Project description:Human BEAS-2B were exposed to whole birch pollen using a Pollen Sedimentation Chamber. The chamber was designed to be able to dose cells to dry whole pollen. The goal was to understand the reaction of human epithelial cells to a human real life pollen exposure.
Project description:BEAS-2B cells, at air-liquid interface, were exposed to Diesel CAST model aerosol in an vitro exposure system and, later, to native birch pollen using a Pollen Sedimentation Chamber stabilished before. The same exposure was performed, without the primed anthropogenic exposure. The goal was to understand the effect of pre-exposure to a model diesel aerosol in allergic sensitization, using a model stabilished that mimics a real life exposure as closer as possible.
Project description:BEAS-2B cells, at air liquid interface, were exposed to birch pollen extract or house dust mite extract in a cloud chamber and, later, to UFP rich combustion aerosols in an in vitro exposure system. As control the same exposure was performed without allergen containing extracts. The goal was to understand the effect of allergenic pre-exposure to a UFP rich combustion aerosol exposed cells and their effect on allergic sensitization, using an established model that mimics more closely real life exposures.
Project description:We performed microarray analysis of miRNA expression in differentiating primary human bronchial epithelial cells. The goal was to identify miRNAs that are dynamically expressed under airway epithelial development. Cells were cultured at air-liquid-interface and were harvested day 4, 6, 8, 11, 13, 15, 18, 20 and 22 for analysis.
Project description:Foxp3+ regulatory T cells (Treg) play a central role for tolerance against self and innocuous environmental antigens. However, the role of antigen-specificity for Treg-mediated tolerance is only incompletely understood. Here we show by direct ex vivo characterization of human CD4+ T cells, that the response against innocuous airborne antigens, such as plant pollen or fungal spores, is dominated by memory-like antigen-specific Treg. Surprisingly, breakdown of tolerance in atopic donors was not accompanied by a quantitatively or qualitatively altered Treg response, but instead correlated with a striking dichotomy of Treg versus Th2 target specificity. Allergenic proteins, are selectively targeted by Th2 cells, but not Treg. Thus human Treg specific for airborne antigens maintain tolerance at mucosal sites and the failure to generate specific Treg against a subgroup of antigens provides a window of opportunity for allergy development. PBMCs from sex and age matched birch pollen allergic patients and healthy controls, were stimulated (7h) with airborne fungal (A. fumigatus) or birch pollen antigen (birch) and sorted into antigen specific conventional and regulatory T cells according to their expression of CD154+ and CD137+ on CD4+ T cells, respectively. Number of samples per group in parentheses: Healthy controls stimulated with A. fumigatus (n=5), allergic patients stimulated with A. fumigatus (n=6), healthy controls stimulated with birch (n=6), allergic patients stimulated with birch (n=4).
Project description:Immunologic response of two patient categories, birch pollen allergic and non-allergic, to natural pollen exposure (spring vs. winter) quantitated at the level of the transcriptome
Project description:In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.
Project description:The hedgehog pathway is crucial during airway epithelial cell differentiation. To assess the transcriptomic print of airway epithelial cells in absence of hedgehog pathway activation, we performed a comparative transcriptomic analysis on air-liquid interface cell cultures. Bronchial epithelial cells from 5 non-COPD subjects (ex- and current smokers) were isolated from bronchial brushes and cultured in air-liquid interface. The total RNA were extracted after 7 days of culture in air-liquid conditions in presence of an antibody targeting the sonic hedgehog ligand (AB5E1) or not (CTL). The libraries were prepared with NEBNext Ultra II directeional RNA Library Prep Kit and sequenced on Illumina.
Project description:Heated tobacco products (HTP) are novel nicotine delivery products with limited toxicological data. HTP uses heating instead of combustion to generate aerosol (HTP-smoke). Physiologically relevant human bronchial and alveolar lung mucosa models developed at air-liquid interface were exposed to HTP-smoke to assess broad toxicological response (n=6-7; ISO puffing regimen; compared to sham; non-parametric statistical analysis; significance: p<0.05). Elevated levels of total cellular reactive oxygen species, stress responsive nuclear factor kappa-B, and DNA damage markers [8-hydroxy-2΄-deoxyguanosine, phosphorylated histone H2AX, cleaved poly-(ADP-Ribose) polymerase] were detected in HTP-smoke exposed bronchial and/ or alveolar models. RNA sequencing detected differential regulation of 724 genes in the bronchial- and 121 genes in the alveolar model following HTP-smoke exposure (cut off: p≤0.01; fold change: ≥2). Common enriched pathways included estrogen biosynthesis, ferroptosis, superoxide radical degradation, xenobiotics, and α-tocopherol degradation. Secreted levels of interleukin (IL)1ꞵ and IL8 increased in the bronchial model whereas in the alveolar model, interferon-γ and IL4 increased and IL13 decreased following HTP-smoke exposure. Increased lipid peroxidation was detected in HTP-smoke exposed bronchial and alveolar models which was inhibited by ferrostatin-1. The findings form a basis to perform independent risk assessment studies on different flavours of HTP using different puffing topography and corresponding chemical characterization.