Characteristics of inflammatory response and repair after experimental blast lung injury in rats.

BACKGROUND AND OBJECTIVES: Blast-induced lung injury is associated with inflammatory, which are characterised by disruption of the alveolar-capillary barrier, haemorrhage, pulmonary infiltrateration causing oedema formation, pro-inflammatory cytokine and chemokine release, and anti-inflammatory counter-regulation. The objective of the current study was to define sequence of such alterations in with establishing blast-induced lung injury in rats using an advanced blast generator. METHODS: Rats underwent a standardized blast wave trauma and were euthanised at defined time points. Non-traumatised animals served as sham controls. Obtained samples from bronchoalveolar lavage fluid (BALF) at each time-point were assessed for histology, leukocyte infiltration and cytokine/chemokine profile. RESULTS: After blast lung injury, significant haemorrhage and neutrophil infiltration were observed. Similarly, protein accumulation, lactate dehydrogenase activity (LDH), alveolar eicosanoid release, matrix metalloproteinase (MMP)-2 and -9, pro-Inflammatory cytokines, including tumour necrosis factor (TNF) and interleukin (IL) -6 raised up. While declining in the level of anti-inflammatory cytokine IL-10 occurred. Ultimately, pulmonary oedema developed that increased to its maximum level within the first 1.5 h, then recovered within 24 h. CONCLUSION: Using a stablished model, can facilitate the study of inflammatory response to blast lung injury. Following the blast injury, alteration in cytokine/chemokine profile and activity of cells in the alveolar space occurs, which eventuates in alveolar epithelial barrier dysfunction and oedema formation. Most of these parameters exhibit time-dependent return to their basal status that is an indication to resilience of lungs to blast-induced lung injury.

Nano-in-Microparticles for Aerosol Delivery of Antibiotic-Loaded, Fucose-Derivatized, and Macrophage-Targeted Liposomes to Combat Mycobacterial Infections: In Vitro Deposition, Pulmonary Barrier Interactions, and Targeted Delivery.

Nontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco-regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline-encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP-1 cells as well as peripheral blood monocyte- and lung-tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry-powder microparticles of spray-dried bedaquiline-loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP-1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.

Dysregulation of cholesterol homeostasis in human lung cancer tissue and tumour-associated macrophages.

BACKGROUND: Based on reports on elevated cholesterol levels in cancer cells, strategies to lower cholesterol synthesis have been suggested as an antitumour strategy. However, cholesterol depletion has also been shown to induce tumour-promoting actions in tumour-associated macrophages (TAMs). METHODS: We performed lipidomic and transcriptomic analyses of human lung cancer material. To assess whether the TAM phenotype is shaped by secreted factors produced by tumour cells, primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype using tumour cell-conditioned medium. FINDINGS: Lipidomic analysis of lung adenocarcinoma (n=29) and adjacent non-tumour tissues (n=22) revealed a significant accumulation of free cholesterol and cholesteryl esters within the tumour tissue. In contrast, cholesterol levels were reduced in TAMs isolated from lung adenocarcinoma tissues when compared with alveolar macrophages (AMs) obtained from adjacent non-tumour tissues. Bulk-RNA-Seq revealed that genes involved in cholesterol biosynthesis and metabolism were downregulated in TAMs, while cholesterol efflux transporters were upregulated. In vitro polarized TAM-like macrophages showed an attenuated lipogenic gene expression signature and exhibited lower cholesterol levels compared with non-polarized macrophages. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro TAM-like macrophages. Modulation of intracellular cholesterol levels by either starving, cholesterol depletion, or efflux transporter inhibition indicated that cholesterol distinctly shapes macrophage gene expression. INTERPRETATION: Our data show an opposite dysregulation of cholesterol homeostasis in tumour tissue vs. TAMs. Polarization of in vitro differentiated macrophages by tumour cell-conditioned medium recapitulates key features of ex vivo TAMs. FUNDING: Deutsche Forschungsgemeinschaft (DFG), Landesforschungsf €orderungsprogramm Saarland (LFPP).

Tobacco Smoke and Inhaled Drugs Alter Expression and Activity of Multidrug Resistance-Associated Protein-1 (MRP1) in Human Distal Lung Epithelial Cells in vitro.

