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Advances in Gastrointestinal Aspects of CF
Drucy Borowitz, MDProfessor of Clinical Pediatrics
State University of New York at Buffalo
“GI”North American Cystic Fibrosis Conference
Orlando 2012
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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
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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
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Gestational Information: The Respiratory and GI Tracts have the same
Embryologic Origins
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Clearance of Obstruction and Infection
LUNGS• ASL hydration and
mucociliary clearance
• Cough
• Antimicrobials from submucosal glands
GI TRACT• Microvillus and luminal
hydration
• Peristalsis
• Antimicrobials from submucosal glands
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Normal GI Tract Physiology
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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
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Liver
Liver and Pancreatic Ducts Secrete Bicarbonate (HCO3
- ) via CFTR to Neutralize Gastric Acid
(HCO3)
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Crypt-villus location of duodenal HCO3
- secretion under cAMP-stimulated conditions:
GI epithelium (especially Brunner’s Glands) also secretes HCO3
- via CFTR
Walker et al, Gastroenterol 2009
Abstract 126
Symposium 15
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Why is HCO3 Secretion Important?
• It neutralizes gastric acid– Needed for pH optima of pancreatic enzymes and micelles
• It allows mucins to unfold / hydrate• It promotes bacterial killing• HCO3 secretion ≡ Fluid secretion
Abstracts # 13 & 131
Abstracts # 99 & 501
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Bicarbonate Drives Fluid Secretion
Novak et al, J Biol Chem 2011
The normal human pancreas secretes
1-2 L / day
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Pancreatic Enzyme and Bicarbonate Secretion have Different Stimuli
Secretin stimulatesduct to secrete
bicarbonate
Cholecystokinin (CCK) stimulatesacini to release enzymes
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How can you measure GI HCO3?
• Measures pH, pressure and temperature
• Single use• Radiofrequency
detector worn outside body
“pH pill”
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Gelfond et al, Dig Dis Sci. 2012
Gastric Acid Neutralization is delayed in Subjects with CF
N=20
N=20
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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
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Small bowel pH changes (1min means)
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120
03
4
5
6
7
8
Pre VX-770Post VX-770
p< 0.05
8 24
Minutes from gastric emtpying
pH
Gastric Acid Neutralization is normalized in Subjects with CF taking Ivacaftor
N=7
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The Earliest Clinical Consequences of CF are Gastrointestinal
• What is the ontogeny of these conditions?• How can we study them?
• Animal models• Human infants
Meconium ileus (MI)Pancreatic Insufficiency
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Google Infants: Human and Otherwise
What have we learned from CF ferrets and pigs?
What have we learned from CF mouse models?
What have we learned from infants with CF ?
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Lessons from CF Mice
Bowel obstructions occur despite normal pancreatic
function
Slide courtesy of P. Durie
Restoration of CFTR in intestinal epithelium eliminates
obstructionHodges et al, Am J Physiol GLP 2011
Restoration of 10-15% CFTR will avoid intestinal obstruction
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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
MI is not caused by pancreatic dysfunction,but is strongly associated with it
Abstract # 180
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Pancreatic Dysfunction in Infants with CF
• Pancreatic insufficiency can be the first clinical manifestation of CF– Occurs in a majority of infants – Occurs prenatally– Treatment with pancreatic
enzyme replacement therapy (PERT) is life-sustaining
Schwachman, Pediatr 1960
Management of complications of steatorrhea can be done by “conservative measures, including strapping of the buttocks, defecating in a reclining position, and measures designed to reduce the frequency and bulk of stool”… (such as ) ...witholding butter, ice cream, peanut butter, potato chips, french fried potatoes, and mayonnaise….”
