanical and structural data were correlated and used to parameterize a physiologically-based computational multi-compartment model of alveolar micromechanics. Main Results: In healthy controls number of open alveoli remained stable in a range of Pao=1-20 cmH2O. Bleomycin challenged lungs demonstrated a considerable alveolar R/D with Pao=1-10 cmH2O. With disease progression at D3, however, approximately 40% of alveoli remained closed even at high Pao while alveolar size heterogeneity dramatically increased at Pao=10 cmH2O. SRT reduced alveolar R/D and alveolar size heterogeneity. In bleomycin challenged lungs alveolar derecruitment at low Pao as well as increase in coefficient of variation of alveolar size at Pao=10 cmH2O correlated with increase in H. Computational modelling showed that individual elastance at a given alveolar size of open alveoli decreased with injury and surfactant dysfunction. Conclusion: After bleomycin challenge alveolar R/D and alveolar size variation dominate alveolar micromechanics at Pao 1-10cmH2O and are linked with increase in H and probably also dynamic tissue strain. Open alveoli are more compliant likely resulting from outward tethering forces generated by adjacent collapsed alveoli making them susceptible for overdistension. SRT attenuates these alterations in alveolar micromechanics thereby reducing dynamic strain.
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Abstract No. 096 Deficiency of Immunoproteasome Subunits Increases Alternative Polarization of Alveolar Macrophages Ilona Kammerl 1, Shanze Chen 2, Ernesto Elorduy Vergara 1, Thomas Meul 1,* , Martin Irmler 1 , Johannes Beckers 1, Tony Muchamuel 3, Christopher Kirk 3, Bogdan Florea 4, Hermen Overkleeft 4, Oliver Eickelberg 1, Silke Meiners 1, and Tobias Stöger 1 1
Helmholtz Zentrum München Sichuan University 3 Kezar Life Sciences 4 Leiden University *Presenting author 2
Introduction: Immunoproteasomes are the main protein degradation system in immune cells. They have been shown to shape adaptive immune responses by generating epitopes for MHC I antigen presentation as well as by modulating inflammatory pathways and cytokine secretion. We have previously demonstrated the impact of the immunoproteasome subunit LMP7 on alveolar macrophage (AM) M2 polarization in vitro indicating that immunoproteasomes also participate in shaping innate immune responses. Here, we analyzed the role of immunoproteasome subunit LMP2 in AM polarization. Methods & Results: Primary AMs from wildtype and LMP2-/- mice were isolated and treated with LPS/IFNγ or with IL-4 for classical (M1) or alternative (M2) polarization, respectively. We did not observe consistent changes in M1 marker gene expression compared to wildtype cells. This was confirmed using LMP2-/- NFκB reporter mice which did not reveal differential activation of NFκB-driven GFP expression. In contrast, LMP2 deficiency clearly augmented alternative AM activation: M2 marker genes were significantly increased in LMP2-/- AMs, which we were able to attribute to increased M2 signaling (STAT6 and AKT) and elevated expression of the key transcription factor IRF4 in response to IL-4 treatment. Microarray analyses confirmed augmented IL-4 signatures upon M2 polarization. In contrast to wildtype and also LMP7-/-, LMP2-/- AMs displayed increased levels of MMP13 under basal and stimulated conditions suggesting that different immunoproteasome subunits have unique functions. We are currently investigating the effect of locally delivered LMP2- or LMP7-specific inhibitors to scrutinize their therapeutic potential in an LPS-induced acute lung injury model. Conclusions: Enhancing the pro-resolution capacity of M2 AMs without affecting M1polarization potential could represent a novel therapeutic option in pulmonary diseases such as pneumonia- or sepsis-related acute respiratory distress syndrome.
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Abstract No. 098 Protein arginine methylation facilitates LPS-triggered enolase-1 exteriorization Dariusz Zakrzewicz 1,* , Miroslava Didiasova 1, Benedetto Daniele Giaimo 1, Tilman Borggrefe 1, Klaus T. Preissner 1, and Malgorzata Wygrecka 1 1
Department of Biochemistry, Faculty of Medicine, Universities of Giessen and Marburg Lung Center *Presenting author
Objectives: Cell surface-associated proteolysis plays a crucial role in embryonic development, tumour cell invasion and inflammatory cell recruitment. The glycolytic enzyme enolase-1 (Eno-1) is translocated from the cytoplasm to the cell surface, where it binds plasminogen to enhance pericellular plasmin production and thereby potentiates lung inflammation and cancer metastasis. Although increased levels of cell surface-bound Eno-1 were reported under above mentioned pathological conditions, the molecular mechanism of Eno-1 exteriorization remains elusive. Here, we investigated the involvement of posttranslational modifications of Eno-1 in its cellular trafficking. Results: Stimulation of human lung epithelial A549 cells with LPS triggered monomethylation of arginine 50 (R50) within Eno-1. Consequently, the substitution of R50 for lysine markedly reduced Eno-1 association with caveolar domains thereby diminishing its exteriorization. The inhibition of protein arginine methylation in A549 cells by AMI-1, an inhibitor of protein arginine methyltransferase (PRMT) activity, significantly attenuated transport of Eno-1 to the cell surface. Interestingly, Eno-1 associated with a Tudorcontaining domain, a “reader” of protein arginine methylation marks. Moreover, in vitro methylation assay demonstrated that Eno-1 is methylated by PRMT5. Hence, overexpression of PRMT5 in A549 cells significantly increased Eno-1 exteriorization; while pretreatment with EPZ01566, a selective inhibitor of PRMT5 activity, markedly reduced the levels of cell surface-bound Eno-1. Conclusions: Blockage of Eno-1 exteriorization by inhibition of Eno-1 methylation may serve as a novel therapeutic option in diseases associated with increased expression of surfaceassociated Eno-1.
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Abstract No. 101 Bone morphogenetic protein-2 regulates expression of protease-activated receptor-2 via Smad and PI3K/Akt signaling pathways in lung epithelial cells Tobias Mawassii 1,* , Dariusz Zakrzewicz 1, and Malgorzata Wygrecka 1 1
Department of Biochemistry, Faculty of Medicine, Universities of Giessen and Marburg Lung Center *Presenting author
Objectives: Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor, which plays an important role in the regulation of cellular processes such as leukocyte migration and epithelial cell integrity. Altered PAR-2 expression has been linked to the pathogenesis of lung injury, which is manifested by elevated levels of bone morphogenetic proteins (BMP), well-known modulators of epithelial barrier function in the inflamed tissue. We hypothesized that activation of the BMP signaling cascade contributes to the regulation of PAR-2 expression under inflammatory conditions in the lung. Results: Exposure of mouse and human alveolar epithelial type II cell lines to BMP-2 significantly downregulated PAR-2 mRNA levels in a time- and dose-dependent manner. BMP-2-triggered attenuation of PAR-2 expression was counteracted by actinomycin D, an inhibitor of transcription. No effect of cycloheximide on BMP-2-dependent PAR-2 downregulation was observed suggesting that the regulation of PAR-2 expression does not required de novo protein synthesis. To study signaling pathways involved in the alteration of PAR-2 mRNA levels by BMP-2, inhibitors of BMP receptor type I, PI3K/Akt, p38 and p44/42 kinases were utilized. Inhibition of the BMP receptor type I in MLE12 cells prevented BMP-2dependent PAR-2 downregulation. While blockage of p38 and p44/42 MAP-kinase activities exerted no effect on the attenuation of PAR-2 levels in response to BMP-2, inhibition of PI3K/Akt signaling cascade reduced the regulatory effect of BMP-2 on PAR-2 mRNA expression. Conclusions: Our findings provide new insights into the molecular mechanisms responsible for the regulation of PAR-2 expression by BMP signaling in the inflamed lung.
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Abstract No. 108 Nasopharyngeal pneumococcal colonization triggers dendritic cell-dependent adaptive immunity against invasive pneumococcal disease in mice Anne Dommaschk 1,* , Meritxell Tort Tarres 1, Lara Bittersohl 1, Regina Maus 1, Jennifer Stolper 1, Danny Jonigk 1, Peter Braubach 1, Torsten Lippmann 1, Tobias Welte 1, and Ulrich Maus 1 1
Hannover Medical School *Presenting author
Nasopharyngeal colonization with Streptococcus pneumoniae (Spn) is an important precondition for the development of pneumococcal pneumonia. At the same time, nasopharyngeal colonization with Spn has been shown to mount adaptive immune responses against Spn in mice and humans. The cellular response of the nasopharyngeal compartment, including the nasal-associated lymphoid tissue (NALT), to pneumococcal colonization and its importance for developing adaptive immune responses is still poorly defined. We here show that exposure of mice to intranasal colonization with Spn triggered Spn-specific antibody responses subsequently protecting mice against invasive pneumococcal disease (IPD). Colonization with Spn led to substantial expansion of the dendritic cell (DC) compartment in nasopharyngeal tissue and NALT, along with elevated PspA-specific antibody levels in serum and nasal wash fluids. Importantly, diphtheria toxininduced selective depletion of classical DC in Spn colonized chimeric zDC+/DTR mice and Spn colonization of Flt3L KO mice lacking major lung DC subsets led to significantly diminished antibody responses together with impaired protective immunity against IPD. Collectively, the data reveal a central role for classical DC of the nasopharyngeal compartment to mediate anti-pneumococcal mucosal immune responses after colonization with Spn and hence protection against IPD as shown in two different mouse models. These data may be of importance for the development of novel nasopharyngeal vaccination strategies against pneumococcal diseases in humans.
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Abstract No. 149 Role of TRAIL in the pathogenesis of Bronchopulmonary Dysplasia (BPD) Tayyab Shahzad 1,* , Cho Ming Chao 1, Saverio Bellusci 1, and Harald Ehrhardt 1 1
JLU Gießen *Presenting author
Background and aims BPD is the chronic lung disease of the newborn infant and is mainly caused by an inflammatory response of the immature lung to mechanical ventilation and oxygen toxicity. The members of the TNF-superfamily are known for their heterogenous impact in different diseases. For BPD, FasL represents a maior contributor to lung damage while in the absence of TNF-α the extent of inflammation and cell death induction is increased by an overweight of TGF-b signaling. The role of TRAIL in the pathogenesis of BPD remains to be determined. Methods: Wildtype (C57BL6) and TRAIL-/- (Nomenklatur) mice were exposed to room air (21% O2) or 85% of oxygen from P1 to P8. Analyses include survival rates, lung histology, determination of the extent of cell death induction and of inflammatory response.
Results: Preliminary data on survival rates, lung histology and changes in cell death induction and inflammation will be available at the time of poster presentation.
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Abstract No. 154 Cleavage and activation of the influenza virus hemagglutinin by non-human primate orthologues of TMPRSS2 Pawel Zmora 1, Paulina Blazejewska 2, Stephanie Bertram 3, Kerstin Walendy-Gnirß 3, Inga Nehlmeier 4, Anika Lins 4, Anna-Sophie Moldenhauer 4, Sebastian Konzok 5, Susann Dehmel 5, Katherina Sewald 5, Constantin Brinkmann 4, Christoph Curths 6, Sascha Knauf 4, Jens Gruber 4, Kerstin Mätz-Rensing 4, Franziska Dahlmann 6,* , Armin Braun 5, and Stefan Pöhlmann 4 1
Max Planck Institute for Dynamics of Complex Technical Systems Boehringer Ingelheim Veterinary Research Center GmbH 3 Heinrich Pette Institute 4 German Primate Center GmbH 5 Fraunhofer ITEM 6 Fraunhofer ITEM, German Primate Center GmbH *Presenting author 2
RATIONALE Influenza A viruses (FLUAV) cause respiratory syndromes and especially young children and elderly patients can develop severe disorders. Virus entry into respiratory target cells requires cleavage and activation of the viral surface glycoprotein, hemagglutinin (HA), by a cellular protease. The type II transmembrane serine protease (TTSP) TMPRSS2 cleaves HA of FLUAV in cell culture and is essential for spread of diverse FLUAV in mice. Besides humans, several other mammals can be infected by FLUAV, including macaques and common marmosets. Here, we investigated whether NHP orthologues of human TMPRSS2 can cleave and activate FLUAV HA and contribute to viral spread in respiratory tissue of these species METHODS Macaque and marmoset orthologues of human TMPRSS2 were cloned jointly with two other members of the TTSP family, TMPRSS4 and HAT. The ability of these proteases to cleave and activate FLUAV HA was assessed in HA transfected and FLUAV infected cells, respectively. In addition, we investigated whether a protease inhibitor active against TMPRSS2, camostate mesylate, inhibits FLUAV spread in precision-cut lung slices (PCLS) of both human and non-human primate (NHP) origin. RESULTS We found that all NHP orthologues of human TMPRSS2, TMPRSS4 and HAT are expressed in transfected cells and can cleave and activate FLUAV HA. Furthermore, TMPRSS2 was endogenously expressed in NHP lung tissue and the serine protease inhibitor camostat mesylate reduced FLUAV spread in PCLS of human, macaque and marmoset origin. CONCLUSION Our results suggest that TMPRSS2 from various NHP species can cleave and activate FLUAV HA. Moreover, our findings indicate that FLUAV depends on serine protease activity for spread in diverse primates and that macaques may serve as models to study FLUAV activation by TMPRSS2 and its inhibition.
