Final Report Summary - P3AGI (Public Private Partnership for Asthma Imaging and Genomics)
The P3AGI project consists of a private-public research network designed to share expertise in the fields of imaging, genomics and bioinformatics to be used for monitoring therapies and disease progression in severe allergic asthma (AA). An interdisciplinary approach combines the expertise from each of the partners including:
1) Development of Novel Optical Imaging Procedures specific to AA,
2) Improvements of current Optical Imaging Methods in AA Models.
3) Monitoring severe AA in Human Severe Combined Immunodeficiency (Hu-SCID) mouse models of AA,
4) Genomics, bioinformatics and knowledge management.
The Scientific and Technical objectives of the project are the following:
The main objectives of WP2 Imaging are to label cells with a fluorescent dye in the NIR range; to design specific optical imaging probes targeting biomarkers during the course of AA and to validate novel optical imaging agents in vitro and in vivo. The final goal is to be able to image the development, progression and treatment course of allergic asthma.
WP4 Biomarkers aims to obtain biomarkers of severe allergic asthma by performing an in-depth genomics analysis of severe AA animal models. It aims to:
* Collect ex-vivo material from AA animal models
* Perform genomics analysis involving expression profiling using microarrays
* Analyse microarray data to identify differentially expressed genes
WP5 Allergic Asthma Model aims to:
* Isolate CD4+GFP+ Th2 memory cells from mouse lungs for tracking purposes with in vivo imaging
* Monitor activated and memory T cells in Hu-SCID models with in vivo imaging
* Use allergic asthma models for testing new treatments compared steroids by imaging and genomics
* Use genomics and in vivo technology for identifying novel biomarkers to follow therapeutic efficacy
The other Wps are related to Knowledge Management (Wp1), Project Management (WP3) and Dissemination (WP6).
Research activities
WP2 Imaging
1. UMG-GOE successfully established an ovoalbumin (OVA) induced murine asthma model in Balb/c mice in collaboration with MUW.
2. Testing several Near Infrared Fluorescent (NIRF) probes for the establishment of an in vivo optical imaging method by using Optix MX2 imager to monitor inflammation within the lung during the course of asthma in Balb/c mice:
3. Successfully testing of the suitability of a new NIRF-labeled antibody in combination with NIRF imaging to monitor the efficacy of treatment of murine asthma using two different treatments, a routinely used treatment for asthma such as dexamethasone, as well as a new treatment produced by project partner Marinomed
4. MicroComputerTomography (MicroCT) has been applied (together with CBM and MUW) to assess morphological alterations in lungs in samples of mice with asthma.
5. Phase contrast CT at the SYRMEP synchrotron beamline (together with CBM) was used to image inflammation and anatomic alterations within the lung of the asthma model at the same time.
6. Together with partner CBM and as part of a secondment from UMG-GOE to CBM, it has been successfully isolated T cells from asthmatic mice, labelled them with a NIRF dye and tracked by optical imaging. These experiments are still in process.
7. UMG-GOE is in the process of testing new NIRF labelled nanoparticles to detect allergic asthma in mice.
8. UMG-GOE is in the process of testing a new NIRF labelled bovine serum albumin (BSA) based probe to detect oedema in asthmatic mice.
9. Finally, all optical imaging results have been validated by immunohistochemistry or immunofluorescence on lung slices, as well as by measurements of the cell counts in the lung fluid.
WP4 Biomarkers
CBM has performed 3 different microarrays experiment to assess the possibility to identify candidate biomarkers of severe allergic asthma. In the first experiment 3 group were analyzed: A) untreated naive mice, B) mice recovered from acute allergic asthma, and C) rechallenged mice at 2 hours after allergen exposure. In the second microarrays experiment, rechallenged mice were assessed at different time points (2,6,14 hrs) after rechallenge and compared to recovered mice. In the third microarray experiment, sensitized mice were challenged with either phosphate buffered saline, bovine serum albumin or chicken-egg ovalbumin, and assessed at 2 and 14 hrs after intranasal challenge. For all samples , RNA was successfully isolated and analysed using Illumina mouse microarrays . All data has been analyzed using the BioConductor collection particularly well suited for microarray experiments
WP5 AA Models
1. MUW characterised Th2 memory cells and isolated memory CD4+ T cells from mice recovered from allergic asthma for the injection and tracking by in vivo imaging. Established OVA induced murine asthma models in BALB/c mice for testing with several Near Infrared Fluorescent (NIRF) probes for in vivo optical imaging using Optix MX2 imager to monitor inflammation within the lung during the course of asthma.
2. MUW monitored the activated and memory T cells in Hu-SCID models with in vivo imaging as well as anti-SiglecF antibody , used to follow eosinophilia in vivo, with imaging in mouse models of allergic asthma.
