|
Work package number |
1 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
Management |
||||||||
|
Participant id |
1 |
2 |
|
|
|
|
|
||
|
Person-months per participant: |
22 |
22 |
|
|
|
|
|
||
|
Objectives
|
|
Description of work Overall
control of the project will be with a Steering Committee, which will appoint
a full time Project Office team, including a director, a project manager and
an assistant manager. An Executive sub-committee from within the Steering
Committee will work closely with the Project Office for the day to day
running of the GABRIEL project. This group will be
responsible for monitoring the progress of the project; for ensuring that the work plan milestones are met and
deliverables are delivered; for financial audit of spending by the consortium
partners; for filing of all documentation accompanying the consortium
arrangement and finances; and preparing reports for the Steering Committee
and the EU directorate. The group will also organise the meetings laid out in
the schedule in 7.5. The
Steering Committee will review the overall progress of the GABRIEL project,
and will plan changes that may become necessary as the project develops. The
Steering Committee will also manage any conflicts that arise between
consortium partners. The Steering Committee will be guided by the General
assembly of all partners, who will contribute to strategic decisions about
the direction and control of change within the project, and ensure
co-ordination between consortium members. Three
Scientific Steering Groups will monitor the workings of the scientific
workpackages, ensuring co-ordination of packages with environmental and
genetic components, and co-ordination between all workpackages through a
central Analysis Steering Group. They will also implement measures to ensure
the quality control of data from the scientific workpackages. Four
internal Advisory Boards will oversee the strategy and management of
communication, exploitation, finances and training activities. They will
report to the Steering Committee. The steering committee will
be advised by an external advisory board, who will be invited to comment on
the progress and organization of the consortium early in the programme, and then
subsequently at yearly intervals. Members of the advisory board will include
leading international authorities from academic backgrounds together with
selected representatives of the biotech and pharmacological industries. Other
Stakeholders (such as Public Health Authorities and Patient Interest Groups)
will also be invited to contribute to the advisory board. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
2 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
14 |
15 |
|
|
|
|
|
||
|
Person-months per participant: |
13 |
18 |
|
|
|
|
|
||
|
Objectives
|
|
Description of work The
WP will establish an ethics committee in close collaboration with the GA2 The
committee’s role will be to formulate policies for ethical conduct for
obtaining data from questionnaires, biological samples, and clinical and
environmental tests. This committee will be closely involved and integrated
into the planning of the study as a whole, particularly the individual
projects in the study that involve interaction with children, to ensure that
best scientific, ethical and legal practice will be followed at all times by
the participants. Principles of data protection will be observed. The
right of children to have their voices listened to and to be engaged in their
own health care decision-making is enshrined in Article 12 of the Convention
on the Rights of the Child. This protection will be incorporated into and
respected in the project by the formulation of clear guidelines for genetic
testing of children for asthma, and in the development of any recommendations
that might result from the project. Focus Group Study Empirical
research will be conducted to explore the views and preferences of target
populations and healthcare providers towards testing children for asthma
susceptibility. Focus
group methodology is an optimal strategy for research on the experiences,
perspectives and needs of specific populations. In the Topics
for discussion will include: the general attitude of parents and children
towards increasing possibilities for genetic testing on asthma; the
requirements for testing they would propose; whether parents and children
should be approached through pro-active or open strategies; the level of
pre-test counselling that parents and children need or want; and an
assessment of the impact on health behaviour. All focus groups will be audiotaped
and transcribed. Content analysis will be used to analyse the transcriptions. Ethical
and Legal Analysis Ethical
and legal research questions will be answered through literature study,
elaboration of existing theoretical accounts of responsibility and autonomy,
and confrontation or testing of theoretical reflection with results from the
focus groups. A
literature review by appropriate members of the committee of policy
documents, guidelines, national and international legal provisions will be
carried out, and the results circulated as a report to all members of the
consortium. The
study will investigate particular difficulties in relation to genetic testing
of children, including: the balancing of any potential negative impact on
families from such testing with the value of early diagnosis in asthma; the
possibility of ‘vulnerable child syndrome’ where parents of a child with a
genetic predisposition become over-protective, restricting that child’s
activities unnecessarily; the issue of whether childhood testing removes the
right of the child to make an autonomous decision, or whether the child’s
best interests are served by an early diagnosis; and whether
confidentiality is applicable to child
patients in the same way as adults. Each
of the workpackages involving contact with human subjects has been given
local ethical approval. The ethics committee will review each of these WPs in
the European context, and report differences in the approach of different
countries, with recommendation for best overall practice for future
pan-European research projects. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
3 |
Start date or starting event: |
7 |
|||||||||
|
Activity Type |
RTD/Innovation |
|||||||||||
|
Participant id |
1 |
2 |
3 |
5 |
7 |
10 |
25 |
26 |
28 |
32 |
||
|
Person-months per participant: |
14 |
1 |
2 |
10 |
13 |
13 |
7 |
14 |
1 |
13 |
||
|
Objectives
|
|
Description of work The
rich existing resource of cross-sectional and cohort studies of asthma in the
European population provides a matchless opportunity to investigate the
combined role of genes and environment in asthma. Population samples to be
studied will include all the most common forms of asthma, including childhood
asthma, adult asthma and occupational asthma. The
samples will include detailed information on key events in the natural
