Gastroenterology and Nutrition: Neonatology Questions and Controversies Series E-Book
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Gastroenterology and Nutrition, a volume in Dr. Polin’s Neonatology: Questions and Controversies Series, offers expert authority on the toughest neonatal gastroenterologic and nutritional challenges you face in your practice. This medical reference book will help you provide better evidence-based care and improve patient outcomes with research on the latest advances.

  • Reconsider how you handle difficult practice issues with coverage that addresses these topics head on and offers opinions from the leading experts in the field, supported by evidence whenever possible.
  • Find information quickly and easily with a consistent chapter organization.
  • Get the most authoritative advice available from world-class neonatologists who have the inside track on new trends and developments in neonatal care.
  • Stay current in practice with coverage on what the controversies are and where the field is moving in terms of basic intestinal development and nutritional requirements for the neonate.


Derecho de autor
Cardiac dysrhythmia
Vitamin D
Autoimmune disease
Gastrointestinal physiology
Systemic disease
Abdominal distension
Blood in stool
Diabetes mellitus type 1
Developmental disability
Atopic dermatitis
Necrotizing enterocolitis
Gastrointestinal perforation
Protein S
Short bowel syndrome
Breast milk
Biological agent
Biliary atresia
Feeding tube
Insulin-like growth factor 1
Iron deficiency anemia
Toll-like receptor
Palmitic acid
Physician assistant
Positive airway pressure
Temperance (virtue)
Glycemic index
Bowel obstruction
Intensive-care medicine
Saturated fat
Medical ventilator
Parenteral nutrition
Gastroesophageal reflux disease
Posttranslational modification
Gene expression
Diabetes mellitus type 2
Intrauterine growth restriction
Medical ultrasonography
Cellular respiration
Human gastrointestinal tract
Mucous membrane
Coeliac disease
Crohn's disease
Large intestine
Vitamin A
Diabetes mellitus
Vitamin K
Transcription factor
Data storage device
Protein biosynthesis
Messenger RNA
Infectious disease
Fatty acid
Amino acid
Maladie infectieuse


Publié par
Date de parution 23 février 2012
Nombre de lectures 2
EAN13 9781455733699
Langue English
Poids de l'ouvrage 2 Mo

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Gastroenterology and Nutrition
Neonatology Questions and Controversies
Second Edition

Josef Neu, MD
Professor of Pediatrics, University of Florida College of Medicine, Gainesville, Florida
Table of Contents
Cover image
Title page
Series page
Series Foreword
Section A: Basic Science of the Intestinal Tract
Chapter 1: Overview of Digestion and Absorption
Chapter 2: Maturation of Motor Function in the Preterm Infant and Gastroesophageal Reflux
Chapter 3: Development of Gastrointestinal Motility Reflexes
Chapter 4: Development of the Intestinal Mucosal Barrier
Chapter 5: The Developing Intestinal Microbiome and Its Relationship to Health and Disease
Chapter 6: The Developing Intestine as an Immune Organ
Chapter 7: The Developing Gastrointestinal Tract in Relation to Autoimmune Disease, Allergy, and Atopy
Chapter 8: What Are the Controversies for Basic Intestinal Development and Where Will the Field Be Moving in the Future?
Section B: Nutritional Requirements and Strategies
Chapter 9: Nutritional Requirements of the Very-Low-Birthweight Infant
Chapter 10: Controversies in Neonatal Nutrition: Macronutrients and Micronutrients
Chapter 11: Regulation of Protein Synthesis and Proteolysis in the Neonate by Feeding
Chapter 12: Lipids for Neonates
Chapter 13: Human Milk Feeding of the High-Risk Neonate
Chapter 14: Nutritional Requirements for the Neonate: What Are the Controversies and Where Will the Field Be Moving in the Future?
Section C: Clinical Conditions
Chapter 15: Necrotizing Enterocolitis
Chapter 16: Special Nutrition of the Surgical Neonate
Chapter 17: Controversies in Short Bowel Syndrome
Chapter 18: Neonatal Cholestasis
Chapter 19: The Neonatal Gastrointestinal Tract as a Conduit to Systemic Inflammation and Developmental Delays
Chapter 20: Adult Consequences of Neonatal and Fetal Nutrition: Mechanisms
Chapter 21: Technologies for the Evaluation of Enteral Feeding Readiness in Premature Infants
Chapter 22: What Are the Controversies for These Clinical Conditions and Where Will the Field Be Moving in the Future?
Series page
Neonatology Questions and Controversies
Series Editor
Richard A. Polin, MD
Professor of Pediatrics
College of Physicians and Surgeons
Columbia University
Vice Chairman for Clinical and Academic Affairs
Department of Pediatrics
Director, Division of Neonatology
Morgan Stanley Children’s Hospital of NewYork-Presbyterian
Columbia University Medical Center
New York, New York
Other Volumes in the Neonatology Questions and Controversies Series