Multidrug resistance-associated protein-1 (MRP1/ABCC1) is highly expressed in human lung tissues. Recent studies suggest that it significantly affects the pulmonary disposition of its substrates, both after pulmonary and systemic administration. To better understand the molecular mechanisms involved, we studied the expression, subcellular localization and activity of MRP1 in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and in the NCI-H441 cell line. Moreover, the effect of cigarette smoke extract (CSE) and a series of inhaled drugs on MRP1 abundance and activity was investigated in vitro. MRP1 expression levels were measured by q-PCR and immunoblot in AT2 and AT1-like cells from different donors and in several passages of the NCI-H441 cell line. The subcellular localization of the transporter was studied by confocal laser scanning microscopy and cell surface protein biotinylation. MRP1 activity was assessed by bidirectional transport and efflux experiments using the MRP1 substrate, 5(6)-carboxyfluorescein [CF; formed intracellularly from 5(6)-carboxyfluorescein-diacetate (CFDA)] in AT1-like and NCI-H441 cell monolayers. Furthermore, the effect of CSE as well as several bronchodilators and inhaled corticosteroids on MRP1 abundance and CF efflux was investigated. MRP1 protein abundance increased upon differentiation from AT2 to AT1-like phenotype, however, ABCC1 gene levels remained unchanged. MRP1 abundance in NCI-H441 cells were comparable to those found in AT1-like cells. The transporter was detected primarily in basolateral membranes of both cell types which was consistent with net basolateral efflux of CF. Likewise, bidirectional transport studies showed net apical-to-basolateral transport of CF which was sensitive to the MRP1 inhibitor MK-571. Budesonide, beclomethasone dipropionate, salbutamol sulfate, and CSE decreased CF efflux in a concentration-dependent manner. Interestingly, CSE increased MRP1 abundance, whereas budesonide, beclomethasone dipropionate, salbutamol sulfate did not have such effect. CSE and inhaled drugs can reduce MRP1 activity in vitro, which implies the transporter being a potential drug target in the treatment of chronic obstructive pulmonary disease (COPD). Moreover, MRP1 expression level, localization and activity were comparable in human AT1-like and NCI-H441 cells. Therefore, the cell line can be a useful alternative in vitro model to study MRP1 in distal lung epithelium.

Partial Anomalous Pulmonary Venous Return Diagnosed by Central Catheter Misplacement.

Anomalous venous connections of the left lung can either affect all of the veins or only the upper lobe. They mostly drain into the innominate vein. We present the case of a patient who underwent a coronary bypass operation and was prepared with insertion of central lines including Swan-Ganz catheter through both the internal jugular veins. Blood gas analysis obtained from these catheters suggested the presence of a left-to-right shunt. CT (computed tomography) imaging confirmed a pulmonary venous anomaly with misplacement of the left-sided catheter in an abnormal pulmonary vein. Such a rare condition can be suspected by obtaining arterialized blood samples and measuring the mean pressure through central catheters.

Impact of Bacterial Toxins in the Lungs.

Bacterial toxins play a key role in the pathogenesis of lung disease. Based on their structural and functional properties, they employ various strategies to modulate lung barrier function and to impair host defense in order to promote infection. Although in general, these toxins target common cellular signaling pathways and host compartments, toxin- and cell-specific effects have also been reported. Toxins can affect resident pulmonary cells involved in alveolar fluid clearance (AFC) and barrier function through impairing vectorial Na+ transport and through cytoskeletal collapse, as such, destroying cell-cell adhesions. The resulting loss of alveolar-capillary barrier integrity and fluid clearance capacity will induce capillary leak and foster edema formation, which will in turn impair gas exchange and endanger the survival of the host. Toxins modulate or neutralize protective host cell mechanisms of both the innate and adaptive immunity response during chronic infection. In particular, toxins can either recruit or kill central players of the lung's innate immune responses to pathogenic attacks, i.e., alveolar macrophages (AMs) and neutrophils. Pulmonary disorders resulting from these toxin actions include, e.g., acute lung injury (ALI), the acute respiratory syndrome (ARDS), and severe pneumonia. When acute infection converts to persistence, i.e., colonization and chronic infection, lung diseases, such as bronchitis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) can arise. The aim of this review is to discuss the impact of bacterial toxins in the lungs and the resulting outcomes for pathogenesis, their roles in promoting bacterial dissemination, and bacterial survival in disease progression.

Evaluating the Use of Circulating MicroRNA Profiles for Lung Cancer Detection in Symptomatic Patients.