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Abstract No. 156 Hypercapnia induces endoplasmic reticulum-associated degradation of the Na,KATPase β-subunit Vitalii Kryvenko 1,* , Werner Seeger 1, and István Vadász 1 1
Justus Liebig University *Presenting author
Elevated levels of CO2 (hypercapnia) are often observed in patients with acute respiratory distress syndrome (ARDS) and directly correlate with impaired alveolar fluid clearance. The key molecule involved in maintaining alveolar fluid homeostasis is the Na,K-ATPase. As a glycoprotein, the enzyme must be properly folded in the endoplasmic reticulum (ER) in order to be delivered to the plasma membrane. Here, we studied the effects of hypercapnia on the regulatory Na,K ATPase β subunit in the ER. Exposing human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid, time- and dose-dependent decrease of the Na,K ATPase β subunit in the ER followed by a significant reduction of the enzyme abundance at the plasma membrane. Of note, stable mRNA levels of the Na,K ATPase β subunit, suggested that posttranslational modification of the enzyme was responsible for the decreased ER and plasma membrane abundance. Knockdown of ER alpha-mannosidase I (MAN1B1) or treatment with the potent MAN1B1 inhibitor, kifunensine prevented the decrease in the ER amount of the Na,K ATPase β subunit, suggesting ER-associated degradation of the enzyme. Moreover, hypercapnia was found to activate unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α). Transfecting cells with a siRNA against IRE1α prevented the decrease of the Na,K ATPase β subunit in the ER. Furthermore, hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism, since treating the cells with thapsigargin or chelating intracellular Ca2+ also resulted in a rapid phosphorylation of IRE1α and promoted quick degradation of the Na,K ATPase β subunit in the ER. Hypercapnia promotes ERassociated degradation of the Na,K ATPase β subunit by activating unfolded protein response in alveolar epithelial cells. Disturbances in ER folding of the Na,K-ATPase may influence the enzyme abundance at the plasma membrane and impair alveolar fluid clearance.
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Abstract No. 165 TGF-β reduces megalin cell surface stability by promoting shedding and transcriptional downregulation of the receptor in alveolar epithelial cells Luciana Mazzocchi 1,* , Werner Seeger 2, and István Vadász 2 1
UGMLC-ECCPS UGMLC *Presenting author 2
Accumulation of protein-rich alveolar edema is a hallmark of acute respiratory distress syndrome (ARDS) associated to worse outcomes. Thus we aimed to investiagate the effect of TGF-β, key player in the pathogenesis of ARDS, on the stability of endocytic receptor megalin, which can be regulated in a Notch-like manner. TGF-β treatment of alveolar epithelial cells resulted in transcriptional downregulation of megalin after 24 to 48 h, which was mediated by shedding of the receptor and a subsequent increase of the megalin c-terminal fragment (MCTF) abundance, a negative regulator of megalin gene expression, with a maximum release between 8 and 12 h after TGF-β exposure. Protein kinase C (PKC) was described as an activator of matrix metalloproteases (MMPs), responsible for megalin shedding.Importantly, MCTF abundance was markedly reduced in the presence of PKC inhibitor (gö6976) and specific knockdown of the kinase prevented TGF-β-induced megalin endocytosis restoring its functionality. Similar results were obtained when γ-secretase activity, which is responsible for regulated intramembrane proteolysis (RIP) of megalin in response to shedding, was inhibited. In order to identify the metalloproteases involved in the shedding of megalin, MMP-2, -9 and -14 expression, release and activation upon TGF-β treatment were assessed. An increase in protein expression of all three MMPs was observed in whole cell lysates, however only MMP-2 release and activity were upregulated in cell culture supernatants. Furthermore, TGF-β increased co-localization of MMP-14 and PKC at the plasma membrane. Finally, genetic inhibition of each of these enzymes rescued megalin cell surface stability and function in the presence of TGF-β. TGF-β downregulates megalin transcriptional expression by increasing shedding and RIP of the receptor in a process dependent on PKC, γ-secretase and MMP activity. As lower alveolar protein concentrations are associated with better outcomes in ARDS, interfering with this newly identified signalling may hold a therapeutic promise.
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Abstract No. 206 Pulmonary aging exacerbates pro-inflammatory response in acute lung injury Christina Brandenberger 1,* , Katharina Maria Kling 1, and Christian Mühlfeld 1 1
Hannover Medical School *Presenting author
Acute lung injury (ALI) is associated with increased morbidity and mortality in the elderly (> 65 years), but underlying mechanisms are still not well understood. Here we investigated the pulmonary and systemic inflammatory response in ALI in young (2-3 months) and old (18-19 months) C57BL/6J mice as well as age-dependent cellular response in vitro. ALI was induced by intranasal application of 2.5 mg lipopolysaccharide (LPS)/kg body weight (n=10-12) or saline (controls; n=7). Animals were sacrificed 24 or 72 hours later. Pulmonary inflammation was assessed in lung tissue and bronchoalveolar lavage fluid (BALF) by analyzing cytokine expression, BALF cytometry and activation of alveolar macrophages. Systemic cytokine response was measured in the blood serum and spleen lymphocytes were characterized. Age-dependent activation of bone marrow derived macrophages (BMDM) was further investigated in vitro. The pathology of ALI was more severe in old compared with young mice and progressing with time. The number of BALF neutrophils and neutrophil chemokine levels were greatly enhanced in the old animals. Concentration levels of other pro-inflammatory cytokines such as IL-6 and TNFalpha were also significantly induced in the BALF of old compared with young mice, but not in the serum. Differences were observed in quantities of spleen regulatory T-cells, CD8+ T-cells and NK cells within control animals, but no age-specific changes to LPS exposure were measured. Analysis of BALF macrophages revealed an increased expression of M1 markers (CD80 and CD86) in old mice 24 and 72 hours postexposure. However, in vitro stimulation of BMDM from young and old mice did result in an increased age-dependent response. The results of this study indicate that the aging lung is prone to an enhanced proinflammatory, neutrophilic response in ALI, which may contribute to the increased severity and mortality in ALI of the elderly.
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Abstract No. 207 Chemosensory cholinergic signaling network in the thymic medullary epithelium A. Soultanova 1,* , C. Cen 1, K. Fleck 1, G. Krasteva-Christ 2, U. Boehm 2, S. M. Wienhold 3, H. Müller-Redetzky 3, M. Witzenrath 3, and W. Kummer 1 1
Justus-Liebig University University of Saarland 3 Charité-Universitätsmedizin Berlin *Presenting author 2
Objective: A subset of medullary epithelial cells in the thymus (mTECs) was previously shown to be cholinergic and to express components of the bitter taste transduction cascade. In this study we set out to further characterize these cells and elucidate their function. Methods: Immunohistochemistry, real-time RT-PCR and intracellular calcium measurements were conducted on thymi from ChAT- (choline acetyltransferase) and Chrna3-eGFP (nicotinic receptor subunit alpha3) reporter mice, mice expressing diphtheria toxin A driven by the TRPM5 promoter (TRPM5: channel in taste transduction signaling), and wild-type mice with streptococcal pneumonia. Newborn human thymi were subjected to immunohistochemistry. Results: Analysis of murine thymi at different age stages revealed that expression of ChAT and chemosensory components in the mTECs starts at birth but not before. The ChATpositive cells in the thymus are in proximity to terminally differentiated mTECs (Hassall-like bodies) carrying Chrna3. In human newborn thymus, these cells closely surround or are integrated in the outer layer of the Hassall’s corpuscles. Hassall-like bodies were not observed in TRPM5-DTA mice lacking chemosensory cells. These cholinergic cells respond to the bitter substance denatonium with an increase in intracellular calcium concentration. Thymic mRNA expression of TRPM5 and alpha-gustducin was up-regulated in murine model of streptococcal pneumonia. Conclusion: We hypothesize that the novel chemosensory cholinergic cell type in the thymic medulla senses bacterial products, presumably coming from the bloodstream, and responds by release of acetylcholine which in turn stimulates Hassall’s corpuscles. Funded by DZL and SFB-TR 84.
Submitted as: Presentation 239 words of 300 possible words
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Abstract No. 255 Hypercapnia induces c-Jun N-terminal kinase activation altering AMPK/Nedd4-2mediated trafficking of alveolar epithelial ENaC Paulina Gwozdzinska 1,* , Werner Seeger 1, and István Vadász 1 1
Justus Liebig University, Universities of Giessen and Marburg Lung Center, Member of the German Center for Lung Research, Giessen, Germany *Presenting author
CO2 retention (hypercapnia) is often observed in patients with acute respiratory distress syndrome (ARDS). Most patients with ARDS have impaired alveolar fluid clearance (AFC) and require lung-protective mechanical ventilation frequently causing further CO2 increase (permissive hypercapnia). Fluid accumulation in the alveolar space during ARDS persists due to impaired lung fluid reabsorption. Vectorial sodium transport through the apicallyexpressed epithelial sodium channel (ENaC) represents a major mechanism of alveolar edema resolution. We have previously shown that the AMP-activated protein kinase (AMPK), the E3 ubiquitin ligase Nedd4-2 and extracellular signal-regulated kinase (ERK)1/2 are involved in CO2-induced ENaC downregulation by impairment the cell surface expression of ENaC subunits. Here, we studied the role of c-Jun N-terminal kinase (JNK)1/2 in ENaC regulation by hypercapnic signaling events. Exposing human alveolar epithelial A549 cells overexpressing α- and β-ENaC to elevated CO2 levels (pCO2 120 mmHg, pH 7.4) for 30 min caused significant increase in Nedd4-2-mediated polyubiquitination of β- (but not α-) ENaC and following the endocytosis of the α/β-ENaC complex. Furthermore, high CO2 levels by rapid and time-dependent activation of AMPK-α1 stimulated phosphorylation of JNK. It has been previously demonstrated that JNK may alter Nedd4-2 activity through phosphorylation of the Thr899 residue of the E3 ligase. Mutation of Thr899 to Ala (T899A) prevented Nedd4-2 phosphorylation and A549 cells co-transfected with T899A mutant of Nedd4-2 had significant decrease in both the hypercapnia-induced polyubiquitination of βENaC and endocytosis of α/β-ENaC. These results show that hypercapnia by activation of JNK may enhance activity of Nedd4-2, promoting downregulation of epithelial sodium transport through increasing β-ENaC polyubiquitination and internalization of α/β-ENaC complex form the cell surface. This mechanism may in part mediate the impaired alveolar edema clearance in patients with ARDS and permissive hypercapnia.
Submitted as: Presentation 282 words of 300 possible words
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Abstract No. 276 A protective role of TRPV4 in ischemia/reperfusion-induced edema formation in the lung Jonas Weber 1,* , Martina Kannler 1, Monika Malczyk 2, Norbert Weißmann 2, Thomas Gudermann 1, and Alexander Dietrich 1 1
Walther-Straub-Institute for Pharmacology and Toxicology, Member of the German Center for Lung Research (DZL), Ludwig-Maximilians-University München 2 Department of Internal Medicine II/V, University of Giessen and Marburg Lung Center (UGMLC), Aulweg 130, 35392 Giessen, Germany *Presenting author
TRPV4 channels are members of the vanilloid family of transient receptor potential (TRP) proteins. Pulmonary expression of TRPV4 has been identified in endothelial cells, epithelial cells and arterial smooth muscle cells (reviewed in 1). The channel is known to be involved in the development of several pulmonary symptoms such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (1). It is a matter of debate however, if TRPV4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for TRPC6 (1, 2). To analyze the potential role of TRPV4 on ischemiareperfusion-(IR)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. We detected a significantly enlarged edema formation after 90 minutes of ischemia in TRPV4-deficient lungs in comparison to wild-type (WT) lungs. Most interestingly, edema formation of TRPV4/TRPC6 double deficient lungs was indistinguishable from WT lungs, indicating antagonizing effects of both channels, because TRPC6 deficiency protected lungs from IR-induced edema (2). Application of a specific TRPV4 inhibitor (3) showed similar increases in edema formation excluding gross compensatory up- or down-regulation in the global knockout lungs as a possible reason. Unraveling the molecular mechanism of TRPV4 function in the endothelial and epithelial barrier will uncover new promising therapeutic options in patients with IR-induced edema.
1. Morty and Kuebler (2014) TRPV4: an exciting new target to promote alveolar capillary membrane function. Am. J. Physiol. Lung Cell. Mol. Physiol 307: L817-L821 2. Weismann, et al. (2012) Activation of TRPC6 channels is essential for lung ischaemia-reperfusion induced oedema in mice. Nature Commun. 3: 649 3. Kevin S. Thorneloe, et al. (2012) An Orally Active TRPV4 Channel Blocker Prevents and Resolves Pulmonary Edema Induced by Heart Failure. Sci Transl Med 4, 159ra148 Submitted as: Presentation 228 words of 300 possible words
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Abstract No. 296 PROGRESS – Prospective Observational Study on Hospitalized Community Acquired Pneumonia Peter Ahnert 1, Petra Creutz 1,* , Norbert Suttrop 1, and The PROGRESS study group 1 1
Charite Berlin *Presenting author
Community acquired pneumonia (CAP) is a high incidence disease resulting in about 260,000 hospital admissions per year in Germany, more than myocardial infarction or stroke. CAP poses numerous medical challenges, which the PROGRESS (Pneumonia Research Network on Genetic Resistance and Susceptibility for the Evolution of Severe Sepsis) network aims to tackle: Operationalization of disease severity throughout the course of disease, outcome prediction for hospitalized patients and prediction of transitions from uncomplicated to severe CAP, and finally, to CAP with sepsis and organ failure as a lifethreatening condition. It is a major aim to understand and predict patient heterogeneity regarding outcome in the hospital and to develop novel treatment concepts. PROGRESS was designed as a clinical, observational, multi-center study of patients with CAP requiring hospitalization. More than 1,600 patients selected for low burden of comorbidities have been enrolled, aiming at a total of 3,000. Course of disease, along with therapy, was closely monitored by daily assessments and long-term follow-up. Daily blood samples allow in depth molecular-genetic characterization of patients. We established a well-organized workflow for sample logistics and a comprehensive data management system to collect and manage data from more than 50 study centers in Germany and Austria, including a central biobank and a central data base which also integrates all data from molecular assessments. We established a comprehensive data base of high quality clinical and molecular data allowing investigation of pressing research questions regarding CAP. In-depth molecular characterization will contribute to the discovery of disease mechanisms and establishment of diagnostic and predictive biomarkers. A strength of PROGRESS is the focus on younger patients with low burden of co-morbidities, allowing a more direct look at host biology with less confounding. As a resulting limitation, insights from PROGRESS will require validation in representative patient cohorts to assess clinical utility.
