Project description:Dendritic cells differentiate from their precursors in the airway mucosa under local environmental instruction. Airway epithelial cells (AEC) are a potent source of both pro- and anti-inflammatory mediators and are in intimate contact with intraepithelial DC and their precursors. Thus, AEC are likely candidates for influencing this differentiation process in order to tailor the DC for optimal function in the airway mucosa. We used Affymetirx microarrays to compare the gene expression profiles of monocyte derived DC (MDDC) populations differentiated with IL-4 and GM-CSF in the presence or absence of 16HBE 14o- epithelial cells. Keywords: Comparison of two cell populations
Project description:We have developed a new model of the human airway epithelial cell by deriving the cell-specific metabolic reactions identified from (i) a draft automated model by Wang et al. 2017 (ii) gene expression datasets of the human airway epithelial cell (Deprez et al., 2020; Braga et al., 2020). (iii) We obtained additional reactions, gene-to-reaction associations and pathways (that were not in the automated model) from HumanCyc (Trupp et al., 2010) and (iv) performed stochastic and dynamic simulations on the model generated including manual curations from primary literature and Recon3D (Brunk et al., 2018). (v) We added the viral biomass maintenance function into the model, previously developed for the macrophage cell (Renz et al. 2020) to develop the new integrated model of the human airway epithelial cell and the SARS-CoV-2 virus, (iBBEC4660).
Project description:murine bone marrow derived dendritic cells (GMCSF) were treated with tracheal epithelial cell conditioned medium (48 h) in the presence or absence of LPS
Project description:<p>This study of three metastatic melanoma patients utilized exome sequencing and RNA sequencing to help identify putative neoantigen peptides that could be further validated by standard immunological assays before design of a personalized vaccine. For each set of neoantigens identified and verified by standard assays, we designed GMP peptides that correspond to the top neoantigens, then used these to condition dendritic cell isolates from each patient. The conditioned dendritic cells were used as the vaccine in three rounds of vaccination, followed by monitoring for T cell memory response to each of the neoantigens.</p>
Project description:Aspergillus fumigatus is the most relevant pathogenic mould in immunocompromised patients. Spores of A. fumigatus are inhaled and reach the lung alveoli, where they interact with the human immune system. In this set of experiments, we co-cultured with defined morphologies of Aspergillus fumigatus (Af293) for 0h, 3h and 6h with either cells representing human alveolar epithelium (A549) or human alveolar endothelium (HPAEC). We included either monocyte-derived or myeloid dendritic cells from volunteer donor blood to the epithelial layer to examine their effect on gene expression. RNA was extracted from both cell lines at each time point and hybridised to microarrays. Microarrays contain probes for approximately 110 selected human immune-relevant genes, including cytokine and chemokine genes, their receptors and downstream innate immunity genes. We used microarrays to detail the gene expression of human alveloar epithelial and endothelials cells after 3h and 6h of co-cultivation with Aspergillus fumigatus and monocyte-derived or myeloid dendritic cells
Project description:Dendritic cells differentiate from their precursors in the airway mucosa under local environmental instruction. Airway epithelial cells (AEC) are a potent source of both pro- and anti-inflammatory mediators and are in intimate contact with intraepithelial DC and their precursors. Thus, AEC are likely candidates for influencing this differentiation process in order to tailor the DC for optimal function in the airway mucosa. We used Affymetirx microarrays to compare the gene expression profiles of monocyte derived DC (MDDC) populations differentiated with IL-4 and GM-CSF in the presence or absence of 16HBE 14o- epithelial cells. Experiment Overall Design: 16HBE 14o- epithelial cells were grown to semiconfluency in EMEM media supplemented with 10% FCS before CD14+ peripheral blood monocytes were added to the AEC with recombinant IL-4 and GM-CSF. In parallel, monocytes were added to empty wells in media with IL-4/GM-CSF without the presence of the AEC. Cells were cultured for 5 days at 37°C and 5%CO2 with media supplementation on day 3. At the end of culture, cells were harvested and viable MDDC populations were isolated using flow cytometric sorting. RNA was extracted from the purified MDDC populations and hybridised to Affymetrix microarrays.
Project description:In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU2AF1, a known transcription co-factor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of up-regulation of POU2AF1 in purified airway basal stem/progenitor cells undergoing differentiation and analysis of differentiating single basal cell clones. Lentivirus-mediated up-regulation of POU2AF1 in airway basal cells induced up-regulation of host defense genes, including MX1, IFIT3, IFITM and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a "host defense tone" even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, suppressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by deregulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium. Methods: Massive parallel RNA sequencing was used to compare the transcriptome of lentivirus mediated POU2AF1 or RFP (control) gene expression in human primary airway epithelial cells (3 samples per group). Uninfected basal cell was used as a further control. Conclusions: The genes up-regulated by POU2AF1 in human airway epithelial cells are mainly related to the intracellular or extracellular anti-pathogen response, suggesting POU2AF1 plays a role in airway epithelial host defense. By genome-wide-based screening, POU2AF1, a known lymphocyte transcription co-factor, was found to be expressed in human airway epithelium and regulate host defense genes. It might be a drug target as smoking-compromised host defense is associated with down-regulation of POU2AF1. In this Series, human airway epithelial cell transcriptomes (3 uninfected without treatment, 3 infected with lenti-RFP virus and 3 infected with lenti-POU2AF1 virus) were compared using massive parallel RNA sequencing (Illumina HiSeq 2000).