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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)!
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