Advances in Gastrointestinal
Aspects of CF
“GI”
North American Cystic Fibrosis Conference
Orlando 2012
Drucy Borowitz, MD
Professor of Clinical Pediatrics
State University of New York at Buffalo
Gain Insights into Gastrointestinal
Aspects of CF
• Recognize similarities between the
respiratory and GI tracts in CF
• Understand CF GI pathophysiology
• Compare findings in CF animal models
and CF infants
• Learn about new technologies to explore
CFTR dysfunction
Disclaimer Statement
• My employers, the State University of New York at Buffalo and University
Pediatric Associates have received payment for my research and
consulting activities; I have not received any personal payments
Gestational Information:
The
Respiratory and GI Tracts have the same
Embryologic Origins
Clearance of Obstruction and Infection
LUNGS
GI TRACT
• ASL hydration and
mucociliary clearance
• Microvillus and luminal
hydration
• Cough
• Peristalsis
• Antimicrobials from
submucosal glands
• Antimicrobials from
submucosal glands
Normal GI Tract Physiology
CF-Related Liver Disease
• “CFLD” is a spectrum :
– Neonatal cholestasis, microgallbladder, cholelithiasis, biliary
tract ductal stones, common bile duct stenosis, sclerosing
cholangitis, hepatic steatosis, nodular regenerative hyperplasia
– focal biliary cirrhosis and portal hypertension are the most
serious complications
• ~ 5 to 10% of CF patients will develop cirrhosis / PHT
in the first 10 years of life
– no correlation between ↑ transaminases and cirrhosis / PHT
• PUSH is a CFF-NIDDK collaboration to explore early
biomarkers of cirrhosis
Liver and Pancreatic Ducts Secrete
Bicarbonate (HCO3- ) via CFTR to Neutralize
Gastric Acid
Liver
(HCO3)
GI epithelium (especially Brunner’s Glands) also
secretes HCO3- via CFTR
Crypt-villus location of
duodenal HCO3- secretion
under cAMP-stimulated
conditions:
Abstract 126
Symposium 15
Walker et al, Gastroenterol 2009
Why is HCO3 Secretion Important?
• It neutralizes gastric acid
– Needed for pH optima of pancreatic enzymes and micelles
• It allows mucins to unfold / hydrate Abstracts # 99 & 501
• It promotes bacterial killing Abstracts # 13 & 131
• HCO3 secretion ≡ Fluid secretion
Bicarbonate Drives Fluid Secretion
The normal human
pancreas secretes
1-2 L / day
Novak et al, J Biol Chem 2011
Pancreatic Enzyme and Bicarbonate
Secretion have Different Stimuli
Secretin stimulates
duct to secrete
bicarbonate
Cholecystokinin (CCK) stimulates
acini to release enzymes
How can you measure GI HCO3?
“pH pill”
• Measures pH, pressure
and temperature
• Single use
• Radiofrequency
detector worn outside
body
Gastric Acid Neutralization is delayed
in Subjects with CF
N=20
N=20
Gelfond et al, Dig Dis Sci. 2012
Could Activation of CFTR with Ivacaftor
↑ GI Bicarbonate?
• Pancreatic tissue
becomes atretic but the
duct is still present
– MRI
– Pathologic studies
– PI patients have ↓ but not
absent HCO3 secretion
Kopelman et al Gastro 1988
• Submucosal glands are
obstructed, but are
still present
Gastric Acid Neutralization is normalized
in Subjects with CF taking Ivacaftor
Small bowel pH changes (1min means)
8
Pre VX-770
Post VX-770
p< 0.05
7
pH
6
5
4
3
0
8
0
5
24
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120
Minutes from gastric emtpying
N=7
The Earliest Clinical Consequences of CF
are Gastrointestinal
Pancreatic Insufficiency
Meconium ileus (MI)
• What is the ontogeny of these conditions?
• How can we study them?
• Animal models
• Human infants
Google Infants: Human and Otherwise
What have we learned
from CF mouse models?
What have we
learned from CF
ferrets and pigs?
What have we learned
from infants with CF ?
Lessons from CF Mice
Restoration of CFTR in
intestinal epithelium eliminates
obstruction
Hodges et al, Am J Physiol GLP 2011
Restoration of 10-15% CFTR
will avoid intestinal obstruction
Bowel obstructions occur
despite normal pancreatic
function
Slide courtesy of P. Durie
Lessons from CF Ferrets & Pigs
• CF Ferrets
• 50% die from MI with perforation in ileum or colon
• Have mild pancreatic histopathology at birth
• CF Pigs
• 100% penetrance for MI & PI
• Pancreatic disease begins in utero & progresses over time
– proinflammatory, complement cascade, proapoptotic, and
profibrotic pathways are activated Abu-El-Aija et al, Am J Pathol 2012
Abstract # 180
MI is not caused by pancreatic dysfunction,
but is strongly associated with it
Pancreatic Dysfunction in
Infants with CF
•
Management of complications of steatorrhea
Pancreatic
insufficiency can be the
can be done by “conservative measures,