history of asthma, in particular the onset of disease in early childhood, the
decline in disease prevalence in late adolescence, and the onset of adult
disease in middle life. The onset of asthma in the workplace will also be
studied. The
samples include detailed measurements of environmental factors influencing
asthma, including active and passive smoking, breast feeding, allergen load,
diet, microbial exposure, family size and parental asthma status. The samples
with occupational asthma have quantification of causal environmental
exposures. Polymorphisms
will be identified from all known asthma genes and genes potentially
interacting with the microbial and smoking environment (WP5.1). Centralised
genotyping of case-control samples (WP5.1) will be carried out in a Phase I
study. Novel polymorphisms identified by a whole-genome association study
(WP5.2) will also be typed in the case-control panels. Genetic effects
verified in the case-control panels will subsequently be typed in a Phase II
study of the industrial samples and the large cohorts. The
Phase II genotyping study may be used to investigate in greater detail gene-environment
interactions that are specific to particular populations or sub-types of
asthma, or to particular environmental exposures. If cost-effective,
genotyping for this phase may be devolved to particular centres. Genotypes
will be returned to the groups for matching with phenotypes and for detailed
analyses. A
limited number of key parameters such as asthma status, total serum IgE
levels and exposure to cigarette smoking will be required from all partners
for meta-analysis in which all consortium members may take part. This data
will be held within a central database at the Genotypic
data will be analysed for G-G and G-E interactions (WP5.4). Measurements of
risk will be made for gene-gene and gene-environment interactions. The public
health implications of these findings will be considered in (WP2). |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
4.1 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
2 |
7 |
8 |
22 |
|
|
|
||
|
Person-months per participant: |
85 |
67 |
38 |
62 |
|
|
|
||
|
Objectives
|
|
Description of work The
study population is recruited through primary schools in Tirol (Austria),
Baden-Württemberg (Germany), Bavaria (Germany), Kanton Luzern and Bern
(Switzerland) and southwest Poland in rural areas with an expected proportion
of at least 10 % farming. In each centre all parents of children attending
school-grade 1-6 will be informed and asked to consent to the study
instruments. All parents will furthermore be asked to fill out a short
questionnaire to assess potential participation-bias. Parents consenting in
all study instruments will receive a comprehensive questionnaire for self-completion
to assess asthma and allergic diseases in the children and their relatives,
as well as life style and environmental determinants. Validated questions
from the ISAAC, ALEX, PARSIFAL and PASTURE studies will be included. In
the schools teachers will be asked to distribute the parental questionnaire
to the children. Based on previous experience we expect a 75% participation
rate for the short questionnaire and approx. 35 % for consent to all study
instruments in the alpine regions and 80% in Blood
samples from children with parental written informed consent will be taken in
the schools and transported to a central laboratory in each study region for
DNA extraction and centrifugation on the same day. In addition, throat swabs
will be taken from the children according to a common standardised protocol
and deep frozen on the same day. Parents will furthermore be contacted to
arrange home visits to collect the environmental samples (WP6.1). All data
will be entered in a central data bank in |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
4.2 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
1 |
|
|
|
|
|
|
||
|
Person-months per participant: |
15 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Recruitment The
study will be based at the National Heart and Lung Institute in Approximately
50 children will be recruited in the first 18 months of the study. Following
the pilot phase the study may be extended to include other European Centres. Approximately
75 adults will be recruited during the same interval from adult clinics at
the NHLI. Severe resistant asthma will be defined according to the standard
European Respiratory Society criteria. Controls for the studies will be
recruited from children undergoing bronchoscopy for non-asthmatic reasons
(e.g. inhaled foreign bodies), and from age matched mild asthmatics (treated
with inhaled β-agonists as required only). Clinical
protocols: children Children
or their parents will answer a standardised respiratory questionnaire as in
WP4.1. Respiratory function assessment will measure bronchodilator response
to β-agonist, exhaled NO concentrations, and combined saline challenge
and sputum induction will be carried out. Venipuncture
will be performed for measurements of total and specific serum IgE
concentration and Fibreoptic
bronchoscopy, bronchoalveolar lavage ( Clinical
protocols: adults Smoking
history, atopic status, lung function tests, exhaled NO, blood eosinophil
count, quality of life scores using Venipuncture
will be performed for measurements of total and specific serum IgE
concentration and Fibreoptic
bronchoscopy, bronchoalveolar lavage ( Investigation
of samples The
samples obtained will be used as a substrate for studies described in WP5. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
4.3 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
2 |
|
|
|
|
|
|
||
|
Person-months per participant: |
6 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work In specialized paediatric
asthma and allergy centers in Blood for DNA extraction has
been taken from all subjects and serum and plasma is stored in multiple
fractions in a central biobank in In
the first 18 months a total of 750 asthma cases and 400 controls will have
been recruited and will have DNA samples available. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
5.1 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
3 |
|
|
|
|
|
|
||
|
Person-months per participant: |
21 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work SNP
identification The
primary driver of SNPs to be typed in the pan-European case-control studies
will be the results of the whole genome study descried in WP5.2. SNPs typed
in WP5.2 will be compared with a list of known or putative asthma genes,
genes involved in innate immunity and genes that interact with microbia,
allergens and cigarette smoke. Any genes from the list that were not covered
in the primary genome screen and any genes of special interest will be included
in the Phase II genotyping study. Between 5 and 10 SNPs will be used per
gene. Hapmap data will be used to exclude the typing of SNPs in complete LD.