Importance: The overall low survival rate of patients with lung cancer calls for improved detection tools to enable better treatment options and improved patient outcomes. Multivariable molecular signatures, such as blood-borne microRNA (miRNA) signatures, may have high rates of sensitivity and specificity but require additional studies with large cohorts and standardized measurements to confirm the generalizability of miRNA signatures. Objective: To investigate the use of blood-borne miRNAs as potential circulating markers for detecting lung cancer in an extended cohort of symptomatic patients and control participants. Design, Setting, and Participants: This multicenter, cohort study included patients from case-control and cohort studies (TREND and COSYCONET) with 3102 patients being enrolled by convenience sampling between March 3, 2009, and March 19, 2018. For the cohort study TREND, population sampling was performed. Clinical diagnoses were obtained for 3046 patients (606 patients with non-small cell and small cell lung cancer, 593 patients with nontumor lung diseases, 883 patients with diseases not affecting the lung, and 964 unaffected control participants). No samples were removed because of experimental issues. The collected data were analyzed between April 2018 and November 2019. Main Outcomes and Measures: Sensitivity and specificity of liquid biopsy using miRNA signatures for detection of lung cancer. Results: A total of 3102 patients with a mean (SD) age of 61.1 (16.2) years were enrolled. Data on the sex of the participants were available for 2856 participants; 1727 (60.5%) were men. Genome-wide miRNA profiles of blood samples from 3046 individuals were evaluated by machine-learning methods. Three classification scenarios were investigated by splitting the samples equally into training and validation sets. First, a 15-miRNA signature from the training set was used to distinguish patients diagnosed with lung cancer from all other individuals in the validation set with an accuracy of 91.4% (95% CI, 91.0%-91.9%), a sensitivity of 82.8% (95% CI, 81.5%-84.1%), and a specificity of 93.5% (95% CI, 93.2%-93.8%). Second, a 14-miRNA signature from the training set was used to distinguish patients with lung cancer from patients with nontumor lung diseases in the validation set with an accuracy of 92.5% (95% CI, 92.1%-92.9%), sensitivity of 96.4% (95% CI, 95.9%-96.9%), and specificity of 88.6% (95% CI, 88.1%-89.2%). Third, a 14-miRNA signature from the training set was used to distinguish patients with early-stage lung cancer from all individuals without lung cancer in the validation set with an accuracy of 95.9% (95% CI, 95.7%-96.2%), sensitivity of 76.3% (95% CI, 74.5%-78.0%), and specificity of 97.5% (95% CI, 97.2%-97.7%). Conclusions and Relevance: The findings of the study suggest that the identified patterns of miRNAs may be used as a component of a minimally invasive lung cancer test, complementing imaging, sputum cytology, and biopsy tests.

Bronchial Fistula and Pneumomediastinum after EBUS-TBNA Following Mediastinoscopy.

Background Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a very useful diagnostic tool for the assessment of enlarged mediastinal and hilar lymph nodes. It is a safe procedure with a low risk of complications. Case Description We report a case of bronchial fistula and pneumomediastinum after EBUS-TBNA, which was performed shortly after a mediastinoscopy. Due to the extent of the bronchial lesion, a surgical closure of the bronchial fistula was necessary. The patient recovered completely. Conclusion The performance of EBUS-TBNA shortly after a mediastinoscopy should not be recommended to avoid possible procedure-related complications.

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Alveolar type II (ATII) cells in the peripheral human lung spontaneously differentiate toward ATI cells, thus enabling air-blood barrier formation. Here, linear Raman and coherent anti-Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated with gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI-like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for three-dimensional visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label-free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.

Medium throughput breathing human primary cell alveolus-on-chip model.

Organs-on-chips have the potential to improve drug development efficiency and decrease the need for animal testing. For the successful integration of these devices in research and industry, they must reproduce in vivo contexts as closely as possible and be easy to use. Here, we describe a 'breathing' lung-on-chip array equipped with a passive medium exchange mechanism that provide an in vivo-like environment to primary human lung alveolar cells (hAEpCs) and primary lung endothelial cells. This configuration allows the preservation of the phenotype and the function of hAEpCs for several days, the conservation of the epithelial barrier functionality, while enabling simple sampling of the supernatant from the basal chamber. In addition, the chip design increases experimental throughput and enables trans-epithelial electrical resistance measurements using standard equipment. Biological validation revealed that human primary alveolar type I (ATI) and type II-like (ATII) epithelial cells could be successfully cultured on the chip over multiple days. Moreover, the effect of the physiological cyclic strain showed that the epithelial barrier permeability was significantly affected. Long-term co-culture of primary human lung epithelial and endothelial cells demonstrated the potential of the lung-on-chip array for reproducible cell culture under physiological conditions. Thus, this breathing lung-on-chip array, in combination with patients' primary ATI, ATII, and lung endothelial cells, has the potential to become a valuable tool for lung research, drug discovery and precision medicine.

Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages.

Activation of toll-like receptors (TLRs) plays a pivotal role in the host defense against bacteria and results in the activation of NF-κB-mediated transcription of proinflammatory mediators. Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory mediator, which inhibits NF-κB activity in macrophages. Thus, we aimed to investigate the regulation and role of GILZ expression in primary human and murine macrophages upon TLR activation. Treatment with TLR agonists, e.g., Pam3CSK4 (TLR1/2) or LPS (TLR4) rapidly decreased GILZ mRNA and protein levels. In consequence, GILZ downregulation led to enhanced induction of pro-inflammatory mediators, increased phagocytic activity, and a higher capacity to kill intracellular bacteria (Salmonella enterica serovar typhimurium), as shown in GILZ knockout macrophages. Treatment with the TLR3 ligand polyinosinic: polycytidylic acid [Poly(I:C)] did not affect GILZ mRNA levels, although GILZ protein expression was decreased. This effect was paralleled by sensitization toward TLR1/2- and TLR4-agonists. A bioinformatics approach implicated more than 250 miRNAs as potential GILZ regulators. Microarray analysis revealed that the expression of several potentially GILZ-targeting miRNAs was increased after Poly(I:C) treatment in primary human macrophages. We tested the ability of 11 of these miRNAs to target GILZ by luciferase reporter gene assays. Within this small set, four miRNAs (hsa-miR-34b*,-222,-320d,-484) were confirmed as GILZ regulators, suggesting that GILZ downregulation upon TLR3 activation is a consequence of the synergistic actions of multiple miRNAs. In summary, our data show that GILZ downregulation promotes macrophage activation. GILZ downregulation occurs both via MyD88-dependent and -independent mechanisms and can involve decreased mRNA or protein stability and an attenuated translation.

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line. METHODS: BCRP expression levels in AT2 and AT1-like cells and in different passages of NCI-H441 cells were determined using q-PCR and immunoblot. Transporter localisation was confirmed by confocal laser scanning microscopy. Efflux and transport studies using the BCRP substrate BODIPY FL prazosin and the inhibitor Ko143 were carried out to assess BCRP activity in the different cell models. RESULTS: BCRP expression decreased during transdifferentiation from AT2 to AT1-like phenotype. Culturing NCI-H441 cells at an air-liquid interface or submersed did not change BCRP abundance, however, BCRP levels increased with passage number. BCRP was localised to the apical membrane and cytosol in NCI-H441 cells. In primary cells, the protein was found predominantly in the nucleus. Functional studies were consistent with expression data. CONCLUSIONS: BCRP is differently expressed in AT2 and AT1-like cells with lower abundance and activity in the latter ones. Nuclear BCRP might play a transcriptional role in distal lung epithelium. In NCI-H441 cells, BCRP is expressed in apical cell membranes and its activity is consistent with the localisation pattern.

Autologous co-culture of primary human alveolar macrophages and epithelial cells for investigating aerosol medicines. Part I: model characterisation.

The development of new formulations for pulmonary drug delivery is a challenge on its own. New in vitro models which address the lung are aimed at predicting and optimising the quality, efficacy and safety of inhaled drugs, to facilitate the more rapid translation of such products into the clinic. Reducing the complexity of the in vivo situation requires that such models reproducibly reflect essential physiological factors in vitro. The choice of cell types, culture conditions and the experimental set-up, can affect the outcome and the relevance of a study. In the alveolar space of the lung, epithelial cells and alveolar macrophages are the most important cell types, forming an efficient cellular barrier to aerosols. Our aim was to mimic this barrier with primary human alveolar cells. Cell densities of alveolar macrophages and epithelial cells, isolated from the same human donor, were optimised, with a focus on barrier properties. The combination of 300,000 epithelial cells/cm² together with 100,000 macrophages/cm² showed a functional barrier (transepithelial electrical resistance > 500Ω.cm²). This cell model was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures. The functionality of the in vitro system was investigated with spray-dried fluorescently labelled poly(lactic-co-glycolic) acid particles loaded with ovalbumin as a model drug.

