JUNB O-GlcNAcylation-Mediated Promoter Accessibility of Metabolic Genes Modulates Distinct Epithelial Lineage in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with substantial unmet medical needs. While aberrant epithelial remodeling is a key factor in IPF progression, the molecular mechanisms behind this process remain elusive. Harnessing a 3D patient-derived organoid model and multi-omics approach, the first inventory of the connection between metabolic alteration, chromatin accessibility, and transcriptional regulation in IPF aberrant epithelial remodeling is provided.

Monocyte-derived alveolar macrophages are key drivers of smoke-induced lung inflammation and tissue remodeling.

Smoking is a leading risk factor of chronic obstructive pulmonary disease (COPD), that is characterized by chronic lung inflammation, tissue remodeling and emphysema. Although inflammation is critical to COPD pathogenesis, the cellular and molecular basis underlying smoking-induced lung inflammation and pathology remains unclear. Using murine smoke models and single-cell RNA-sequencing, we show that smoking establishes a self-amplifying inflammatory loop characterized by an influx of molecularly heterogeneous neutrophil subsets and excessive recruitment of monocyte-derived alveolar macrophages (MoAM).

Application of human iPSC-derived macrophages in a miniaturized high-content-imaging-based efferocytosis assay.

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages.

Antifibrotic Drug Nintedanib Inhibits CSF1R to Promote IL-4-associated Tissue Repair Macrophages.

Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both and , impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining and bulk and single-cell RNA-sequencing approaches.

Time and phenotype-dependent transcriptome analysis in AAV-TGFβ1 and Bleomycin-induced lung fibrosis models.

We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFβ1. Here, we provide an in-depth characterization of phenotypic and transcriptomic changes (mRNA and miRNA) in a head-to-head comparison with Bleomycin-induced lung injury over a 4-week disease course. The analyses delineate the temporal state of model-specific and commonly altered pathways, thereby providing detailed insights into the processes underlying disease development.

Functional human iPSC-derived alveolar-like cells cultured in a miniaturized 96‑Transwell air-liquid interface model.

In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells (hiPSCs) into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays. In this work, a unique, miniaturized 96-Transwell microplate system is described where hiPSC-derived alveolar-like cells were cultured at an air-liquid interface (ALI). To this end, hiPSCs were differentiated into lung epithelial progenitor cells (LPCs) and subsequently matured into a functional alveolar type 2 (AT2)-like epithelium with monolayer-like morphology.

Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure.

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls.

Development of a miniaturized 96-Transwell air-liquid interface human small airway epithelial model.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced.

Fecal MicroRNAs Show Promise as Noninvasive Crohn's Disease Biomarkers.

Background: Short non-coding microRNAs (miRNAs) are involved in various cellular processes during disease progression of Crohn's disease (CD) and remarkably stable in feces, which make them attractive biomarker candidates for reflecting intestinal inflammatory processes. Here we investigated the potential of fecal miRNAs as noninvasive and translational CD biomarkers.

Methods: MiRNAs were screened in feces of 52 patients with CD and 15 healthy controls using RNA sequencing and the results were confirmed by PCR.

Linagliptin and telmisartan induced effects on renal and urinary exosomal miRNA expression in rats with 5/6 nephrectomy.

Dipeptidyl peptidase 4 inhibitors and angiotensin II receptor blockers attenuate chronic kidney disease progression in experimental diabetic and non-diabetic nephropathy in a blood pressure and glucose independent manner, but the exact molecular mechanisms remain unclear. MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of nephropathy. miRNAs are present in urine in a remarkably stable form, packaged in extracellular vesicles.

Erratum to Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia).

DMSO cryopreservation is the method of choice to preserve cells for droplet-based single-cell RNA sequencing.

Combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has recently become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomic changes associated with cell preservation and how they may bias further downstream analysis remain unknown.

Differentiation of hiPS Cells into Definitive Endoderm for High-Throughput Screening.

In drug discovery, there is an increasing demand for more physiological in vitro models that recapitulate the disease situation in patients. Human induced pluripotent stem (hiPS) cell-derived model cells could serve this purpose. To date, several directed differentiation approaches have been described to generate definitive endoderm (DE) from hiPS cells, but protocols suitable for drug development and high-throughput screening (HTS) have not been reported yet.