50 unlinked non-disease SNPs will be included, in order to quantify the
genetic structure of the European
population. SNP
Genotyping Phase
II Genotyping will be performed at the CNG in Verified
candidates, which are estimated to contain 300-400 SNPs in total, will
subsequently be genotyped in the cohort studies using the best available technology.
Data
handling The
data will be subject to strict quality controls through diverse statistical
measures. The large-scale genotyping systems at the CNG are controlled
through a laboratory management information system, with bar coding of and
robotic handling of samples. Error checking for the process is carried out by
the inclusion of appropriate controls. Genotype
information will be returned to data centres for analyses as described in
4.1.3 (WP3) above, as well as to the data centre at Partner 21. A common
statistical approach to the large volume of data will be developed in WP5.4. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
5.2 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
3 |
12 |
29 |
30 |
31 |
|
|
||
|
Person-months per participant: |
21 |
7 |
2 |
12 |
2 |
|
|
||
|
Objectives
|
|
Description of work The
study will investigate 750 clinic asthmatics and 750 age, sex and
geographically matched controls. The
results will build on studies already being carried out in the laboratory of
Partner 1, who will complete whole-genome association studies of 600 families
with severe asthma or asthma and eczema by 1Q06. Data from these earlier
studies will be made available to the consortium Analysis
of data Association
analyses are described in WP5.4, and will include strategies for reducing
data complexity and procedures to establish empirical probability levels.
Partners 1, 3 and 21 are experienced in the analyses of this type of complex
data. Confirmation
of results Confirmation
of potentially positive results will be sought by genotyping cases and
controls with childhood asthma recruited through the Kinderklinik in Integration
of information The
mapping information will be integrated with proteomic and expression
profiling data from asthmatics and non-asthmatics generated from WP5.3 and
WP5.5. The
study will provide an extensive dataset of novel genes involved in asthma.
These will be available for the investigation of gene-environment effects in
populations (WP3.1) and (WP5.1) and in the models (WP7.1). The results will
also serve as a database of novel targets for subsequent commercialisation in
WP8. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
5.3 |
Start date or starting event: |
7 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
3 |
|
|
|
|
|
|
||
|
Person-months per participant: |
12 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Proteomic
experiments will be utilised to identify novel proteins that differ in their
expression between individuals with different phenotypes and different
environmental exposures. Plasma
samples will be tested from children with severe asthma, mild asthma and
controls. Plasma will also be taken from children in the PARSIFAL study of
the rural environment. Comparisons will be made between asthmatic and
non-asthmatic and farmers’ children and non-farmers’ children. Twenty samples
will be used to represent each group. Specialised
samples to be tested will include saliva from children and adults with severe
asthma and controls, and broncho-alveolar lavage from the same individuals. Twenty
samples will be used to represent each group. The
technology will be based on ClinProt from Bruker, and is in operation at the
CNG in Antibodies
will be raised against proteins identified by the experiments above, or from
WP6 or WP7, and investigated for diagnostic utility. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
5.4 |
Start date or starting event: |
|
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
4 |
21 |
|
|
|
|
|
||
|
Person-months per participant: |
12 |
22 |
|
|
|
|
|
||
|
Objectives
|
|
Description of work Genetic
epidemiology It
is intended that much of the data from the genotyping in WP5.1 will be
analysed at the level of the individual population samples. However, a joint
analysis of key phenotypic and environmental factors will be necessary for an
overall synthesis of the results, and to place the results in the full
European context by taking into account genetic variation between European
continental ancestry subgroups. The
common methodology for data analysis and the interpretation of the results of
the joint analysis will be developed through a series of workshops. These
will be held in different centres during the 18 months. Smaller meetings will
be held for the analyses of specific genetic and environmental factors. The
underlying population substructure in Multi-locus
analyses will be performed to take account of background patterns of linkage
disequilibrium between SNPs and the complex genetic architecture of asthma.
Novel methods to perform appropriate haplotype-based analyses are under
development in the statistical group of Partner 21. Whole
genome association The
study will identify 1000 SNPs with P< 0.01, and with an estimated prior
probability that 100-200 of these will represent real genetic effects.
Statistical analyses will follow the approach of Hoh and Ott 17, and will include strategies for reducing data
complexity and nested bootstrap procedures with repeat re-sampling to
establish empirical probability levels. Ultimately, potential associations
will be confirmed by genotyping in the large case-control samples, described
in 4.1.5.3 (WP5.3) above. Data
from Bioinformatics The
results may be expected to identify 103 genes that are
differentially expressed between normal and asthmatic cells of different
types. Function of novel genes will be inferred from the Gene Ontology (GO)
databases, by homology comparisons, and by the analyses of protein domain
composition. Integration
of results In
order to maximise the use of this complex dataset, the results of the various
components of 4.1.4 (WP5) will be stored in the central database at the A
relational database will be developed to manage the expression, genotypic and
phenotypic data gathered by this project. To leverage the greatest use from
the data the database will be integrated with other genomics resources, such
as the Ensembl and UCSC genome browsers, by ensuring that Affymetrix
expression tags and SNPs are mapped onto the current genome build. Software
will be written to query the genome browsers and extract annotations of
regions identified by our analyses. The database will use common protocols
developed by the BIOSAPIENS NoE, of which Partner 21 is a member. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
5.5 |
Start date or starting event: |
7 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
1 |
|
|
|
|
|
|
||
|
Person-months per participant: |
28 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Human
genomics Many
different types of cells may be recruited in airway inflammatory reactions,
and it is not possible to study all of them. However, two permanently
resident cell types are central to the initiation of such inflammation, and
these will be investigated as a priority. Airway
epithelial cells are very active immunologically, producing a wide range of
cytokines, and express functional pattern-recognition receptors such as CD14
and TLR-4. Dendritic cells (DCs) are specialised antigen presenting cells
present in the dermis and epidermis that play a key role in initiating and
modulating adaptive immune responses. It is proposed therefore to build a detailed
understanding through expression arrays of the functioning of airway
epithelial cells and dendritic cells stimulated under controlled conditions.