first
clinical
manifestation
CF
including
strapping
of the buttocks,of
defecating
in a reclining position, and measures designed
–
Occurs
in
a
majority
of
infants
to reduce the frequency and bulk of stool”…
(such
as ) ...witholding
butter, ice cream,
– Occurs
prenatally
peanut butter, potato chips, french fried
– Treatment
with pancreatic
potatoes,
and mayonnaise….”
enzyme replacement therapy
(PERT) is life-sustaining
Schwachman, Pediatr 1960
Advances in Pancreatic Enzyme
Replacement Therapy (PERT)
• New Drug Application process improved safety
• no longer overfilled
• improved stability
• free of enveloped viruses
Abstract # 447
• 5 delayed release PERTs approved by US - FDA
•
•
•
•
•
Creon (Abbott)
Zenpep (Aptalis)
Pancreaze (Janssen / J&J)
Pertzye (Digestive Care)
Ultresa (Aptalis)
Normal
vs. Fibrosing
Colonopathy
Smyth et al,
Lancet 1994
Schwarzenberg et al
J Pediatr 1995
Addressing Issues with PERT Dosing
20
18
# of cases
18
16
14
14
12
10
11
10
9
8
8
4
0
7
5
6
2
13
11
12
4
1
8
6
3
2
2
0
6
Phase IV
surveillance
study for
fibrosing
colonopathy
is underway
Years
Baby Observational Nutrition Study (BONUS)
sub-study to help answer questions about
dosing in infants
Growth Investigations
• BONUS will also help us understand factors that
influence growth in the first year of life
• FIRST (Feeding Infants Right -- from the Start)
• Explores breastfeeding, respiratory infections and growth
• Docosahexaenoic acid study
• Explored the effects of DHA on pancreatic function as
measured by monthly fecal elastase (FE)
» FE is unaffected by oral PERT
» FE > 200 μg/g and consistent with PS
Schematic Pattern of Evolution of
Pancreatic Function in Infants in the
1st Year of Life
O’Sullivan et al, J Pediatr (in press)
Why Do Fecal Elastase Levels Change
Early in Life?
• High levels  low because pancreatic
dysfunction evolves over the 1st year
• An opportunity to modify disease evolution
• Low levels high
o
• Initial levels may be 2 PI
– “false positive”
• Other factors
• Re-measure FE at 1 year of age
Garner Intelligence
Poohed
Microbiomics: Interactive Symbiosis
• Human intestine is colonized by an
estimated 100 trillion bacteria
–
–
–
–
Bronchi
Promote optimal digestion
Maintain epithelial homeostasis
Modulate fat metabolism
Promote angiogenesis and enteric
nerve function
– Support resistance to infection
• Dysbiosis in patients or animal
models is associated with
–
–
–
–
–
inflammatory bowel disease
obesity
cancer
diabetes
allergy
Microbiomic Techniques
• Culture independent using 16S ribosomal RNA
• Data are analyzed in terms of:
•
•
•
•
•
Relative abundance
Diversity / Richness
Presence or absence of taxa
Evenness (distribution)
Total bacterial load QPCR
Symposium # 10
Reference genomes from the human microbiome
Bacterial Communities in Mammalian
Intestine
Adapted from Hill et al, Mucosal Immunol 2009
Abstract # 326
Correlation between Fecal and
Respiratory Microbiomes in CF
• 7 infants with CF diagnosed by NBS, followed for
9-21 months
• 14 of 16 genera increasing in the gut were also
increasing in the respiratory tract
• For 7 of these 16 genera, gut colonization
presages appearance in the respiratory tract
Madan et al, mBio 2012
Heat Map and Simpson’s Diversity Index of Respiratory and Fecal Microbiomes
Abstract # 279
a= microbes with increased abundance in the intestines early in life  b= later in life
GI and Respiratory Tract
Commonalities
• Selective epithelial barrier + mucus-gel layer
• Protects against bacteria, pathogens and foreign antigens
• Mucosa-associated lymphoid tissue (MALT)
• Regulates antigen sampling, lymphocyte trafficking and
mucosal host defense
• “ It is most likely that it is the similar inflammatory and
immune components of these organs that are the cause of
the overlap in pathological changes in respiratory and
intestinal mucosal diseases”.
Keely et al, Mucosal Immunol. 2012
Go Inside
• Immune responses to intestinal
bacteria  inflammation:
•
•
•
•
Bacterial signals
Toll-like receptors
NOD-like receptors
G protein-coupled receptors
• Measureable clinically with
Abstract # 522
lab tests
and video
endoscopy
Hill & Artis, Ann Rev Immunol 2010
Capsule Video Endoscopy shows
Inflammation in CF Intestine
Healthy jejunum
Videos courtesy of M. Wilschanski, Jerusalem
Patient with CF
Abstract # 510
Grab Intestine
• New preclinical model systems and clinical
trial biomarkers
• Rectal short circuit current
measurements (ICM)
• Organoids
Rectal tissue as a biomarker and preclinical
model system for CFTR