Autologous co-culture of primary human alveolar macrophages and epithelial cells for investigating aerosol medicines. Part II: evaluation of IL-10-loaded microparticles for the treatment of lung inflammation.

Acute respiratory distress syndrome is linked to inflammatory processes in the human lung. The aim of this study was to mimic in vitro the treatment of lung inflammation by using a cell-based human autologous co-culture model. As a potential trial medication, we developed a pulmonary dry powder formulation loaded with interleukin-10 (IL-10), a potent anti-inflammatory cytokine. The inflammatory immune response was stimulated by lipopolysaccharide. The co-culture was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures )PADDOCC), to deposit the IL-10-loaded microparticles on the inflamed co-culture model at the air-liquid interface. This treatment significantly reduced the secretion of interleukin-6 and tumour necrosis factor, as compared to the deposition of placebo (unloaded) particles. Our results show that the alveolar co-culture model, in combination with a deposition device such as the PADDOCC, may serve as a powerful tool for testing the safety and efficacy of dry powder formulations for pulmonary drug delivery.

Paired proteomics, transcriptomics and miRNomics in non-small cell lung cancers: known and novel signaling cascades.

High-throughput omics analyses are applied to elucidate molecular pathogenic mechanisms in cancer. Given restricted cohort sizes and contrasting large feature sets paired multi-omics analysis supports discovery of true positive deregulated signaling cascades. For lung cancer patients we measured from the same tissue biopsies proteomic- (6,183 proteins), transcriptomic- (34,687 genes) and miRNomic data (2,549 miRNAs). To minimize inter-individual variations case and control lung biopsies have been gathered from the same individuals.Considering single omics entities, 15 of 2,549 miRNAs (0.6%), 752 of 34,687 genes (2.2%) and 141 of 6,183 proteins (2.3%) were significantly deregulated. Multivariate analysis also revealed that effects in miRNA were smaller compared to genes and proteins indicating that expression changes of miRNAs might also have limited impact of pathogenicity. However, a new algorithm for modeling the complex mutual interactions of miRNAs and their target genes facilitated precise prediction of deregulation in cancer genes (92.3% accuracy, p=0.007). Lastly, deregulation of genes in cancer matched deregulation of proteins coded by the genes in 80% of cases.The resulting interaction network, which is based on quantitative analysis of the abundance of miRNAs, mRNAs and proteins each taken from the same lung cancer tissue and from the same autologous normal lung tissue confirms molecular pathological changes and further contributes to the discovery of altered signaling cascades in lung cancer.

Integrated quantitative proteomic and transcriptomic analysis of lung tumor and control tissue: a lung cancer showcase.

Proteomics analysis of paired cancer and control tissue can be applied to investigate pathological processes in tumors. Advancements in data-independent acquisition mass spectrometry allow for highly reproducible quantitative analysis of complex proteomic patterns. Optimized sample preparation workflows enable integrative multi-omics studies from the same tissue specimens.We performed ion mobility enhanced, data-independent acquisition MS to characterize the proteome of 21 lung tumor tissues including adenocarcinoma and squamous cell carcinoma (SCC) as compared to control lung tissues of the same patient each. Transcriptomic data were generated for the same specimens. The quantitative proteomic patterns and mRNA abundances were subsequently analyzed using systems biology approaches.We report a significantly (p = 0.0001) larger repertoire of proteins in cancer tissues. 12 proteins were higher in all tumor tissues as compared to matching control tissues. Three proteins, CAV1, CAV2, and RAGE, were vice versa higher in all controls. We also identified characteristic SCC and adenocarcinoma protein patterns. Principal Component Analysis provided evidence that not only cancer from control tissue but also tissue from adenocarcinoma and SCC can be differentiated. Transcriptomic levels of key proteins measured from the same matched tissue samples correlated with the observed protein patterns.The applied study set-up with paired lung tissue specimens of which different omics are measured, is generally suited for an integrated multi-omics analysis.

DrugTargetInspector: An assistance tool for patient treatment stratification.