1600 John F. Kennedy Blvd.
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Philadelphia, PA 19103-2899
Gastroenterology and Nutrition: Neonatology Questions and Controversies second edition ISBN: 978-1-4377-2603-9
Copyright © 2012, 2008 by Saunders and imprint of Elsevier Inc.
All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: .
This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.
Library of Congress Cataloging-in-Publication Data
Gastroenterology and nutrition: neonatology questions and controversies / [edited by] Josef Neu. — 2nd ed.
p. ; cm.
Includes bibliographical referencse and index.
ISBN 978-1-4377-2603-9 (hardback)
I, Neu, Josef.
[DNLM: 1. Gastrointestinal Disease. 2. Infant, Newborn, Diseases. 3. Infant Nutritional Physiological Phenomena. 4. Infant, Newborn. WS 310]
Content Strategist: Stefanie Jewell-Thomas
Content Development Specialist: Lisa Barnes
Publishing Services Manages: Peggy Fagen and Hemamalini Rajendrababu
Project Manager: Deepthi Unni
Designer: Ellen Zanolle
Printed in The United States of America
Last digit is the print number: 9 8 7 6 5 4 3 2 1

Kjersti Aagaard-Tillery, MD, PhD
Assistant Professor Baylor College of Medicine Obstetrics and Gynecology Houston, Texas
Adult Consequences of Neonatal and Fetal Nutrition: Mechanisms

Joel M. Andres, MD
Professor, Pediatrics University of Florida College of Medicine Gainesville, Florida
Neonatal Cholestasis

Tracy Gautsch Anthony, PhD
Associate, Professor Department of Biochemistry and Molecular Biology Indiana University School of Medicine Evansville, Indiana
Regulation of Protein Synthesis and Proteolysis in the Neonate by Feeding

Carolyn Berseth, MD
Director, Medical Affairs North America Mead Johnson Company Evansville, Indiana.
Development of the Gastrointestinal Motility Reflexes

Ricardo A. Caicedo, MD
Associate Professor Pediatrics, Gastroenterology and Nutrition Levine Children’s Hospital Carolinas Medical Center Charolotte, North Carolina
Development of the Intestinal Mucosal Barrier

Ashish N. Debroy, MD
Department of Pediatrics Divisions of Gastroenterology and Neonatology University of Texas Medical School at Houston Houston, Texas
Controversies in Short Bowel Syndrome

Clotilde desRobert-Marandet, MD
Neonatal Intensive Care Unit University Hospital of Nantes Nantes, France
Adult Consequences of Neonatal and Fetal Nutrition: Mechanisms

Frank R. Greer, BS, MD
Professor, Pediatrics University of Wisconsin School of Medicine and Public Health Madison, Wisconsin
Controversies in Neonatal Nutrition: Macronutrients and Micronutrients

Allah B. Haafiz, MD
Assistant Professor of Pediatrics University of Florida College of Medicine Division of Gastroenterology and Hepatology Gainesville, Florida
Neonatal Cholestasis

William W. Hay, Jr., MD
Professor Department of Pediatrics (Neonatology) University of Colorado School of Medicine Aurora, Colorado
Nutritional Requirements of the Very-Low-Birthweight Infant

Anna Maria Hibbs, MD, MSCE
Assistant Professor, Pediatrics Case Western Reserve University Cleveland, Ohio Director Nutrition and Metabolism Child and Family Research Institute Scientific and Professional Staff Division of Neonatology B.C. Children’s and Women’s Hospitals Vancouver, Canada
Maturation of Motor Function in the Preterm Infant and Gastroesophageal Reflux

Essam Imseis, MD
Department of Pediatrics Divisions of Gastroenterology and Neonatology University of Texas Medical School at Houston Houston, Texas
Controversies in Short Bowel Syndrome

Sheila M. Innis, PhD
Professor, Pediatrics University of British Columbia Director Nutrition and Metabolism Child and Family Research Institute Scientific and Professional Staff Division of Neonatology British Columbia Children’s and Women’s Hospitals Vancouver, Canada
Lipids for Neonates

Sudarshan Rao Jadcherla, MD, FRCPI, DCH, AGAF
Professor, Department of Pediatrics The Ohio State University College of Medicine Sections of Neonatology and Pediatric Gastroenterology & Nutrition Columbus, Ohio
Development of Gastrointestinal Motility Reflexes

Tom Jaksic, MD
W. Hardy Hendren Professor Surgery Harvard Medical School Vice Chairman Department of Pediatric General Surgery Children’s Hospital Boston Boston, Massachusettes
Special Nutrition of the Surgical Neonate