Opposing effects of in vitro differentiated macrophages sub-type on epithelial wound healing.

Inappropriate repair responses to pulmonary epithelial injury have been linked to perturbation of epithelial barrier function and airway remodelling in a number of respiratory diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. We developed an in vitro mechanical scratch injury model in air-liquid interface differentiated primary human small airway epithelial cells that recapitulates many of the characteristics observed during epithelial wound injury in both human tissue and small animal models. Wound closure was initially associated with de-differentiation of the differentiated apical cells and rapid migration into the wound site, followed by proliferation of apical cells behind the wound edge, together with increases in FAK expression, fibronectin and reduction in PAI-1 which collectively facilitate cell motility and extracellular matrix deposition.

Olodaterol shows anti-fibrotic efficacy in in vitro and in vivo models of pulmonary fibrosis.

Background And Purpose: Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by excessive fibroblast activation ultimately leading to scarring of the lungs. Although, the activation of β -adrenoceptors (β -AR) has been shown to inhibit pro-fibrotic events primarily in cell lines, the role of β -adrenoceptor agonists has not yet been fully characterized. The aim of our study was to explore the anti-fibrotic activity of the long-acting β -adrenoceptor agonist olodaterol in primary human lung fibroblasts (HLF) and in murine models of pulmonary fibrosis.

Clinical imaging in anti-atherosclerosis drug development.

The development of novel drugs for the treatment of atherosclerosis faces many challenges, particularly caused by the need for large and costly outcome trials. When predictive biochemical biomarkers are not available, clinical imaging data can serve as intermediate Phase II endpoints to demonstrate mechanistic and anti-atherosclerotic activity of new compounds. These data can support risk mitigation before continuing development in large Phase III outcome trials.

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells.

Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g.

Modeling Pulmonary Disease Pathways Using Recombinant Adeno-Associated Virus 6.2.

Viral vectors have been applied successfully to generate disease-related animal models and to functionally characterize target genes in vivo. However, broader application is still limited by complex vector production, biosafety requirements, and vector-mediated immunogenic responses, possibly interfering with disease-relevant pathways. Here, we describe adeno-associated virus (AAV) variant 6.

Development of a novel severe triple allergen asthma model in mice which is resistant to dexamethasone and partially resistant to TLR7 and TLR9 agonist treatment.

Severe asthma is characterised by persistent inflammation, hyperreactivity and remodeling of the airways. No efficient treatment is available, this is particularly the case for steroid resistant phenotypes. Our aim therefore was to develop a preclinical model showing characteristics of severe human asthma including steroid insensitivity.

Molecular basis for the long duration of action and kinetic selectivity of tiotropium for the muscarinic M3 receptor.

Antagonizing the human M3 muscarinic receptor (hM3R) over a long time is a key feature of modern bronchodilating COPD drugs aiming at symptom relief. The long duration of action of the antimuscarinic drug tiotropium and its kinetic subtype selectivity over hM2R are investigated by kinetic mapping of the binding site and the exit channel of hM3R. Hence, dissociation experiments have been performed with a set of molecular matched pairs of tiotropium on a large variety of mutated variants of hM3R.

Induced Syk deletion leads to suppressed allergic responses but has no effect on neutrophil or monocyte migration in vivo.

The spleen tyrosine kinase (Syk) is a key mediator of immunoreceptor signaling in immune cells. Thus, interfering with the function of Syk by genetic deletion or pharmacological inhibition might influence a variety of allergic and autoimmune processes. Since conventional Syk knockout mice are not viable, studies addressing the effect of Syk deletion in adult animals have been limited.

Functional and biochemical rationales for the 24-hour-long duration of action of olodaterol.

β(2)-Adrenoceptor (β(2)-AR) agonists are powerful bronchodilators and play a pivotal role in the management of pulmonary obstructive diseases, such as asthma and chronic obstructive pulmonary disease. Although these agents first were used many years ago, progress in drug development has resulted in better tolerated, long-acting β(2)-AR agonists (LABAs), such as formoterol and salmeterol. Although LABAs have been on the market for several years, relatively little is known on the rationale(s) behind their long duration of action.