Inflammatory Bowel disease

Delaying the onset of the disease as well as finding novel potential targets for intervention strategies would dramatically increase the likelihood of healthy, active and independent lives until old age

Inflammatory bowel diseases are characterized by severe chronic intestinal inflammation with huge impact on quality of life. Current treatment is expensive and lacks in therapeutic precision. This reduces efficacy and safety and the disease progress throughout life as well as causes un-necessary side effects from patients receiving treatment they do not respond to.   Delaying the onset of the disease as well as finding novel potential targets for intervention strategies would dramatically increase the likelihood of healthy, active and independent lives until old age.

How inflammatory bowel disease is manifested depend on the area of the intestinal tract involved. Many of the symptoms, however, are not specific for this disease. The World Gastroenterology Organization indicates the following symptoms may be associated with inflammatory damage in the digestive tract

• Diarrhea

• Bowel movement abnormalities:

• Abdominal cramping and pain:

• Nausea and vomiting:

If you suspect yourself of having IBD please consult your local physician.

Patients included in this study are recruited via the normal admission to hospitals within the regions where project partners are active. No recruitment will be made outside those regions.

 Protein profiling

Proseek Multiplex – measure 92 biomarkers in 1µl of sample without compromising data quality

To enable identification and understanding of molecular and cellular pathways involved in initiation of the diseases we will develop novel and improved methods for large scale sensitive affinity proteomics based on partner Olink’s proprietary Proximity Extension Assay (PEA) technology. Initially developed in the FP7 program Proactive it is now made available and sold under the trade name Proseek Multiplex. In this project we will build dedicated multiplexed panels targeting proteins of interest for IBD.

Our hypothesis is that (i) large numbers of protein biomarkers must be screened in order to find the best panels of relevant diagnostic markers, (ii) individual protein biomarkers will not be sufficient as diagnostic marker and (iii) informative tissue leakage protein biomarkers will be present at low concentration in circulation. Current multiplexed protein formats are limited either by their low sensitivity, low throughput or applicability for clinical testing, or all three. Therefore we aim to utilize Proseek multiplex, a multiplexed protein assay capable of detecting low abundant protein biomarkers in high throughput format.

Proseek Multiplex is based on the PEA developed at Olink. PEA has a major advantage over conventional multiplex immunoassays in that only correctly matched antibody pairs give rise to a signal. PEA is a homogeneous assay that uses pairs of antibodies equipped with DNA reporter molecules. When binding to their correct targets, they give rise to new DNA amplicons each ID-barcoding their respective antigens. The amplicons are subsequently quantified by high throughput real-time PCR.

Proseek® Multiplex can provide accurate quantification of up to 92 proteins in one run at low to sub picogram per milliliter levels from1 µl samples such as serum or tissue lysates.

 Microbial profiling

The GA-map allows screening of many samples using a panel tailor-made for IBD analysis

It is now well recognized that understanding microbial content of the gut is relevant for inflammatory bowel diseases .  However little is known about the aetiology – if changes in microbial content of the gut are causative or an effect of disease progression. Furthermore there are lacking large collaborative studies coupling knowledge about gut microbial content and host genetic factors as well as identification of differences in gut microbiota early on in the IBD. Therefore, a comprehensive study of the gut microbiota will be important for this project. In order to allow screening of large numbers of patients for presence of many different microbes, we will develop an IBD-relevant microbe bead-array (called GA-map™). By performing 16S rRNA amplicon pyro-sequencing of collected samples from IBD patients and healthy controls, a catalogue of microbes of interest in IBD will be established. This in combination with other public projects for microbe cataloguing, e.g. and the Human Microbiome project  and MetaHIT , partner GA will design unique and specific DNA probes for the GA-map platform, capable of reporting the presence of the various selected microbes. Based on the observations that there is high intra-individual variation of intestinal microbiomes in the human population, we hypothesize that characterizing IBD microbiomes could disclose bacterial and fungal species implicated in IBD aetiology. To better understand how microbiomes relate to human disease, we will undertake a comparative sequencing survey of the microbiomes present in faces in 40 IBD patients and 40 unaffected individuals using whole metagenomic sequencing in order to verify data from GA-map and look for additional microbial markers outside the 16sRNA amplicon.

Currently pyro-sequencing of the 16S rRNA gene is the most commonly used method for studying diversity in the gut microbiota . Although the method is useful as a discovery tool, the output of the sequence analysis requires several complex computational steps, as well as some subjective decisions as to sequence identity. Reducing the number of samples in each run will reduce the complexity of analysis, but this will drastically increase the cost per sample. Therefore, in order to be able to do a quality comparison of all samples, a novel technology (GA-map™) will be adapted that utilizes a combination of specific DNA probes to profile the gut microbiota. The GA-map™ technology platform is also included in another ongoing FP7 project (KBBE.2011.2.2-01, grant no 289517), and will here be further developed and tailor-made for IBD analysis. To our knowledge, this will be the first time a microbial profile will be obtained and compared from this many clinical samples. In addition, whole genome metagenomic sequencing of a sub-set of samples is expected to give additional information on biological markers and phenotypic traits of important bacteria contributing to disease. These various microbial profiling approaches will be performed on a unique sample material, namely from patients just diagnosed with IBD and before any treatment has commenced. Using this unique material will help us to understand the mechanism of initiation of disease, as well as identify markers or microbial profiles that can be used for early diagnosis of disease.