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Characterizing Intestinal Inflammation in Fanconi
Anemia Mutants
Characterizing Intestinal Inflammation in Fanconi
Anemia Mutants
Sam CarpentierUniversity of Oregon SPUR
2009
Sam CarpentierUniversity of Oregon SPUR
2009http://blogs.nature.com/nm/spoonful/zebrafish.jpg
Zebrafish Models of Intestinal
Inflammation
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?Why Fanconi Anemia? Fanconi Anemia is a DNA-repair disease that leads to problems such as cancer and blood-related diseases.
Fanconi Anemia is a DNA-repair disease that leads to problems such as cancer and blood-related diseases.
http://wpcontent.answers.com/wikipedia/commons/thumb/7/78/Fanconi%27s_anemia_101.jpg/190pxFanconi%27s_anemia_101.jpg
Inflammatory Bowel Disease Symptoms have been associated with some cases of Fanconi Anemia.
AimAim
Test the hypothesis that Fanconi mutants have increased intestinal inflammation.
Test the hypothesis that Fanconi mutants have increased intestinal inflammation.
We will count Myeloperoxidase (MPO) positive neutrophils in the zebrafish intestine.
We will count Myeloperoxidase (MPO) positive neutrophils in the zebrafish intestine.
Assay
Wild-Type Mutant
Fanconi D1 Mutants Do Not Have Elevated Inflammation
Fanconi D1 Mutants Do Not Have Elevated Inflammation
Data from 2 Independent trials
p=.64
WT FancD1 Mutant0
5
10
15
20
MPO Positive Cells Per Fish
Fanconi G Mutants Do Not Have Increased Inflammation
Fanconi G Mutants Do Not Have Increased Inflammation
p= .40
WT Fanc G Mutant0
5
10
15
20
25
MPO Positive Cells Per Fish
Fanconi C Mutants May Have Increased Inflammation
Fanconi C Mutants May Have Increased Inflammation
0123456789
101112
MPO Positive Cells Per Fish
WT
1
WT
2
Mu
t 2
Mu
t 1
p=.007
p=.70
Modifications of MPO Counting Protocol
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.
Fanconi C Mutants May Have Increased Inflammation
Fanconi C Mutants May Have Increased Inflammation
P=.18
WT FanC C Mutant0
5
10
15
20
MPO Positive Cells Per Fish
Fanc G Appear to Have Increased InflammationFanc G Appear to Have Increased Inflammation
P=.05
WT FancG Mut0
5
10
15
20
MPO Positive Cells Per Fish
Why is There Increased Inflammation in the Fanconi C Mutants?
Why is There Increased Inflammation in the Fanconi C Mutants?
•Immune imbalance, independent of microbiota.
•Increased inflammatory response to the microbiota.
inflamed mutant
derive germ-free
?
Raise Fanc C Mutants Germ- Free
(Without Microbiota)
Fanc C Mutant Inflammation is a Response to the
Microbiota
Fanc C Mutant Inflammation is a Response to the
Microbiota CV WT
Fanc C
CV Mut
GF WT
Fanc C
GF Mut
0
5
10
15
20
MPO Positive Cells Per Fish
WT p=.0003
Mutant p=.0001
Do Fanc C Mutants Exhibit Defective Peristalsis and
Bacterial Imbalance?
Do Fanc C Mutants Exhibit Defective Peristalsis and
Bacterial Imbalance?
5dpf 6dpf 7dpf 8dpf
Fanc C fish were fed fluorescent green food for three days, followed by a fluorescent red food.
Fanc C WT MovieFanc C WT Movie
QuickTime™ and aH.264 decompressor
are needed to see this picture.
Fanc C Mutant MovieFanc C Mutant Movie
QuickTime™ and aH.264 decompressor
are needed to see this picture.
ConclusionsConclusions 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.
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 DirectionsFuture Directions
Gut Microbiota Reciprocal Transplantation
Transfer CV Fanc C Mutant microbiota into GF Wild-Type Fish
Gut Microbiota Reciprocal Transplantation
Transfer CV Fanc C Mutant microbiota into GF Wild-Type Fish
inflamed mutantdonor
AcknowledgementsAcknowledgements
Erika MittgeKaren Guillemin Kate WoronowiczKristin Alligood Judith Eisen
The Guillemin Lab Peter O’Day Chelsie Fish SPUR Program Catharine WilsonTom Titus Poh Kheng Loi Rose UO Zebrafish Facility
