DiAgRaMs- RAAK PRO Standardization of the bicycle ergometer test as challenge model to assess nutritional effects on intestinal health and the immune system Shirley Kartaram Department Innovative Testing in Life Sciences and Chemistry Institute for Life Sciences and Chemistry DAS_Conference_14112013 1
Content Introduction intestinal health, epithelial integrity and permeability Translational Strategy In vitro experiments DiAgRaMs: aim, study design, relevance Protégé study Wageningen Acknowledgements DAS_Conference_14112013 2
Intestinal health and diseases Epithelial integrity vs permeability Passage macromolecules Antigens Gut-immune system Inflammation Gut-nervous system Nervus vagus Microbiota Inflammatory bowel diseases(ibd) Arthritis Food allergy Obesity Diabetes Alzheimer Cancer DAS_Conference_14112013 3
Epithelial integrity - Tight Junctions DAS_Conference_14112013 4
Translational Research Strategy In vitro Caco2 cells In vivo Human study DiAgRaMs Stressor model Biomarker epithelial integrity (TEER) Distribution TJ proteins Gene expression and pathways Effects food compounds and probiotics Stressor model Biomarkers intestinal permeability Biomarkers inflammation Effects food compounds and probiotics DAS_Conference_14112013 5
In vitro experiments TEER measurement Caco2 cells EVOM - Epithelial Voltohmmeter DAS_Conference_14112013 6
Day 14: In vitro experiments TEER measurement : t=0h 2 h preincubation with/without probiotic strain Stressor model: + cytomix (IFN-γ, TNF-α, IL-1β) Day 15: TEER measurement: t=16h Collection supernatants, Cell lysates for WB Membrane filters for IF staining DAS_Conference_14112013 7
TEER development In vitro experiments experiment TEER (Ω/cm2) strain Days after seeding DAS_Conference_14112013 8
DiAgRaMs Standardization of the bicycle ergometer test as challenge model to assess nutritional effects on intestinal and immune system DAS_Conference_14112013 9
Main aim: Standardize exercise as challenge model to test health products Primary Objective: To determine the effect of exercise on a selection of relevant biomarkers of intestinal permeability and inflammation in healthy young men Secondary Objective: To standardize the exercise model with the selected relevant biomarkers of intestinal permeability and inflammation (within three laboratories) Tertiary Objective: To study the effects of natural food compounds on biomarkers of intestinal permeability and inflammation DAS_Conference_14112013 10
Health Health Human study Bicycle ergometer Intestinal barrier & Immune system Health- Inflammation status Biomarkers Protocol Challenge model Standardization Nutritional effects Inflammation Inflammation DAS_Conference_14112013 11
Biomarkers Intestinal permeability Sugartest lactulose/rhamnose Zonulin Inflammatory responses Cytokines TNF-α, IFN-γ, IL1β IL6, IL7, IL8, IL4, IL5, IL13, IL18, IL10, IL2, IL33 I-FABP BLOOD SALIVA FAECES Urine CRP Cellular responses (macrophages, neutrophils, NK cells) Microbial components (LPS) Calprotectin DAS_Conference_14112013 12
Study design Phase 1 1a: Protégé - study, WUR 1b: DiAgRaMs - study, HU/WUR, set up challenge model Phase 2 2a: Standardization challenge model HU, WUR, HANZE 2b: Nutritional intervention probiotica mix cross over study design DAS_Conference_14112013 13
Phase 1a Phase 1a WUR: Protégé-study protein digestion and uptake after intense physical exercise To determine interpersonal variation power calculation phase 1b/2 final selection parameters Final timetable samples DAS_Conference_14112013 14
Phase 1b dag 1 dag 2 dag 3 tijdstip conditie bepalingstest 1 week 9.00 u 10.00 u geen inspanning nacht met vasten 10.00 u 11.00 u inspanning nacht 11.00 u bloed suiker drankje 11-13-15 u 12-14-16 u 9.30 u 10.30 u urine elk uur tot 5 u na test van 5 tot 24 u na test elk uur tot 5 u na test van 5 tot 24 u na test faeces DAS_Conference_14112013 15
Z Z Phase 2 WUR, HU and Hanze 2a: Standardization of the exercise challenge model by determining inter- and intralab variation 2b: Nutritional intervention cross over study design darm permeabiliteits test uithoudings test W darm permeabiliteits test uithoudings test C darm permeabiliteits test uithoudings test W C darm permeabiliteits test uithoudings test 14 weken voedingsinterventie (N) of placebo (C) W wash out periode van 4 weken, zonder inname probiotica mix DAS_Conference_14112013 16
Partners Partners DAS_Conference_14112013 17
Relevance human study A standardized test allows to: Support health claims of food products and natural compounds (e.g. EFSA) Evaluate health in specific patient groups (e.g. obese, alzheimer) DAS_Conference_14112013 18
Acknowledgements Ir. Laura M Rabet Lonneke Janssen Duijghuijsen http://www.innovativetesting.nl/ Prof. Johan Garssen DAS_Conference_14112013 19