Epithelial cells will be studied by induction of differentiation, stimulation
by heat killed gram-negative and gram-positive bacteria, and stimulation by
the protease Der p I. The study of dendritic cells obtained from
peripheral blood will also be carried out under similar conditions. In
addition to epithelial cells and DCs, it has been shown that EBV transformed
PBLs are an excellent substrate for mapping and identifying differences in
gene expression between diseased and normal subjects. EBV lines will be
established from 50 severe asthmatics taken from WP4.2 above, and cultured
and harvested for RNA extraction under standardised conditions. The results
of these studies will be compared with 200 arrays from EBV lines of asthmatics
and normal controls currently being generated in the laboratory of partner 1. Whole-genome
expression levels will be measured with the Affymetrix or Illumina
technology. Data will be analysed as described in WP5.4. In general, the
number of passages of experimental lines will be controlled ± 2 for all
comparisons. Each experimental point will be repeated in triplicate. This
data will be used to inform studies of expression in biopsies of children and
adults with severe asthma and normal controls, as well as to guide the choice
of positional candidates in the mapping of specific loci identified in the
whole genome association study described in WP5.3. Bacterial
genomics An
individual’s commensal microflora is established in the first year of life,
both within the colon and upon the skin, and the pattern of flora remains
relatively stable thereafter. Studies
of microbial populations have been hampered by the technology for conducting
comprehensive ecological analyses of bacterial flora and of environmental
bacteria. The development of genomic approaches offers new possibilities for
the systematic investigation of bacterial populations in a systematic manner.
Sequencing of small sub-unit rRNA genes has become a standard procedure
in the identification of isolates, and it is now impossible to
adequately describe microbial communities without SSU rRNA sequence
data. There are currently, >79,000 16S rRNA sequences available in It
is therefore proposed to use genomic techniques to study the childhood
environment, as well as the intestinal flora of children in differing
environments. Multiple samples will be collected from children with and
without disease as described in WP4 and WP6.1. Global |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
6.1 |
Start date or starting event: |
7 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
11 |
17 |
18 |
19 |
20 |
|
|
||
|
Person-months per participant: |
13 |
14 |
8 |
8 |
13 |
|
|
||
|
Objectives
|
|
Description of work Generation
of a data bank with existing environmental information. It
is intended that much of the data will be analysed at the level of the
individual population samples. However, a meta-analysis of key environmental
factors will be necessary for an overall synthesis of the results, and to
place the results in the full European context. Therefore, the available
information about active and passive smoking, air pollution, early life
infections, pet exposure, farm characteristics and allergen exposures will be
collected from each responsible partner under strict observation of data
confidentiality and ethical policies. Dust
samples Measurements
of exposure to microbial contaminants in epidemiologic studies among children
have traditionally focused on collection and analysis of settled dust from
floors and mattresses. Very little research has been done to validate such
measures against (more) personal measurements of exposure. As part of the
GABRIEL project we will in a first step validate the exposure assessment
against a set of (more) personal exposure measurements. Samples
will be collected by: a) dust deposition samples using deposition plates; b)
floor & mattress dust samples taken during home visits. a)
Indoor deposition measurements can be taken with surfaces such as a large
petri dish, that can be located in an unobtrusive location for periods of
weeks to a month. Fairly large amounts of dust (compared to airborne) can be
collected this way. b)
The ‘sock’ method which was successfully used in the PARSIFAL study. Socks
are being mailed to participants, who then, using a photo-instruction which
can be augmented by a internet based video instruction, collect their own
samples with their own vacuum cleaners. After sampling, the socks are
returned by mail to the laboratory. The
validation study will be performed among 75 subjects (25 atopic asthmatic, 25
non-atopic asthmatic and 25 controls living on a farm) from the rural areas
to identify the most relevant exposure and the best measure of a subject’s
personal exposure. In the farm homes 4 different samples and in the control
homes 2 different samples will be taken on two occasions in order to analyse
components of variance within and between locations. Sample
analysis Substances
which already have been measured in large scale studies (but never rigorously
validated) will be included such as endotoxin, muramic acid and extracellular
polysaccharides (EPS). Depending on sensitivity of the assays, we will add to
this Heat shock proteins for which antibodies have been developed at In the rural alpine regions
and South-West As substrates for WP 6.4
additional pooled dust samples from stables, barns and mattresses,
respectively, will be sampled according to the ALEX protocol during the dust
validation study. Milk samples. Consumption of farm milk has been shown to be strongly
protective against the development of asthma. However, very little is known
about various milk components (microbial load, proteins such as lactoferrin
or immunoglobulins, etc) responsible for such an effect. Along with the dust
validation study a sample of the milk usually consumed by the child will be
collected at each of the two home-visits. Milk
samples will first be tested for total aerobic bacterial counts based on
BACTOSCAN analyses. In parallel, the somatic cell counts will be assessed
using automated methodology. To estimate the degree of Gram-negative
contamination, the lipopolysaccharide levels will be monitored by means of
microtiter assays. The raw milk status will be confirmed based on enzymatic
assays (lactoperoxidase, alkaline phosphatase). In
addition, a more in-depth microbiological characterisation of the milk
samples will be performed by selective viable counts (total plate count,
psychrotrophic count, enterobacteria count, micro- and staphylococci,
endospore count, yeast and mould count). To assess other biochemical
compounds with potential immunomodulatory properties fractions of
immunoglobulins will be determined (with chromatographic methods) and,
contents of lactoferrin (known to exert immunomodulatory as well as
antimicrobial effects) will be assessed using optimized chromatographic
methods. In the rural alpine regions
and South-West |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
6.2 |
Start date or starting event: |
7 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
20 |
|
|