Cancer is a large class of diseases that are characterized by a common set of features, known as the Hallmarks of cancer. One of these hallmarks is the acquisition of genome instability and mutations. This, combined with high proliferation rates and failure of repair mechanisms, leads to clonal evolution as well as a high genotypic and phenotypic diversity within the tumor. As a consequence, treatment and therapy of malignant tumors is still a grand challenge. Moreover, under selective pressure, e.g., caused by chemotherapy, resistant subpopulations can emerge that then may lead to relapse. In order to minimize the risk of developing multidrug-resistant tumor cell populations, optimal (combination) therapies have to be determined on the basis of an in-depth characterization of the tumor's genetic and phenotypic makeup, a process that is an important aspect of stratified medicine and precision medicine. We present DrugTargetInspector (DTI), an interactive assistance tool for treatment stratification. DTI analyzes genomic, transcriptomic, and proteomic datasets and provides information on deregulated drug targets, enriched biological pathways, and deregulated subnetworks, as well as mutations and their potential effects on putative drug targets and genes of interest. To demonstrate DTI's broad scope of applicability, we present case studies on several cancer types and different types of input -omics data. DTI's integrative approach allows users to characterize the tumor under investigation based on various -omics datasets and to elucidate putative treatment options based on clinical decision guidelines, but also proposing additional points of intervention that might be neglected otherwise. DTI can be freely accessed at http://dti.bioinf.uni-sb.de.

High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer.

Validation of biomarkers is essential to advance the translational process to clinical application. Although there exists an increasing number of reports on small non-coding RNAs (microRNAs) as minimally-invasive markers from blood, serum or plasma, just a limited number is verified in follow-up studies. We used qRT-PCR to evaluate a known miRNA signature measured from blood that allowed for separation between patients with non-small cell lung cancer (NSCLC), COPD and healthy controls.From the data of our previous microarray studies we selected a panel of 235 miRNAs related to lung cancer and COPD. We observed a high concordance between the AUC values of our initial microarray screening and the qRT-PCR data (correlation of 0.704, p < 10-16). Overall, 90.3% of markers were successfully validated. Among the top markers that were concordant between both studies we found hsa-miR-20b-5p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-106a-5p. The qRT-PCR analysis also confirmed that non-small cell lung cancer patients could be accurately differentiated from unaffected controls: a subset of five markers was sufficient to separate NSCLC patients from unaffected controls with accuracy of 94.5% (specificity and sensitivity of 98% and 91%). Beyond differentiation from controls, we also succeeded in separating NSCLC patients from patients with COPD. MiRNAs that were identified as relevant for the separation between lung cancer and COPD by both qRT-PCR and the array-based studies included hsa-miR-26a-5p, hsa-miR-328-3p and hsa-miR-1224-3p. Although for differentiation between NSCLC patients from COPD patients more markers were required, still high accuracy rates were obtained (5 markers: 78.8%; 10 markers: 83.9%; 50 markers: 87.6%).

Organic cation transporter function in different in vitro models of human lung epithelium.

Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air-blood barrier. OCT activity was investigated by measuring [(14)C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1µM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02µl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.

Mono- and Cocultures of Bronchial and Alveolar Epithelial Cells Respond Differently to Proinflammatory Stimuli and Their Modulation by Salbutamol and Budesonide.

The aim of this study was to investigate the changes in transport and effectiveness of salbutamol sulfate (SAL) and budesonide (BD) following stimulation with transforming growth factor-ß (TGF-ß) in mono- and coculture models of bronchial and alveolar epithelium. Primary bronchial and alveolar epithelial cells, grown at air interface on filters, either as monocultures or in coculture with airway smooth muscle cells or alveolar macrophages, respectively, were stimulated with TGF-ß. The biological response was modulated by depositing aerosolized SAL and BD on bronchial and alveolar models, respectively. Barrier integrity, permeability to fluorescein-Na, transport of the deposited drug, and the pharmacological response to SAL (cAMP and IL-8 levels) or BD (IL-6 and -8 levels) were measured. While stimulation with TGF-ß did not have any significant effect on the transepithelial electrical resistance and permeability to fluorescein-Na in mono- and coculture models, transport of SAL and BD were affected in cultures from some of the patients (6 out of 12 for bronchial and 2 out of 4 for alveolar cells). The bronchial coculture showed a better responsiveness to SAL in terms of cAMP release than the monoculture. In contrast, the difference between alveolar mono- and cocultures to TGF-ß mediated interleukin release and its modulation by BD was less pronounced. Our data point to intrinsic differences in the transport of, and responsiveness to, SAL and BD when epithelial cell cultures originate from different patients. Moreover, if the biological responses (e.g., IL-8, cAMP) involve communication between different cell types, coculture models are more relevant to measure such effects than monocultures.