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Advances in Pancreatic Enzyme Replacement Therapy (PERT)
• New Drug Application process improved safety• no longer overfilled• improved stability• free of enveloped viruses
• 5 delayed release PERTs approved by US - FDA• Creon (Abbott)• Zenpep (Aptalis)• Pancreaze (Janssen / J&J)• Pertzye (Digestive Care)• Ultresa (Aptalis)
Abstract # 447
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Smyth et al, Lancet 1994
Schwarzenberg et al J Pediatr 1995
Normal vs. Fibrosing Colonopathy
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Addressing Issues with PERT DosingPhase IV surveillance study for fibrosing colonopathy is underway
Years
Baby Observational Nutrition Study (BONUS) sub-study to help answer questions about
dosing in infants
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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
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Schematic Pattern of Evolution of Pancreatic Function in Infants in the
1st Year of Life
O’Sullivan et al, J Pediatr (in press)
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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• Initial levels may be 2 o PI
– “false positive”• Other factors
• Re-measure FE at 1 year of age
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Garner Intelligence
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Poohed
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Microbiomics: Interactive Symbiosis
• Human intestine is colonized by an estimated 100 trillion bacteria– 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
Bronchi
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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
Reference genomes from the human microbiome
Symposium # 10
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Bacterial Communities in Mammalian Intestine
Adapted from Hill et al, Mucosal Immunol 2009
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Abstract # 326
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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
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a= microbes with increased abundance in the intestines early in life b= later in life
Heat Map and Simpson’s Diversity Index of Respiratory and Fecal Microbiomes
Abstract # 279
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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
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Go Inside• Immune responses to intestinal
bacteria inflammation:• Bacterial signals• Toll-like receptors• NOD-like receptors• G protein-coupled receptors
• Measureable clinically with lab tests
Hill & Artis, Ann Rev Immunol 2010
and videoendoscopy
Abstract # 522
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Capsule Video Endoscopy shows Inflammation in CF Intestine
Healthy jejunum Patient with CF
Videos courtesy of M. Wilschanski, Jerusalem Abstract # 510
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Grab Intestine
• New preclinical model systems and clinical trial biomarkers
• Rectal short circuit current measurements (ICM)
• Organoids
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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:
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.
Abstract # 210
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2K+
3Na+
~
Na/K/2Cl
K+
K+Cl-
cAMP Ca++
Na+Amil
Bum
Cl-
NKCC1
NBC1
Na+HCO3-
KvLQT
IK
CaCCCFTR ENaC
IK BKSKCa++
cAMP
KvLQT
cAMP
Ca++
K+
Adapted from H. DeJonge
Colonocyte Ion Transport
• Differences from respiratory epithelial ion transport:– Presence of K+
secretory pathway– Colon is an
absorptive cell (i.e. has more Cl- secretion )
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0
-100
-50
Time (min)
20 40 60 80
50
100
150
200
250
300
Amil CChIndo
AmilFsk/IBMX Bum
Indo
1B. CF
0
50
150
250
300
350
400
Amil
CCh
Bum
Indo
20 40 60 80
1A. Non-CF
Time (min)
100
200
Bum
Fsk/IBMXIsc
(uA/
cm2)
Standard Rectal ICM Recordings
CFTR response CFTR response
↓ response to F/IBMX, mixed response to CCh b/o K+ transport in absence of CFTR
Abstracts # 175 & 256
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Hirtz, S. et al. Gastroenterol, 2004
More CFTR function
Controls Carriers CF-PS CF-PI
CF
TR
cur
rent
-180
-150
-120
-90
-30
-60
30
Intestinal current measurements:genotype/phenotype relationships
Solid line, median; dashed line, 25th and 75th percentiles
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Organoids / Enteroids / Colonoids*
• Are 1o 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
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Crypt Culture in 3D Gels – “Enteroids”
Sato et al, Nature 2009
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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 2012With additional work from L. Clarke lab, Missouri
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Goblet Cell Degranulation after Carbachol
Wild Type CF Knock-out Mouse
Normal Degranulation Granules go into the lumen without undergoing dissolution
lumen lumen
Videos courtesy of L. Clarke lab -Missouri
Abstract # 122
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“The submucosal gland contains the elixir of airway health “ – Dr. Jeff Wine
Abstracts # 86, 87, 89
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Healthy human organoids F508del / F508del organoids
F508del / F508del organoids treatedwith VX-809 + VX-770
Videos courtesy of J. Beekman lab, Utrecht NL
Forskolin induces rapid swelling of organoids |--30μm --|
|--40μm --|
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Forskolin-induced swelling in intestinal human organoids can be quanitated
N=8
N=2
N=11
J. Beekman lab, Utrecht NL
Abstract # 191
![Page 54: Advances in Gastrointestinal Aspects of CF Drucy Borowitz, MD Professor of Clinical Pediatrics State University of New York at Buffalo “GI” North American](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9d5503460f94a861b0/html5/thumbnails/54.jpg)
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
![Page 55: Advances in Gastrointestinal Aspects of CF Drucy Borowitz, MD Professor of Clinical Pediatrics State University of New York at Buffalo “GI” North American](https://reader036.vdocuments.mx/reader036/viewer/2022062321/56649d9d5503460f94a861b0/html5/thumbnails/55.jpg)
Gee, I think this is The End(s)!