|
|
|
|
||
|
Person-months per participant: |
13 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Cigarette
smoke is the most significant cause of respiratory disease in Even non-smoking children have significant
respiratory exposure to adults who smoke. The sidestream smoke, which burns
hotter and is more toxic than the smoke inhaled by the tobacco user, is
particularly irritating to the respiratory mucosa. The established health
effects of involuntary exposure to tobacco smoke include increased lower
respiratory symptoms in children (cough, phlegm, and wheeze) as well as an
increased risk for asthma and exacerbations of
asthma. Effects on normal lung development are also observed, with boys
possibly being at higher risk than girls. This exposure has its most marked
effects in the first two years of life, and the highest risk is transferred
through mothers who smoke. The WP will therefore
carry out a meta-analysis of the effects of active and passive smoking in the
case-control and cohort and prospective studies available to the studies in
WP3. The WP will
additionally test for gene-environment interactions following Phase II and
Phase III genotyping studies described in WP5.1 above. This very large
dataset will permit a precision of risk quantitation that will be of unique
public health importance. The use of this infomation will be explored through
the involvement of the JRC, and through WP2 above. The G-E interactions
will be further integrated with other significant exposures, from the
domestic and rural environments in WP6.1 and industrial exposures in WP6.3. Genes identified as important in
the WP will be assessed for their functional effects by RNAi knockdown in
appropriate cellular systems as described in WP7.1. Selected genes will be
knocked out and investigated in murine models of disease in WP7.3. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
6.3 |
Start date or starting event: |
7 |
||||||
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Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
25 |
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Person-months per participant: |
9 |
|
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Objectives
|
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Description of work Occupational
asthma is attributable to, or is made worse by, environmental exposures in
the workplace. It has become the most prevalent occupational lung disease in
developed countries, is more common than is generally recognized, and can be
severe and disabling. The
WP will investigate working populations that are at high risk for developing
asthma with a range of causative agents ranging from wood dust and
agricultural dusts and isocyanates and wheat allergens and enzymes. These
populations are of particular interest because the underlying mechanisms that
lead to adult onset asthma are different. Classical Type I sensitization
occurs in some circumstances, such as bakers asthma, but less well understood
mechanisms lead to asthma after isocyanate and other small molecule
exposures. In addition, interactions between industrial exposures and common
allergens and pro-inflammatory agents such as endotoxins may be important. All
studies in the WP (except agricultural populations) have had detailed
exposure assessments, so that exposure levels can be described
quantitatively. Bio-allergens have been measured with earlier described
immunoassays (wheat, enzymes) and GC-MS analyses have been used to quantify
monomer or prepolymer levels of aromatic or aliafatic isocyanates. The
WP will undertake analysis of gene-environment (and G-G and E-E)
interactions, following genotyping of SNPs in multiple genes in WP5.1,
following the analysis protocols described in WP3 and WP5.4. Plasma
from affected and unaffected subjects in the WP will also be submitted for
proteomic analyses in WP5.3. |
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Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
6.4 |
Start date or starting event: |
7 |
||||||
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Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
6 |
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||
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Person-months per participant: |
11 |
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||
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Objectives
|
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Description of work Dust samples will be
collected by standardized techniques according to established protocols
during the dust validation study described in WP6.1 above. The
first 18 months of the project will be centered around the optimisation of
dust fractionation methods to produce
samples amenable to structural analysis by mass spectrometry. The biological
activity of the fractionated samples will be screened in series of rapid
assays that involve binding to receptors (TLRs and GPCRs) and a list of
compounds for further characterization for immunomodulatory activities will
be made. Various
extraction methods involving polar (e.g., chloroform, methanol, water)
solvents will be applied to dust samples and extracts will be profiled. In
the first phase of the project, the analysis will be restricted to molecules
that carry lipid moieties, as recent studies have indicated that lipids and
lipo-conjugates from microbial origin can have strong effects on innate
immunity and thereby modulate the wiring/development of the developing immune
system. Thus, extraction procedures with organic solvents as well as polar
solvents that are particularly suitable for lipo-conjugates will be used. Here, samples will
be suspended, stirred and left overnight for phase separation. Or, in the
cases where no phase separation occurs, samples are suspended in the
solvent(s), centrifuged and the obtained supernatant is evaporated. We have
already established that extraction by phase separation results in samples
with a wide range of hydrophobicity/hydrophilicity. All samples will be
characterized by mass-spectrometry to provide the consortium with a database
of profiles for protective and non protective samples. The extracted samples will
be further divided into fractions, which will be screened for
immunomodulatory actions in WP7.1 below. For this purpose, silica-gel and
reversed-phase (RP) chromatography methods will be utilized, the latter will
employ among others RP-8 or RP-18 surfaces. Also, chromatography on
octylsepharose will be performed. Solvents used will include inter alii chloroform, methanol,
propanol, and also apolar solvents such as hexane. Detected fractions will be
isolated by evaporation of the solvent and their mass will be determined
followed by their biological and immunomodulatory activities as described in
workpackage 7. The overall activity is
characterized by farm sample profiling and fractionation. Fractions will be
characterized by mass-spectrometry (MS) profiling and the results stored with
software to deal with large data sets and specific lipid databases (http://lipidbank.jp; http://www.lipidat.chemistry.ohio-state.edu).