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Abstract No. 030 Vascular RASSF1A: HIF-1 feed-forward loop triggers hypoxia induced pulmonary hypertension Swati Dabral 1,* , Christian Muecke 1, Rajkumar Savai 1, Chanil Valsarajan 1, Astrid Wietelmann 1, Norbert Wiessmann 2, Werner Seeger 1, and Soni Savai Pullamsetti 1 1
MPI for heart and lung research University of Giessen *Presenting author 2
Objective: The pulmonary circulation responds uniquely to hypoxia with proliferation of vascular cells and pulmonary hypertension (PH). The mechanisms underlying the vascular responses and regulation of hypoxia-inducible factor 1 (HIF-1) remain to be elucidated. Results: We report here that Ras association domain family 1A (RASSF1A), functions as a stabilizing factor for HIF-1α by inhibiting its hydroxylation, leading to increased HIF-1α mediated transactivation of target genes (PDK1, LDHA, HK2), and promoting proliferation and glycolytic shift in hypoxia-exposed pulmonary vascular cells. Hypoxia initially induces reactive oxygen species (ROS) and stabilizes RASSF1A by protein kinase C (PKC)mediated phosphorylation. RASSF1A transcription is later upregulated by HIF-1. Thus, RASSF1A and HIF-1 participate in a positive feedback loop that amplifies the upregulation of known HIF-1 target genes (PDK1, LDHA, HK2). Consistently, RASSF1A knockout mice (Rassf1a-/+; Rassf1a-/-) do not develop PH in response to hypoxia. Notably, we observed high expression levels of RASSF1A, HIF-1α and HIF-1 target genes in the pulmonary vasculature of patients with PH (IPAH or PH associated with COPD) and in rodent models of PH (hypoxia and hypoxia+SU5416). In addition, we observed that siRNA mediated inhibition of RASSF1A expression reverts the hyper-proliferative and glycolytic phenotype of ex-vivo cultured human PH-PASMCs. Conclusions: We propose a novel feed-forward regulatory interaction of RASSF1A and HIF1 drive hypoxia-induced PH. These findings support inhibition of RASSF1A as well as inhibition of RASSF1A-HIF signaling as a potential therapeutic option for PH.
Submitted as: Presentation 233 words of 300 possible words
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Abstract No. 041 Applicability of the SU5416/hypoxia rat model to study human pulmonary hypertension Lavinia Maegel 1,* , Britta Ludewig 1, Mark Kuehnel 1, Paul Borchert 1, Olga Rafikova 2, Gregor Warnecke 3, Axel Haverich 3, Sabina Janciauskiene 4, Marius M. Hoeper 5, Stephen M. Black 2, and Danny Jonigk 1 1
Institute of Pathology Center for Lung Vascular Pathobiology 3 Department of Cardiothoracic, Transplantation and Vascular Surgery 4 Department of Experimental Pneumology 5 Department of Pneumology *Presenting author 2
Pulmonary Hypertension is a complex and debilitating disease with a high mortality. Severe disease is characterized by progressive and irreversible remodeling of the pulmonary vasculature with characteristic concentric and plexiform lesions. Studies on human pulmonary hypertension are challenging due to limited availability of fresh lung tissue. Therefore an appropriate (animal) model, which closely mimics the pathophysiologic changes seen in human disease, is required. The SU5416 (sugen)/hypoxia model has been put forward as the best model for pulmonary hypertension, because rodents develop vascular changes which resemble human plexiform vasculopathy. Here, we addressed similarities and differences between sugen/hypoxia induced vasculopathy in rats and human pulmonary hypertension by comparing histomorphologic and molecular aspects using compartment-specific gene expression analysis including laser microdissection, RT-PCR and immunohistochemistry. Our data indicate that plexiform lesions in sugen rats consist of well-structured endotheliallined vascular channels supported by an interstitium of smooth muscle cells, comparable to corresponding human lesions. In human end-stage lungs we observed identical gene expression patterns in men and women, while rats showed molecular differences between sexes. In mild vascular remodeling we found most of our target genes to be regulated similarly in rats and humans. However, severely remodeled vasculature, represented by plexiform lesions, exhibited a rather different expression profiles in humans compared to rats. In conclusion, our data indicates that the sugen rat model of pulmonary hypertension reflects relevant key aspects of the human disease on a morphological and molecular level. However, human plexiform lesions appear to have a more diverse angiogenetic microenvironment. Furthermore, we describe distinct molecular differences between male and female rats that do not appear to be recapitulated in humans, which need to be taken into consideration in further studies.
Jonigk D, Hoeper MM, Kreipe H, Länger F. Histopathological aspects of pulmonary hypertension. Pathologe. 2012 May; doi: 10.1007/s00292-011-1560-x. Sakao S, Tatsumi K, Voelkel NF. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation. Respir Res 2009; 10:95. Ryan JJ, Marsboom G, Archer SL. Rodent models of group 1 pulmonary hypertension. Handb Exp Pharmacol. 2013; doi: 10.1007/978-3-642-38664-0_5. Review.
DZL Annual Meeting 2017 Jonigk D, Golpon H, Bockmeyer CL, Maegel L, Hoeper MM, Gottlieb J, Nickel N, Hussein K et al. Plexiform lesions in pulmonary arterial hypertension composition, architecture, and microenvironment. Am J Pathol. 2011 Jul; doi: 10.1016/j.ajpath.2011.03.040. Rafikova O, Rafikov R, Meadows ML, Kangath A, Jonigk D, Black SM. The sexual dimorphism associated with pulmonary hypertension corresponds to a fibrotic phenotype. Pulm Circ. 2015 Mar; doi: 10.1086/679724. Submitted as: Presentation 277 words of 300 possible words
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Abstract No. 055 MicroRNAs are regulators of PDGFRβ expression in pulmonary arterial hypertension Astrid Weiss 1,* , Akylbek Sydykov 1, Ingrid Henneke 1, Lavinia Maegel 2, Danny Jonigk 2, Ardeschir Ghofrani 1, Norbert Weissmann 1, Friedrich Grimminger 1, Werner Seeger 1, and Ralph Schermuly 1 1
JLU Giessen - Internal medicine MHH - Institute for pathology *Presenting author 2
In several diseases, microRNAs serve as translational repressors of key signaling proteins leading to their differential expression compared to healthy conditions. In pulmonary arterial hypertension (PAH), an up-regulation of PDGFRβ in hyper-proliferative smooth muscle cells of the lung vasculature has been implicated in the pathobiology of this devastating disease. Therefore, we aim to define the role of miR-9, miR-29a and miR-34c in the aberrant PDGFRβ expression and their impact on the onset, progression and therapy of PAH. Western blot analysis from human lung homogenates and realtime-PCR from lasermicrodissected pulmonary arteries were performed for expression profiling. Thus, we identified miR-9 as a potential translational repressor of PDGFRβ mRNA. Our expression analyses confirmed the over-activation of PDGFRβ and demonstrated its inverse correlation to endogenous miR-9, miR-29a and miR-34c levels in lung homogenates of hypoxia-treated mice which resemble the pathological phenotype of structural remodeling observed in the pulmonary system of idiopathic PAH patients. We also evaluated the binding of the most promising microRNA (e.g. miR-9) to the 3´UTR of the PDGFRβ mRNA followed by an inhibition of translation into PDGFRβ protein. Next, we investigated the effects of forced up-regulation of microRNAs on the PDGFRβ expression in vivo after intratracheal nebulization of adeno-associated viruses (AAVs) encoding miR-9, miR-29a or miR-34c as well as a reporter gene (EGFP). Successful and targeted infection was confirmed by PCR for the viral EGFP locus. During our analyses, we focused mainly on functional parameters of the lung and the right ventricle which would reveal signs of pulmonary vascular remodeling and subsequent development of pulmonary hypertension (PH). Our preliminary data demonstrate a significant improvement of PH symptoms, especially of right ventricular function, upon miR-9 re-constitution. In summary, this project allows us to understand the role of microRNAs (miR-9, miR-29a, miR-34c) as negative regulators of PDGFRβ expression in this life-threatening disease. The emerging role of epigenetics in pulmonary arterial hypertension: an important avenue for clinical trials (2015 Grover Conference Series). Huston JH, Ryan JJ. Pulm Circ. 2016 Sep;6(3):274-84. The emerging role of microRNAs in hypoxia-induced pulmonary hypertension. Mohsenin V. Sleep Breath. 2016 Sep;20(3):1059-67. Mechanisms of disease: pulmonary arterial hypertension. Schermuly RT, Ghofrani HA, Wilkins MR, Grimminger F. Nat Rev Cardiol. 2011 Jun 21;8(8):443-55. Hypoxic pulmonary hypertension in mice with constitutively active PDGFRβ. Dahal BK, Heuchel R, Pullamsetti SS, Wilhelm J, Ghofrani HA, Weissmann N, Seeger W, Grimminger F, Schermuly RT. Pulm Circ. 2011 Apr-Jun;1(2):259-68. The AAV vector toolkit: poised at the clinical crossroads. Asokan A, Schaffer DV, Samulski RJ. Mol Ther. 2012 Apr;20(4):699-708.
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Abstract No. 141 The role of mitophagy in development of pulmonary hypertension Alireza Saraji 1,* , Natascha Sommer 1, Oleg Pak 1, Katharina Schäfer 1, Akylbek Sydykov 1, Hossein Ardeschir Ghofrani 1, Ralph T. Schermuly 1, and Norbert Weissmann 1 1
Excellence Cluster Cardio-Pulmonary System (ECCPS), University of Giessen and Marburg Lung Center *Presenting author
Background: Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling leading to increased pulmonary arterial pressure and ultimately right heart failure. Mitochondrial dysfunction plays an important role in the development of PH. Mitophagy serves to remove dysfunctional mitochondria mostly via phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and parkin mediated pathways. However, the role of mitophagy in PH development is still unknown. Methods: Expression analysis of PINK1 and parkin was performed using western blot. PH was quantified in wild type (WT) and PINK1 knockout (PINK1 ko) mice after exposure to hypoxia (10% O2) for 4 weeks by hemodynamics, morphometry and echocardiography. Proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC) were investigated by EdU incorporation and caspase 3 assay, respectively. Results: Protein expression of PINK1 was increased in lung homogenate from mice with chronic hypoxia-induced PH as well as in PASMC exposed for 5 days to 1% O2. The increase of right ventricular systolic pressure and degree of right heart hypertrophy after chronic hypoxic exposure were similar in both WT and PINK1 ko mice. Moreover, echocardiography showed no significant differences in right ventricular function or morphometry between the genotypes. However, hypoxia-induced proliferation of PINK1 ko PASMC was decreased while their apoptosis was increased after incubation in a hypoxic atmosphere (1% O2) for 5 days compared to WT PASMC. Conclusion: Our preliminary data suggests that global knock-out of PINK1 does not alter development of chronic-hypoxia induced PH, although PINK1 may regulate hypoxia-induced alterations of PASMC proliferation and apoptosis.
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Abstract No. 148 The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling Oleg Pak 1,* , Susan Scheibe 1, Azadeh Esfandiary 1, Mareike Gierhardt 1, Akylbek Sydykov 1 , Athanasios Fysikopoulos 1, Matthias Hecker 1, Ralph T. Schermuly 1, Michael P. Murphy 2, Norbert Weissmann 1, and Natascha Sommer 1 1
ECCPS MRC Mitochondrial Biology Unit *Presenting author 2
Introduction: Increased release of mitochondrial superoxide has been suggested to mediate acute and sustained hypoxic pulmonary vasoconstriction (HPV) as well as chronic hypoxiainduced pulmonary hypertension (PH) and right heart remodeling. Thus, we investigated the superoxide release during HPV, chronic hypoxia-induced PH and after pulmonary arterial banding (PAB), as well as the effect of the mitochondria-targeted antioxidant MitoQ on these processes. Measurements and Main Methods: Superoxide release in isolated pulmonary arterial smooth muscle cells (PASMC) and the right ventricle (RV) was measured by electron spin resonance spectroscopy. The effect of MitoQ or its inactive carrier substance, triphenylphosphonium (TPP+), on acute and sustained HPV (1% O2 in the inhaled air for 10 or 180 minutes, respectively) was investigated in isolated mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after PAB were treated with MitoQ or TPP+ (50 mg/kg/day) by gavage to investigate the effect of MitoQ on chronic hypoxia-induced PH and right heart remodeling. Results: Superoxide levels were increased in PASMC during acute hypoxia, not altered during sustained hypoxia, and decreased after 5 days of hypoxia. In parallel MitoQ, but not its inactive carrier substance, significantly inhibited acute HPV and the rise in superoxide concentration induced by acute hypoxia. However, MitoQ application did not affect the strength of sustained HPV, hypoxia-induced proliferation of PASMC or the development of chronic hypoxia-induced PH. In contrast, MitoQ application attenuated right ventricular remodeling after chronic hypoxic exposure as well as after PAB with regard to development of right heart hypertrophy and dilatation. Accordingly, superoxide levels were increased in the RV after PAB. Conclusion: Increased superoxide concentration mediates acute HPV, while decreased superoxide levels were detected in chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV and to prevent the development of right heart remodeling.
