Predicta follows three interconnected workflows: models, mechanisms and translational output. Models are designed to test the hypothesis, identify mechanisms and subsequently evaluate translational outputs. They include a longitudinal cohort (WP1), mouse models of virus infection (WP2), in-vitro models of primary bronchial and nasal epithelial cells (WP3), viral-bacterial interaction models (WP4), and models of epithelial-T-cell-dendritic cell interactions (WP5). Using this wide range of models we will look into disease persistence (WP1, WP2 and WP7), inflammation patterns (WP2, WP3 and WP5), dysbiosis (WP4), and mechanisms of immune regulation (WP5) and resolution of inflammation (WP6). Translational outputs include prognostic use of antiviral antibodies and development of a high throughput spot chip for the differential detection of RV species (WP7), a clinically relevant targeted delivery technology to the bronchial epithelium (WP8) and DNAZymes for therapeutic use (WP8).
WP1: Longitudinal cohort
Disease persistence will be evaluated in a longitudinal cohort (WP1) of an age group in which disease is expected to transform (resolve or become persistent) in a significant number of patients. The effect of specific infections will be quantified, in parallel to cytokine profiling and gene array analysis (WP5), levels of pro-resolution mediators (WP6) and bacterial colonization (WP4). Antibodies against RV will be measured and their prognostic value evaluated (WP7).
WP2: Mouse models
Mouse models of repeated RV infection and parallel exposure to allergens will be developed in WP2, based on experience with single RV infection models. The major outcome in these models will be the inflammation pattern and duration (WP2), also evaluating immune regulation (WP5) and resolution of inflammation (WP6), as well as novel treatments in WP8. Knock-out mice will be used to elaborate into the mechanisms of inflammation persistence.
WP3: Epithelial models
Epithelial responses (WP3) will be studied in primary cells from normal, atopic, asthmatic and combinations thereof. The molecular basis of defective type-I IFN production (transcription factor recruitment, IFN enhanceosome assembly and interchromosomal associations), response to infection, contribution to inflammation resolution (WP6) and immune regulation (WP5) will be evaluated. These models are also going to be used in evaluating in-vitro effects of DNAzymes (WP8).
WP4: Virus-Bacteria interactions
Dysbiosis will be evaluated in WP4, in an ex-vivo mucosal model of parallel exposure to virus (RV) and bacteria (S.aureus). These interactions will be explored in patients with upper airway diseases.
Mechanisms of immune regulation will be studied in WP5, including the influence of infection in breaking tolerance in patients, in tonsil dendritic cells and under the influence of the epithelial response to virus.
WP6: Resolution of inflammation
Mediators of resolution of inflammation will be measured in WP6, using state-of-the-art technologies. Material from WP1, WP2, WP3 and WP4 will be evaluated, as well as modulation by therapeutic interventions (WP8).
The development of recombinant viral proteins will permit antibody measurements (WP7), allowing for evaluation of cumulative exposure to specific virus groups and consequently, association with disease persistence (WP1). These proteins will be spotted on an array for high-throughput, low volume diagnosis. In parallel, detailed information about infectious exposures is going to be available in WP7 using multiplexed PCR-based approaches.
RV specific DNAzymes, targeted to the epithelium with advanced amphoteric liposome-based innovative carriers, with the intention of reducing or preventing viral replication and consequent inflammation, is an exciting possibility to be evaluated in WP8, using epithelial cells from WP3 and mouse models developed in WP2. Alternative strategies with anti-inflammatory DNAzymes will also be evaluated.
All activities will be coordinated through an effective management framework that will also ensure the efficient dissemination, IPR protection and exploitation of new knowledge (WP9).