Moreover, there will be search activity to find structures within the samples
and fractions thereof that correspond to already known compounds with
modulatory activity; this will be done by Tandem MS. In a deliver and feedback
system between this workpackage and those described in WP7, it will be
possible to form an extended database of biological activity, immunological
activity and structural motifs of compounds found in protective farm samples. The activity within the first 18 months can
establish a new line of research the combines detailed chemistry to the
immune activation that can be extended to highly hydrophilic samples in farm
samples. |
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Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
7.1 |
Start date or starting event: |
13 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
1 |
|
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||
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Person-months per participant: |
9 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Airway epithelial cells are
highly active immunologicaly. They contain a wide range of pattern
recognition-receptors that recognize microbial components and the presence of
cellular damage, and are responsible for initiating events in the innate
immune response to danger. As a standard model cells
will be stimulated with IL-1β (as a positive control), LPS or and the
effect on cytokine (IL-8 and GM-CSF) release measured by ELISA. RNA will be
extracted from cells and examined for global gene expression, as described in
WP5. The effect of an environmental oxidative stress (cigarette smoke) on
this model will initially be examined. The effects of other environmental
agents identified in WP6 on basal and stimulated cytokine release will be
measured subsequently. An extension of the model
will use siRNA and overexpression vectors to knockdown or overexpress genes
associated with severe asthma such as DPP10
and PHF11 and also those associated
with protection against asthma and identified in WP3 or WP5. Transfection of siRNA and vectors will be
performed with jetPEI™ (Polyplus transfection, |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
7.2 |
Start date or starting event: |
13 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
13 |
|
|
|
|
|
|
||
|
Person-months per participant: |
9 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work We will test the effect of
farm samples that have been extracted and fractionated within WP6.4 on
dendritic cell function to identify fractions in protective and non
protective farm samples that are responsible for Th1, Th2 and Treg
skewing. Molecules that deviate the
immune system away from Th2 will be of particular interest within the context
of protection from allergic disorders.
Within the first 18 months,
the optimization of methodology for application of environmental samples will
be followed by identification of fractions that skew away from Th2. The most promising fractions will be sent
back to WP6.4 for structural analysis and further fractionation and
purification. These new more specified
samples will re-enter DC experimentation to determine structure function
relationship. The first experiments
will start using fractions from 5 farm samples that have shown the presence
of compounds/masses specific to protective farms. These farm samples will be
prioritized based on the chemical profile information as well as data on
their ability to stimulate TLRs and GPCR (fast biological assays from WP6.4)
with priority given to samples with activity in these assays; however testing
will not be restricted to these samples only. The toxicity and dose response
curves will be assessed in the DC culture system and then more elaborate
tests looking at T cell polarization will be started. If in 3 independent experiments similar
results are obtained in terms of skewing to non-Th2 response, the fractions
will be selected and sent for further structural characterization. This approach will be repeated with another
5 farm samples and depending on the progress made we will aim to characterize
immunomodulatory compounds from a total of 20 protective farm samples in this
period. The assay is well
established and involves the incubation of dendritic cells derived from
monocytes (after culture with IL-4 and GMCSF) with positive controls (known
agents that induce Th1, Th2 and Treg) and test samples obtained from farms
and fractionated in WP6.4. The activity will be tested in the presence and
absence of maturation factors to understand the activity of compounds under
conditions of no inflammation (iDC) and inflammation (mDC). Moreover the
assay will be set up in such a way that not only the induction of Th1, Th2 or
Treg by farm compounds is tested but also modulation of responses in the
positive controls. Production of cytokines by dendritic cells, particularly
IL-12 and IL-10 will be assessed as well as the expression of surface
molecules such as CD40, OX40L, CD80, CD86, PD1 and TLRs. These DCs will then
be co-cultured with naďve T cells and the phenotype of the induced T cells
will be determined by their cytokine production. Compounds that drive on their own the
generation of TH1 and Treg cells will be selected for further
characterization. Moreover, compounds that prevent the induction of Th2
response will also be included in the selection of compounds that enter a
second round of characterization and purification in WP6.4. Depending on how the DC
experiments progress, we will also start to focus on the molecular
characterization of events downstream of DC modulation. Gene expression levels will be examined
with particular emphasis placed on genes that are identified in WP3 or WP5 as
important in asthma severity or protection against asthma. Thus depending on the rate of progress, it
may also be possible to get an insight into the mechanism of the action of
promising compounds at the gene expression level, capitalizing on knowledge
generated elsewhere in the project. The experiments will take two
approaches. One, the expression levels
of candidate genes such as TLRs/CARDs, GPRA, chemokines, cytokines, SOCSS and
STATS (depending on info generated during the project in WP3 and WP5) will be
compared in DC exposed to protective and non protective compounds. This can if necessary be combined with gene
arraying. By identification of genes
up or down regulated in DC exposed to protective environmental samples a
model can be constructed on how allergies are prevented. Two, dendritic cells from individuals with
known genotypes (prioritized from work in WP3 and WP5) will be cultured with
the protective compounds and their activity will be assessed. This will
