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Final Activity Report Summary - IMMUNOBIOTA (Tools for integrated studies of the immunoregulatory properties of commensal microbiota)

Our hypothesis is that the infantile microbiota of Western societies is deficient in microbes that support development of tolerance to innocuous antigens and that this is the cause of the rising incidence of allergies. The purpose of this TOK was to build a method- and knowledge-based platform focussed on identifying microbes in the infantile gut flora and testing their immunoregulatory properties in people and animals. To enable this goal, we have developed a common protocol for studies of infantile gut microbiota, immune development and protection from clinical allergy shared between the host and the training partners Marmara University Hospital in Istanbul and University of Medicine and Pharmacy, Cluj-Napoca, Romania. Using this protocol, we have identified differences in early colonisation pattern and immune maturation between the cohorts.

To enable non-culture dependent characterisation of the infant microbiota, T-RFLP (terminal restriction fragment length polymorphism) was modified and a database containing intestinal bacteria was constructed, now containing 130 species. This method development speeds up the process of identification enormously and also permits the use of frozen faecal samples. S. aureus was identified as a microbe with potential beneficial immune regulating properties. In IMMUNOBIOTA, we have assembled techniques and knowledge necessary to elucidiate the role of superantigens produced by this bacterium for immune maturation and protection from allergy development.

A series of multiplex PCRs were set up for identification of 47 virulence genes in S. aureus, including >10 superantigens through exchange with Centre National de Référence de Staphylococces in Lyon. Mutant and transformant strains isogenic for the superantigen genes sea and egr were created, first in a laboratory strain background, then from a clinical isolate retrieved from a healthy infant's microbiota during an exchange visit at Trinity College, Dublin, Ireland. To our knowledge, this is the first time isogenic mutant of egr and the first S. aureus sea mutant in a clinical strain background.

Suitable mouse models were developed, including models of oral tolerance, airway and gastrointestinal allergy, as well as gnotobiotic animal technology. Germfree animals were then colonised with wild-type strains of S. aureus differing in superantigen production, or with superantigen-negative S. aureus (controls).

These studies showed that certain superantigens promoted oral tolerance development and accumulation of Foxp3+ regulatory T cells in lymph nodes draining the gut. Neonatal peroral treatment with S. aureus superantigen also promoted oral tolerance in adult life shown as improved protection in experimental allergy models. For in vitro studies on microbe-immune interactions, a gene encoding a dietary protein (ovalbumin) was cloned into E. coli and several Gram-positive lactococci and lactobacilli through exchange with Norwegian Food Research Institute. Antigen-presenting cells were pulsed with the constructs, and with soluble ovalbumin, and ovalbumin-specific T-cells (DO11.10) were added and their proliferation and cytokine production was studied. Important differences between Gram-positive and Gram-negative bacteria were demonstrated. A volunteer study was performed in which the immune activating effects of intake of different species of lactobacilli were studied.

In summary, IMMUNOBIOTA has successfully achieved transfer of knowledge and the build-up of a platform enabling studies on how commensal bacteria shape the infant's immune system and protect against allergy development. The project has helped in defragmenting the European Research Area and increased its competitiveness by integrating researchers from EU member and Associated States, basic and clinical scientists and academic and company-driven research. It may form the basis for further studies both in infants, animal models and healthy adult volunteers.

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