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Abstract No. 162 Vascular cell-specific epigenome-wide profiling uncovers novel gene regulatory networks in human pulmonary arterial hypertension Prakash Chelladurai 1,* , Carsten Künne 1, Christine Weber 2, René Reiner Nötzold 2, Werner Seeger 1, Mario Looso 1, Uta-Maria Bauer 2, and Soni Pullamsetti 1 1
Max-Planck-Institute for Heart and Lung Research, Germany Institute of Molecular Biology and Tumor Research, Philipps-Universität Marburg *Presenting author
2
Hypertensive stimuli-induced phenotypic transformation facilitates normal vascular cells to acquire and exhibit pro-proliferative, apoptotic-resistant, pro-inflammatory, pro-fibrotic responses in pulmonary arterial hypertension (PAH). Recent observations indicate that epigenetic mechanisms may coordinate the maintenance of hypertensive phenotype outside the vascular tissue, which remains unexplored in PAH, at a genome-wide scale. Therefore, we employed next generation sequencing approaches to identify disease-specific transcriptome profile, chromatin signatures and signaling networks deregulated between pulmonary artery adventitial fibroblasts (PAAF) isolated from donor and IPAH patients ex vivo. Cell-specific transcriptome profiling(RNA-seq) revealed differential expression of 2069 genes (2-fold) in IPAH-PAAF, which indicates the existence of genome-scale alterations in transcription regulatory mechanisms. To profile the epigenetic signatures that arbitrates aberrant transcriptional responses in IPAH, we employed ChIP-seq (chromatin immunoprecipitation) to generate genome-wide maps for RNA polymerase-II, histone modifications associated with euchromatin(H3K9/K14Ac, H3K4me3, H4K5/8/12/16ac), heterochromatin(H3K27me3, H3K9me3) and enhancers(H3K27ac, H3K4me1). Analysis confirmed massive epigenetic alterations in histone acetylation and methylation pattern in IPAH-PAAFs, which can be attributed to aberrant expression and recruitment of histone modifying enzymes in IPAH. Integrative analysis revealed strong correlation between epigenetic alterations and differential transcriptome in IPAH. KEGG analysis of epigenetically regulated genes including transcription factors revealed association with smooth muscle contraction, ABC-transporters, cGMP-PKG, CAMP, NF-κB, WNT, TGF-β signaling pathways. Transcription factor prediction analysis also indicated significant likelihood of euchromatin state at promoters of differentially expressed genes with HIF1A or NF-kB binding sites, which can favor expression of pro-proliferative and pro-inflammatory genes in PAH. Overall, integrative analysis provided insights into the mechanistic link between aberrant recruitment of histone modifiers that coordinately alter the chromatin landscape and DNA accessibility leading to aberrant transcriptional responses in PAH. This study confirmed the existence of genome-scale epigenetic alterations that is central for the maintenance of hypertensive phenotype in human PAH, and also uncovered novel regulatory networks as prospective targets for intervention.
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Abstract No. 176 Proangiogenic and wound healing molecular and histological fingerprint of chronic thromboembolic pulmonary hypertension Dijana Iloska 1,* , Jochen Wilhelm 2, Stefan Guth 3, Rajkumar Savai 4, Ralph Schermuly 2, Ludger Fink 2, Eckhard Mayer 3, Werner Seeger 4, and Soni Pullamsetti 4 1
Max Planck Institute for Heart and Lung Research Universities of Giessen and Marburg Lung Center 3 Kerckhoff-Klinik, Bad Nauheim 4 Max Planck Institute for Heart and Lung Research *Presenting author 2
Objective: Chronic thromboembolic pulmonary hypertension (CTEPH) is a multifactorial disease initiated by pulmonary thromboembolism, leading to death if unrecognized and untreated. We intend to characterize and compare the distal remodeling in CTEPH with idiopathic PH (IPAH), in terms of histological and molecular phenotyping, employing transplanted CTEPH, IPAH and donor human lungs and material from pulmonary endarterectomy (PEA). Methods and Results: Transplanted CTEPH, IPAH and donor lung tissues (n=12) were proceeded to laser capture microdissection (LCM) of vessels, followed by RNA isolation and microarray screening. Assessment of vascular remodeling, inflammatory cell composition and collagen quantification was followed after histochemical stainings. Similar analysis was performed from PEA material (distal and proximal). Additionally, total microvessel density was calculated from vWF, CD31 and CD34 stainings. Morphometric analysis confirmed similar extent of medial hypertrophy, but differences in collagen deposition and vascularization between CTEPH and IPAH lungs, as compared to donors. Bioinformatics analysis of the microarray data revealed differentially (607) and similarly (366) regulated genes and gene networks. Our in vitro data suggest that two of the differentially regulated genes, CHI3L1 and ENPP2 play an important role in neovascularization and migration of smooth muscle cells and fibroblasts. In the PEA tissues, angiogenic markers are elevated, most probably resulting in neovascularization, supported by immunofluorescent staining and microarrays screening. Wound healing response in the distal PEA material induced increase in the insoluble collagen. Important transcription factor networks (FoxO3, GATA6 and KLF2) are altered in the PEA tissue, suggesting their contribution to neovascularization. Conclusions: These studies highlight the similarities and difference in terms of histological and molecular networks between different groups of PH. Further in vitro and in vivo studies will provide more knowledge into the molecular mechanism playing a role in the disease and can possibly contribute to identify novel therapeutic targets.
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Abstract No. 179 Role and Regulation of Jumonji C domain-containing histone demethylases 1A and 2B in Pulmonary Hypertension Christian Mücke 1,* , Swati Dabral 1, Stefan Günther 1, Mario Looso 1, Carsten Künne 1, Werner Seeger 1, and Soni Savai Pullamsetti 1 1
Max-Planck-Institut für Herz-und Lungenforschung *Presenting author
Objective: Pulmonary hypertension (PH) is characterized by increased proliferation and apoptosis resistance of pulmonary vascular cells. Jumonji C domain-containing histone demethylases (JMJDs) are a novel class of epigenetic regulators, implicated in chromatin regulation and often possess the ability to demethylate lysine residues on histones. Given that JMJDs and histone methylation levels are important for the proliferative capacity of cells, we postulated that JMJDs contribute to excessive vascular remodeling underlying PH. Results: Expression of JMJDs was investigated in vascular cells exposed to different time points of hypoxia. Increased expression of JMJD1A and JMJD2B were observed in all three vascular cell types (pulmonary arterial –smooth muscle cells (PASMCs), -fibroblasts (PAFBs), and –endothelial cells (PAECs). Further, increased nuclear localization and activity of JMJD1A and JMJD2B were observed in PASMCs. The increased expression of JMJD1A and JMJD2B was dependent on hypoxia induced factor 1α (HIF1α). Importantly, selective knockdown of these JMJDs and isoform selective JMJD inhibitors led to inhibition of proliferation and induction of apoptosis in vascular cells exposed to hypoxia. Furthermore, in laser micro-dissected vessels and pulmonary arteries from idiopathic pulmonary arterial hypertension (IPAH) patients, increased expression of JMJD1A and JMJD2B was observed compared to donors. Furthermore, next generation RNA-sequencing (RNA-Seq) of hypoxia treated PASMCs with JMJD1A and JMJD2B knockdown lead to identification of promising JMJD target genes, which are involved in biological processes like proliferation, apoptosis, metabolism and inflammation. Based on the gene enrichment analysis and the top upregulated genes, lactate dehydrogenase A (LDHA) and baculoviral IAP repeat containing 5 (BIRC5) were identified as JMJD2B target genes. Further, chromatin imunoprecipitation (ChIP) studies confirmed enrichment of JMJD2B on the both gene promoters confirming them as direct targets of JMJD2B demethylase activity. Conclusions: In conclusion, our results suggest that JMJDs are regulated in PH vasculature and play an important role in PH pathogenesis.
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Abstract No. 185 Wnt-Signaling Pathway drives right ventricular remodeling Alexandra tretyn 1, Sreenath Reddy Nayakanti 1,* , Swati Dabral 1, Mario Böhm 2, Baktybek Kojonazarov 2, Wiebke Janssen 1, Werner Seeger 3, Ralph Schermuly 2, and Soni Savai Pullamsetti 3 1
Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany 3 Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany. Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany. *Presenting author 2
Introduction: The right ventricle (RV) is the major determinant of functional state and prognosis in pulmonary hypertension (PH). Although much is known about mechanisms triggering left ventricular hypertrophy, little is known about signaling cascades leading to RV hypertrophy and RV failure. Since Wnt signaling was shown to be important for the development of RV, we hypothesize that it may play a role in the RV hypertrophy, RV fibrosis and RV failure. Methods and Results: In this study, we employed pulmonary artery banding (PAB) and monocrotaline (MCT) animal models to induce RV hypertrophy and RV failure in rats and mice. Cardiac MRI and invasive hemodynamic measurements were performed after 3 weeks in PAB- and sham-operated mice/rats respectively. The PAB and sham mice were randomized and treated for 2 weeks with LGK974 ( -catenin activity inhibitor) or vehicle treatment. Subsequently, the RV were investigated for fibrosis assessment and analyzed for gene expression. We found significant upregulation of several Wnt signaling molecules: GSK3β, β-catenin, Frizzled1 in the RV- and fibroblasts- isolated from RV of PAB and MCT rats. Immunohistochemical analysis of hypertrophic human hearts also showed an increased βcatenin expression compared to human donor hearts. Genetic or pharmacological inhibition (LGK974) of β-catenin, in vitro significantly regulated collagen synthesis and fibroblast proliferation. Importantly, MRI data revealed significant increase in the RV mass, end diastolic volume (EDV), end systolic volume (ESV) and decrease in ejection fraction of RV of PAB mice, upon LGK treatment they could recover significantly. mRNA expression of βmyosin heavy chain, natriuretic peptides, collagen family members were increased in PAB, that were significantly decreased in LGK treated PAB mice. Conclusion: It seems reasonable to suggest that therapeutic agents specifically targeting the WNT receptors and its downstream signaling molecules could offer potential treatment strategies for RV fibrosis and failure
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Abstract No. 189 The response of the pulmonary vasculature to hypoxia in isolated lungs of mice expressing the alternative oxidase (AOX) from Ciona intestinalis Nasim Alebrahimdehkordi 1, Oleg Pak 1, Susan Scheibe 1, Alireza Saraji 1, Karin Quanz 1, Werner Seeger 2, Hossein A. Ghofrani 1, Norbert Weissmann 1, Thomas Braun 3, Howard Jacobs 4, Marten Szibor 5, and Natascha Sommer 1 1
Excellence Cluster Cardio-Pulmonary System (ECCPS), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany 2 Excellence Cluster Cardio-Pulmonary System (ECCPS), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany 3 Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany 4 Institute of Biotechnology, 00014 University of Helsinki, P.O. Box 56, Helsinki 00014, Finland; BioMediTech and Tampere University Hospital, University of Tampere, Tampere 33014, Finland 5 Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany; Institute of Biotechnology, 00014 University of Helsinki, P.O. Box 56, Helsinki 00014, Finland; BioMediTech and Tampere University Hospital, University of Tampere, Tampere 33014, Finland
Introduction: Hypoxic pulmonary vasoconstriction (HPV) is essential to match local blood perfusion to ventilation and thus to prevent life-threatening hypoxemia. Increased production of superoxide originating from mitochondrial complex III has been suggested as underlying oxygen sensing mechanism. The mitochondrial alternative oxidase (AOX) which is only expressed in some non-vertebrates can bypass complex III and IV when electron transfer at these complexes is inhibited. We thus examined if expression of the AOX of Ciona intestinalis in mice affects HPV. Methods: HPV was measured in isolated ventilated and perfused lungs from mice expressing AOX (AOXtg) and compared to WT mice. HPV was induced by ventilation with hypoxic gas (1% O2, 5% CO2, rest N2) for 10 minutes. Hypoxia-independent vasoconstriction was measured by application of the thromboxan analogon U46619. Hypoxia-induced superoxide release was measured in pulmonary arterial smooth muscle cells (PASMC) by electron spin resonance spectroscopy. Development of chronic hypoxiainduced pulmonary hypertension (PH) was determined after exposure of mice to hypoxia (10% O2) for 4 weeks. Results: Isolated lungs from AOXtg mice specifically lacked acute HPV, while the response to the unspecific vasoconstrictive stimulus U46619 was preserved. Inhibition of AOX by npropyl-gallate (nPG) could restore HPV. Accordingly, isolated PASMC from AOXtg mice showed no hypoxia-induced increase of superoxide which was found in WT mice. In contrast, development of chronic hypoxia-induced PH was not different in AOXtg and WT mice. Conclusion: We conclude that mitochondrial superoxide production acts as trigger mechanism in acute, but not chronic hypoxic signaling in the pulmonary vasculature. AOX might have been abandoned during evolution to allow acute oxygen sensing processes in organisms which depend on oxidative metabolism.