3. Prepared and trained secondments at partners site to induce allergic asthma in mice for further assessment of treatment and for use in gene expression profiling. Generated severe asthma in Hu-SCID models for the purpose of testing treatments. Tested the suitability of a new NIRF-labeled antibody in combination with NIRF imaging to monitor the efficacy of treatment of murine asthma upon treatment with dexamethasone and beta escin. Isolated CD4+ T cells from asthmatic mice, labelled them with a NIRF dye and tracked them by optical imaging. Validated optical imaging results with immunohistochemistry and immunofluorescence of lung sections and by evaluation of airway inflammation. Established a severe asthma model. Pre-screened a panel of marine-derived natural compounds for efficacy and toxicity in vitro for the selection of candidates. Identified three compounds as potent anti-inflammatory candidates for further in vivo testing.
4. Prepared and trained secondments and on site at partner sites to induce allergic asthma in mice for further assessment of treatment and for use in gene expression profiling for biomarker search. Generated severe asthma in Hu-SCID models. Established co-staining with macrophage labelling.
The above research activities are integrated by 3 horizontal work-packages such as: WP1 Knowledge Management, WP6 Dissemination and WP3 Project Management. WP1 provided bioinformatics support in the acquisition, management, archiving, and analysis of imaging and genomics data. CBM implemented a database to store the data from all genomic microarray experiments has been developed for according to MIAME protocol. Standard data acquisition procedures were established for CT, Optix imaging and microarray and microCT imaging analysis to evaluate alterations in lung morphology.
Within the network, the researchers have acquired experience in conducting animal experimentation and have undergone specific training to perform research in accordance with the respective national Law for Protection of Laboratory Animals which has been harmonized with the existing EU legislation. All animal experiments meet the ethical guidelines and protocols that are used for breeding mice and different kinds of treatment were also approved by the ethical committees where animal experiments are conducted.
Overall, P3AGI is progressing lively, the intense network of collaborations among partners with very different backgrounds is one of the main driving forces of the Project. The achievement of objectives is still realistic within the planned time and with the resources of the project. We believe that the secondments and necessary interaction between the teams are a strong stimulus towards the presentation of further research joint collaborative research activities.
As regards the expected impact of the project, estimates have shown that the number of people with asthma could grow to as many as 400-450 million people worldwide by 2025. Therefore, any advancement in terms of biomarkers of disease monitoring, treatment and care of this disorder provides a huge economic benefit for European society. The identification of biomarkers that play a fundamental role in function in AA will provide a list of potential drug targeted for therapeutic treatments. P3AGI is contributing to:
* improving European Translational Research by pooling sharing data, protocols and conducting joint experiments,
* training experienced and early stage researchers providing them with inter-sectorial cutting edge research experiences
* inspiring European Research and translational advancements in the field of Allergic Asthma
* motivating European Research Collaborations and
* providing access to cutting-edge research infrastructures.
1) Development of Novel Optical Imaging Procedures specific to AA,
2) Improvements of current Optical Imaging Methods in AA Models.
3) Monitoring severe AA in Human Severe Combined Immunodeficiency (Hu-SCID) mouse models of AA,
4) Genomics, bioinformatics and knowledge management.
The Scientific and Technical objectives of the project are the following:
The main objectives of WP2 Imaging are to label cells with a fluorescent dye in the NIR range; to design specific optical imaging probes targeting biomarkers during the course of AA and to validate novel optical imaging agents in vitro and in vivo. The final goal is to be able to image the development, progression and treatment course of allergic asthma.
WP4 Biomarkers aims to obtain biomarkers of severe allergic asthma by performing an in-depth genomics analysis of severe AA animal models. It aims to:
* Collect ex-vivo material from AA animal models
* Perform genomics analysis involving expression profiling using microarrays
* Analyse microarray data to identify differentially expressed genes
WP5 Allergic Asthma Model aims to:
* Isolate CD4+GFP+ Th2 memory cells from mouse lungs for tracking purposes with in vivo imaging
* Monitor activated and memory T cells in Hu-SCID models with in vivo imaging
* Use allergic asthma models for testing new treatments compared steroids by imaging and genomics
* Use genomics and in vivo technology for identifying novel biomarkers to follow therapeutic efficacy
The other Wps are related to Knowledge Management (Wp1), Project Management (WP3) and Dissemination (WP6).
Research activities
WP2 Imaging
1. UMG-GOE successfully established an ovoalbumin (OVA) induced murine asthma model in Balb/c mice in collaboration with MUW.
2. Testing several Near Infrared Fluorescent (NIRF) probes for the establishment of an in vivo optical imaging method by using Optix MX2 imager to monitor inflammation within the lung during the course of asthma in Balb/c mice:
3. Successfully testing of the suitability of a new NIRF-labeled antibody in combination with NIRF imaging to monitor the efficacy of treatment of murine asthma using two different treatments, a routinely used treatment for asthma such as dexamethasone, as well as a new treatment produced by project partner Marinomed
4. MicroComputerTomography (MicroCT) has been applied (together with CBM and MUW) to assess morphological alterations in lungs in samples of mice with asthma.
5. Phase contrast CT at the SYRMEP synchrotron beamline (together with CBM) was used to image inflammation and anatomic alterations within the lung of the asthma model at the same time.
