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January 29, 2018 | Author: Anonymous | Category: Science, Health Science, Immunology
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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


• 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


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



6 5 4 3 0





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


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)


vs. Fibrosing Colonopathy Smyth et al, Lancet 1994

Schwarzenberg et al J Pediatr 1995

Addressing Issues with PERT Dosing 20


# of cases

18 16






10 9












4 1

8 6

3 2

2 0


Phase IV surveillance study for fibrosing colonopathy is underway


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


• Initial levels may be 2 PI – “false positive”

• Other factors

• Re-measure FE at 1 year of age

Garner Intelligence


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-


Na+ Amil ENaC






Na/K/2Cl Na+HCO3-


2K+ cAMP

Ca++ NBC1




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)





250 200 Indo

CFTR response













-100 20







40 60 Time (min)






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


CFTR current

-120 -90 -60 -30


Controls Carriers



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


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



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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