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Abstract No. 197 Histone Deacetylase 7 regulates master transcription factors through modulation of mitochondrial function Elisabetta Gamen 1,* , Prakash Chelladurai 1, Friederich Grimminger 2, Rajkumar Savai 1, Evangelos Michelakis 3, Werner Seeger 1, and Soni Savai Pullamsetti 1 1
Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany Universities of Giessen and Marburg Lung Center 3 University of Alberta, Edmonton, Alberta, Canada *Presenting author 2
Reprogramming of the cellular metabolism towards glycolysis (“Warburg effect”), increased mitochondrial membrane potential and apoptosis resistance is known hallmarks of cancer. Similarly, pulmonary arterial smooth muscle cells derived from pulmonary arterial hypertensive patients (PAH-PASMCs) have hyperpolarized mitochondria, suppressed glucose oxidation and reduced mitochondria-dependent apoptosis. Signals from suppressed mitochondria may regulate the nuclear trafficking of master transcription factors (e.g. HIF1α, STAT3, FOXOs) through modulation of their acetylation and/or phosphorylation. We have identified the pathophysiological role of HDAC7 in the context of apoptosis and apoptosis-associated metabolic and mitochondrial remodeling and characterized the mechanism by which HDAC7 exerts its function, using hypoxia exposed primary culture of healthy PASMCs (control-PASMCs), PAH-PASMCs and human non-small cell lung cancer cells (A549). We observed that HDAC7 is the only commonly upregulated class IIa HDAC in PH and lung cancer (LC). In vivo, HDAC7 upregulation is confined majorly to the medial layer of the pulmonary vasculature in PH lungs and cancer cells in LC lungs. Pharmacological inhibition and genetic ablation of HDAC7 leads to mitochondrial depolarization, increased glucose oxidation and induces cell apoptosis. Interestingly, we identified that specific transcription factors already known to play an important role in both PH and LC, such as FOXOs, HIF-1α and STAT3, are regulated by HDAC7. Moreover, HDAC7 influences the phosphorylation of the AMP-activated protein kinase (AMPK), a highly conserved sensor of energy stress, activated in both cancer and PH. Taken together these findings sustain the hypothesis that HDAC7 may act as a regulator of transcription factors through modulation of mitochondrial function. It might represent a link to the several molecular abnormalities so far described in both PH and cancer and its targeting may affect the diverse signals associated with the two conditions.
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Abstract No. 208 The role of BDNF in chronic hypoxia-induced pulmonary hypertension Katharina Schäfer 1, Oleg Pak 1, Mareike Gierhardt 1, Azadeh Esfandiary 1, Akyl Sydykov 1, Werner Seeger 1, Grazyna Kwapiszewska 2, Ludger Fink 3, Ralph T. Schermuly 1, Natascha Sommer 1, and Norbert Weissmann 1 1
ECCPS Ludwig Boltzmann Institute for Lung Vascular Research 3 Institut für Pathologie, Justus-Liebig-University Giessen 2
Objective: The brain derived neurotrophic factor (BDNF) is strongly upregulated in mouse lungs of chronic hypoxia-induced pulmonary hypertension (PH). BDNF stimulation increased proliferation of pulmonary arterialsmooth muscle cells (PASMCs) in normoxia. However, the role of BDNF in chronic hypoxia-induced PH is still unknown. Therefore, we aimed to investigate the effect of BDNF deletion in smooth muscle cells (SMCs)on the development of chronic hypoxia-induced PH. Methods and Results: In vitro experiments showed that exposure of PASMCs to hypoxia (1% O2)for 5 days increased the release of BDNF into the medium.Mice with SMC specific inducible deletion of BDNF(SMMHC-BDNF-/-) were exposed to normoxia or hypoxia (10% O2) for 4 weeks. The deletion of BDNF was confirmed by real-time PCR. Right ventricular systolic pressuredetermined by in vivohemodynamics was not different between (SMMHCBDNF-/-) -and respective control mice after exposure to chronic hypoxia. However, BDNF deletion in SMCs attenuatedthe development of chronic hypoxia-induced pulmonary vascular remodelingas well asright ventricular remodeling. Histological analysisrevealed that(SMMHC-BDNF-/-) showed a significant lower amount offully muscularized arteries after hypoxic exposure compared to respective control mice. Additionally, BDNF deletionattenuated the dilatation of the right ventricle (right ventricle internal diameter (RVID): 1.20 ± 0.06 vs 1.40 ± 0.06, respectively, p<0.001) andthe decrease of RV function (tricuspid annular plane systolic excursion (TAPSE): 1.10 ± 0.03 vs. 0.96 ± 0.03, respectively, p<0.001) after chronic hypoxic exposure measured by echocardiography. Conclusion: Our data demonstrate that regulation of BDNF in PASMCs could play an important role in development of chronic hypoxia-induced PH.
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Abstract No. 214 Gas6/Axl Axis: a Potential Therapeutic Target for Pulmonary Arterial Hypertension Tatyana Novoyatleva 1,* , Svenja Tiede 1, Sebastian Herpel 1, Nabham Rai 1, Baktybek Kojonazarov 1, Manuel Richter 1, Norbert Weissmann 1, Henning Gall 1, Hossein A Ghofrani 1 , Werner Seeger 2, and Ralph T Schermuly 1 1
ECCPS, Justus Liebig University ECCPS, Justus Liebig University, Max Planck Institute for Heart and Lung Research *Presenting author
2
Objective: The interaction between Axl receptor tyrosine kinase and its ligand Growth arrest-specific 6 (Gas6) plays a role in proliferation, migration, differentiation, resistance to apoptosis and survival and has been implicated in the progression of a wide number of malignancies. Axl overexpression has emerged as a key molecular determinant underlying the development of acquired resistance to targeted anticancer agents. While many studies have focused on the role of Gas6/Axl in inflammation and cancer, the role of this axis for Pulmonary Arterial Hypertension (PAH) is unknown. Results: An upregulated expression of both Axl and Gas6 in pulmonary arteries (PAs) and Pulmonary Arterial Smooth Muscle Cells (PASMCs) of experimental Su/Hox, MCT and Hypoxia experimental models of PH have been determined. Axl and Gas6 enhanced expression has been confirmed in PAs and SMCs of patients with idiopathic pulmonary arterial hypertension (IPAH). Axl inhibition, by clinically applicable R428, caused a deterioration of proliferation, migration and increase of apoptosis in hPASMCs. Gas6 stimulation of hPASMCs outcomes in Axl phosphorylation, PI3K-Akt-PTEN-mTOR and ERK activation, augmentation of migration and resistance to apoptosis. Furthermore, Gas6 repressed, R428 enhanced apoptosis, through a caspase-dependent mechanism. Gas6 and sAxl levels were tested in plasma from PH patients: IPAH (30), associated with connective tissue disease (CTD) (34), pulmonary venous hypertension (PVH) (25), and Chronic Thromboembolic PH (CTEPH) (30). The non-PH control group (33) undergoing invasive exclusion of PH (mPAP<25 mmHg). Although sAxl and Gas6 levels were not significantly different between all groups, patients with Gas6 above the median have a poor survival prognosis (multivariate cox regression p=0.107, hazard ratio=1.855), suggesting Gas6 as a novel prognostic biomarker in PH. Conclusion: Our studies are the first to demonstrate the importance of Gas6/Axl in PAH pathogenesis and highlight its potential, as a novel therapeutic target for preclinical interventions.
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Abstract No. 222 The physiological significance of GPCRs in the development and treatment of PAH: the role of the P2Y2 receptor Mazen Shihan 1,* , Julia Stockburger 1, Stefan Offermanns 2, H. Ardeschir Ghofrani 1, Friedrich Grimminger 1, Norbert Weissmann 1, Werner Seeger 1, and Ralph Schermuly 1 1
Universities of Giessen and Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL), Justus-Liebig University 2 Max Planck Institute for Heart and Lung Research *Presenting author
Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and increased blood pressure in the pulmonary arteries. The elevated blood pressure is a result of complicated cellular processes that are mediated by distinct signaling pathways. The disease mechanism, however, is not yet well understood at the molecular level. Crucial mediators of vasodilation and blood pressure reduction, such as nitric oxide (NO), are released from endothelial cells in response to elevated blood pressure. Recent studies have shown the relevance of G protein–coupled receptors (GPCRs) in PAH progression and have demonstrated the participation of purinergic receptor P2Y2/G-protein interactions in NO secretion that results in systemic vasodilation. Despite the fact that more than 80% of newly developed medications partially or entirely target GPCRs, an effective GPCR-associated therapy for PAH has not yet been developed. The aim of our investigation is to examine a possible physiological involvement of the P2Y2 receptor in the development and mechanism of PAH. This receptor will be investigated as a potential pharmacological target in primary human pulmonary arterial endothelial cells. Results of studies using of the selective P2Y2 agonist MRS2768 and of fluid shear stress to activate mechanosensing pathways in vitro as well as in vivo should increase our knowledge about PAH mechanisms and potentially offer new prospects and approaches to overcoming the disease.
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Abstract No. 224 Non-invasive prediction of pulmonary hypertension based on automated 3D volumetry of pulmonary CT angiography. Claudius Melzig 1,* , Stefan Wörz 2, Benjamin Egenlauf 3, Sasan Partovi 4, Karl Rohr 2, Ekkehard Grünig 3, Hans-Ulrich Kauczor 1, Claus Peter Heussel 3, and Fabian Rengier 1 1
Heidelberg University Hospital BioQuant at Heidelberg University 3 Thoraxklinik at Heidelberg University Hospital 4 University Hospitals Case Medical Center, Case Western Reserve Univerity *Presenting author 2
Purpose: To non-invasively predict the presence of pulmonary hypertension by estimating the mean pulmonary arterial pressure (mPAP) using automated 3D volumetry of central pulmonary arteries based on CT pulmonary angiography (CTPA) in patients being evaluated for pulmonary hypertension (PH). Methods and Materials: 92 consecutive patients undergoing CTPA, right heart catheterisation (RHC) and echocardiographic pulmonary arterial systolic pressure measurement (PASP) for suspected PH in our institution were retrospectively reviewed. Patients with chronic thromboembolic PH were excluded (n=19). Patients in the final study cohort (n=73, mean age 66.3 years, 50 female) were randomised to a derivation (n=36, mean age 66.4 years, 25 female) and a validation cohort (n=37, mean age 66.1 years, 25 female). Automated segmentation of CTPA data was performed using in-house developed software. Volumes of the main as well as right and left pulmonary artery were calculated and corrected for vessel length and body surface area (MPA-volume and RLPA-volume). A linear regression model was established in the derivation cohort to estimate mPAP and diagnostic accuracy compared to gold standard RHC assessed in the validation cohort. Results: Mean mPAP was 29.2 mmHg in the derivation cohort and 28.9 mmHg in the validation cohort. Regression analysis yielded the following formula: estimated mPAP = -2.353 + (11.04 x MPA-volume) + (13.82 x RLPA-volume) + (0.489 x PASP) (r=0.92). When applied to the validation cohort sensitivity, specificity, positive and negative predictive value for prediction of PH (mPAP >= 25 mmHg) were 86%, 100%, 100% and 82% respectively. Conclusion: Non-invasive estimation of mPAP using automated 3D volumetry of central pulmonary arteries based on CTPA can predict the presence of PH as confirmed by RHC with high diagnostic accuracy.
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Abstract No. 261 Epigenetic changes might be induced by exercise training in a hypoxia pulmonary hypertension mouse model Christina Eichstaedt 1,* , Izabela Tuleta 2, Ekkehard Grünig 1, and Dirk Skowasch 2 1
Thoraxclinic at the University Hospital Heidelberg University Hospital Bonn *Presenting author 2
Background: Exercise and respiratory training in pulmonary hypertension patients has been shown to be an effective add-on to medical therapy improving exercise capacity, hemodynamics and quality of life. The molecular mechanisms of the exercise training benefits are not fully understood. Therefore, we propose a mouse pulmonary hypertension model for voluntary exercise training to measure epigenetic changes. Methods: Mice will be split into five groups with 10 animals each and followed for 5 weeks. The first group will be kept as a control at normoxia. The other four groups will be kept under constant hypoxia (10% O2) and subsequently develop pulmonary hypertension. The first group will receive the phosphodiesterase 5 inhibitor sildenafil in week 3-5. The second group will conduct voluntary exercise training by using a freely accessible running wheel from week 3-5. In the fourth group both treatments will be combined and the last group will not receive any treatment. Tissue samples will be taken from heart, lung and pulmonary artery to analyze DNA methylation and microRNA profiles of the five groups. Hypotheses: We suspect to see the least degree of pulmonary hypertension in the group of mice which received both treatments. The degree of cell proliferation and hypertrophy will correlate with the epigenetic profile. Hypo- and hyper-methylated genes and the level of expressed microRNA will be different between groups.
Submitted as: Presentation 220 words of 300 possible words
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Abstract No. 262 Plasma drug-concentrations in patients with pulmonary arterial hypertension on combination treatment Ekkehard Grünig 1, Johanna Ohnesorge 1, Nicola Benjamin 1, Jürgen Burhenne 2, Yeliz Enderle 2, Christina Eichstaedt 1,* , Benjamin Egenlauf 1, Christine Fischer 3, Satenik Harutyunova 1, Andrea Huppertz 2, Hans Klose 4, and Walter Haefeli 2 1
Thoraxclinic at the University Hospital Heidelberg University Hospital Heidelberg 3 University of Heidelberg 4 University Hospital Hamburg *Presenting author 2
Introduction: Combination therapy of the phosphodiesterase-type 5 inhibitors (PDE-5i) sildenafil or tadalafil and endothelin receptor antagonists (ERA) bosentan, ambrisentan, or macitentan may cause mutual pharmacokinetic interactions in patients with pulmonary arterial hypertension (PAH). The objective of this study was to analyze plasma drug concentrations in different combination treatments. Methods: PAH patients receiving a stable combination treatment with ERA and PDE-5i with targeted dosage for at least one month were routinely assessed, including clinical parameters and plasma drug concentrations. Concentrations were normalized considering dose and time from last medication intake and presented as multiples of the expected mean of the respective monotherapies. Results: 125 PAH-patients (84 female, 57% idiopathic/heritable) were included. Sildenafil and tadalafil concentrations were lowest in combination with bosentan (MOM 0.44±0.42, CI 0.30-0.57 and 0.89±0.53, CI 0.50-1.28, respectively) compared to the combination with ambrisentan (MOM 1.3±0.97, CI 0.86-1.73 and 1.67±0.63, CI 1.40-1.94) and macitentan (MOM 1.16±0.87, CI 0.86-1.46 and 1.59±0.99, CI 0.80-2.38). Combination of sildenafilbosentan led to double of the expected concentrations of bosentan in 53.8%. Patients from sildenafil-bosentan to macitentan showed a significant increase in sildenafil concentrations (p<0.001). Conclusions: Only the combination with macitentan or ambrisentan led to targeted mean PDE-5i plasma concentrations and should therefore be preferred to bosentan. Sildenafilbosentan showed the strongest interaction with low sildenafil and high bosentan concentrations. The study was not powered to analyze if lower PDE-5i concentrations cause unsatisfying clinical response. However, plasma concentrations within a targeted range are desirable and may become of increasing importance.
