In vitro Methods for the Development and Analysis of Human Primary Airway Epithelia

Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies

A Modified Implementation Mapping Methodology for Evaluating and Learning From Existing Implementation

When empirically supported interventions are implemented in real-world practice settings, the process of how these interventions are implemented is highly relevant for their potential success. Implementation Mapping is a method that provides step-by-step guidance for systematically designing implementation processes that fit the respective intervention and context. It includes needs assessments among relevant stakeholders, the identification of implementation outcomes and determinants, the selection and design of appropriate implementation strategies, the production of implementation protocols and an implementation outcome evaluation. Implementation Mapping is generally conceptualized as a tool to prospectively guide implementation. However, many implementation efforts build on previous or ongoing implementation efforts, i.e., “existing implementation.” Learnings from existing implementation may offer insights critical to the success of further implementation activities. In this article, we present a modified Implementation Mapping methodology to be applied when evaluating existing implementation. We illustrate the methodology using the example of evaluating ongoing organized colorectal cancer screening programs in Switzerland. Through this example, we describe how we identify relevant stakeholders, implementation determinants and outcomes as well as currently employed implementation strategies. Moreover, we describe how we compare the types of strategies that are part of existing implementation efforts with those that implementation science would suggest as being suited to address identified implementation determinants. The results can be used for assessing the current state of implementation outcomes, refining ongoing implementation strategies, and informing future implementation efforts

Implementation of infection prevention and control for hospitalized neonates: A narrative review

Background The most prevalent infections encountered in neonatal care are healthcare-associated infections. The majority of healthcare-associated infections are considered preventable with evidence-based infection prevention and control (IPC) practices. However, substantial knowledge gaps exist in IPC implementation in neonatal care. Furthermore, the knowledge of factors which facilitate or challenge the uptake and sustainment of IPC programmes in neonatal units is limited. The integration of implementation science approaches in IPC programmes in neonatal care aims to address these problems. Objectives The aim of this narrative review was to identify determinants which have been reported to influence the implementation of IPC programmes and best practices in inpatient neonatal care settings. Sources A literature search was conducted in PubMed, MEDLINE (Medical Literature Analysis and Retrieval System Online) and CINAHL (Cumulative Index to Nursing and Allied Health Literature) in May 2022. Primary study reports published in English, French, German, Spanish, Portuguese, Italian, Danish, Swedish or Norwegian since 2000 were eligible for inclusion. Included studies focused on IPC practices in inpatient neonatal care settings and reported determinants which influenced implementation processes. Content The Consolidated Framework for Implementation Research was used to identify and cluster reported determinants to the implementation of IPC practices and programmes in neonatal care. Most studies reported challenges and facilitators at the organizational level as particularly relevant to implementation processes. The commonly reported determinants included staffing levels, work- and caseloads, as well as aspects of organizational culture such as communication and leadership. Implications The presented knowledge about factors influencing neonatal IPC can support the design, implementation, and evaluation of IPC practices

IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro

AbstractRecent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6–48 h causes a marked inhibition of the amiloride-sensitive Na+ channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca2+-dependent Cl− channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Rα. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the γ amiloride-sensitive Na+ channel mRNA, one of the subunits of the amiloride-sensitive Na+ channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid

Click-tambjamines as efficient and tunable bioactive anion transporters

A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design.European Union’s Horizon 2020 research and innovation programme (TAT-CF project, grant agreement 667079), Instituto de Salud Carlos III (Grant PI18/00441) (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and “La Caixa” Foundation and Caja Burgos Foundation (CAIXAUBU004

IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

BACKGROUND: Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. METHODOLOGY/PRINCIPAL FINDINGS: Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rbeta2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R(+) neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/beta2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/beta2(+) cells inhibited angiogenesis in vitro. Tumors formed by Calu6/beta2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. CONCLUSIONS: This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector in vivo and in vitro only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR

Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins

The TMEM16A chloride channel as an alternative therapeutic target in cystic fibrosis

Cystic fibrosis (CF), a multiorgan genetic disease, is caused by loss of function of CFTR, a cAMP-regulated anion channel. In CF airway epithelia, defective Cl(-) and bicarbonate secretion impairs mucociliary clearance and other innate defense mechanisms, favoring the colonization of the lungs by highly virulent bacteria. The airway epithelium expresses TMEM16A, a second type of Cl(-) channel that is activated by cytosolic Ca(2+). TMEM16A is particularly expressed in goblet cells. This specific localization could be important in the release and hydration of mucins. Activation of TMEM16A with pharmacological agents could circumvent the primary defect in CF. This strategy needs to be carefully designed and tested to avoid possible undesired effects due to the expression of TMEM16A in other cell types such as bronchial smooth muscle cells. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances