The impact of human breast milk components on the infant metabolism

Background & aims Breastfeeding is beneficial for mothers and infants. Underlying mechanisms and biochemical mediators thus need to be investigated to develop and support improved infant nutrition practices promoting the child health. We analysed the relation between maternal breast milk composition and infant metabolism. Methods 196 pairs of mothers and infants from a European research project (PreventCD) were studied. Maternal milk samples collected at month 1 and month 4 after birth were analysed for macronutrient classes, hormone, and fatty acid (FA) content. Phospholipids, acylcarnitines, and amino acids were measured in serum samples of 4-month old infants. Associations between milk components and infant metabolites were analysed with spearman correlation and linear mixed effect models (LME). P-values were corrected for multiple testing (PLME). Results Month 1 milk protein content was strongly associated with infant serum lyso-phosphatidylcholine (LPC) 14:0 (PLME = 0.009). Month 1 milk insulin was associated to infant acetylcarnitine (PLME = 0.01). There were no associations between milk protein content and serum amino acids and milk total fat content and serum polar lipids. Middle- and odd-chain FA% in breast milk at both ages were significantly related to serum LPC and sphingomyelins (SM) species in infant serum (all PLME<0.05), while FA% 20:5n-3 and 22:6n-3 percentages were significantly associated to serum LPC 22:6 (PLME = 1.91×10−4/7.93×10−5) in milk only at month 4. Other polyunsaturated fatty acids and hormones in milk showed only weak associations with infant serum metabolites. Conclusions Infant serum LPC are influenced by breast milk FA composition and, intriguingly, milk protein content in early but not late lactation. LPC 14:0, previously found positively associated with obesity risk, was the serum metabolite which was the most strongly associated to milk protein content. Thus, LPC 14:0 might be a key metabolite not only reflecting milk protein intake in infants, but also relating high protein content in milk or infant formula to childhood obesity risk.


Project summary
Coeliac disease (CD) is a chronic disorder caused by hypersensitivity to some of the most common proteins (gluten) in the diet of the European population. CD affects as much as 1% of the Europeans (2.5 million people) and is the most common food intolerance in Europe. If recognised, CD patients have only limited access to safe foods and there is not causal therapy available. The general objective of this STREP is to significantly reduce the number of people suffering from CD in Europe, by developing primary prevention strategies for CD. By European collaboration of leaders from disciplines beyond those traditionally used a.o. from academia, patient organisation and industry, we will carry out innovative research to examine the hypothesis that it is possible to induce tolerance for gluten in genetically predisposed Through European collaboration of leaders from disciplines beyond those traditionally used, among others from academia, patient organisations and industry, we will carry out innovative research on primary prevention of CD by studying the effect of the dietary history in the induction of early and later (in)tolerance for gluten and other auto-immune phenomena. Taking full advantage of genomics techniques we will elucidate (some of) the genetic and immunological mechanisms involved in this process.
The proposed project will help to identify vulnerable population groups, genetically predisposed to CD, and to assess how improved food and early nutrition guidelines might reduce the prevalence of CD in the future. A better understanding of the influence of the dietary history on human health will establish the basis to provide safer, and health-promoting early nutrition guidelines so as to improve the health and well-being of the European citizens.
The proposed project represents a significant step forward in the integration of knowledge from basic science into the prevention and treatment of food-related diseases in Europe, in this case CD. Normally insert "month 1 (start of project)" and "month n (end of project)" These columns are needed for possible later contract revisions caused by joining/leaving participants *** Partners will accede to the contract in accordance with the 1 st contract amendment **** Partner removed from consortium due to its nonaccession to the contract

Relevance to the objectives of the specific programme and/or thematic priority
The proposed project contributes to the objectives of the 6 th Framework Programme, priority "Food Quality and Safety", and its scientific objectives strictly comply with those of the area "Epidemiology of food-related diseases and allergies". Coeliac disease (CD) is the most common food intolerance, its prevalence being approximately 1:100 in the European population. If recognised, CD patients have only limited access to safe foods and there is no causal therapy available. This project is expected to add significantly to the present knowledge on the possibility of identifying vulnerable individuals within the population; it will provide information concerning the natural history of this condition and about the possible protective role of specific early dietary regimens. Moreover, the influence of genetic variability will be studied, linking it to the measurement of the immune response to gluten.

Influence of early nutrition in relation to genetic factors
The main objective will be to study the influence of dietary history, including early nutrition, on the development of CD in relation to genetic factors. These aims will be accomplished both at general population level and in a selected "high-risk" group. In the first case, the long-term influence of dietary regimes in the first year of life on the development of CD will be investigated, taking advantage of the epidemic of CD experienced in Sweden in the mid-80s. Potential contributing environmental risk factors were identified, as it was found that the risk of developing CD was possibly related to the dietary history and reduced when gluten-containing foods were introduced into the diet while the infant was still being breast-fed. Now, we wish to determine the long-term effects of these different dietary regimens. In the second arm of this part of the project, these conditions will be reproduced in a prospective randomised controlled study, through an active intervention based on the administration of small amounts of gluten in a period when the infant is still being breast-fed. This second part of the study will be realised in subjects genetically at risk; i.e. first-degree relatives of celiac patients. It is expected that the potential results of this effort will go well beyond the limit of the project. This cohort could be followed for a longer period of time, adding further relevant information on the natural history of this condition and on its relation with the genetic make-up of the individual. In fact, from the genetic point of view, this project will contribute to verifying prospectively whether the genetic factors identified already are really associated with the risk of developing CD and, if so, to what extent.

Identification of molecules involved in the expression of the disease and genes they are linked to.
This project aims to elucidate the role of HLA and non-HLA genes in relation to early feeding in the development of CD. It will take advantage of the knowledge available so far (data obtained mostly by the same researchers involved in this project).
Importantly, the information, here validated in a prospective study, is expected to reinforce the role attributed to HLA and non HLA genes The identification of molecules coded by the latter genes, in some cases in a very advanced phase, will significantly contribute to the better comprehension of the pathogenesis of the disease.

Early nutrition and autoimmunity
The main outcome evaluated in this project is the development of CD. This condition is strictly associated with autoimmune conditions and it is considered to be

Potential Impact
Screening studies for coeliac disease (CD) in Europe have revealed prevalence as high as 1.0% and in first-degree relatives of CD patients the frequency of the disease is as By European collaboration among leaders from academia, patient organisations and industry, the PREVENTCD project will carry out innovative research on primary prevention of CD by studying the effect of the early dietary history in the induction of (in)tolerance for gluten in genetic predisposed children. The proposed project represents a significant step forward in the integration of knowledge from basic science into the prevention and treatment of food-related diseases in Europe, in this case CD.

Innovative impact
The results of PREVENTCD may have an innovative impact in the European Food Industry, concerning the scientific basis for the composition of bottle-feeding, aimed to prevent CD in children in whom breast-feeding is not possible. In this context, the studies on gluten content in breast-feeding samples from mothers from high risk families from different European countries with different nutritional practices, will offer novel data to the European Industry in its effort to develop food for health.
Morover, the commercial diagnostic tools used in PREVENTCD, i.e. serological markers for CD (AtTG, AGA) and autoimmunity (TPO), and also for determining the genetic vulnerabilty (HLA-typing) for CD, will have a marketing advantage as being used in high quality international research.

Risk assessment and related communication strategy
WP1: the management and governance structure of PREVENTCD is described in detail, including the risks and contingency plans, under "6.1 Project management". WP2: no contingencies or risks are expected.
WP3: the main activity in WP3 is represented by the recruitment of newborns belonging to at-risk families for CD. The number of newborns expected from each participating group has been carefully evaluated on the basis of the number of families followed-up in that group and of the birth rate of the local general population.
Deliverables set at the end of each year of the project will allow following strictly the progression of the recruitment. Already at month 12 it will be possible to have a first evaluation of the recruitment. If at this moment it turns that the recruitment of newborns is significantly lower than expected, all efforts will be done to increase the recruitment rate. This will mainly be done by increasing the awareness about the project by, among others, the members of the local Coeliac Patients Associations.
Reinforcement of the call to participate may be done by additional information on the Journals and web-sites of the AOECS and local associations, as well as on the local newspapers and radio and television programmes. Those strategies have previously proved to be successful in increasing the participating rate by target groups in research projects.
Although the project clearly states that breast feeding will be strongly encouraged for all the participating children, and it is expected that families with coeliac cases will strictly comply with this prescription, it is possible that a proportion of infants at the age of 4 months will turn out not to be breast fed. To avoid bias they will be in any case enrolled. Retrospectively, the impact of the length of breast feeding on the outcome of the intervention will be evaluated. It must be emphasised that no evident risk are foreseen for deliverable 3.1, which may be considered as one of the most important deliverables of the whole project. This project is the first large prospective study that will describe the natural history and the development of the specific immune response of subjects belonging to "at-risk" families, in relation to a very precisely defined genetic background. It is also important to stress that the work planned in two other important WPs (genetics and immunology) although certainly done on the infants recruited in WP3 and WP4, are not strictly dependent on the deliverables 3.4, 3.5 and 3.6.
WP4: the population based study doesn't involve any intervention with respect to infant feeding, but takes advantage of a previous "experiment" in Sweden with extensive changes with respect to breast-feeding and early gluten exposure. Thus, for WP 4 risks no contingencies or risks are expected.
WP5: this part of the project relies on existing knowledge on the genetics of CD and the technology available for high-throughput genotyping. This implies that the feasibility is good. The genotyping will be performed on an Illumina platform which is an extremely powerful technology currently in use by numerous groups and also fully operational the UMCG. No contingencies or risks are expected to execute this WP. WP6:

Gluten analysis
Characterization of the gluten preparation will be done with T cell reagents already established by partners 1 and 13 and with monoclonal antibodies established by partner 1. These tests have already proved to yield reliable results. We therefore see no risks associated with this part of the work package.

Breast milk analysis
For the breast milk analysis we will primarily use the monoclonal antibody based assays. In a pilot study performed in the Netherlands our preliminary findings indicate that gliadin fragments can be reliably detected in the breast milk samples and that these levels remain stable over extended periods of breast milk feeding. We therefore see no risks associated with this part of the work package.

Detection of gluten specific T cells HLA tetramers during gluten exposure.
Detection of gluten specific T cells in peripheral blood by HLA tetramers has so far been successfully undertaken in adult coeliacs in remission on day 6 after a 3 day oral gluten challenge. Detection of HLA tetramer positive T cells has so far not been tested in children and it is unclear whether gluten reactive T cells can be detected in peripheral blood when CD is about to precipitate. The amount of blood needed to detect tetramer positive T cells in children is also uncertain because there is no data available on the frequency of gluten reactive T cells in peripheral blood under these conditions. Initially we will therefore test 10 patients for tetramer staining. If no positive results emerge, we will terminate this approach and explore alternatives, in particular ELIspot analysis of peripheral blood samples or CFSE dilution experiments in which responding T cells are visualized by the CFSE dilution as the results of cell division. Again we will test 10 patients in each group. If all approaches fail, this part of the workpackage will be terminated.

Repertoire analysis
Intestinal biopsies taken from children at partner site 1 will require no transport and can be analyzed directly. Biopsies taken at sites different from that of partner 1, will require transportation before the culturing of gluten reactive T cells. It is uncertain how well biopsies will maintain their ability to produce functional T cell reagents.
This will be tested in simulation experiments before large scale transfer of biopsies will take place. In these simulation experiments we will define the best conditions for transport. If transportation of biopsies does affect T cell outgrowth, we will develop alternative protocols through which the start of the biopsy cultures can be initiated at sites other than partner 1 and 13. Such sites could include partners 2, 4, 6 and 7. After the initial outgrowth of the T cells these could be frozen and shipped to partners 1 and 13 for further analysis.

Regulatory T cell compartment
There is now a large body of literature of markers though which regulatory T cell can be identified through FACS-analysis. Markers include Fox-p3, CTLA-4, CD4CD25 and characteristic cytokine secretion profiles. The techniques for this analysis are all well established. We therefore see no risks associated with this part of the work package.
WP7: no contingencies or risks are expected in this WP.

Project management and exploitation/dissemination plans
The management and governance structure of PREVENTCD ensures: -Effective and optimised management; -Effective and optimised decision making; -Enhanced networking between the participants and stakeholders (like patient organisations, public health authorities, research initiatives on coeliac disease (CD)).
The public participation and awareness of the project will be raised by widely dissemination of its activities and results to other directly interested parts, such as food industry, CD patients and patients associations.
The PREVENTCD project will have a website and a newsletter both to facilitae internal communication, and to make the process and results easily accessible  She/he will be responsible for setting up the Project Office, for management, financial accounting and organizational aspects, and for maintaining the web-based activities and the networking activities. They will receive assistance within the LUMC from a financial project officer, an officer of European research grants, from IPR and legal experts, and from ICT specialists and communication experts. The coordinator will ensure that reports are prepared and submitted to the EC project officer in time and according to instructions (i.e. periodic-mid-term-and final reports, including cost statements and audit certificates).
The Project Council, which is composed by the representatives from all the collaborating organisations and is chaired by the co-ordinator, is responsible among others for: 1. Deciding upon the allocation of the project's budget in accordance with the EU Contract, and reviewing and proposing budget re-allocations; 2. Deciding on the plan for using and disseminating knowledge arising from the project.
The major decision making body in the PREVENTCD project is the Management Team, which is composed by the leaders of the 7 Work Packages and is chaired by the coordinator. The PREVENTCD Management Team will be responsible for the day-to day direction of the project and will be responsible, among others for:

Planning and Control
PREVENTCD is a project in which decisions on activities to be undertaken will be made centrally. On the other hand, the execution of these activities will be decentralized. The activities are subdivided in 7 Work Packages. For each Work Package one of the partners is made responsible. The responsible partner or Work Package Leader coordinates a taskforce designated to achieve the goals set out in the Work Package description. The overall planning of the PREVENTCD project is given in part 7 (Workplan) and in the Gantt charts. For the planning the following procedure will be followed. Every three months, progress will be reported at the Management Team meetings. In case of changes in planning or research, the Work Package Leaders will propose measures needed to realise the Project's goals. The management will decide minor changes in the execution of the project. The Project Consortium will decide on major changes. However, it is expected that there will be no large deviations from the overall work plan submitted in this proposal.
Every three months the Management Team will make an evaluation of the progress of the project based on material that has been submitted to the project database and on short progress reports submitted to the coordinator. These reports should at least contain descriptions of the deliverables and milestones achieved and a self-evaluation of the progress and difficulties encountered. Because of the close coupling of planning and control within a three months cycle, the Management Team will be able to control the project closely and to adjust the various building blocks where needed, or shift activities between partners.

Role of the Partners
Each partner forms part of a larger network of related institutes and research centres in Europe and has a track record in performing and coordinating large nationally projects, funded on a national or European level. A partner will participate in several other Work Packages as described in the work plan (part 7). Each partner has the right to re-allocate resources in the Work Package, subject to the prior permission of the management, the Project Council and the EC representative. Adjustments in the Work Package resulting in other and/or delayed milestones and deliverables require prior permission of the Management Team.

Communication
The communication in the PREVENTCD project will first of all be based on the Internet. A website will be maintained in which all information is brought together.
This information is accessible to the partners and to the research community at large whenever appropriate. An area with restricted access will be used for communication on: 1. Management issued and storage of used documents (i.e. reporting formats); 2. Research data, results and discussions (see WP 7).
Twice a year progress meetings will be organized in which all the partners are expected to participate. These progress meetings will also have a formal role in the management of the project, because at these meetings the progress report will be presented and decisions will be made with respect to the plans for the next period. The partners will be obliged to submit their contributions for this report in time. Other means of communication, like e-mail, telephone calls or telephone conferences, will also be used when useful or necessary to fulfil the obligations of the project.

Intellectual Property Rights and Exploitation
The PREVENTCD partnership functions as an European Interest Group in the sense that the partners strive to realise joint objectives in order to establish scientific bases for effective prevention of the most common food sensitivity in Europe (CD). There may be a need to explore the role of intellectual property rights (IPR) protection for research results derived from the project. The partners are members of larger research organizations and universities which have gained considerable experience in protecting IPR and in the exploitation of knowledge. It should not be difficult to involve experts from within the partners' institutes. The results of PREVENTCD will be scanned (yearly and before publication of results) for IPR potential by the IPR specialists employed by the participants. The involvement of three industrial companies and patient organisations ensures that results will be implemented and commercialized in (baby) food and diagnostics. Through national networks several participants have contacts with other industrial stakeholders. These will be contacted if the use of results is foreseen in other areas than the present industrial participants are working. IPR issues will be arranged in a Consortium Agreement.

Plan for using and disseminating knowledge
The results of PREVENTCD will be disseminated beyond the consortium as The results of PREVENTCD will, if appropriate, take the form of policy statements on a European Common Strategy to prevent CD. PREVENTCD will offer new, evidence-based, European Guidelines for early feeding practices, including gluten exposure and breast-feeding. Such results will be presented to the scientific community as a position paper and to the coeliac patients and the Europeans in general in the form of flyers and on-line publications. Regulations, rights and obligations with respect to prior knowledge (prior defined as preceding the date of contract signature) will adhere to standard regulations defined in the model contract.

Raising public participation and awareness
The results of the project will be widely disseminated to other directly interested parts, such as food the industry and patients' associations. Once the strategies for primary prevention of CD will have been identified, the ongoing integrated research collaboration network will facilitate the dissemination of the new findings, in that it promptly will reach and be of benefit to the European citizens' health and well-being.

Introduction -general description and milestones
Our hypothesis is that early dietary history, i.e. the introduction of small quantities of gluten during the period of breast-feeding, may prevent coeliac disease (CD) in genetically predisposed individuals by induction of tolerance for gluten and for other related auto-antigens.
Through integration of novel collaborative research among European leaders from different and crosscutting areas we will examine the complex interactions and molecular mechanisms involved in the development of CD including: To achieve our objectives we propose the following structure and methodology: 1. A prospective early dietary intervention study in 1000 young children from high-risk families for CD with at least one case of CD among the siblings and /or the parents, to assess the possibilities to induce immune tolerance for gluten in genetically predisposed children; 2. A prospective dietary intervention population study in 16.000 Swedish children aged 12 years, born during and after the Swedish epidemic of CD, to assess the late effect of dietary history, concerning gluten exposure and breastfeeding, on the development of CD and related autoimmune phenomena.
The work of the proposed STREP will be integrated in the following Work Packages    7 Deliverable numbers in order of delivery dates: D1 -Dn 8 Please indicate the nature of the deliverable using one of the following codes: R = Report P = Prototype D = Demonstrator O = Other 9 Please indicate the dissemination level using one of the following codes: PU = Public PP = Restricted to other programme participants (including the Commission Services). RE = Restricted to a group specified by the consortium (including the Commission Services). CO = Confidential, only for members of the consortium (including the Commission Services). 10 Month in which the deliverables will be available. Month 1 marking the start of the project, and all delivery dates being relative to this start date.
D3.1 European databank on the interaction between early gluten intake and breastfeeding in children from highrisk families for CD, their early immune response to gluten and their genetic background 3   Objectives  Coordination by ensuring effective day-to-day work affecting the project  Management of the project facilitating the roadmap of activities necessary to achieve the objectives, milestones and deliverables.  Production of reports containing accounting information, descriptions of the achieved deliverables and milestones and a self-evaluation of the progress and difficulties encountered.

Description of work
 A project office, with a project assistant and a data manager, will be open.  The project assistant and the data manager will support the coordinator in her tasks and will be responsible for maintaining the web-based and networking activities.  The coordinator, ML Mearin, is responsible for the financial management, facilitating the allocation of the funding to achieve the objectives of the project. She produces accounting information. The coordinator is responsible for the operational management, chairs the Management Team, is the contact person for the Advisory Board and for the EU Commission and produces reports of the work achieved. Deliverables D.1.1 Project office with a project assistant and a data manager (m1) D.1.2 Bi-annual progress reports (m6, m18, m30, m42, m54) D.1.3 Annual progress reports (m12, m24, m36, m48) D.1.4 Quarterly progress management reports (m3, m9, m15, m21, m27, m33, m39, m45, m51, m57) D.1.5. Mid term review (m 24) D.1.6 Final report (m60) D1.7 Mid-term assessment report (m25)

Milestones and expected result
M1. Opening project office: m 1 The final report will contain an overall description of the work, the results achieved and accounting information.

Introduction
This WP is necessary to get the infrastructure ready to carry out the project and it ends with the "Kick-off meeting". During this WP the project-office and website will be open, the personnel involved in the project will be contracted and the digital standardized forms and databanks will be developed. During the start-up phase, the frequency food questionnaires (FFQ's) will be adapted in each participating country to their own eating pattern and their own brands of food products. No contingencies or risks are expected.

Work package number 2
Start date or starting event: Month1 Activity Type RTD/Innovation Participant id 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Person-months per participant: Objectives  To get the project started  Development of the infrastructure to carry out the project

Introduction
We will carefully assess the early immune response to gluten in 1.000 infants from high-risk families for CD from different European countries and with different degrees of genetic predisposition for the disease, under different nutritional conditions, including gluten introduction, gluten exposure and breast-feeding practices. (www.dzg-online.de) will invite their members with CD expecting a newborn (child or sibling) during the next 18 months to participate. Partner 9, the AOECS, is able to easily link the project to each country in Europe, in particular those member states with large populations, in order to ensure the necessary number of infants. (table 2).


Informed consent: during pregnancy, the families will be asked informed consent for participation by the local responsible physician.
 Enrolment: after informed consent is given, the families will be enrolled in the study. Breast-feeding during at least six months will be STRONGLY encouraged to all the families.
 Genetic risk stratification: the genetic risk for CD will be assessed and stratified as described in WP 5 "Genetics".
 Randomisation to "tolerance induction for gluten" or to the "control" group.
Shortly before the dietary intervention, the children bearing HLA-DQ2 and/or DQ8, and who consequently may at some time develop CD, will be randomised to the group for "tolerance induction for gluten" or to the "control" group (table 1).
The randomisation is performed by partner 1 (LUMC, dept. of Medical Statistics).
The randomistion codes will be kept by the department of Medical Statistics LUMC.

 Intervention
At the age of 4 months the blind intervention will take place. This age is chosen because it is known to represent a "window of opportunity" to introduce grains into the diet, with respect to development of autoimmune phenomena. The present evidence over this "window of opportunity" is based on recent publications covering, among others, two studies in Germany and USA. The American study concerns the first prospective observational study that attempts to outline a link between early infant diet and development of CD (19). The study was performed on a cohort of 1560 children derived from the DAISY project (Diabetes Autoimmunity Study in the Young), who had an increased risk of developing CD or type1 diabetes, as defined by possessing either HLA-DR3 or DR4 alleles, or having a first-degree relative with type 1 diabetes. At a mean follow-up of 4.8 years, the authors concluded that: 1) there is a "window of opportunity" of introducing gluten into the diet when the child is aged between 4 and 6 months with regard to the risk of developing CD, and 2) that the contribution of breastfeeding was to be disregarded in this respect. However, the authors did not make specific attempts to calculate the gluten amount ingested by the children or to correlate this important early nutrition event with the presence or absence of breastfeeding. The German BABYDIAB study followed 1610 newborn children of parents with type 1 DM and found that food supplementation with glutencontaining foods before age 3 months was associated with significantly increased islet autoantibody risk (18). A systematic review and meta-analysis of observational studies published between 1966 and June 2004 that examined the association between breast feeding and the development of CD has been published (20). This study gives also support for the concept that breast feeding during the o Tolerance induction will attempted in the randomised group of children by blind daily intake of 1g wheat flour (100 mg gliadin, approximately 7-10 % of the gluten intake at age 1 year) during 8 weeks. Gluten is a common name used for some proteins (prolamins and glutenins) of wheat, barley, rye, and oats. Being gluten a naturally occurring component of food, it should be considered a food that is supplemented. Compliance will be assessed by 2 weekly visits or interviews.
The control intervention will take place by blind daily intake of 1g. lactose during 8 weeks.
Danone will provide the placebo (lactose, Pharmatose) and the intervention product (Glutival obtained from Cargill). Danone has outsourced the production of the identical sachets with the gluten intervention / placebo products to NIZO Food Research, Ede, the Netherlands.
NIZO will prepare and package the gluten intervention product as well as the placebo according to quality criteria adequate for infants aged 4-6 months..
Every intervention sachet will be filled 1.8 gr Pharmatose and 0.2 gr Glutival (gluten intervention product); the placebo sachets will be filled with 2 gr of Pharmatose.
NIZO will pay special attention to the microbiological assessment of the products and will make sure that the results will be in accordance with the standards of Danone. Also it will be analytically verified whether the blending of the materials has been adequate and resulted into a homogeneous mixture.


Monitoring of early immune response to gluten in all the children: see Description of WP 6 "Immunological studies on early response to gluten introduction" and table 1.

 Nutritional assessment:
After the intervention, at the age of 6 months, all the families will be advised to gradually introduce gluten into the diet of their children. The parents will be advised to give the children 500 mg of gluten at the age of 7 months (10 g of ordinary biscuit ), 1000 mg at 8 months (10 g semolina and 10 g biscuits), 1500 mg at 9 months (20 g semolina, or pasta, and 10 g biscuits). After that, the children will consume "ad libitum" quantities of gluten-containing products. The use of breast-feeding and the quantities of gluten ingested will be assessed using Follow-up 0-4 years: All the children will be frequently controlled for clinical and nutritional assessment and for the presence of immunological markers of gluten (in)tolerance, including determination of serum IgA antibodies against gliadin and tissue trans-glutaminase (tTGA) (table 1). In case of health complaints extra controls will take place and, if indicated, the intervention will be stopped.
 Diagnosis of CD: The parents of the children with tTGA or symptoms of CD will be offered a small bowel biopsy for the diagnosis of CD. Biopsie will only be performed when medically indicated, that is: only in these children with CD antibodies in their serum that indicate gluten sensitivity and are highly suspect for active CD, and NOT just for purpose of the study. Such children would undergo a biopsy also in non-study circumstances.
A reduction of 50% of CD among the intervention group at the age of 3 will be considered as an effective prevention.  Objectives Assessment of the possibilities to achieve primary prevention for CD in genetically predisposed children by early dietary intervention consisting of introduction of small quantities of gluten during the period of breastfeeding and Germany (www.dzgonline.de) will collaborate in the study.  The Societies will invite to participate their members with CD expecting a newborn (child or sibling) during the next 18 months. Informed consent to participate will be asked from the families by the local responsible physician.  The children bearing HLA-DQ2 and/or DQ8 will be blindly randomised to either a group for "tolerance induction for gluten" or to a "control" group. At least 6 months of breast-feeding will be STRONGLY encouraged for all the children. At the age of 4 months tolerance induction will be attempted by the daily intake of 1g wheat flour (100 mg gliadin) during 8 weeks while continuing breast-feeding. No gluten will be given in these 8 weeks to control infants, but 1g. lactose as a placebo intervention. Danone will provide the placebo (lactose, Pharmatose) and the intervention product (Glutival obtained from Cargill). Danone has outsourced the production of the sachets with the gluten intervention / placebo products to NIZO Food Research, Ede, the Netherlands. NIZO will prepare and package the gluten intervention product as well as the placebo according to quality criteria adequate for infants aged 4-6 months. Every intervention sachet will be filled 1.8 gr Pharmatose and 0.2 gr Glutival (gluten intervention product); the placebo sachets will be filled with 2 gr of Pharmatose. NIZO will pay special attention to the microbiological assessment of the products and will make sure that the results will be in accordance with the standards of Danone. Also it will be analytically verified whether the blending of the materials has been adequate and resulted into a homogeneous mixture. Compliance will be assessed by visit or interview.

Description of work
 After the 8 weeks intervention period, all the families will be advised to gradually introduce gluten into the diet of their children, and to increase it gradually till the age of 12 months, for which simple recommendations will be provided to the parents.  The infants will be strictly followed up (Table 1). Blood (5 ml) is sufficient to allow for the screening of CD-specific antibodies, phenotypic characterization of 20 markers indicating lymphocyte activation and regulatory T cell induction, and monitoring for the occurrence of glutenspecific T cell responses typical for CD. At age 3 years 10 ml blood will be obtained to test for additional (auto)immune phenomena related to the development of CD.  Children with positive antibodies strongly indicating CD or with clinical suspicion of CD will be offered a small bowel biopsy for the definitive diagnosis a reduction of 50% of CD among the intervention group at the age of 3 will be considered as an effective prevention.

Introduction
We will take advantage of the "experiment" in the Swedish population with extensive changes in infant feeding practices within a few years, which resulted in a unique epidemic of symptomatic CD in children below 2 years of age. The incidence rate of symptomatic CD increased four-fold to levels higher than ever previously reported, and after a ten-year period on this high level, suddenly returned back to the previous level. A representative sample of 16.000 Swedish children aged 12 years, 8.000 born during the peak of the epidemic (1993) and 8.000 born after the epidemic (1997), will participate in the study. This study is based on a long-standing collaboration with paediatric units in different parts of Sweden, and also a well-developed collaboration with the school health services.
 Design: prospective population based intervention study with a long-term follow-up  Study base: the study on the 8.000 children from the epidemic will be done before the project, but the results will be used to be compared with the data from the cohort from the post-epidemic period.
 Invitation to participate: requirement for inclusion is an informed consent from the child and its parents.
 Symptomatic CD cases diagnosed before the age of 12-years will be identified through the National Register of CD in Swedish children.
 Screening for CD: all children will be invited for a blood sample during the schoolyear 2009-2010 for determination of tissue transglutaminase antibodies type IgA (AtTG), and those with s-IgA-deficiency also for AtTG type IgG. When CD is suspected the child is referred for a diagnostic small bowel biopsy. Biopsies will only be performed when medically indicated, that is: only in these children with CD antibodies in their serum that indicate gluten sensitivity and are highly suspect for active CD, and NOT just for purpose of the study. Such children would undergo a biopsy also in non-study circumstances.
 Double the numbers of screening-detected cases in the epidemic cohort are expected compared to the cases in the post-epidemic cohort.
 HLA and non-HLA genetic characteristics and autoimmune serological markers will be assessed in all the children with CD (symptomatic and screening-detected) and in 5 population based controls per case (n=1800).
 Early dietary history: The parents will be asked to fill in a questionnaire about the child's history with respect to breast-feeding and early gluten exposure.

Description of work 
The study base is defined by a representative, population based sample of children born in 1993 and 1997, 8.000 from each cohort, using the Swedish population register. The study on the first cohort (1993) will be finalized before this project starts. However, the results will be brought into the project.
The study on the second cohort (1997) falls within the project.  All cases of symptomatic CD diagnosed before the age of 12 will be identified through the National Register of CD in Swedish children.  All children will be invited to a screening for CD at the age of 12 including blood sampling and parental questionnaires (on e.g. early dietary history and autoimmune diseases).


Blood samples of all children will be analysed for tissue transglutaminase antibodies (TTGA), and when CD is suspected a diagnostic small intestinal mucosa will be evaluated we expect to find double the number of screening-detected cases in the cohort of 1993 compared to the one of 1997.  Blood samples from all children with CD (symptomatic and screening-detected), from those with an isolated aberrant TTGA response, and from five population based controls per case (n=1800) will be used for HLA and non-HLA genetic analyses (WP5), and for determination of auto-antibodies.  Questionnaire data on early dietary history and auto-immune diseases will be compared between the CD cases (symptomatic and screening-detected), and population based controls, as differences between these groups and also between the cohorts of 1993 and 1997 are expected.  The prevalence of CD, both symptomatic and screening-detected, will be compared between the two birth cohorts, and it will be related to differences in early dietary history and genetics (WP5). This population based study exploring birth cohorts with different early dietary history, and with a followup of twelve years, gives an opportunity to determine the late effects of breast-feeding and early gluten exposure involved in the primary prevention of CD.

Introduction
There is a strong inherited predisposition to CD susceptibility. Around 90% of CD patients possess the HLA-DQ2 and/or HLA-DQ8 molecules. However, the HLA-DQ2 allele is common in the healthy population, carried by approximately 30% of Caucasians.
Therefore, genetic factors outside the HLA region contribute to disease susceptibility or resistance. To date, at least three non-HLA loci have been identified that are likely to large-scale genetic testing of some 100 functional candidate genes, and a genome-wide genetic association studies. The data generated through these ongoing efforts will benefit this proposal. Therefore, it is expected that in the next few years additional loci/genes will be added to the current list of CD susceptibility genes and these will then also be included without jeopardizing the proposed plan of work and the deliverables.
In this proposal we will study the effect of the currently known genetic risk factors on CD and the early dietary intervention, in two cohorts: a Swedish population-based cohort (a representative sample of approximately 1.800 individuals from a total cohort of 16.000 individuals) and a European-based family cohort (approx. 3.500 individuals). Each individual will be genotyped for the four genes listed above: HLA-DQ, CTLA4, CELIAC2, MYO9B. DNA will be isolated from peripheral blood from all participants according to standard protocols. Genotyping will be performed using 384 single nucleotide polymorphisms (SNP) covering these four regions and analyzed on an Illumina Bead Array system. All samples will be blindly coded to prevent any biases in data-analysis. Appropriate controls will be included to control for genotyping and sample errors. Statistical analysis will be performed separately for the population-based cohort and the family-based cohort.
Evaluating HLA and non-HLA susceptibility genes in the family-based cohort Since the families will be collected from different geographic areas we can also study if this affects their genetic risk profile. Upon the early dietary intervention study the glutentreated CD cases (randomized to tolerance induction) will be compared to the placebotreated CD cases for their genetic profile to identify differences in distribution of the four genetic risk factors in high risk families.

Evaluating HLA and non-HLA susceptibility genes in the population-based cohort
The population study encompasses in total 16 000 children divided among the birth cohorts of 1993 and 1997. DNA will be available for all CD cases diagnosed before the age of 12, both symptomatic (expected n= 93) and screening-detected (expected n=69), and a representative sample of five controls per identified CD case. Therefore, this cohort is excellently suitable to properly assess the effect of HLA and non-HLA genes at both the individual and the population level.
Genetic testing for the HLA and non-HLA loci will allow evaluation of these genetic factors in relation to the prevalence and the phenotype (symptomatic/screening-detected) of CD. This will allow us to determine the role of both HLA and non-HLA genes on disease-risk in patients and their possible predictive value in the population. In addition, we can determine if these genes are differentially involved in symptomatic versus screening-detected CD subjects. Interestingly, since the 1993 and 1997 birth cohorts experienced different exposure with respect to early feeding we will also be able to assess the effect of genetic risk factors on the effect of different early feeding patterns.

Work package number 5
Start date or starting event: Month 1 Activity Type RTD/Innovation Participant id

Gluten analysis
For the early dietary intervention in the family study (WP3), a standardized gluten preparation will be used. There is strong evidence that particular gluten peptides are more frequently recognized by patient derived T cells than others. It is therefore imperative that the composition of the gluten used for the intervention is determined. Recently, we have developed novel methods to carry out such a quantitative and qualitative analysis of the presence of harmful gluten fragments in gluten and we will use these for this purpose.
At random, monsters of the gluten provided for the dietary intervention will be solubilized, treated with pepsin at acid pH, followed by trypsin cleavage at neutral pH.
The resulting preparation will be divided in two portions. One for determination of T cell stimulatory gluten fragments from gliadin and glutenin and the other for tests with a panel of patient derived T cell clones specific for gliadin and glutenin, as described previously.

Breast-milk analysis
Breast-milk contains low but measurable amounts of gluten that might have an impact on the early dietary intervention in children from high risk families for CD (WP3).
Therefore, we will use the above described monoclonal antibody-based assays to determine the level of gluten exposure of the children enrolled into the dietary intervention due to the presence of gluten in breast-milk. For this purpose breast-milk samples will be collected monthly after birth for quantitative and qualitative analysis of gluten content using an established protocol.

Detection of alterations in gene expression profiles that correlate with gluten introduction and/or disease development.
As discussed extensively during the midterm review the limited amount of blood available for analysis precludes the use of HLA tetramers to analyze the presence of gluten reactive T cells in peripheral blood. Moreover, alternative protocols using IFNgamma and IL-10 ELISPOT assays that have been developed by partners 2 and 13 (UNINA and UO) are also likely to be inadequate for the reliable detection of glutenspecific T cells in the samples available. In agreement with the discussion with the reviewers of the midterm review, we have therefore decided to use the samples for gene expression analysis. As until now the blood samples have been used to isolate serum and lymphocytes we will continue to collect samples in an identical fashion to allow comparison of samples in the future. For the gene expression analysis a series of samples from children that did develop CD and control samples from children that did not develop CD will be thawed and stimulated with PHA in a standard protocol in order to obtain sufficient amounts of cells for isolation of good quality RNA. Subsequently gene expression analysis will be performed with the use of the latest available technology.
While gene expression microarrays have been the default technology for transcriptome analysis, the introduction of deep sequencing technology now enables the simultaneous sequencing of up to millions of different DNA molecules. This has been proven to be a powerful technique for the identification of differentially expressed transcripts and allows the detection of differential expressed low-abundant transcripts that are well beyond the reach of classical micro-array analysis. For this approach we will collaborate with partner 5 who has well established expertise in this field.

Repertoire analysis
During the course of the family study, between 50 and 100 children are expected to develop CD. This is by far the largest group of paediatric patients to have been included in a study to determine the repertoire of gluten-specific T cells. Moreover, due to the setup of the study, it can be expected that the development of symptoms associated with CD will be noted very early in the disease development. The repertoire analysis will thus give a clue to which T cell responses are the earliest to develop. Finally, the repertoire analysis will reveal whether the early dietary intervention will have an impact on the repertoire development. For this purpose we will make use of established protocols to generate gluten-specific T cells from biopsies of children who developed CD. In short, polyclonal gluten specific T cell lines will be generated from these biopsies by a three day specific stimulation with pepsin/trypsin (+/-tissue transglutaminase) treated gluten followed by one round of a-specific expansion with IL-2. These polyclonal T cell lines will subsequently be tested against synthetic peptides representing previously characterized gluten derived T cell epitopes. T cell proliferation and IFNgamma production will be measured. In particular cases we will generate gluten specific T cell clones to analyse further the fine specificity of the gluten response. Furthermore, we will use ELISPOT to determine the (relative) frequency and specificity of the gluten reactive T cells in these polyclonal T cell lines.

Regulatory T cell compartment
Given the immunogenic nature of gluten, the maintenance of tolerance towards gluten is similar to the results of active regulation of gluten specific T cell responses in healthy individuals, a mechanism that fails in CD patients. In murine models regulatory cells have been identified in the mucosa and were found to differentiate from naive T-cells in the periphery, to be antigen-specific and to suppress irrespective of ongoing Th1 or Th2 cytokine polarization. The mechanism underlying suppression by these mucosal regulatory T cells may include secretion of TGF- or IL-10, but remains debated. Based on these observations we will test whether the early dietary intervention in children from high risk families induces regulatory T cells that suppress the development of an inflammatory T cell response to gluten. In particular, we will determine whether glutenspecific, IL-10 and/or TGF- producing cells are present in peripheral blood and biopsy material from individuals included in the early dietary intervention. In these studies we will pay particular attention to the possibility that these cells will be present within the CD4CD25 regulatory T cell compartment. For this purpose, peripheral blood and biopsy samples from the children who develop CD will be collected and tested against gluten.
Subsequently, cytokine secretion by T cell subsets will be measured using established protocols.

Genetic background and mucosal immune response to gliadin
In relation to the early dietary intervention towards "tolerization for gluten" planned in the cohort of newborns from coeliac families, the main outcome will be the development of CD. Its recognition will be based on standard diagnostic criteria (i.e. enteropathy in presence of serum anti-tissue transglutaminase antibodies, possibly in a symptomatic child). Nonetheless, the alterations elicited by the ingestion of gluten are now considered to be a spectrum, ranging from the sole presence of immune abnormalities to minor enteropathy, to the full blown picture of flat jejunal mucosa. We will take advantage of this large cohort of subjects at risk. By means of this cohort we wish to evaluate 1) the relationships between their genetic backgrounds and the extent of the mucosal immune response to gliadin; 2) the natural immunopathological history of gluten sensitivity. In

Description of work 
Gluten analysis The composition of the gluten used for the family intervention study will be determined by using our recent developed method to carry out quantitative and qualitative analyses of harmful gluten fragments.  Breast-milk analysis We will use monoclonal antibody-based assays to determine the level of gluten exposure in the children enrolled into the dietary intervention due to the presence of gluten in breast-milk. Breast-milk samples will be collected monthly after birth.  Detection of alterations in gene expression profiles that correlate gluten introduction/or disease development Gene expression profiles visualize gluten specific T cells. Using this technological breakthrough, we will be determinedanalyse the presence of gluten reactive T cells in the peripheral blood samples collected from the children that did develop CDrandomised to tolerance induction for gluten and compared with similar expression profiles of from the children that did not develop CD. randomised to the group of controls.  Repertoire analysis We will make use of established protocols to generate polyclonal specific T cells from biopsies of children who develop CD. These polyclonal T cell lines will subsequently be tested against synthetic peptides representing previously characterized gluten derived T cell epitopes. T cell proliferation and IFNgamma production will be measured. We will use ELISPOT to determine the (relative) frequency and specificity of the gluten reactive T cells in these polyclonal T cell lines.  Regulatory T cell compartment We will test whether the early dietary intervention in children from high risk families induces regulatory T cells that suppress the development of an inflammatory T cell response to gluten. We will determine whether gluten-specific, IL-10 and/or TGF- producing cells are present in peripheral blood and biopsy material. We will pay particular attention to the possibility that these cells will be present within the CD4CD25 regulatory T cell compartment. Peripheral blood and biopsy samples from the children who develop CD will be collected and tested against gluten. Subsequently, cytokine secretion by T cell subsets will be measured using established protocols.  Genetic background and mucosal immune response to gliadin We will evaluate 1) the relationships between their genetic background and extent of the mucosal immune response to gluten; 2) the natural immunopathological history of gluten sensitivity. Tests to measure the degree of mucosal inflammation in relation to the exposition to gluten include immunohistochemical analysis of the jejunal mucosa, the mucosal cytokine pattern and the detection of intestinal auto-antibodies.

Objectives
The objective of this WP is to ensure that the results of PREVENTCD will be widely known to the scientific and general community and that potential users such as industry, regulation bodies and patients' associations will have access to the results.

Description of work
 The project's newsletter will be published quarterly to give information about actual project proceedings and the results, as well as general information about the project  Website (EXTRA-net) of the project  Publications in journals  Presentations at relevant national and international conferences and meetings  Organization meetings with regulatory bodies, governments, and the European Commission  Preparation of a multimedia presentation of the project, which will include a slide presentation, a brochure, and a CD/DVD leaflet.  Preparation of educational information on CD for health professionals (nurses, midwifes, dieticians, paediatricians, etc.) and for the general public (teachers, pupils, journalists, etc.) which will include a slide presentation, a brochure, and a CD/DVD leaflet.

Milestones and expected result
M6. Opening web-site of the project (m3) M7. Submission of 2 papers for publications in scientific journals (m60) M8. Submission of guidelines on optimal gluten introduction (m 60)

Project resources and budget overview 8.1 Efforts for the project (STREP/STIP Efforts Form )
PROJECT EFFORT FORM person months  Twice a year a progress meeting will be organized in which all the partners (approximately 30 persons) are expected to participate. The meetings are expected to have place on months: 3, 9,15,21,27,33,39,47,53 and 60. The place of the meetings will be the working place of one of the European partners. Eight of the management meetings will take place at the same place, before the progress meeting. The rest of the management meetings will take place as telephonic conferences.

Justification of the requested budget for travels and meetings
Budget for meetings (10 total expected)  (2001) concerning the strict prohibition of economic, employment, social or insurance discrimination on the grounds of genetics.

Budget for travel/transport
Important EU directives, which have a significant impact on the work of our project, are: In addition, each partner will comply with its relevant national legislation. A list of applicable laws and regulatory texts follows for each country involved.

-Implementation of EU directive 2001/20/EC into national legislation in The
Netherlands is aimed by changing the WMO and the WOG. All medical research that involves humans (including personal identifiable information and human material) need to be approved by a regional ethics committee.
All research that includes handling of personal data, including de-identified information (i.e "avidentifisert"), need to be presented and approved either by an ombud for protection of privacy or the Data Inspectorate.

Israel:
- PREVENTCD confirms that the project will require the approval of the Independent Ethic Committee that governs clinical studies for each participating study site and that they will inform the Commission whether the local, regional or national ethical approval has been obtained before the research to which it relates is carried out.
The Ethics Committee approval or authorisation of a competent body will be specified as a project deliverable.
The population based multicenter study will only involve Swedish participants, and has already been approved by the relevant Regional Ethical Review Board.

Description of the sensitive ethical issues of the proposed research
Identification of the potential ethical aspects of the proposed research:

9.3.a.I. Early feeding intervention family study
A prospective early dietary intervention study of 1000 young children from high-risk families for CD, to assess the possibilities to induce immune tolerance for gluten in genetically predisposed children.

Explanation and justification of the research design
The proposed project must necessarily involve infants, and cannot be performed in any other age group, because it is during infancy that the first exposure to gluten takes place and that the immunological response to this antigen occurs and may be modulated. The only way to find out whether the introduction of small quantities of gluten into the diet of infants during the period of breast-feeding does indeed protect against CD is to conduct a double blind prospective randomised study, as the one we here we herewith propose.
Informed consent: Informed consent to participate will be obtained from the parents, guardians or other legitimate representative, as it is usual in the case of young children. The AOECS or the local Coeliac Disease Societies will invite their members with CD expecting a newborn (offspring or sibling) during the next 18 months to participate. All parents, guardians or other legitimate representative of the children, interested to collaborate will be given full information and explanations about the trial and asked to sign a written informed consent form (annexes 2 and 3) by the local responsible physician according to the national legislation. Each parent, guardians or other legitimate representative of the children will receive detailed information about the study procedures, the potential risks and benefits of the intervention, as well as information about the fundamental rights of a patient included in an experimental study. In addition, care will be taken to inform the patient about the potential benefits of the experimental intervention in a manner that avoids an overestimation of these benefits, by explicitly mentioning that the intervention is fully experimental, that it has no proven efficacy, and that no guarantee can be given that it will work against CD.
These information's will be provided by a physician in charge of the trial, and an informed consent document, preliminarily approved by the relevant ethics committee, will be given to the parents, guardians or other legitimate representative of the children.
In case of acceptance by the parents, guardians or other legitimate representative of the children, they/he/she will sign the informed consent document, which will be countersigned by the responsible physician. The signed informed consent form will be archived in the Case Report Form (CRF) at the project office, and a copy will remain with the parents, guardians or other legitimate representative of the child. The parents, guardians or other legitimate representative of the children will always give informed consent by their free will. Only after informed consent is signed will the families be enrolled in the study. The family physician will be duly informed and his standing as the family's health provider will be respected -and relied on for his/her cooperation. The participants may retract from their participation at any moment, also without giving their reasons to do so and without any adverse consequences regarding their relationship with their paediatrician/responsible physician. A copy of both the informed consent form and of the parent's information sheet will be submitted to the pertinent Independent Review Board. No financial reward will be given to participating families at all. Only their real expenses (primarily travel costs), which are related to their accurate participation in the studies, shall be reimbursed. The only exception will be to give a small present to the children: this will not be told to the families in advance, to avoid influencing them in their decision to participate or not.

Breast-feeding during at least six months will be STRONGLY encouraged to all the families.
Care for the children and their families during the study The children and their families will receive frequent and careful medical follow up during the study (table 1), to ensure their support and their welfare, to ascertain that any undesired effects or symptoms are rapidly detected and that adequate measures are promptly undertaken. In case of health complaints, extra observation will be provided and, if indicated, the participation will be stopped. The main discomfort for the children concerns the blood sampling and the clinical follow up examinations, as detailed in table 1. The risk element involved with this trial concerns that of the performance of small bowel mucosal biopsy in the children with suspected CD. It is again to be stressed that such a biopsy is still the "gold standard" for diagnosing CD, and would be performed only when medically indicated, that is: only in these children with CD antibodies in their serum that indicate gluten sensitivity and are highly suspect for active CD, and NOT just for purpose of the study. Such children would undergo a biopsy also in non-study circumstances. Each centre where the research takes place will make the necessary treatment and infrastructure available to patients who develop trial-related injuries and will provide sufficient insurance to cover subjects for adverse effects directly arising from participation in the trial. All adverse and serious adverse events will be reported according to the regulations cited above.
Implications of the results.
The implications of the proposed study may be important, because its results may contribute to the development of new European guidelines for early infant feeding practices, aimed at the primary prevention of CD and related autoimmune diseases.
In all the countries involved in the project there is clear legislation concerning the strict prohibition of economic, employment, social or insurance discrimination on the grounds of genetics. In the participating countries is illegal for prospective employers to demand genetic testing or to demand to receive the results of genetic testing. Genetic issues may be considered in very limited instances of LIFE insurance. In this context it must be taken into account that testing for HLA in a child of a family with coeliac disease may be regarded as a clinical service, meant to detect a subject that needs to be followed up for an early recognition of the disorder and timely institution of adequate management. In this respect, the HLA testing of infants in this study might constitute an advantage for the participating infants. The information thereby obtained is of a nature that is similar, if not better, than the knowledge that this is an infant of a "coeliac" family.
The results will have no potential ethical adverse implications, concerning the protection of dignity, autonomy, integrity and privacy of persons, and may lead to healthier early feeding recommendations for infants. Finally, the results may lead to a decline in the incidence of CD in the European population. Explanation and justification of the research design The "Swedish case" as it is called among scientists internationally, provides a unique opportunity to increase our knowledge concerning CD aetiology. Notably, this study can only be carried out in Sweden, and only for a limited period. The study takes advantage of the recent Swedish epidemic of CD, which has no likeness anywhere else in the world.
The epidemic of CD was most likely initiated by changes in infant feeding, which, while considered appropriate at the time, were in retrospect probably quite unfortunate.
However, now that the epidemic has occurred it provides a unique opportunity to increase our knowledge concerning CD aetiology. The results may contribute to primary prevention strategies for CD such as changed infant feeding guidelines. Thus, the results may lead to a decline in the incidence of CD in the European population.
-Possible disadvantages for participants: Invited children and parents might feel anxiety when reading the folder inviting them to the study (annex 3), as it describes CD as a rather common disease, that a blood sample is needed as the first step to identify currently undiagnosed cases, etc. Families, in which the child has increased values of the serological marker (AtTG) suggesting undiagnosed CD (2%), are also likely to experience some anxiety when waiting for further examinations, thus this time period will be kept as short as possible. The small intestinal biopsy necessary for confirming or excluding CD is a well-established method for diagnosis and complications are extremely rare, however, during the procedure some discomfort isn't uncommon. Biopsie will only be performed when medically indicated, that is: only in these children with CD antibodies in their serum that indicate gluten sensitivity and are highly suspect for active CD, and NOT just for purpose of the study. Such children would undergo a biopsy also in nonstudy circumstances. With the CD screening strategy and serological tests that are used a high sensitivity and specificity are combined, thus, almost all CD cases will be identified, and the number of "unnecessary" small intestinal biopsies will be kept to a minimum. A database system is used for the storage of all findings, e.g. results of blood sample analyses and replies in the questionnaires, which might worry some families. However, informed consent is required before inclusion of any child into the study and the database, only a small group of researchers have access to the database, and if the child and/or parents express a wish to end their participation their identity code will immediately be erased.
Advantages for participants: Children with newly diagnosed CD, determined by the study (1%), who follow a gluten-free diet, will gain a lot as their health and quality of life will increase, and the risk for long-term health complications will decrease. All children with a normal blood-sample result, and their families, will most likely experience a relief that CD has been excluded.

Risk-benefit evaluation for participants:
The participating children are expected to fall into one of the three different groups with respect to risk-benefit evaluation: A) AtTG with a normal value. The child and parents might experience some anxiety when reading the folder with information about the study and when the child takes part in the blood sampling. These children will incur minimal risk/burden and gain the knowledge that CD can be excluded for them. Risk and benefit are considered balancing each other.
B) AtTG is elevated and the follow-up confirms the CD diagnosis. These children will benefit considerably from the study as an early diagnosis of a chronic and treatable disease (CD) should allow improved future health and well being for all years coming. C) AtTG is elevated but CD is excluded by further examinations. These children willwithout any apparent benefit to themselves -undergo a small bowel biopsy in addition to the minimal risks/burdens experienced by others (blood sample, anxiety). These children and parents are "unnecessarily" exposed to anxiety. Also the small intestinal biopsy has to be done, which causes some discomfort, but no medical risks. The burden may be considered larger than the benefits.

Informed consent
The population based study invites children who are 12-years old, in the sixth grade of basic school, to participate in a screening for CD. These children are at an age approaching adolescence. They have the right to be informed about the study, and their wish to participate or not should be taken into account. However, their parents or other legitimate guardian should make the final decision after having informed and discussed it with the child. Informed consent from both the child and their parents or other legitimate guardian is required before participation is accepted. Only children and parents who give informed consent will be approached regarding participation. Information and an invitation to participate are sent to the child's home through their school (annex 4). This folder with information has several parts; one for the parents (Dear Parents!), one for the child (Greetings to you in the 6 th grade!), and another to read and discuss jointly.
Information is given about the study procedures, the potential risks and benefits of the intervention, as well as information about the fundamental rights of any person taking part in a research study. The informed consent will always be given by free will. The written information will be kept by the child and their parents, while the signed consent form will be archived at the project office. The participants will not receive any payment or other benefit for their participation, except jus, fruit and/or a small biscuit at the blood sample occasion, and a small present (value 2 Euro), which is not notified beforehand as it should not influence the decision to participate or not. The participants are clearly informed that they may retract from participation at any moment, also without giving their reasons to do so and without any consequences regarding their relationship with their school, school health service or paediatrician. By the PREVENTCD project, combining the family intervention approach and the population based survey, we will be able to clarify whether primary prevention of CD is possible by means of a change in infant feeding practices, or whether it is merely the symptoms and thereby the chance of being diagnosed that are influenced. By the population based screening at 12 years of age we will also determine whether infant feeding practices delay the development of CD enteropathy, or if the reduced risk also remains later in life. We will also evaluate whether the risk for autoimmunity can be reduced in CD and non-CD subjects by a reduction in the amount of gluten ingested during infancy. Moreover, we can determine whether CD subjects will have fewer additional autoimmune diseases if their CD is detected and treated early in life. Thus, PREVENTCD may contribute to the development of new European guidelines for early feeding, aimed at the primary prevention of CD and related autoimmune diseases. It may lead to healthier early feeding recommendations for infants. Finally, the results may lead to a decline in the incidence of CD in the European population.

Identification of four possible problems concerning compliance with the Convention on Human Rights and Biomedicine Article 17 -Protection of persons not able to consent to research.
Executive summary: Problem I: Gluten will be given to the infants at the age of 4-6 months, which is against the current recommendations of WHO and ESPGHAN.
The project is -in principal -strongly backing the general principle of breast feeding, which is the only generally accepted prevention strategy with respect to CD. This leading principle is clearly in line with current recommendations.
The current WHO and ESPGHAN recommendations refer to the general population, but not to the high risk group predisposed to CD as within the family study of this project.
For this sub-population, there are no guidelines at all.
The strong recommendation of ESPGHAN not to feed gluten before month 4 will strictly be followed. The current advice for the age between months 4 and 5 (and later) is less strict in the current recommendations.
Gluten is given only in the lowest possible amount expected to have an effect on the immune system (max. 100 mg), which is much lower (aprox. 3%) and not comparable to the amount of gluten introduced as complementary food after weaning (grams).
There are several studies on epidemiology and basic immunological principles supporting the "window of opportunity concept". Scientific studies by the Swedish partner of PREVENTCD exploring in detail the "CD epidemic" in Sweden, strongly support introduction of gluten during the 4 th to the 5 th month of age not to increase the risk for CD, and possibly also to decrease the risk. Notably, the CD epidemic started in 1983 when gluten introduction was postponed from month 4 to 6 by changed national recommendations to parents, and also the baby food producers taking out existing gluten from products aimed for this age-group. Moreover, the epidemic subsided when gluten introduction in 1996 was once again "allowed" from 4 months of age.

Problem II: the bowel biopsies in the intervention study go beyond the minimum burden/risk.
A small bowel biopsy will only be performed on children participating in the study if there is a medical indication to do so. A small bowel biopsy is the only tool to provide a diagnosis of CD. The biopsies are part of the normal clinical diagnostic routine and are not exclusively used for research purposes in this study. Early identification of CD in high risk populations is an accepted recommendation and should be offered to these high risk children also if they do not participate in the project. This is a crucial aspect, since a diagnostically/medically indicated intervention is not an issue of the Convention. For individuals with elevated immunological blood markers, a biopsy is strongly recommended and medically indicated.There are no limiting recommendations with respect to the children's age for diagnostic biopsies. Because of their high risk for CD 8-10 % (n = 80-100) of the children would undergo a biopsy even without the study.
Problem III: The dense blood sampling scheme for infants in the intervention study was regarded to be beyond minimum burden/risk.
The ethically justified blood sampling scheme should not only avoid and limit burden, but should also be of high diagnostic value for the patients and to allow valid scientific results. The consortium will reduce the number of blood samples to seven blood punctures in total (Table 1) and the quantity of blood needed to 5 ml. The number and timing of the blood punctures is such that it will enable to monitor the short and longtime effects of the intervention. At the age of 3 years 10 ml blood will be obtained from the children to allow for determination of additional (auto)immune phenomena related to the development of CD.

Problem IV: The Swedish population study imposes biopsies on an estimated
subgroup of 160 children without direct benefit to them.
Biopsies in the population study will only be performed when medically indicated, which means only in children with CD antibodies in their serum implying a high suspicion of active CD. The first screening round, funded mainly by Swedish government research agencies, has revealed that undiagnosed and, thus, untreated CD is more common than expected (aprox. 2%). A second screening round might therefore be considered as a benefit to all the children involved since: 1) 2% of children will benefit considerably from the study as an early diagnosis of CD should allow improved future health; 2) 97.6% of them will incur minimal risk/burden and gain the knowledge that CD can be excluded for them and 3) the small group (aprox. 0.4%, 58 children) with signs of gluten sensitivity, as evidenced by the presence of CD antibodies in serum, but without evident small intestinal lesions, will benefit from the planned follow-up by a paediatric gastroenterologist. Preliminary results already indicate that most of these children already have health problems as in undiagnosed CD, and will develop evident CD within short.

National/local ethical approval of the studies
The second round of the Swedish population study has already been approved by the regional ethical committee. For the family intervention study the approval of the local ethical committees is a necessary step before the research can start in any of the countries.
A more detailed explanation of the reasons involved in the Executive Summary is exposed below.

Children with a genetic predisposition for CD will be exposed to small amounts of gluten at ages 4-6 months, contrary to current dietary advice.
This point represents indeed a possible ethical concern, because the current dietary advice in most countries is to not introduce gluten into the diet before the age of six months.

This is based on recommendation issued by the European Society for Paediatric
Gastroenterology, Hepatology and Nutrition (ESPGHAN 1982) and by the World Health Organisation (WHO 2003). ESPGHAN states that: "Gluten containing foods should not be introduced before 4 months of age. Even further postponement until the age of 6 months may be advisable". Moreover, WHO recommends exclusive breast-feeding for six months, with introduction of complementary foods and continued breast-feeding thereafter. However, it is necessary to clarify the following aspects that should be taken into account: -It is important to emphasize that the current advice with respect to the introduction of gluten into the diet of young children is meant for the general population, but there is not current dietary advice for children genetically predisposed for CD.
-PREVENTCD concerns the administration of very small amounts of gluten (100mg) after the age of 4 months, which may be interpreted as in accordance with the ESPGHAN recommendations. PREVENTCD is fully aware that the project, where plans to give a subgroup of infants 100 mg gluten from the 4 th to the 6 th month of age, somewhat contradicts the WHO guidelines recommending exclusive breast-feeding for six months with introduction of complementary foods and continued breastfeeding thereafter. However, it should be noted that PREVENTCD strongly encourages breast-feeding for at least 6 months and that we do not propose weaning at the age of 4 months, but to give very small amounts of gluten.
CD is mainly a genetically-based immunologically-mediated disorder, whose major feature concerns a destructive immunologic/inflammatory process of the upper small intestine in genetically predisposed individuals who consume gluten. According to the immunological principles regarding treatment protocols whereby tolerance to an offending antigen can be induced by very small quantities of this same antigen, PREVENTCD considers that these principles can be employed in order to produce a benefit on the health of the coeliac-prone infant: that is, to render him/her tolerant to gluten by administration of very small quantities of this antigen, to avert -or at least minimize -the manifestation of frank CD. The dose of 100 mg, which is less than 3% of the amount of gluten introduced in the diet at weaning time (Hopman, 2007) is the lowest that can be expected to have an effect on the immune system (Catassi 1993), which is a requirement for immunomodulatory effects and for the possible induction of tolerance to avoid the development of CD. PREVENTCD does not propose to advance the current introduction of gluten into the diet of the children prone to CD: that is the administration of (relatively) large quantities of gluten at once, but to administer 100mg gluten per day, an amount not comparable to those usually given at the weaning time. Therefore, it should not be considered "gluten introduction", but rather a gluten tolerance-inducing procedure. Notably, the current dietary advice with respect to gluten introduction to infants is not evidence-based. The only international guideline available is the one mentioned above by ESPGHAN, and the studies behind this recommendation do not at all fulfil today's requirements for valid scientific evidence. This is important to consider because, since this recommendation was launched, the incidence of CD in Europe has not decreased, but increased (Auricchio 1992;Catassi 2001) suggesting that the current dietary advice does not necessarily represent a benefit for the participating children.
-The available scientific evidence of today shows that gluten introduction within the interval of 4-6 months of age is not a risk factor for the development of CD, since none of the studies performed on this topic have ever identified the age of the infant at introduction of gluten as an independent risk factor for developing the disease -The experience with the Swedish "CD epidemic", and studies that followed thereafter, strongly support that introduction of gluten during the 4 th to the 5 th month of age does not increase the risk for CD, and possibly also to decrease the risk. Notably, the CD epidemic started in 1983 when gluten introduction was postponed from month 4 to 6 by changed national recommendations to parents, and also the baby food producers taking out existing gluten from products aimed for this age-group. Moreover, the epidemic subsided when gluten introduction in 1996 was once again "allowed" from 4 months of age. However, scientific studies by the Swedish partner of PREVENTCD exploring the epidemic in detail suggest that the true causal factors were whether breast-feeding was ongoing or not while gluten was introduced and also the amount of gluten then given (Ivarsson 200, 2002(Ivarsson 200, , 2005. Thus, when introduction of gluten in 1983 was postponed it also implied that more infants had ended breast feeding, and that gluten was introduced by larger amounts. When gluten once again in 1996 was given from the age of 4 months, more of the infants were still breast-fed and gluten was introduced by smaller amounts. Thus, the Swedish studies strongly support to introduce gluten in small amounts and while breast-feeding is ongoing, as proposed in PREVENTCD.
For the above named reasons the proposed dietary intervention may be considered in the category of "not more than minimal risk".
As stated above there is strong scientific evidence suggesting that introduction of gluten in the age interval of 4-6 months is not a risk factor to develop CD, and it might even be beneficial to seize this "window of opportunity" for developing oral tolerance towards gluten. Also, by introducing very small quantities of gluten earlier than commonly done today, a larger proportion of infants will still be breast feeding and this is likely to increase there capability to develop oral tolerance to gluten and avoid CD ).
Notably, studies performed by the Swedish partner of our consortium, Anneli Ivarsson, strongly indicate that the introduction of small amounts of gluten during the period of breast-feeding may indeed prevent the development of as much as 50% of the cases of CD . In these studies the consequences of early exposure to gluten were also explored in detail with conclusions emphasising the importance of introducing gluten to infants in small amounts. A recent systematic review and a metaanalysis of observational studies published between 1966 and 2004 , also concludes that breast-feeding during the introduction of dietary gluten, and increased duration of breast-feeding are associated with a reduced risk of developing CD. Because the majority of the European infants are not breast-fed after the age of 6 months, when current (not evidence based) advice is to introduce gluten, the European populations is presently not benefiting from the possible tolerogenic effect that breast milk might have on gluten, but would have to a larger extent if gluten introduction was done earlier in life, e.g. between 4-6 months of age as suggested in PREVENTCD.

The bowel biopsies in the intervention study go beyond the minimum burden/risk.
-A small bowel biopsy is the only tool to provide a diagnosis of CD. The biopsies are part of the normal clinical diagnostic routine and are not exclusively used for research purposes in this study. Early identification of CD in high risk populations is an accepted recommendation and should be offered to these high risk children also if they do not participate in the project. This is a crucial aspect, since a diagnostically/medically indicated intervention is not an issue of the Convention. The small intestinal biopsy necessary for confirming or excluding CD is a well-established method for diagnosis and complications are extremely rare, however, during the procedure some discomfort isn't uncommon. In this context we would like to clarify the following aspects:It is important to emphasize that the small bowel biopsies in PREVENTCD will be performed only when medically indicated, that is: only in these children with CD antibodies in their serum that indicate gluten sensitivity and are highly suspect for active CD, and NOT just for purpose of the study. Such children would undergo a biopsy also in non-study circumstances.
In addition PREVENTCD wishes to make clear that the -Furthermore, there will be a direct health benefit for all the participating children since: 1) In the immunologically identified and diagnosed sub-group of individuals, the clinical manifestation will be detected at an early stage allowing the prompt institution of a gluten-free diet; 2) the children who, thanks to the study, can be classified as early as possible as not being prone to CD, will be spared the anxiety of vigilance and expectation toward the development of CD and 3) the small group of children with signs of gluten sensitivity, as evidenced by the presence of CD antibodies in serum, but without evident small intestinal lesions, will benefit from the planned follow-up by a paediatric gastroenterologist. Preliminary results already indicate that most of these children will develop CD later in life. It is important to take into account that the positive predictive value of blood testing is very high, so that there is only a small chance for "false positive" cases; in particular as this study is working with a high risk cohort.
-The dietary intervention study of PREVENTCD deals with families where at least one member has CD. They are conscious that their infant has a risk of 10% to develop CD. These families have extensive knowledge of the disease, its problems, complications and management. These families, by experience, will be able to judge if the burden from obtaining blood samples for the purposes indicated in PREVENTCD is in balance to the burden derived from the work-up that their infant would undergo whenever he/she has a possible diarrhoeal bout.
In addition, and to diminish the burden of PREVENTCD as much as possible we make clear the following aspects: -Because to our knowledge there are not guidelines about the frequency of blood sampling in young children ethically acceptable in research settings, we have reduced the number of blood sampling (7) and the amount of blood to be obtained (5 ml) to the minimum acceptable to obtain scientific valid results. The amount of blood is sufficient to allow for the screening of CD-specific antibodies, phenotypic characterization of 20 markers indicating lymphocyte activation and regulatory T cell induction, and monitoring for the occurrence of gluten-specific T cell responses typical for CD. At the age of 3 years 10ml blood will be obtained to allow for determination of additional (auto) immune phenomena related to CD.
-The pain caused by venapunctures will be diminished as much as possible by previous administration of a local anaesthetic crème.
-The "frequent controls" consist of fairly routine infant health care evaluations (weight, height, well-being, development, etc).
-The anthropometric data will be, as much as possible, obtained from the files of the Well-baby Clinics attended frequently by most of the babies from the participating countries.
-The data necessary to fill in the standardized forms on Clinical History and Food Questionnaire will be obtained as much as possible by telephonic interview with the parents or guardians.
-If the organisation of the postal system in the participating country allows it, the mothers will be helped to send their samples of breast milk at months 1,2,3, and 6, by post, following written instruction and using "ready to use" material provided by the project.
For the above named reasons the proposed dietary intervention may be considered in the category of "potential to produce health benefits" and "minimal burden".

The Swedish population study imposes biopsies on an estimated subgroup of 160 children without direct benefit to them.
The 1) In about 9800 (98%) of the families child and guardian agreed on whether or not to participate, and then it is evident how to proceed.
2) For about 150 children (1.5%) an informed consent to participate was received, however, when it was time for blood sampling the child hesitated, the process was halted and the guardian informed about the non-participation.
3) About 50 children (0.5%) expressed an interest to participate but the guardian hadn't signed the consent form, and when the guardian was approached with this information they mostly consented (47/50). and although many felt some worry before the blood sampling afterwards they stated that they experienced less pain then expected.
It is important to emphasize here again that the small bowel biopsies in PREVENTCD will be performed only when medically indicated, that is: only in children with CD antibodies in their serum indicating gluten sensitivity and highly suspected for active disease, and NOT just for purpose of the study. Such children would have a biopsy also in non-study circumstances.
The participating children are expected to fall into the following three different groups with respect to risk-benefit evaluation: A) 98% Children who will incur minimal risk/burden and gain the knowledge that CD can be excluded for them.
B) 1% Children who will benefit considerably from the study as an early diagnosis of CD should allow improved future health.
C) 1% Children who will undergo a small bowel biopsy because elevated serological markers strongly indicating CD, but without evident small bowel lessions. Interestingly, out of the 55 biopsies so far performed, as many as 47 (85%) reveal villous atrophy, thus confirming the suspicion of active CD. Thus, this first screening round (2005)(2006) has revealed that undiagnosed, and thereby also untreated CD, is more common than expected and as frequent as about 2% (group B). Notably, the group of children with elevated serological markers but without evident CD (group C) was less than half of expected and as infrequent as 0,4% (29/7400). In addition, most of them report considerable health problems commonly seen in CD .
A second screening round might therefore be considered as a benefit to all children involved since: 1) 2% of children will benefit considerably from the study as an early diagnosis of CD should allow improved future health (group A); 2) 97.6% of them will incur minimal risk/burden and gain the knowledge that CD can be excluded for them -Baby's: cord blood, whole venous blood and small bowel biopsies (table 1).
-Mothers: Breast milk (table 1) and 1 whole venous blood sample (10 ml) -Fathers: 1 whole venous blood sample (10 ml) -Siblings: 1 whole venous blood sample (5 ml) In the population based study only the children are involved and the sampling will be limited to whole venous blood, saliva and a small intestinal biopsy. The samples will be used to analyse biological characteristics relevant to the project, such as presence and quantity of gluten peptides, immunological mechanisms triggered by exposure to gluten (e.g. appearance of gluten peptides-specific lymphocytes in the blood or in small bowel mucosa), and to identify differences in distribution of the genetic risk factors for CD. The amount of material obtained will be limited to the minimum required for these investigations. The risk caused by the procedure will be kept to a minimum.
Collected biological material will either be used immediately for in vitro tests, or will be kept frozen until used in further tests. In order to allow later scientific analyses with newer techniques that are not yet available, frozen samples may be kept stored for 10 years after completion of the project, after which unused material will be destroyed. The biological samples will only be used in the scientific experiments described in PREVENTCD. No commercial use of this material will be made. The samples pertaining to a clinical trial will be owned by the coordinating investigator responsible for that trial. Biological material will only be collected and stored after specific informed consent by the parents, guardians or other legal representatives of the children has been obtained. The informed consent information relating to the collection and storage of biological material will include a description of the procedures, amounts and risks related to the collection of samples. It will mention that these samples will only be used for the genetic and immunological tests associated with the present research project, and that no commercial use will be made thereof. This informed consent will be an integral part of the informed consent document that the parents, guardians or other legal representatives of each child will sign before inclusion in the corresponding trial.

9.4.b. Protection of personal data
Each centre participating in PREVENTCD will be responsible for protecting the confidentiality of the children and the families that it enrols. This protection will comply with the local, national and European standards for protection of privacy and confidentiality. In all cases, this will include the anonymisation of each child and his/her first degree family members (parents and siblings), by replacing his/her name with a code that will make any direct identification impossible. This code will be used in all exchange of information related to this subject. Sensitive hard copy records such as medical and trial-related records will be kept in a key locked, access-restricted place and computer access to sensitive electronic data will be password protected. Access to this information and to the identity of the study participants will be strictly restricted to the physicians in charge of the patient, data managers in charge of the study, study monitors and authorized regulatory bodies. The name or any other identifying information of participating children will not be used in publications resulting from the study. PREVENTCD will study the effect of the currently known genetic risk factors on CD and the early dietary intervention, in two cohorts: a Swedish population-based cohort and a European-based family cohort. Each individual will be genotyped for the four genes known to be associated to CD: HLA-DQ, CTLA4, CELIAC2 and XX. DNA will be isolated from peripheral blood from all participants according to standard protocols.
Genotyping will be performed using 384 single nucleotide polymorphisms (SNP) covering these four regions. A specific ICF for genetic studies will be obtained, with due explanations about DNA and what the genetic study involves. children and on human material, including DNA, and protection of personal data. It will remain active during the entire period of the research, and will work in close collaboration with the advisory board.
The tasks of the ethical management will be to: -Ensure that all research activities within PREVENT CD are carried out in full respect with the ethical and safety rules described above.
-Ensure that each partner has the required knowledge on the ethical and safety issues of the project, and that this knowledge is constantly updated. The members of the ESC will be independent from the research teams, chosen among the members of the ethics committee/institutional review board (EC/IRB) of each centre participating in the project. Each EC/IRB will be asked to propose one of its members, or select another faculty member to join the ESC. However, two or more EC/IRB from the same country may designate one single representative member. Thus, the ESC will be composed of at least one representative from each participating country. This will ensure a good coordination between the ESC and local EC/IRB, and will promote ethical cooperation and exchanges between EU countries.

Gender dimension
The proposed project agrees with the European policy to integrate the gender dimension in FP6 projects, because: 1. The topic to be studied

The participants in the project
The topic of the project.
The proposed project involves coeliac disease (CD), a disorder that, as most others with autoimmune character, affects women at least twice as men. Also many of the long-term complications of CD, such as retarded menarche age, infertility, premature births, early menopause and osteoporosis are disorders concerning directly health women's needs.

Appendix A -Consortium description
A.1 Participants and consortium In order to design and carry out the scientifically most robust studies and achieve the best The consortium presented in Table 1 consists of 13 research centres, 3 industrial partners and the Association of European CD Societies (AOECS). One of the participants is based outside the EU, in Israel.

Complementarity
The participants complement to each other concerning both the expertises necessary to

InCo Countries
There is no participation of the so called International Collaboration (InCo) countries.

SME
One of the partners involved, namely partner 11, Eurospital, is SME dedicated, among others, to the design and marketing of rapid, affordable and patient oriented diagnostic tests of CD  designing and executing nutrition based clinical studies, his participation will be of significant value to the project. He will specifically contribute by designing and manufacturing the intervention products (versus placebo) for WP3 and arrange for the blinding and randomisation process in WP3. He will be the independent guardian of the randomisation code, using appropriate techniques and procedures to ensure maximal blindness of study participants and investigators to the assigned study products. Yet, he will have a system in place to quickly reveal the identity of the study product / group assignment in cases of adverse events. Danone will provide the placebo (lactose, Pharmatose) and the intervention product (Glutival obtained from Cargill). Danone has outsourced the production of the sachets with the gluten intervention / placebo products to NIZO Food Research, Ede, the Netherlands. NIZO will prepare and package the gluten intervention product as well as the placebo according to quality criteria adequate for infants aged 4-6 months.. Every intervention sachet will be filled 1.8 gr Pharmatose and 0.2 gr Glutival (gluten intervention product); the placebo sachets will be filled with 2 gr of Pharmatose.
NIZO will pay special attention to the microbiological assessment of the products and will make sure that the results will be in accordance with the standards of Danone. Also it will be analytically verified whether the blending of the materials has been adequate and resulted into a homogeneous mixture.

Relevant publications; a selection
Its membership base consists of 30 regional or national societies across 25 European countries, representing over 200,000 coeliac families. It is thus ideally situated to recruit coeliac subjects from its membership base, as well as to desseminate the results and recommendations.
Dr Christian A. Scerri, (PhD) is a member of the board of directors of AOECS and presently its chair. He has a vast experience in subject recruitment for epidemiological and genetic studies, having been responsible for the Maltese WP in the Eurobiobank project as well as in the recently terminated Geoparkinson project.
Phadia GmbH (Partner 10), is a world leader in the development and production of diagnostics kits in autoimmunity. The Phadia Biotechnikum II in Freiburg was established as a Centre of Excellence for autoimmune test research and production. All processes are performed using GMP guidelines and are ISO approved. Using human recombinant tissue transglutaminase (tTG), produced in a eukaryotic cell system, Phadia has established the Celikey range of products for the diagnosis and monitoring of CD.
The Celikey products provide exceptional sensitivity and specificity in this area and their performance is well recognised in many publications. Phadia will contribute to the study by supplying Celikey test kits for detection of IgA and IgG anti tTG antibodies as well as IgA and IgG anti gliadin kits. The company will also provide their expertise, manpower and the facilities of their Application laboratory to perform all the testing. Centre for HLA typing for CD patients.

Prof. Raanan Shamir (MD, PhD, associated professor Technion -Israel Institute of
Technology) will bring his experience in conducting large studies aimed at identifying subjects with CD, such as the blood donor study to identify the prevalence of CD in Israel. Through his commitment to the CD community in Israel, serving as a medical advisor to the Israel Coeliac Association, the Coeliac Society in Israel is already committed to this study and Raanan Shamir will enrol families on a National basis.
Raanan Shamir will also contribute to preparing and conducting nutrition surveys and will assist in the development of preventive education and treatment strategies for CD. As a member of the ESPGHAN Committee on Nutrition (CoN), and as the CoN representative at the ESPGHAN Council, he will also take care of the dissemination of the results at different levels: i.e. the scientific community, industry, regulation bodies and patients' associations.

Relevant publications; a selection
projects on CD (BMH4983087 and QLK1-CT-2000-00657). He and his group have made many key findings in relation to the genetics and immunology of CD. He identified DQ2 and DQ8 to be responsible for the HLA effect in CD. His group made the seminal findings that gluten reactive T cells can be cultured from intestinal biopsies of CD patients (but not controls), and that these T cells recognize gluten peptides in the context of the disease associated DQ2 or DQ8 molecules. These findings have paved the road for many subsequent findings of CD pathogenesis. His group played a central role in identifying the first sequence of a DQ2 restricted gluten T cell epitope. Importantly, Ludvig Sollid and his co-workers reported that this epitope is deamidated. They later showed that the deamidation of this and other gluten T cell epitopes is mediated by the enzyme tissue transglutaminase. His group was able to produce soluble DQ2 molecules.
These molecules have been used to stain and visualize gluten specific T cells with HLA tetramers, and to solve the crystal structure of DQ2 in complex with a deamidated gluten peptide. Sollid is the member of advisory boards of several foundations, companies and research centres dealing with CD both in the US and in Europe.

A.2 Sub-contracting
Danone will provide the placebo (lactose, Pharmatose) and the intervention product (Glutival obtained from Cargill). Danone has outsourced the production of the sachets with the gluten intervention / placebo products to NIZO Food Research, Ede, the Netherlands.
NIZO will prepare and package the gluten intervention product as well as the placebo according to quality criteria adequate for infants aged 4-6 months. Every intervention sachet will be filled 1.8 gr Pharmatose and 0.2 gr Glutival (gluten intervention product); the placebo sachets will be filled with 2 gr of Pharmatose.
NIZO will pay special attention to the microbiological assessment of the products and will make sure that the results will be in accordance with the standards of Danone. Also it will be analytically verified whether the blending of the materials has been adequate and resulted into a homogeneous mixture. The costs made by NIZO will be paid by the budget of Danone, the estimated costs are € 60.000,-.