Initially based on European experience, now
TDN-sponsored SOP

Can do direct or suction biopsy

Advantages:



Abstract # 210
Accessible
High expression
Multiple ex vivo assessments



Can apply reagents not suitable in vivo
Can apply agents to apical or basal side
ICM , biochemistry, metabolomics, etc.
Colonocyte Ion Transport
Cl-
ClCFTR
Na+
Amil ENaC
K+
CaCC
KvLQT
SK IK BK
cAMP
Ca++
cAMP
Na/K/2Cl
Na+HCO3-
Ca++
2K+
cAMP
Ca++
NBC1
~
NKCC1
Bum
KvLQT
IK
3Na+
K
+
Adapted from H. DeJonge
K
+
• Differences from
respiratory epithelial
ion transport:
– Presence of K+
secretory pathway
– Colon is an
absorptive cell (i.e.
has more Cl- secretion )
Standard Rectal ICM Recordings
Abstracts # 175 & 256
1A. Non-CF
1B. CF
CFTR response
300
400
350
Isc (uA/cm2)
250
CCh
300
Bum
250
200 Indo
CFTR response
200
150
Fsk/IBMX
AmilFsk/IBMX
CCh
Bum
50
100
0
50
-50
0
-100
20
Indo
Amil
100
Amil
150
Indo
40
60
Time (min)
80
20
40
60
80
Time (min)
↓ response to F/IBMX, mixed response to CCh b/o K+ transport in absence of CFTR
Intestinal current measurements:
genotype/phenotype relationships
-180
More
CFTR function
-150
CFTR current
-120
-90
-60
-30
30
Controls Carriers
CF-PS
CF-PI
Solid line, median; dashed line, 25th and 75th percentiles
Hirtz, S. et al. Gastroenterol, 2004
Organoids / Enteroids / Colonoids*
o
• Are 1 cultures that can express
crypt and/or luminal domains
• Lgr5+ cells at base of crypts generate all
cell types in crypt-villus axis
• Can study
– Crypt secretory physiology in an
integrated cell culture environment
– Luminal absorptive physiology
• Have been created from
• Mouse and human intestine
• Mouse and human embryonic stem cells
* See Stelzner et al Am J Physiol GI Liver 2012 for NIH nomenclature
Crypt Culture in 3D Gels – “Enteroids”
Sato et al, Nature 2009
Enteroids isolate intestinal epithelium from
microbial population and systemic factors
• Changes caused by microbial environment:
• Goblet cell hyperplasia
• Changes intrinsic to epithelium with CFTR
dysfunction:
• Hyperproliferation:
– May be caused by alkaline pH
– May give clues to ↑ incidence of GI cancers in CF
• Goblet cell degranulation defect:
– Mucus that is released stays attached to goblet cells
Liu J et al, Am J Physiol Cell 2012
With additional work from L. Clarke lab, Missouri
Goblet Cell Degranulation after
Carbachol
Wild Type
CF Knock-out Mouse
Abstract # 122
lumen
lumen
Normal Degranulation
Videos courtesy of L. Clarke lab -Missouri
Granules go into the lumen
without undergoing dissolution
“The submucosal gland contains the elixir of
airway health “ – Dr. Jeff Wine
Abstracts # 86, 87, 89
Forskolin induces rapid swelling of organoids
Healthy human organoids
F508del / F508del
organoids treated
with VX-809 + VX-770
|--40μm --|
F508del / F508del organoids |--30μm --|
Videos courtesy of
J. Beekman lab,
Utrecht NL
Forskolin-induced swelling in intestinal
human organoids can be quanitated
Abstract # 191
N=8
N=2
N=11
J. Beekman lab, Utrecht NL
Generalize Insights
• There are similarities between the respiratory
and GI tracts
• CFTR dysfunction causes pathology in both via
obstruction-infection-inflammation
• Animal models and techniques used to explore one
system lend insight to the other
• Treatments that focus on CFTR modulation are likely to
improve GI as well as respiratory tract function
Gee, I think this is The End(s)!