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Abstract No. 275 Influence of CYP2c gene deletion on hypoxic pulmonary vasoconstriction in isolated, ventilated and perfused mouse lungs Alexandra Erb 1,* , Natascha Sommer 1, Ralph Theo Schermuly 1, Jens Bier 1, Marisa Heipel 1 , Dorothea Peters 1, Karin Quanz 1, Werner Seeger 1, Friedrich Grimminger 1, Norbert Weissmann 1, and Ladislau Kiss 1 1
University of Giessen *Presenting author
Rationale: Hypoxic pulmonary vasoconstriction (HPV) is an essential physiological mechanism to optimize gas exchange under conditions of local alveolar hypoxia. It matches perfusion to ventilation by redistributing the blood flow to well ventilated areas, thereby improving systemic arterial oxygenation. Arachidonic acid (AA)-derived mediators are known to be potent vasoregulators in different organs in health and disease, however their exact role in HPV remains under debate. Cytochrome P450 enzymes metabolize AA to different mediators. Especially epoxyeicosatrienoic acids (EETs) are mainly produced by CYP2c and CYP2j isoforms. The aim of this study was to investigate the effect of CYP2c gene deletion and the resulting reduce of EET levels on acute and sustained HPV. Methods: Investigations were performed in isolated ventilated and blood free perfused lungs of CYP2c-deficient (CYP2c-/-) mice. The increase of pulmonary arterial pressure (ΔPAP) in response to acute hypoxic ventilation (1% O2, 10 minutes; normoxia: 21 % O2 , 15 minutes) or sustained hypoxic ventilation (1% O2, 3h), as well as after application of the thromboxane A2 agonist U46619 and potassium chlorid (KCl) was recorded. Results: Isolated lungs of male and female CYP2c-/- mice did not show any difference in vasoconstriction during acute hypoxic ventilation or in presence of U46619 or KCl compared to WT mice. However, during inhibition of EET degradation by application of the soluble epoxide hydrolase inhibitor N-[1-(1-oxopropyl)-4-piperidinyl]-N’-[4-(trifluoromethoxy)phenyl]urea (TPPU) isolated lungs from male CYP2c-/- mice showed significantly less inhibition of HPV than WT lungs. During 3h of hypoxic ventilation of CYP2c-/- lungs no differences to WT lungs could be detected. Conclusion: Deletion of CYP2c did not affect HPV suggesting that CYP2c-dependent production of EETs under physiologic conditions is too low to exert vasoactive effects. However, enhancing EET levels by application inhibited HPV which was dependent on presence of CYP2c.
Submitted as: Presentation 292 words of 300 possible words
Page 286 of 286
DZL Annual Meeting 2017
Author Index Firstn ame
Lastna me
KORA Study Group Micha Bustin el Stefani Höppner e The PROGR ESS study group Rainer Pepperk ok Poona Sarode m Ralph Schermu T. ly Georgi Tsiavalia os ris Andre Weigert as Norber Weissm t ann All (ALLIAN Age CE) Asthm study a group Cohort Marcu A. Mall s Shariq Abid Mania Ackerma nn Maxim Ackerma ilian nn Jerzy Adamski Jirmo Adan Heiko Adler Serge Adnot Rama Agrawal n Marjan Ahmadi Peter Ahnert Nancy Ahrendt
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Abstract No. 246 194
087 257 296
090 037,232 195,141,148,208 020 037,232,234 181,192,195,256,270,268,055,141,148,189,208,214,222,275 193
254 134 095,274 226 173 273 249 134 254 083 296 061 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Saket Micha ela Reza
Melani e Tobias Lorenz Nasim
Hani Sandr o Peter Christi na M Oana V. Ole
Ahuja Aichler
136 239
Akbarza deh Najar Albrecht
081
Albrecht Aldung Alebrahi mdehkor di Alsafadi Altamura
089 291 181,189
Alter Alvira
012,060,183 124
Amerie
283
Ammerp ohl Valerie Amselle m Ilias Angelidis Adrian Anklam m Hossei Ardeschi n r Ghofrani Jennif Arndt er Stefan Arnhold Bernd Arnold Paola Arnold Alina Asafova Franzi Aschenb ska renner Yasse Assenov n Katrin Audrit Ines Aumann Sigrid Auweter Murat Avsar Chidie Awah bere Malik Aydin Hoeke Baarsma Hoeke Baarsma A. Thoma Bahmer s
25.01.2017
057
182 254
029,074,077 134 049 094 141
191 181 252 167,145,178 234 109 295 264 054,091 278,279 008 020 150,220,273 202 212,066,239 007,150,282,006
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DZL Annual Meeting 2017 Caroli na Caroli na Carme n Emel Robert Anita Fabian Rösler Guiller mo Sabin e Jörg Jürgen Yannic Jason HT Nadja Nadja V. UtaMaria Rudolf
Ballester Lopez Ballester López Ballester osMerino Baloglu Bals Balázs Bamberg Barbara Barreto
256
Bartel
088,106,115,119,138,158,196
Bartel Barton Bartsch Bates
116 167 168 085
Batora Batora
035 161
Bauer
162
Bauerfei nd Gregor Bauman Isabell Bauman n Ulrich Bauman n Ingo Bauman n Sandr Baus a Johan Beckers nes Erik Beckma nn Mariol Bednorz a Friede Behlerrike Janbeck Jürgen Behr Jurgen Joche n AnnKathri n
103 203
250 012,052,060,143,183,248,146 090 278 288 084,062
095 019 150,152 014 089,280 180,216 116,249,096 215 181,192,256,270 018 060,167,183,036,050,058,059,099,105,155,213,236,239,073,145,178,2 41 025 064,082
Behr Behrend s Behrendt 272
25.01.2017
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DZL Annual Meeting 2017 Christ oph Alexan der Saveri o Johan nes Nicola Dietric h Frank Lenna rt Inga
Beisswe nger Belenko v Bellusci
248
Beneke
289
Benjami n Berdel
262
Berger Berndt
203,244 085
268 192,003,133,062,149
053
Bernema 147,240 nn Thoma Bertero 002 s Steph Bertram 154 anie Wilhel Bertrams 046,047,044,045 m Anita Bhandari 139 Luisa Biebach 124 Jens Bier 275 Frank Biertz 060,143,183 Carlo Bifulco 203 B. Heike Biller 160 Leonh Binzenh 058,059 ard öfer Helge Bischoff 258 Lara Bittersoh 108 l Emilie Bizard 134 Robert Blach 094 Steph Black 041 en M. Sasch Blanken 047 a burg Paulin Blazejew 154 a ska Caroli Boden 085 ne Martin Bodenbe 075 nnerTürich Johan Bodner 062 nes Andre Boeck 246 as U. Boehm 207
25.01.2017
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DZL Annual Meeting 2017 Laura Martin David David Madel eine Philipp e Andre a Paul Tilman
Boge Bohm Bonders son Bondess on Bonert
286 291 169
Bonniau d Bopp
097
Borchert Borggref e Judith Bossen Sébast Boutin ien Sebast Boutin ien Farast Bozorgm uk ehr Ken R. Bracke Christi Branden na berger Peter Braubac h Armin Braun Dieter Braun Thoma Braun s Mariell Breau e Anna Brichkin a Kerstin Brinkert Paul Brinkma nn Const Brinkma antin nn Ryan Brown David Brunn Eva Brunnem er Guy Brussell G. e Irene Brüske Theres Buchegg a er Bernd Bufe Lara Buhl Dmitry Bulavin Gerald Burgstall
25.01.2017
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135 094,041 098 043 252,251 011 161,258 212 254,206 219,286,174,227,094,108 159,200,209,219,286,107,174,227,154 123 084,133,062,189 134 002 095 166 154 111 184 006 212 194 095 070 105 002 087,028,127,130,204 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 er Burhenn e Kerstin Burmest er Hauke Busch Andre Bush w Mandy Busse Laure Byrnes n Andre Bähr a Andre Böck as Martin Böhm Mario Böhm Deniz Bölükba Ali s Melani Börries e Marisa Böttger Elaine Cabral Serrao Brian Caffrey E. Monic Campillo a s Alfons Carleo o Suna Cebesoy Felix Ceelen C. Cen Adelh Cerwenk eid a Adam Chaker ChoChao Ming Cho Chao Ming Cather Charron ine Kanch Chauhan an Prakas Chelladu h rai Shanz Chen e Shashi Chillapp Pavan agari Shashi Chillapp agari Joann Chorosto Jürgen
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DZL Annual Meeting 2017 a
Sandr a Petros Ruppe rt Thoma sM Thoma s M. Thoma s Adrian a Julio Rita Milene Nina Bruno Petra Steve Christ oph All memb ers of the
Swati Edgar Franzi ska Alexan der Kathle en Reinh ard Reinh ard H Olga Theres e Farshi d Fabio
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030,179,185 001 209,154 252,011,110,251
062 159,219 123
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DZL Annual Meeting 2017
David S. David Klaus
Marco DeLuca
193
DeLuca Deckma nn Dehmel
220,015 075
Deluege
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Delventh al Mario Deng Sofia Depner Tushar Desai J. Tushar Desai J Martin Dichgan s Mirosl Didiasov ava a Hendri Dienema k nn Oliver Dietrich Alexan Dietrich der Steffe Dietz n Julien Dinkel Daniel Dipresa e Anna- Dittrich Maria Anna Dittrich Maria Susan Dittrich ne A. Dittrich Susan ne Oliver DittrichBreiholz Micha Dmoche ela witz Steph Dobersc anie h Gerga Dobreva na Christ Doelling oph er Anne Dommas chk
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25.01.2017
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274 278 084,062 062 135 108
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DZL Annual Meeting 2017 Christi an Marie Julian Fotios
Daniel Ilona Katarz yna Julia Anja Dmytr o Shero n Julia Simon e Martin Ralf Beate Evgeni ia Matthi as Marku s Benja min Johan na C. Marc Harald Maxim ilian Monik a Christi na Oliver Elie Mei Alexan der Nageh an
Dopfer
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Dorda Dorer Drakopa nagiotaki s Dröman n Du Bois Duda
014 087 097
Duerr Dutschk e Dvorniko v Dzoro
086,198 267
Dürr Ebener
226 230
Eberhar dt Eberhar dt Eckes Edeleva
046,045
Eder
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Ege
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Egenlauf
224,262
Ehlers
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029 045 102
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259,260,265,266,267,188 050 123
Ehlers 168 Ehrhardt 114,124,125,149 Ehrmann 151 Eichinge r Eichstae dt Eickelbe rg El Agha Elsayed Emelyan ov Emiralio uglu
25.01.2017
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Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Yeliz Volker Christi ne Theres a Lena Rita
Alexan dra Alexan dra Ezgi Birgit Zeyne p Azade h Christi ne Martin Heinz Iven Zipei Isis Isis E. Joachi m Susan ne HansGerd Alexan der A. Christi ne Lutger Bijan Ludge r Fred Siokou Daniel
Enderle Endris Engel
262 004 120
Engelma nn Engels Engenha rtCabillic Epp
198
Erb
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ErmisKaya ErtlWagner Ertüz
250
Esfandia ry Falk
148,208
Fallenbu echel Fehrenb ach Fellhaue r Feng Fernand ez Fernand ez Ficker
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Fiedler
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Fieguth
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170,180,205 190
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Finkelma 168 n Fiona 244 Firnkorn 112,117
25.01.2017
