13 research outputs found

    Validation and application of human in vitro models for investigating bronchial response to cigarette smoke

    Get PDF
    Cigarette smoke (CS) is the single most deadly and preventable cause of death. It profoundly affects smokers’ lungs, by altering genes expression profiles, epigenetic modifications, causes DNA damage and changes cell function and morphology [1]. The respiratory tract is the main lung compartment exposed to CS. The bronchial epithelium lining the airway tracts subjected to chronic smoking often leads to incurable diseases such as lung cancers or chronic obstructive pulmonary disease (COPD). Due to its complicated nature and profound impact on human health, the response to CS has been extensively studied in both in vivo and in vitro settings. While CS effects can be investigated in numerous distinct experimental set-ups, little has been done in terms of standardization or validation of these methods. Here, a comprehensive direct comparison between different in vitro CS exposures has been established and analyzed. The cell-delivered dose and the expression profile of genes typically upregulated among smokers were assessed between models, also in relation to expression profile in human lungs. Three surprisingly dissimilar models, namely acute submerged basal cells exposure to cigarette smoke extract (CSE), chronic basolateral CSE ex-posure and acute whole cigarette smoke exposure, yielded responses that were substantially better than any other investigated experimental set-up. Despite the cell-delivered doses varying substantially between these three models, each of them significantly upregulated at least six of out 10 analyzed genes in the primary human bronchial epithelial cells (phBECs). Conclusions from validation study helped choosing the right model, which was later used in the next study, in the proteomic differential expression analysis. Chronic basolateral CSE exposure was the only model that successfully upregulated seven out of 10 genes typically upregulated in smokers. Results of the proteomic analysis further validated the physiological relevance of the model by identifying activation of the molecular pathways characteristic for the CS exposure, such as activation of xenobiotic metabolism pathways and inhibited sirtuin 1 pathway [2]. Interestingly, by using advanced pathway analysis software, a new potential ferroptotic regulator was found, namely nuclear factor 1 (NUPR1). Overall, this study reported a first evidence of critical ferroptosis repressor being aberrantly changed by the CS in phBECs derived from healthy donors. The final topic of this study addressed the question whether the differentiated phBECs from ex- and current smokers exhibit transient and persistent changes caused by smoking that can be seen in vivo. Here, the advantage was taken from proteomic study performed on bronchoalveolar lavage samples, which were derived from never-, ex- and current smokers. After identifying sev-eral genes which expression changes were either transient or persistent after smoking cessation, the basal expression levels of these genes was analyzed on transcript level in differentiated phBECs in vitro, derived also from never-, ex- and current smokers. Surprisingly, the in vitro anal-ysis revealed lower constitutive expression of analyzed genes in phBECs from patients from his-tory of smoking, which did not reflect changes seen in BALF study. Taken together, this thesis presents a successful validation of the several CS exposure models on phBECs. One of them, namely chronic basolateral CSE exposure, was further validated by the proteomic analysis, which, for the first time, revealed NUPR1, a crucial ferroptosis regulator [3], as a CS-regulated gene. This study establishes practical technique of validating CS exposure models, which can be used in in vitro studies, despite possibly different basal genes expressions of CS-regulated genes

    Drug development progress in duchenne muscular dystrophy

    Get PDF
    Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space

    Membrane vesicles: Examination of biophysical properties with atomic force microscopy

    Get PDF
    Extracellular vesicles (EVs) are not only intensively studied to increase our fundamental knowledge on their functioning, but also for diagnosis, therapeutics and drug delivery purposes. To improve the current and potential applications of EVs, a fundamental understanding of their stability, structure, and function is crucial. Such studies can be conducted at the single particle level to gain biological and physical information about the vesicles and the particle to particle variability. A suitable technique to investigate EVs under near- to physiological conditions is atomic force microscopy (AFM). Operated in liquid, it provides images of the EVs while mechanical properties of the particles can be obtained as well. Here we present our approach and the latest results in studying the structure and mechanics of these particles

    Hardware and software integration and testing for the automation of bright-field microscopy for tuberculosis detection

    Get PDF
    Automated microscopy for the detection of tuberculosis (TB) in sputum smears would reduce the load on technicians, especially in countries with a high TB burden. This dissertation reports on the development and testing of an automated system built around a conventional microscope for the detection of TB in Ziehl-Neelsen (ZN) stained sputum smears. Microscope auto-focusing, image analysis and stage movement were integrated. Images were captured at 40x magnification
    corecore