generate information on the gene by environment interaction at the molecular
level in vitro. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
7.3 |
Start date or starting event: |
13 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
16 |
20 |
|
|
|
|
|
||
|
Person-months per participant: |
5 |
3 |
|
|
|
|
|
||
|
Objectives ·
To investigate the effects of environmental factors
on airway hyperresponsiveness, airway inflammation and sensitization in
murine models of asthma. |
|
Description of work In
vivo models will be used to assess the biological relevance of
environmental factors identified in WP6.1 and WP6.4. Female BALB/c mice aged 7-8
weeks are used and acclimated to the animal facility for 14 days prior to the
experiments. Mice will be sensitized according to a standardized protocol by
injecting intraperitoneally on days 1 and 14 20 µg OVA (ovalbumin GradeV;
Sigma) emulsified in 2.2 mg aluminium hydroxide (ImjectAlum, Pierce) in a
total volume of 200 µl. On days 28 and 38 mice will be challenged with OVA
aerosol for 20 minutes. OVA aerosol will be generated with a PARI-Boy aerosol
generator from a 1% OVA solution. A control group of mice will be injected
with aluminium hydroxide and challenged with PBS instead of OVA
(non-sensitized control). Previous experiments have
shown that 14 inhalations of dust extracts concomitant to the sensitization
process are sufficient to down regulate sensitization and airway
responsiveness. The dust extracts will be given as aerosols generated from a
10 mg/ml solution. For the inhalation mice are placed in a Plexiglas chamber
with 11 l volume. The box is connected to a jet stream aerosol generator
(PARI-Boy Turbo) and mice are allowed to inhale the aerosol for 20 min. Dust
inhalation begins at the day of the first OVA injection and the last
treatment six days before the second OVA-aerosol challenge. One control group
of mice is sensitized as described above and treated 14 times with PBS during
sensitization, a second control group will inhale only dust extract without
sensitization to characterize any immunological or inflammatory reaction
towards the dust under study. Twenty-four hours after the
last aerosol challenge, mice will be evaluated for airway hyperresponsiveness
(AHR). AHR is measured in conscious, unrestrained mice by
whole-body-plethysmography using an established method. The Penh (enhanced
pause) value measured after provocation with 0, 6, 12, 25 and 50 mg/ml
methacholine is correlated to the corresponding methacholine concentration.
From the resulting curve the area under the curve (AUC) is taken and used as
a value expressing the grade of airway hyperreactivity of mice. Inflammatory reactions are ascertained by
cell counts particularly eosinophils, by measuring mediators in the
bronchoalveolar lavage fluid (BAL) and by histology of the bronchial mucosa.
Sensitization is measured by systemic serum antibodies and local antibodies
in the BAL fluid (IgE, IgG1 and IgG2). To determine T-cell reactivity of
activated T-cells splenocytes will be stimulated with OVA and mediators
(IL-4, IL-5, IL-10, IL-12 and IFN-γ) will be measured in the
supernatants. We will in a first step test
4 to 6 different candidate dust extracts or environmental factors selected
through the work in WP6.1 and WP6.4. |
|
Deliverables
|
|
Milestones and expected result
|
|
Work package number |
8.1 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
24 |
|
|
|
|
|
|
||
|
Person-months per participant: |
36 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Lipoproteins
are part of the outer membrane of Gram negative bacteria, Gram positive
bacteria, Rhodopseudomonas viridis, and mycoplasma. Bacterial lipoproteins
have no shared sequence homology but are characterized by the N-terminal
unusual amino acid S-(2,3-dihydroxypropyl)-L-cysteine acylated by two or
three fatty acids. Lipoproteins
and their synthetic analogues are strong immune modulators of the early host
responses and initiate adjuvant effects on the adaptive immune system.
Lipoproteins from different bacteria activate NF-κB and cytokine
production through their interactions with cellular PRRs. Several PRRs have
been identified by genetic studies as having a role in asthma susceptibility,
and offer a link between the microbial environment and the immune system in
asthmatics. Partner 23 has a strong track
record in the use of Lipopeptides as
adjuvants, and have shown for example that the built-in lipotripeptide
adjuvant Pam3Cys-SS and of a B- and T-cell epitope of the
foot-and-mouth disease virus leads to a long-lasting protection against virus
challenge. The company also have extensive experience of the usage of
lipopeptides in murine models. The safety, reproducible production and ease
of storage and handling of lipopeptide vaccines suggest that they have
significant potential for the development of vaccines for humans and domestic
animals. Partner 23 also has a strong track
record on the use of lipopeptides to modify the responses of
PRRs. It is therefore proposed that Partner 23 develop a range of
lipopeptides to interact with key PRRs, including TLR-2 and TLR-9. These
compounds will be investigated for their effects in the models systems
described in WP7, with particular attention to their ability to modify the
responses of epithelial and dendritic cells to inflammatory stimuli, an for
their effects on murine models of allergic asthma. After
isolation and purification of lipopeptides using e.g. liquid-liquid
extraction (craig extraction), membrane filtration, chromatography (size
exclusion chromatography (HPSEC), RP chromatography and HPLC, affinity
chromatography, flash chromatography) and free flow electrophoresis as well
as proprietary methods, compounds will be characterized by biological
methods, chemical methods and instrumental analytics. Enrichment of
lipopeptides will be investigated by cellular assays concerning their ability
to activate cells via TLR2 (IL-8 and/or TNF-a determination). Quantitative
determination of the unusual amino
acid dihydroxypropylcysteine (Dhc) of homogeneous TLR2 agonist will be
carried out by microchemical and microanalytical methods. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
8.2 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
23 |
|
|
|
|
|
|
||
|
Person-months per participant: |
36 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Previous workpackages WP3 and WP5 have described
the use of genetic polymorphisms in the identification of disease
susceptibility genes for different types of asthma at different times of
life. A further important use of genetic polymorphism
and other biomarkers becomes anticipation of the response to therapy (pharmacogenetics).
Partner
21 (GeneOs)
is in the process of receiving Ethical and Regulatory approval in The whole process is
voluntary and will be governed by appropriate ethical and regulatory
reviews. Historically, people in The company is developing
IT- infrastructure, proprietary statistical and data mining tools to
generate, assemble and analyze our vast sets of genealogical, disease and
biomarker information. We believe we have unique informatics tools for
utilizing large datasets to identify correlations between individual
variation and disease. The company will develop GeneOs will start by the
recruitment of patients with asthma and COPD (a disease due to the
combination of asthma and cigarette smoking). The company will recruit not
less than 500 patients in each disease arm, in 10-12 subcohorts, giving a
total of 6000 patients). Retrospective health care data and all the samples
will be collected and restored according to the GLP and GCP standards. The company will type
polymorphisms and biomarkers in the patient samples and will identify predictors
of the response of standard asthma therapies (inhaled β-adrenergic
agonists and inhaled or oral glucocorticoids) as well as the response to
novel therapies identified in WP7 and WP8.1. Factors anticipating
non-response to therapy will also be identified. The results will be
integrated with WP2 to ensure ethical use of this diagnostic information,
taking into account the likely determinants of cost-effective therapeutic
intervention. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
8.3 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
RTD/Innovation |
||||||||
|
Participant id |
27 |
|
|
|
|
|
|
||
|
Person-months per participant: |
36 |
|
|
|
|
|
|
||
|
Objectives
|
|
Description of work Modern
therapeutics includes the use of biological products, such as proteins,
antibodies and anti-sense RNA. These
biotherapeutics are making a significant impact in the biotech sector and are
close to impacting on clinical management of disease. Most potential targets
identified by genetic or genomic studies are not amenable to small molecule
(drug) therapy. Biotherapeutics considerably widen the range of therapeutic
possibilities, as deficient proteins may be replaced, and over-expressed or
overactive proteins may be nullified with antibodies. In order to maximise the
therapeutic potential of the results of genetic and genomic studies, the
therapeutic use of antibodies against particular targets will be developed. Targets will be identified from
the results of WP3, and assay systems for therapeutic effects will be
developed from the results of WP5 and WP7. |
|
Deliverables
|
|
Milestones and expected
result
|
|
Work package number |
9 |
Start date or starting event: |
1 |
||||||
|
Activity Type |
Training |
||||||||
|
Participant id |
1 |
2 |
3 |
13 |
20 |
21 |
22 |
||
|
Person-months per participant: |
1 |
1 |
1 |
1 |
1 |
0 |
1 |
||
|
Objectives
|
|
Description of work A
great strength of the GABRIEL consortium is the participation of a large
number of qualified scientists from a variety of European centres of
excellence. The partners bring to the project cutting edge expertise of
genetics, molecular biology, immunology, microbiology, epidemiology,
biostatistics, bioinformatics, genomics and proteomics. The ability to do
research in the post-genome environment depends on the establishment of
cross-disciplinary links. This is to be achieved through studentships,
visiting fellowships, and workshops. The workshops will be open to junior and
senior European scientists who are indirectly linked to the project through
GA2LEN or other networks. Studentships GABRIEL
will provide funding for three 3-year PhD studentships. Competitive
applications for these studentships will be invited early in the project. The
application process will be handled by the training subcommittee, who will
make recommendations to the Steering Committee. Successful applications will
be judged on the high ability of students, the scientific value of the
proposed project, and the track record of supervisors. Visiting
fellowships GABRIEL
will provide funding for six 3-month visiting fellowships. These will enable
scientists at the middle level to visit other laboratories within the
consortium to learn specific techniques, or to work on collaborative
projects. Competitive applications for these fellowships will be invited at
yearly intervals. The application process will be handled by the training
subcommittee, who will make recommendations to the Steering Committee.
Successful applications will be judged on the quality of the individuals
applying for the fellowships, the scientific value of the proposed project,
and the degree with which the consortium would gain added value from the
visit. Funding for longer term
fellowships will be sought through the existing EC Marie Curie program Workshops The
consortium will fund analysis workshops, particularly to advance the
meta-analysis of data from WP3, WP5 and WP6. The workshops will contain
approximately 30 participants, but attendance will not be withheld to any
interested individuals. GABRIEL
will also fund tutorial workshops to present cutting-edge methodologies to a
limited group of approximately 30 participants. The
program will also run training courses on selected themes related to the
project such as advanced genetics, biostatistics, epidemiology, and
immunology. Two of these courses will be organised in the new EU member
states with the aim of recruiting participants from The timing of the workshops and courses will be planned
by the training subcommittee, and their organization delegated to local WP
leaders in the centre in which the meetings will be held. |
|
Deliverables
|
|
Milestones and expected
result
|