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DZL Annual Meeting 2017 Sebast ian Christi ne Paul K. Andre as W. Claudi a Veit Bogda n Kai Alistair Bernar d A. Andre Andre as Marion Simon Julia Urs Andre as Eva Oliver Helmu t Oliver Thoma s Elaine Manue la Takas hi Athan asios Sebast ian Kai Thoma s Karolin e Vincen tD Cedric
Fischer
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Fischer
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Flechsig Fleck Flemmer
161 207 125
Flexeder
053,173
Flockerzi Florea
277 096
Foerster Forrest Fox
124 236 203
Franke Franke
068 291
Franken berger Frauman n Freise Frey Frey
167,025,124,236,239,145,178,241
Fritzschi ng Fuchs Fuchs
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Fuchs** Fuehner
166 018
Fuertes Funke
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Furusaw a Fysikopo ulos Fähndric h Förster Gaaß
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Gaertner
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Gaggioli
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25.01.2017
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056,126,129,150,151,152,193,210,220,273,282,288 087
148 143 125,285 169
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DZL Annual Meeting 2017 Arne Gaida Valerie GailusDurner Luis Galietta Henni Gall ng Elisab Gamen etta Virajith Garapati Claudi Garcia a Castro Ferna nda Holger Garn Stefan Gattenlö hner Jack Gauldie Gemm Gaya Jordi Sonja Gehlen Micha Gercken el s Vasiliki Gerovasi li Hossei Ghofrani n A. Hossei Ghofrani n Ardesc hir Ardesc Ghofrani hir Hossei Ghofrani nA H. Ghofrani Ardesc hir Bened Giaimo etto Daniel e Christi Gieger an Mareik Gierhard e t Silke Glage Torste Glomb n Sven Gläser Lutz Goldbec k Torste Goldman n n
25.01.2017
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238 062 109 017 019 204,213 265 195,189 270,268
055 214 222
098
173 148,208 274 274 012 042 010,029,065,074,077,082
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Heiko Daniel a Nestor Olga Jens Simon Anke Marco Ulrich Veroni ka Flavia Niklas Tim Matthi as Friedri ch Friede rich Kathri n Lukas Judith Roma n Jens Ina Simon Simon Y. Ekkeh ard Thoma s Andre as Stefan Marcel Stefan Ernest o Paulin a Mira Yasem
Golpon Gompel mann Gonzale z Roldan Gorlanov a Gottlieb Graeber Graessel Gramm Grandel Grau
091,161 259,260,265,266
Greiffo Gremke Greulich Griese
025,236 033 052 042,257
Grimmin ger Grimmin ger Griss
195,034,037,092,100,184,190,232,234,268,055,222,275
Groman n Gronbac h Grothau smann Gruber Gruh Gräber Gräber
278
Grünig
224,261,262
Guderm ann Guenthe r Guenthe r Gutberle t Guth Guzmán -Díaz Gwozdzi nska Gökyildir im
277,276
25.01.2017
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197 046
125 021 154 205 009,019,251 201
292,293,133,136,163,237 037 160,269,281,284 176 062 255 190
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DZL Annual Meeting 2017 in Jessic a Andre as Stefan Frank Ferdin and Amal Alexan dra Anika Stefan Walter Wolfga ng Lars
Götzfried 123 Günther
032,036,038,084,097,099,191,221,062,161,117,146
Günther Günther Gürth
084,062,179 051 071
HOUSS AINI Haase
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25.01.2017
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DZL Annual Meeting 2017 Selina Katja Rudolf Stefani e Axell Axel Ying Matthi as Silke Jan Lena Holger Joachi m Femke Anous ka Kathar ina Marisa Kathar ina Andre as Gudru n Meike Marku s Ingrid Lisa Elisab eth Christi ane Christi na Sebast ian DirkPeter Felix JF Felix Julia Christi an
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25.01.2017
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DZL Annual Meeting 2017 Christi na Nina Claus Peter ClausPeter ClausPeter Claus Peter Gudul a Stefani e Anne Anne Stefan Steph anie Antho ny Julian e Marius M. Thoma s Paul Kathar ina Simon Jens Jens M Stefan Garn Rolf Olaf Carme n Heinz Alexan der HeinzGerd HeizGerd
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Hoyman n Hoyman n
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25.01.2017
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174
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DZL Annual Meeting 2017 Martin
Jin Rudolf Maria Magda lena Rudolf M Rudolf M. Martin Simon e Andre a Cornel ia Rebec ca Martin a Sigfrie d Linda Christ oph Martin Gereo n Marius Olga N Thoma s Dijana Martin Kazuhi ro Fabio Nicole Howar d Peter Const anze Heger mann Sabin a Silke Wiebk
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25.01.2017
073 185 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 e Kathar ina Uta Micha el Gregor André Dieter Jie Sharo n Adan Chari Bertra m Gerrit Danny Brend a Anna Lena David Christ opher Bened ikt Rudolf A. Rudolf Micha el Hans Joachi m Sajo Nicola s Kathri n Till Kimbe rly Panag iotis Nona Naftali Ilona Marian
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Jobst
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060,143,183 056
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DZL Annual Meeting 2017 Hadia n Martin a Julia Annika Florian M. Stefan Brigiite Brigitte Gabi HansUlrich Hans Ulrich Diego
Dagm ar Daniel Kanny Agilo Luitger Agilo Marku s Vidya sagar Rose marie Susan na Nural Christ opher AnneMarie Anne Detlef Ladisl au Bastia n Steph an Björn Walter Filip Kathar ina
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25.01.2017
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036 004,271 134 160
104
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DZL Annual Meeting 2017 Maria Ursula Ursula Edda Jens Norma n Hans Christi na Elisab eth Oleksi y Sasch a Petra Nikola us Mikael Juerge n Lars AnnKathri n AnnKathri n Lariss a Armin Andre a Ina Thoma s Cordul a Bakyt
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25.01.2017
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009 181 270 268,185,214 109 053 226 259,260,265,266 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 ntina Sebast ian Matthi as Matthi as V. Annett e Martin a Sotiris Sotirio s Cathar ina Marku s Marku s Melina Dragin ja Tom Bernh ard AnnaMaria Gabrie la G. Thorst en Susan ne Simon e Simon e Hans Katarz yna Helen e Micha el Thoma s Mark
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004,271
25.01.2017
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DZL Annual Meeting 2017 Gabrie le A. Florian Jan Lena Vitalii Maren Marcu s Arne Katrin Mark Christi an Alexan der Skadi Wolfga ng W. Sonja Johan na David Grazy na Inke Peter Melani e Melani e Günter Iris Christi an Mark Christi ane Carste n Lucas Christi an Nico Karl-
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249
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Lachma nn Ladwig
274,170
25.01.2017
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064 264,070,075
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194 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Heinz Xin Marko Katja Joann a Felix Susen Philipp Matthi as Bianca Lisa Andre as Mareik e Wolf
Lai Lampe Landgraf -Rauf Lange
047,044 090 106,246
Lasitsch ka Latterma nn Latzin Lauth
252
LavaeMokhtari Laßwitz Lechner
063,051
Lehman n Lehman n Reiner Leidl Domini Leitz k Gabrie Leuschn la er Patrici Leutz a Lariss Lewitan a Reinh Liebers ard Micha Lindner el Heidru Lingner n Anika Lins Torste Lippman n n Xiaoqi Liu ng Zhongj Liu uan Hui Loh Mun Mario Looso Elena LopezRodrigue z Tanja Lucke Britta Ludewig
25.01.2017
042
291 056 076
159 060 038,066,105,182,213,239 291 052,054,036,042 086,198,254 167,073,145,178 019,263 069 026 038,105,182,213,203,146,241 054 154 108 104 120 002 162,179 061,017,230,085
060,183 041 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Christi na Joni Valesk a Lars Agnes Florian Ingo Nodir Lavini a Helgo Poorni ma Mandy Heimo Bernar d Rapha el W. Rapha el Monik a Rainer Marcu s A. Marcu s Marcu sA Anna Samu el Matthi as Elisab eth Philipp Ramo n Annali sa Ulrich Aina
Lukas
134
Lund
088
Lunding Luzak Länger Maack Madrahi mov Maegel
252 053 094 278 008
Magnuss en Mahava di Mai Mairbäur l Maitre
247,006
Majeed
117
Majeed
147
Malczyk
192,276
Malik Mall
123 048,027,039,090,110,113,201,263,280
Mall
026,256,009,011,019,024,072,089,111,218,226,251,036,086,198
Mall
079
Malmgre n Mang
010
Mann
050
Marcos
134
Markart Marrade s Marsico
036,076 120
Martin Martin Medina Sebast Marwitz ian
25.01.2017
041,055
292,293,133,136,163,191 043 250 134
174
045 170,180,205,216 239 010,029,065,077,082
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Sandh ya Regin a Ulrich A. Ulrich Tobias
Matthes
167,145,178
Maus
109,018,108
Maus
109,018
Maus Mawassi i Eckhar Mayer d Christ Mayr oph Lucian Mazzocc a hi J. McIntosh Micha el Aditi Mehta Felix Meinel Silke Meiners Christ Meisinge a r Micha Meister el Liz Meister Claudi Melzig us Sandr Menke a Cléme Mercé ntine Hayan Merhej Olivia Merkel Sylvia Merkert Ruth Merkle Julian Merle Pham Marco Mernber ger Lisa Merthan Thoma Meul s Almut MeyerBahlburg Sven Michel Evang Michelak elos is Katrin Milger Pamel Millara Büchner Anja Mirenska Nodir Mirsaido
25.01.2017
108 101 176 049,050 165 067
084,062,153 279 134,032,130,096 173 188,001,016,023,080,118,250,253,258,135 135 224 205 001 215 153 170,180,216 291 032,059 005 087 032,096 200,272 056 197 073,243 079 274 075 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017
Harald Kirstin Irene Tomof umi Juerge n Pia AnnaSophi e Rafael Patrici a Franco Thoma s Carme la Daphn eS Daphn e S. MarieChristi n Adele Tony Christi an Shrika nt R. Thoma s Antje Dieter Micha el P. Kathri n Lavini a Kerstin
v Mischak Mittelstra ss Mitterma nn Miyamot o Mohr
233 240 057 018 278
Moinzad eh Moldenh auer
050
Molina Moran Losada Moritz Moritz
120 014
Morrone
187
Mous
114,124
Mous
125
Moßner
005
Mucci Mucham uel Muecke
274 096
Mulay
032
Muley
188,001,004,005,016,035,080,120,253,258,295,112,135,146,147
Munder Munker Murphy
095 167,145,178 148
Mutze
212,038
Mägel
094
MätzRensing Winfrie Möller d Christi Mücke an
25.01.2017
154
249 274
030
154 283 179
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Christi an Meike Ullrich
Mühlfeld
061,021,230,254,206
Müller MüllerLisse MüllerRedetzk y Nahrlich Nakagiri
286 161,244
Nassens tein Nattenm ueller Nayakan ti
264
Nehlmei er Micha Neuberg el er Karolin Neuman an Theres a Claus Neurohr Petra Neuser Joana Neves Dao Nguyen Thi Nguyen Minh Nguye t Anna Nickel Monik Niehof a Andre Nist a Nina Noskovic ova Nina Noskovic ovà Ettore Novellin o Tatyan Novoyatl a eva Dennis Nowak Peter Noël B. Lutz Nährlich René Nötzold Reiner
154
H.
