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Characterizing Intestinal Inflammation in Fanconi Anemia Mutants Sam Carpentier University of Oregon SPUR 2009 http://blogs.nature.com/nm/spoonful/zebrafish.jpg Zebrafish Models of Intestinal Inflammation •Intestinal Alkaline Phosphatase Mutant •Unable to detoxify LPS from Gram-negative bacteria. •Sox 10 Mutant •Defective gut peristalsis that leads to increased intestinal inflammation. Why Fanconi Anemia? Fanconi Anemia is a DNA-repair disease that leads to problems such as cancer and bloodrelated diseases. Inflammatory Bowel Disease Symptoms have been associated with some cases of Fanconi Anemia. http://wpcontent.answers.com/wikipedia/commons/thumb/7/ 78/Fanconi%27s_anemia_101.jpg/190pxFanconi%27s_ane mia_101.jpg Aim Test the hypothesis that Fanconi mutants have increased intestinal inflammation. Assay We will count Myeloperoxidase (MPO) positive neutrophils in the zebrafish intestine. Wild-Type Mutant MPO Positive Cells Per Fish Fanconi D1 Mutants Do Not Have Elevated Inflammation 20 15 p=.64 10 Data from 2 Independent trials 5 0 WT FancD1 Mutant MPO Positive Cells Per Fish Fanconi G Mutants Do Not Have Increased Inflammation 25 20 15 10 p= .40 5 0 WT Fanc G Mutant MPO Positive Cells Per Fish Fanconi C Mutants May Have Increased Inflammation 12 11 10 9 8 7 6 5 4 3 2 1 0 p=.007 p=.70 Modifications of MPO Counting Protocol • MPO-positive cells were counted that may have been outside of the gut. •Further studies quantified the MPO-positive cells in sectioned tissue. MPO Positive Cells Per Fish Fanconi C Mutants May Have Increased Inflammation 20 15 10 P=.18 5 0 WT FanC C Mutant MPO Positive Cells Per Fish Fanc G Appear to Have Increased Inflammation 20 15 10 P=.05 5 0 WT FancG Mut Why is There Increased Inflammation in the Fanconi C Mutants? •Immune imbalance, independent of microbiota. •Increased inflammatory response to the microbiota. Raise Fanc C Mutants Germ- Free (Without Microbiota) derive germ-free inflamed mutant ? MPO Positive Cells Per Fish Fanc C Mutant Inflammation is a Response to the Microbiota 20 15 10 5 0 WT p=.0003 Mutant p=.0001 Do Fanc C Mutants Exhibit Defective Peristalsis and Bacterial Imbalance? Fanc C fish were fed fluorescent green food for three days, followed by a fluorescent red food. 5dpf 6dpf 7dpf 8dpf Fanc C WT Movie QuickTime™ and a H.264 decompressor are needed to see this picture. Fanc C Mutant Movie QuickTime™ and a H.264 decompressor are needed to see this picture. Conclusions There does not appear to be increased intestinal inflammation in the Fanc D1. Intestinal Inflammation may be increased in both the Fanc C and G mutant fish. Intestinal inflammation in the Fanc C mutants is a response to the microbiota. Gut motility appears to be unimpaired in the Fanc C mutant fish. Future Directions Gut Microbiota Reciprocal Transplantation Transfer CV Fanc C Mutant microbiota into GF Wild-Type Fish inflamed mutant donor Acknowledgements Erika Mittge Karen Guillemin Kate Woronowicz Kristin Alligood Judith Eisen The Guillemin Lab Peter O’Day SPUR Program Tom Titus Rose Chelsie Fish Catharine Wilson Poh Kheng Loi UO Zebrafish Facility