Andrianaki A.,Center for Basic Research |
Siapati E.K.,Center for Basic Research |
Hirata R.K.,University of Washington |
Russell D.W.,University of Washington |
And 2 more authors.
Gene Therapy | Year: 2010
Several gene therapy applications require the transfer and simultaneous expression of multiple genes in the same cell. In this study, we analyzed the potential for coordinated expression of an endogenous bidirectional promoter located on chromosome X, which controls the expression of the heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2) and alpha-galactosidase (GLA) genes. The promoter was cloned in both transcriptional orientations in a foamy virus (FV) vector backbone, whereas the enhanced green fluorescent protein (EGFP) and low-affinity nerve growth factor receptor (ΔLNGFR) reporter genes were cloned in the 5′-3′ and 3′-5′ transcriptional orientations, respectively. In all the cell lines tested, both vectors showed high levels of transgene coexpression that reached 76% of total positive cells (range from 76 to 18%). Comparison of EGFP and ΔNGFR levels revealed that the side of the promoter that drives the expression of the HNRNPH2 gene in the genome was stronger and in accordance to its in situ activity. When tested with CD34 cells, transgene coexpression reached 35.3% of all positive cells in progenitor assays and 16.8% of all positive cells after transplantation in NOD/severe combined immunodeficient mice. In summary, we show that the endogenous promoter used in this study holds bidirectional activity in the context of FV vectors and can be used in gene therapy applications requiring synchronized expression of two genes. © 2010 Macmillan Publishers Limited All rights reserved.
Samitas K.,Asthma Center |
Samitas K.,Center for Basic Research |
Samitas K.,Gothenburg University |
Zervas E.,Asthma Center |
And 11 more authors.
European Respiratory Journal | Year: 2011
Recent studies have associated osteopontin (OPN) with allergic inflammation; however, its role in human asthma remains unclear. The aim of this study was to measure OPN levels in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of healthy controls and asthmatics, identify cellular sources of OPN and examine possible correlations between OPN expression, disease severity and airway remodelling. Serum samples were obtained from 35 mild-to-moderate asthmatics, 19 severe asthmatics and 17 healthy controls in the steady state and in cases of exacerbation. Of these subjects, 29 asthmatics and nine controls underwent bronchoscopy with endobronchial biopsy and BALF collection. OPN expression was determined by ELISA and immunohistochemistry/immunofluorescence. Reticular basement membrane thickness and goblet cell hyperplasia were also determined. Serum and BALF OPN levels were significantly increased in all asthmatics in the steady state, whereas serum levels decreased during exacerbations. OPN was upregulated in the bronchial tissue of all patients, and expressed by epithelial, airway and vascular smooth muscle cells, myofibroblasts, T-lymphocytes and mast cells. OPN expression correlated with reticular basement membrane thickness and was more prominent in subepithelial inflammatory cells in severe compared to mild-to-moderate asthma. OPN expression is upregulated in human asthma and associated with remodelling changes, and its subepithelial expression correlates with disease severity. Copyright©ERS 2011.
Papafotiou G.,Center for Basic Research |
Paraskevopoulou V.,Center for Basic Research |
Vasilaki E.,Center for Basic Research |
Kanaki Z.,Center for Basic Research |
And 2 more authors.
Nature Communications | Year: 2016
The urothelium is a specialized epithelium that lines the urinary tract. It consists of three different cell types, namely, basal, intermediate and superficial cells arranged in relatively distinct cell layers. Normally, quiescent, it regenerates fast upon injury, but the regeneration process is not fully understood. Although several reports have indicated the existence of progenitors, their identity and exact topology, as well as their role in key processes such as tissue regeneration and carcinogenesis have not been clarified. Here we show that a minor subpopulation of basal cells, characterized by the expression of keratin 14, possesses self-renewal capacity and also gives rise to all cell types of the urothelium during natural and injury-induced regeneration. Moreover, these cells represent cells of origin of urothelial cancer. Our findings support the hypothesis of basally located progenitors with profound roles in urothelial homoeostasis.