Lutz Tomoy uki Christi na Johan na Sreen ath Reddy Inga
25.01.2017
207
292,293 131
035 185
203 231
167,073,145,178 099 254 251 002
054 174 005 028 213 212 214 053 278 009,125 162
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Elfried e Micha el
Nößner
228
O. 253 Breckwol dt Prajakt Oak 114,124,125 a Helen Obernolt 159,200,219 a e Matthi Ochs 017,021,136,198,230,085 as Stefan Offerma 222 nns Bettina Oherle 127 Johan Ohnesor 262 na ge Yoshih Ohno 019 aru Sebast Ohse 080 ian Gregor Ojak 164 Till Olchers 074,112 Olga Olejnick 247 a Brain Oliver 068 G. Ruth Olmer 170,180,205,216 Eric Olson 237 Elzbiet Opdazait 280 a e Davia- Optazait 161 Elzbiet e a Almud Ortega025 ena Gomez Herme Overklee 096 n ft Marga P. 253 reta Correia Winfrie Padberg 067 d Oleg Pak 181,141,148,189,208 Steph Papenm 225 anie eier Klaus Parhofer 183 Sasan Partovi 224 Helen Pasterna 074 ck Linda Pauksch 008,131 Bettina Paul 097 Michell Paulsen 026,027 e Martin Pech 068,288
25.01.2017
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Frauke Pederse n Rolan Penzel d Sven Perner Alexan Perniß der Britta Peschel Annett Peters e Doroth Peters ea Frank Petersen AnnPetersen Kristin Nicole Pfarr Petra Pfefferle Ina Micha Pfeifer el Franz Pfeiffer Olaf Pfennig Micha Pflaum el Thoma Pfluger s Wolfga Pfützner ng Gary. Piazza A. Alexan Pichl dra Katarz Pienkow yna ska Mario Pieper Katarz Piskulak yna Christ Plass oph Jessic Plum a Andre Pomsch as ar Thoma Pralow s Ruben Prange Antje Prasse Cornel Prehn ia Stefan Preisend örfer Gerhar Preissler d
25.01.2017
282,010 004 029,074,077 070 182,147,241 173,194 275 081,083 173 004 146 036 278,279 219,174,227 186,205,215 244 168 100 195,270 014 218,223 133 026,295 132 125,285 278 175,139 015,099,294,018 173 058 228
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Klaus T Klaus T. Mareik e Immo Tina Jana Rolan d Micha el Micha el U. Soni Soni Savai Stefan Kun Yu Karin Klaus F. Klaus F Klaus Ellen Olga Amir Nabha m Ulf R Bereni ce Andre as Martin Tobias Niels Max Maxim ilian Maxim ilian F. Fabian Harald Simon e
Preissne r Preissne r Price
084
Prinz Pritzke Prokein Proksa
290 114,124 240 278
Puderba ch Puderba ch Pullams etti Pullams etti Pöhlman n Qian Qiang Quanz Rabe
019,201,263
Rabe
150,029
Rabe Raddatz Rafikova Rafiq Rai
247,099 020 041 075 214
Rapp Rath
062 094
Raue
291
Reck Reicherz er Reinmut h Reiser Reiser
065,074,077,082,161 114
Reiser
278
Rengier Renz Reu
224 193 145,203,243,244
25.01.2017
098 220,273
280 084,162,176 237,037,100,234,030,179 154 120 023 189,275 007,282,010,006
077,161 132 169,279
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Sebast ian Kai Katrin Manue l Veren a
Reuter
088,115,119,231,158,196
Richter Richter Richter
010 067 214
RickertZacharia s Domini Rideau que Anja Riediger Lisa Ernst Rietsche l Karste Rindt n Felix Ringsha usen Angel Risch a Agusti Rodrigue n zGonzale z Thoma Roeder s Karl Rohr Addi J RomeroOlmedo Marcu Rosenbl s att Ina Rothenai ger Sandr Rothkirc a h Robbe Rottier rt J. Robert Rottier J Elsa T. Roxlau Riti Roy Karla Rubio Roma Rubtsov n Ernst Rummen J. y Cleme Ruppert ns Kai Ruppert Cleme Ruppet ns Niels Röckend
25.01.2017
072
134 001 150 278 057 295 291
175,139,043,223 023,224 062 080 202 069 114,125 124 195 236 084 267 278 195,136,163,191,221,230,117,146,147 160 085 210 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017
Barbar a Ylia Jawad Johan na J Johan nna Johan na J. Florian
orf Rösler
150
Salazar Salman Salomon
034 008 079
Salomon
089
Salomon
090
Salopiat a Christ Samako os vlis Lea Samija* Rachel Sanborn E. Alireza Saraji Rim Sarker Sabrin a Jahan Gaura Sarode v Pouya Sarvari Steffe Sass n Christi Sattler ne Annett Sauere Heilborn Rajku Savai mar Soni Savai Pullams etti Carme Schaden Brittinger Liliana Schaefer Ulrich Schaible Andre Schamb a erger Esther Schams chula Jolant Schatter he ny Fee Schatz Bianca Schaub Dirk Schaudi en Micha Schedel ela
25.01.2017
023,291 163 166 203 141,189 087
293 084,237 125 249 057 034,037,062,065,092,100,184,232,234,030,176,197 034,232,185,197
099 076 158 059 295 048,027,110,111,113,086 007 106,220,238,246,273 107 246
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Susan Max Aloys Ralph Ralph T Ralph Theo Heike Swetla na Maxim ilian Sabin e Herber t B. Herber t Marcel Ulrike Julia Uwe Kathar ina Anja Bernd Sabrin a Otmar Simon e Anne Felicit as Susan ne LarsHenni ng Carste n Philipp e Lea Marc Marc
Scheibe Schelker Scheper s Schermu ly Schermu ly Schermu ly Scheuer mann Scheufel e Schieck
148,189 291 058
Schild
095
Schiller
022
Schiller
038,049,050
Schilling Schindlb eck Schipke Schirmer Schleret h Schmall Schmec k Schmec kebier Schmid Schmidt
291 257
Schmidt Schmidt
056 128
Schmidt
246
Schmidt
248
SchmidtWeber SchmittKopplin Schmoll Schneid er Schneid
069,071,246,282
25.01.2017
192,270,268,055,176,185,222 214 275 089 077 056
061 001 005 034 046,047,044,045 186,205,215 283 048,024,027,110,111,086
249 033 188,023,080,147 016,135 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 A. Jennif er Kenji Rene Tobias Madlin e Sven Maren Carste n Holger Christi ne Rainer Hinner k Andre as Christi an Lariss a Michal Nicola us Kathar ina Moritz André Aruni ma Oliver Werne r Kerstin Micha el Nora Anna Kather ina Tayya
er Schnied er Schorpp Schram m Schroete r Schubert
093 202 073,228 278 170
Schucha rdt Schuhm ann Schultz
063,015
Schulz Schulz
053,173,012,052,194 044
Schulz SchulzHildebra ndt Schulze
181 218,223
Schwag er Schwarz kopf Schweig er Schwerk
064,210
Schäfer
181,141,208
Schäfer Schütte Seal
191 110 127
Sedlacz ek Seeger
161
Seidel Seimetz
259,260,266 039,072,113
125
042,040 045 042
192,195,270,076,084,133,163,237,034,037,062,065,092,100,184,190,2 32,234,268,156,165,255,030,055,162,176,179,185,189,197,208,214,22 2,275 046 192,256,270
Semren SerranoMollar Sewald
032 017
Shahzad
149
25.01.2017
159,200,209,219,286,107,174,227,154
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 b Irina Mazen Hoenoh Ulf Robin Reiner Thierry Lukas Rajeev Indrab ahadu r Heike Alexan dra Chrys anthi Dirk
Shalash ova Shihan Shin
133,163,221
Sibelius Siebers Siebert Siemeni Simon Singh Singh
190 067 077 008 049 076 084,062
Sinneck er SittkaStark Skevaki
210
Skowasc h Wiolett Skronsk a a-Wasek Jelena Skuljec Natalia Smirnov a Maia Smith Bradfo Smith rd Oliver Soehnlei n Olga Solyanik Natasc Sommer ha Wiebk Sommer e Wielan Sommer d Olaf Sommer burg Rocio Sotillo A. Soultano va Steph Spahn an Claudi Staaba Weijnitz Jakob Stadler Micha Stadler el Jerem Stadter
25.01.2017
222 294
046,044,045 193 036,261 212 200,272,290 187 194 085 025 144,169 181,141,148,189,208,275 008,131,094 132 019,201,263,280 258 207 079 050,058,059,155,182 167 094 164 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 y Mirjam Marie Frauke Astrid
Stahl Standl Stanke Stasche wski Georgi Stathopo os lous Beatrix Steer Lilian Steffen Bernh Steiert ard Karina Stein Tobias Steingrü ber Eva Steinke Martin Steins Andre Stelzl as Albrec Stenzing ht er Marku Stepath s Peter Sterk J. Susan Stiebeler ne Christi Stielow na Thorst Stiewe en Wolfra Stiller m Julia Stockbur ger Florian Stocker Tobias Stoeger Erich Stoelben Christ Stolp a Jennif Stolper er Ursula Storch Marian Stornaiu o olo Nicole Strobl Maxim Strunz ilian Beate Stubbe the Study ALLIA Group NCE ALLIA Study
25.01.2017
009,011,019,201,251,263,280 053 020,095 202 153 249 230,085 291 078 061 201 035 283 004,258 291 166 161 046 005,033,100 226 222 178 249 211 135 109,018,108 277 212 228 049,050 060,183 126,288
166,220,273 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 NCE Julia Sophi a Tobias Mark R. Prema
Group Stump 203,244 Stöcklein 285 Stöger Stöhr
Subbara yal Florent Subtil ine Karste Suhre n Laila Sultanse i Leila Sultanse i Kristin Surmann Norber Suttorp t Norber Suttrop t Akylbe Sydykov k Akyl Sydykov Zulfiya Syunyae va Magda Szczygie lena l Magda Szczygie lena ł Boglár Szentes ka Mónik Szepes a Marten Szibor Sandr Söhler a Holger Sültman n Cliffor Taggart d C. Tomot Takano sugu Stefani Tamm e Steph Tamm anie Helmu Teschler t Birgit Teucher Natalie Thaens Fabian Theis
25.01.2017
096 117 100 190 173 150 152 047 046 296 055,141,148 208 243 118 080 042 205 189 012,060 001,016,092,258 039 018 020 095 143 035 169 116,049 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Fabian J. Marcu s Domini k Carl Maxim ilian Elisab eth Micha el Barbar a Thoma s Fei Svenja Jens Irina Antoa neta A Meritx ell Meritx ell Achim Marina Alexsa ndra Simon Juerg Igor Aman da Izabel a Kati Selina Stefan Burkh ard Martin a Mohib
Theis
125
Thiedma nn Thiele
139,043
Thielma nn
253
Thiering
173
Thomas
001,016,080,161,258,112
Thorand
194
Thum
273
Tian Tiede Timmer Titkova Tonchev a Tort Tarres Tort Tarrés Tresch Treskov a Tretyn
235 214 023,080,291 080 056
Triphan Tschirre n Tudorac he Tufman
137,287 260
Tuleta
261
Turkows ki Tümkay a Tümmer s* Tümmler
065,092
U. Muckent haler Uddin
254,253
25.01.2017
288
018,108 109 062 091 232
008,289 040,161,203,243,244
251 166 014,020,031,057,170,295
010 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Franzi ska Karin Saskia István Rudolf Pierre Chanil Carlo Tobias Astrid Julio Ernest o Elordu y Janika Sarah Heidru n Martin Christi na Jens
Claus F. Claus Vanes sa Anja Evelyn Ekkeh ard Andre as Uwe Marga rethe E. Marga rethe Frank Christi na Darcy Darcy E.
Uhl
199
Uliczka Ulrich Vadász Valenta Validire Valsaraj an Vancheri Veit Velroyen Vera Vergara
231,043 170,180,216 156,165,255 057 134 030
Viereck Vierkotte n VillenaHermoza Vingron Vock
273 130,239,277
VogelClausse n Vogelme ier Vogelme ier Vogelsa ng Vogt Vollmeist er Vollmer
091,161,160,269,281,284
Voskreb enzev Völker Wacker
160,269,281,284
Wacker
054,060,183,036
Wacker Wagner
161,160,269,281,284 231
Wagner Wagner
199,202,038,105,130,182 212,066,239
25.01.2017
097 167,145,178 279 046,047,044,045 096
167 045 138
012 052,060,143,183,292 246 183 047 077
047 052
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Claudi us Willi Jasmi n MarieChristi ne Axel Kerstin Doroth ee Julia Zhe Rui
Wagner
113
Wagner Wagner
226 097,099,117,147
Wagner
291
Walch Walendy -Gnirß Walter
239 154
Walter Wang WangSattler Wanzel
040 235 173
Micha el Gregor Warneck e Arne Warrth Arne Warth Benaj Waschki min Benja Waschki min Anita Wasik A. Roxan Wasnick a Iris Waterma nn Henrik Watz Jonas Weber Rebec Weber ca Christi Weber ne Marku Weckma s nn Marcu Weckma s nn Marku Weckma s nn** Micha Wegman el n Birgit Wehnl Wilko Weichert Steph Weiding an er Thoma Weig s
25.01.2017
107
033,100 219,286,174,008,131,228,227,094,041 004 001,080,250,271 007 010,247,006 022 133,163,221 074,077,082 007,282,010,012,029,060,183,247,006 277,276 016 162 068,150,193,273,282,288 220 166 252 120 004 173 073
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Bettina Weingar d Oliver Weinhei mer Daniel Weiss Astrid Weiss Micha Weitnau el er Norber Weißma t nn Vanes Welk sa Athol Wells Tobias Welte Franzi Wert ska André Wesener Martin Wetzke Sarah Weustho ff Joche Whilhel n m Nataly Wiebern eit Christi Wiedem ane ann Conra Wiederh d old Silke Wiegand Bettina Wiegma nn Lutz Wiehlma nn Mathia Wieland s Mark Wielpütz Mark Wielpütz Oliver Marc Wielpütz O. Mark Wielpütz O. S. M. Wienhol d Norber Wiessm t ann Astrid Wietelm ann Rainer Wiewrod t Joche Wilhelm n Konsta Willer
25.01.2017
147 164,267,287 199 055 252,110,251 276 032 097 012,054,143,233,247,109,091,094,284,018,108 273 047 274 289 133 278 161 036 264 121,186,215 014,031 135 137,019,226,035,287 164,267 201 263,280 207 030 030 248 046,195,076,163,176 278 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 nttin Konsta ntin Hauke Patrick Joachi m Sabin e Heiko Thoma s M. Raoul Lisa Martin Wim Susan Emiel Christ oph Sabin e Sabin e Patrick Lukas z Malgor zata Marvin Stefan Ke Sho Andre Ali Ö. Ali Önder Önder Youbi ng Xinhu a Peter Darius z Anna
Willer
279
Winter Wintrode Wiskem ann Witt
073,228,203,244 123 035
Witt Wittman n Witzenra th Wochner Wolf Wolff Wolkers Wood Wouters Wrede
133 257
Wrenger
233
Wronski
159,219,286
Wuchter Wujak
291 076,093
Wygreck a Wäsch Wörz Xiao Yamasa ki Yaroshe nko Yildirim Yildirim
076,093,098,101
Yildirim Yin
187 266
Yu
081,083
Zabel Zakrzewi cz Zakrzewi cz Konsta Zangntina Pappa
25.01.2017
042
207 035 248 119,196 215 266 060 021
291 224 273 018 278,279 283,212 087,103,116,256,114,279
007,077 076,098,101 067 051
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 Ralf Micha el Xiaoto ng Xiang Xiu-yi Zhe
Zarbock Zemlin
257 150
Zhang
120
Zheng Zhi ZhouSuckow Jana Zimmer Ulrich Zissler Pawel Zmora Frank Zufall Wolfga Zwicken ng pflug Anja S chmall Martin de a Zwaan Jeann de la e Roche Rose kiefl marie all of the memb Platform ers Biobanki ng & Data Manage ment all of the memb Platform ers Biobanki ng and Data Manage ment the study DIABI group MMUN E the study MAS groups and PAST URE Alexan tretyn dra Erika v. Mutius Micha van den el Heuvel Hendri van der k Heijden Andre von Berg
25.01.2017
037,232,234 120 256,110,113 252 069,071,282 154 070 277 234 289 170 235 147
146
128
129
185 068 120 278 053 Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
DZL Annual Meeting 2017 a Nikola s Erika Daniel J.Matthi as Yildiri m
von Bubnoff von Mutius von der Beck von der Schulen burg Önder
25.01.2017
001 126,128,129,150,151,152,166,193,210,220,246,273,282,288 133 054
277
Supported by SciSerTec Your Virtual Congress Manager Web: http://www.scisertec.de
