Developmental control of CFTR: from bioinformatics to novel therapeutic approaches.

miRNA-binding blocker oligonucleotides offer an appealing option for developing tools to correct CFTR function http://ow.ly/AjS8

Myeloid-Derived Suppressor Cells as a Potential Biomarker and Therapeutic Target in COVID-19

Clinical presentations of COVID-19 are highly variable, yet the precise mechanisms that govern the pathophysiology of different disease courses remain poorly defined. Across the spectrum of disease severity, COVID-19 impairs both innate and adaptive host immune responses by activating innate immune cell recruitment, while resulting in low lymphocyte counts. Recently, several reports have shown that patients with severe COVID-19 exhibit a dysregulated myeloid cell compartment, with increased myeloid-derived suppressor cells (MDSCs) correlating with disease severity. MDSCs, in turn, promote virus survival by suppressing T-cell responses and driving a highly pro-inflammatory state through the secretion of various mediators of immune activation. Here, we summarize the evidence on MDSCs and myeloid cell dysregulation in COVID-19 infection and discuss the potential of MDSCs as biomarkers and therapeutic targets in COVID-19 pneumonia and associated disease

Chemokinrezeptorexpression auf T-Zellen in bronchoalveolärer Lavage und peripherem Blut bei Kindern mit chronischer Bronchitis und interstitiellen Lungenerkrankungen

Background: Lymphocytes are recruited to sites of inflammation by chemokines. Accordingly, a number of chemokine receptors are differentially expressed on effector T cells. We hypothesized that selected T cells accumulate in inflammatory lung diseases involving different pulmonary compartments. To test this hypothesis the frequencies of chemokine receptor expressing T cells were compared in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) of children with chronic bronchitis and interstitial lung diseases. Methods: BAL was performed in 70 children. According to clinical, macroscopic and cytological findings 37 children were selected for the study and classified as chronic bronchitis (CB, n=17, m=7, mean age 6.6 yrs.) or interstitial lung diseases (ILD, n=20, m=13, mean age 7.0 yrs.). Patients (n=33) with other diagnoses or without cells in BALF were excluded. CD4+ and CD8+ T cells were analyzed in PB (n=30) and BALF (n=37) by flow cytometry. The percentages of CCR5+and CXCR3+ cells were determined within each T cell subset. Results are expressed as medians. For statistical analyses non-parametric tests (Wilcoxon, Mann-Whitney U) were applied. Results: In peripheral blood, the percentage of CXCR3+ T cells (16.4%, range: 0-35.2%) was higher than the percentage of CCR5+ T cells (3.9%, range: 0-19.1%; p<0.001). The percentages of CCR5+ and CXCR3+ PB T cells did not differ between the patient groups. The proportions of CCR5+CD4+ and CCR5+CD8+ cells were clearly higher in BAL (7.8% and 12.4%) than in PB (1.6% and 2.3%) of all patients (p<0.001). The percentage of BAL CXCR3+CD8+ cells was significantly higher in patients with CB (25.2%, range: 0-74.6%) compared to patients with ILD (1.2%, range: 0-16.7%; p<0.05). Conclusions: The results indicate that CCR5+ T cells are a regular constituent of the bronchoalveolar space in pediatric pulmonary diseases. The high frequency of pulmonary CCR5+ T cells may be due to a specific recruitment or alternatively to a local expansion of these cells. Furthermore, CXCR3+CD8+ T cells in bronchoalveolar lavage seem to be characteristic for chronic bronchitis in children.Wir untersuchten 37 Kinder aus einer Gruppe von 70 Kindern, bei denen in den letzten zwei Jahren an unserer Kinderklinik eine bronchoalveoläre Lavage durchgeführt wurde. Um die beiden Kompartimente des pulmonalen Immunsystems, die Bronchialmukosa und den interstitiell-alveolären Raum, zu charakterisieren, untersuchten wir Lymphozyten in der bronchoalveolären Lavage von Kindern mit chronischer Bronchitis und interstitiellen Lungenerkrankungen. Die Gruppe „chronische Bronchitis“ umfasste 17 Kinder, darunter sechs Kinder mit chronischer Bronchitis ohne Obstruktion, sechs Kinder mit chronischer Bronchitis mit Obstruktion und fünf Kinder mit Asthma bronchiale. Die Gruppe „interstitielle Lungenerkrankungen“ enthielt 20 Kinder mit verschiedenen Krankheiten des Interstitiums. Durchflußzytometrisch wurden Lymphozyten in der bronchoalveolären Lavage bestimmt. Die Prozentsätze CD4-, CD8- und CD19-positiver Lymphozyten wurden in der bronchoalveolären Lavage und im Vollblut bestimmt. Weiter wurde die Häufigkeit CXCR3-, CCR5-, CCR4- und CCR3-positiver Zellen innerhalb der CD4- und CD8-positiven T-Zellpopulationen bestimmt. In der bronchoalveolären Lavage waren CD8-positive Zellen signifikant häufiger enthalten als CD4-positive T-Zellen. Die mediane CD4/CD8-Ratio in der bronchoalveolären Lavage betrug 0,4 in der chronischen Bronchitis-Gruppe und 0,8 in der interstitiellen Lungenerkrankungen-Gruppe. Im Vollblut überwogen CD4-positive T-Zellen signifikant über CD8-positive Zellen mit einer medianen CD4/CD8-Ratio von 2,1. CD19-positive B-Zellen waren in der bronchoalveolären Lavage sehr selten. Im Vollblut fanden wir signifikant häufiger CD19-positive B-Zellen. CCR5-, CXCR3- und CCR4-positive T-Zellen waren in der bronchoalveolären Lavage häufiger als im peripheren Blut. CCR3 war auf T-Zellen nicht nachweisbar. CCR5 wies ein signifikant höheres bronchoalveoläres Lavage- zu Vollblut-Verhältnis auf. Es fanden sich signifikant mehr CXCR3- und CCR5-CD8-positive als CD4-positive Zellen, wogegen signifikant häufiger CCR4-CD4-positive T-Zellen gefunden wurden. In der bronchoalveolären Lavage der chronischen Bronchitis-Gruppe wurde ein signifikant erhöhter Prozentsatz an CXCR3-CD8-positiven Zellen im Vergleich zur interstitiellen Lungenerkrankungen-Gruppe gefunden. Innerhalb der chronischen Bronchitis-Gruppe fand man in der bronchoalveolären Lavage von Kindern ohne Obstruktion signifikant häufiger CXCR3-CD8-positive Zellen im Vergleich zu Kindern mit Obstruktion

