ObjectivesWe have discovered that early-onset inflammatory bowel disease (IBD) can be caused by mutations in IL10 or its receptor. This project aims to identify further patients suffering from IL10/IL10 receptor deficiency and to clarify the pathomechanism causing the severe phenotype of the patients.DesignThe project will investigate patients with early-onset IBD. Patients will have blood samples taken, white blood cells will be separated, DNA isolated and functional assays and genetic analyses carried out. Intestinal tissue samples will be stained by using immunohistochemical methods.Methodology:We will investigate different stages of IL10/STAT3 signalling in patients' white blood cells using ELISAs and/or Western blot; abnormalities will be confirmed by gene sequencing.Treg cells and Th17 cells will be analysed using flow cytometry or immunohistochemistry; function of Treg cells will be examined using commercially available tests. The role of cytokines other than TNF will be examined by challenging cells with different stimuli and subsequent analysis using chemiluminescence-based cytokine arrays. Neutrophil function and apoptosis will be examined using cytokine arrays, expression of pro- and anti-apoptotic proteins using Western blot and real-time PCR. Investigation of therapy resistance to immunosuppressive drugs includes analysis of NF-kB and NFAT pathways and, if necessary, the use of gene expression assays.Scientific and medical opportunities of the studyThis project may (i) identify further patients with IL10- or IL10 receptor deficiency; (ii) clarify the role of Th17 and Treg cells in the intestine of IL10/IL10 receptor deficient patients; (iii) investigate the role of cytokines other than TNF in the pathogenesis of the disease, (iv) shed light on the impact of neutrophils on the clinical phenotype; and (v) discover the mechanism why patients' cells are resistant to therapy with immunosuppressive drugs.