6. Together with partner CBM and as part of a secondment from UMG-GOE to CBM, it has been successfully isolated T cells from asthmatic mice, labelled them with a NIRF dye and tracked by optical imaging. These experiments are still in process.
7. UMG-GOE is in the process of testing new NIRF labelled nanoparticles to detect allergic asthma in mice.
8. UMG-GOE is in the process of testing a new NIRF labelled bovine serum albumin (BSA) based probe to detect oedema in asthmatic mice.
9. Finally, all optical imaging results have been validated by immunohistochemistry or immunofluorescence on lung slices, as well as by measurements of the cell counts in the lung fluid.
WP4 Biomarkers
CBM has performed 3 different microarrays experiment to assess the possibility to identify candidate biomarkers of severe allergic asthma. In the first experiment 3 group were analyzed: A) untreated naive mice, B) mice recovered from acute allergic asthma, and C) rechallenged mice at 2 hours after allergen exposure. In the second microarrays experiment, rechallenged mice were assessed at different time points (2,6,14 hrs) after rechallenge and compared to recovered mice. In the third microarray experiment, sensitized mice were challenged with either phosphate buffered saline, bovine serum albumin or chicken-egg ovalbumin, and assessed at 2 and 14 hrs after intranasal challenge. For all samples , RNA was successfully isolated and analysed using Illumina mouse microarrays . All data has been analyzed using the BioConductor collection particularly well suited for microarray experiments
WP5 AA Models
1. MUW characterised Th2 memory cells and isolated memory CD4+ T cells from mice recovered from allergic asthma for the injection and tracking by in vivo imaging. Established OVA induced murine asthma models in BALB/c mice for testing with several Near Infrared Fluorescent (NIRF) probes for in vivo optical imaging using Optix MX2 imager to monitor inflammation within the lung during the course of asthma.
2. MUW monitored the activated and memory T cells in Hu-SCID models with in vivo imaging as well as anti-SiglecF antibody , used to follow eosinophilia in vivo, with imaging in mouse models of allergic asthma.
3. Prepared and trained secondments at partners site to induce allergic asthma in mice for further assessment of treatment and for use in gene expression profiling. Generated severe asthma in Hu-SCID models for the purpose of testing treatments. Tested the suitability of a new NIRF-labeled antibody in combination with NIRF imaging to monitor the efficacy of treatment of murine asthma upon treatment with dexamethasone and beta escin. Isolated CD4+ T cells from asthmatic mice, labelled them with a NIRF dye and tracked them by optical imaging. Validated optical imaging results with immunohistochemistry and immunofluorescence of lung sections and by evaluation of airway inflammation. Established a severe asthma model. Pre-screened a panel of marine-derived natural compounds for efficacy and toxicity in vitro for the selection of candidates. Identified three compounds as potent anti-inflammatory candidates for further in vivo testing.
4. Prepared and trained secondments and on site at partner sites to induce allergic asthma in mice for further assessment of treatment and for use in gene expression profiling for biomarker search. Generated severe asthma in Hu-SCID models. Established co-staining with macrophage labelling.
The above research activities are integrated by 3 horizontal work-packages such as: WP1 Knowledge Management, WP6 Dissemination and WP3 Project Management. WP1 provided bioinformatics support in the acquisition, management, archiving, and analysis of imaging and genomics data. CBM implemented a database to store the data from all genomic microarray experiments has been developed for according to MIAME protocol. Standard data acquisition procedures were established for CT, Optix imaging and microarray and microCT imaging analysis to evaluate alterations in lung morphology.
Within the network, the researchers have acquired experience in conducting animal experimentation and have undergone specific training to perform research in accordance with the respective national Law for Protection of Laboratory Animals which has been harmonized with the existing EU legislation. All animal experiments meet the ethical guidelines and protocols that are used for breeding mice and different kinds of treatment were also approved by the ethical committees where animal experiments are conducted.
Overall, P3AGI is progressing lively, the intense network of collaborations among partners with very different backgrounds is one of the main driving forces of the Project. The achievement of objectives is still realistic within the planned time and with the resources of the project. We believe that the secondments and necessary interaction between the teams are a strong stimulus towards the presentation of further research joint collaborative research activities.
As regards the expected impact of the project, estimates have shown that the number of people with asthma could grow to as many as 400-450 million people worldwide by 2025. Therefore, any advancement in terms of biomarkers of disease monitoring, treatment and care of this disorder provides a huge economic benefit for European society. The identification of biomarkers that play a fundamental role in function in AA will provide a list of potential drug targeted for therapeutic treatments. P3AGI is contributing to:
* improving European Translational Research by pooling sharing data, protocols and conducting joint experiments,
* training experienced and early stage researchers providing them with inter-sectorial cutting edge research experiences
* inspiring European Research and translational advancements in the field of Allergic Asthma
* motivating European Research Collaborations and
* providing access to cutting-edge research infrastructures.