Design principles of promoter and enhancer activity in mammalian genomes

Correct gene expression patterns are central for cellular function and the development of organisms. This is controlled by regulatory elements such as enhancers and promoters. In this thesis, I present work from two projects with the goal to identify design principles of promoter and enhancer activity in mammalian genomes. In the first part of the thesis, I focused on CpG island promoters. This promoter type represents the majority of mammalian promoters and is characterised by a high density of the CpG dinucleotide. However, to what extent and how this characteristic dinucleotide contributes to promoter activity is still unclear and is one central question of this project. By monitoring binding of transcription factors (TFs) assumed to play a role in CpG island activity and quantifying the activity of promoter mutants and artificial promoters, we gained insight into the role of CpGs in transcriptional activity. The generated data suggests that high CpG density is not sufficient for transcriptional activity, yet necessary when combined with more complex TF binding motifs. We could further show that DNA methylation decreases activity of promoter mutants with low CpG density. Our experiments led us to hypothesise that high CpG density is required to generate a chromatin environment permissive for transcriptional activity. In the second part of the thesis, I focused on cell type and tissue specific regulatory elements. To illustrate an experimental workflow to identify and test regulatory elements for transcriptional activity in specific cell types, we used the mouse retina, a very specialised tissue comprised of ~50 cell types. To identify regulatory elements, we combined transcriptome and epigenome profiling to map the regulatory landscape of four distinct cell types isolated from mouse retinas (rods, cones, horizontal and starburst amacrine cells). This data also revealed sequence determinants and candidate TFs that control cellular specialisation. We tested previously identified regulatory regions using a parallelised reporter assay for their ability to autonomously control transcriptional activity in the four cell types. We were able to generate a catalogue of cis-regulatory regions active in retinal cell types and further demonstrate their utility as a potential resource for cellular tagging and manipulation. Taken together, the work presented here advances our knowledge about location and regulation of regulatory regions that function in specialised cell types and also provides insight into the regulation of CpG island promoters that tend to be ubiquitously expressed

Current Concepts of Hyperinflammation in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is the most common inherited disorder of phagocytic functions, caused by genetic defects in the leukocyte nicotinamide dinucleotide phosphate (NADPH) oxidase. Consequently, CGD phagocytes are impaired in destroying phagocytosed microorganisms, rendering the patients susceptible to bacterial and fungal infections. Besides this immunodeficiency, CGD patients suffer from various autoinflammatory symptoms, such as granuloma formation in the skin or urinary tract and Crohn-like colitis. Owing to improved antimicrobial treatment strategies, the majority of CGD patients reaches adulthood, yet the autoinflammatory manifestations become more prominent by lack of causative treatment options. The underlying pathomechanisms driving hyperinflammatory reactions in CGD are poorly understood, but recent studies implicate reduced neutrophil apoptosis and efferocytosis, dysbalanced innate immune receptors, altered T-cell surface redox levels, induction of Th17 cells, the enzyme indolamine-2,3-dioxygenase (IDO), impaired Nrf2 activity, and inflammasome activation. Here we discuss immunological mechanisms of hyperinflammation and their potential therapeutic implications in CGD

Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

BACKGROUND: Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma. METHODOLOGY/PRINCIPAL FINDINGS: Corticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser²²⁶, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2A(C)-Tyr³⁰⁷ phosphorylation compared with those in healthy volunteers. As the results, GR-Ser²²⁶ and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser²²⁶. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells. CONCLUSIONS/SIGNIFICANCE: PP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser²²⁶ via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma

The Calpain, Caspase 12, Caspase 3 Cascade Leading to Apoptosis Is Altered in F508del-CFTR Expressing Cells

In cystic fibrosis (CF), the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER). We previously showed that the unfolded protein response (UPR) may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt) and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF

Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions

E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells

Suspicion of respiratory tract infection with multidrug-resistant Enterobacteriaceae: epidemiology and risk factors from a Paediatric Intensive Care Unit

Enterobacteriaceae distribution. Distribution of Enterobacteriaceae isolates (n = 167) in lower respiratory tract material, MDR (n = 51) vs susceptible (n = 116) organisms during the study period. (XLSX 14 kb