Lisa A. Joss-Moore, PhD
Assistant Professor, Pediatrics University of Utah Salt Lake City, Utah
Adult Consequences of Neonatal and Fetal Nutrition: Mechanisms

Jamie Kuang Horn Kang, MD
Research Fellow Department of Surgery Harvard Medical School Research Fellow Department of Surgery Children’s Hospital Boston Boston, Massachusettes
Special Nutrition of the Surgical Neonate

Ee-Kyung Kim, MD
Department of Pediatrics Seoul National University Children’s Hospital Seoul, Korea
Technologies for the Evaluation of Enteral Feeding Readiness in Premature Infants

Robert H. Lane, MD, MS
Professor, Neonatology University of Utah Neonatology University Health Care Neonatology Primary Children’s Medical Center Intermountain Healthcare Salt Lake City, Utah
Adult Consequences of Neonatal and Fetal Nutrition: Mechanisms

Patricia Lin, MD
Assistant Profesor of Pediatrics Pediatrics Emory University School of Medicine Atlanta, Georgia
The Developing Intestine as an Immune Organ

Volker Mai, PhD
Assistant Professor Microbiology and Cell Science University of Florida Gainesville, Florida
The Developing Intestinal Microbiome and Its Relationship to Health and Disease

Camilia R. Martin, MD, MS
Assistant Professor of Pediatrics Department of Pediatrics Division of Newborn Medicine Harvard Medical School Associate Director Neonatal Intensive Care Unit Department of Neonatology Director For Cross Disciplinary Research Partnerships Division of Translational Research Beth Israel Deaconess Medical Center Boston, Massachusettes
Development of the Intestinal Mucosal Barrier

Nicole Mitchell, MD
Adult Consequences of Neonatal and Fetal Nutrition: Mechanisms

Susan Hazels Mitmesser, PhD
Manager, Global Medical Communications Medical Affairs Mead Johnson Nutritiion Evansville, Indiana
Regulation of Protein Synthesis and Proteolysis in the Neonate by Feeding

Ardythe L. Morrow, PhD
Professor Environmental Health Nutrition University of Cincinnati College of Medicine Cincinnati, Ohio Director Center for Interdisciplinary Research in Human Milk and Lactation Perinatal Institute Cincinnati Children’s Hospital Cincinnati, Ohio
Human Milk Feeding of the High-Risk Neonate

Fernando Navarro, MD
Department of Pediatrics Divisions of Gastroenterology and Neonatology University of Texas Medical School at Houston Houston, Texas
Controversies in Short Bowel Syndrome

Ursula Nawab, MD
Department of Pediatrics Divisions of Gastroenterology and Neonatology University of Texas Medical School at Houston Houston, Texas
Controversies in Short Bowel Syndrome

Andrew S. Neish, MD
Epithelial Pathobiology Unit Department of Pathology and Laboratory Medicine Emory University School of Medicine Atlanta, Georgia
The Developing Intestine as an Immune Organ

Josef Neu, MD
Professor of Pediatrics University of Florida College of Medicine Gainesville, Florida
Overview of Digestion and Absorption
The Developing Intestinal Microbiome and Its Relationship to Health and Disease
What Are the Controversies for Basic Intestinal Development and Where Will the Field Be Moving in the Future?
Nutritional Requirements for the Neonate: What Are the Controversies and Where Will the Field Be Moving in the Future?
Necrotizing Enterocolitis
The Neonatal Gastrointestinal Tract as a Conduit to Systemic Inflammation and Developmental Delays
Technologies for the Evaluation of Enteral Feeding Readiness in Premature Infants
What Are the Controversies for These Clinical Conditions and Where Will the Field Be Moving in the Future?

Sungho Oh, MD
Division of Neonatology Department of Pediatrics University of Florida
Technologies for the Evaluation of Enteral Feeding Readiness in Premature Infants

Ravi M. Patel, MD
Assistant Professor of Pediatrics Department of Pediatrics Division of Neonatal-Perinatal Medicine Emory University School of Medicine Attending Neonatologist Children’s Healthcare of Atlanta Atlanta, Georgia
The Developing Intestine as an Immune Organ

J. Marc Rhoads, MD
Professor, Pediatrics University of Texas Houston, Texas
Controversies in Short Bowel Syndrome

Renu Sharma, MD
Neonatal Biochemical Nutrition and GI Development Laboratory Department of Pediatrics Division of Neonatology University of Florida Gainesville, Florida
Necrotizing Enterocolitis

Patti J. Thureen, MD
Professor Department of Pediatrics (Neonatology) University of Colorado School of Medicine Aurora, Colorado
Nutritional Requirements of the Very-Low-Birthweight Infant

Outi Vaarala, MD, PhD
Professor of Pediatric Immunology Immune Response Unit National Institute for Health and Welfare Biomedicum1 Helsinki Helsinki, Finland
The Developing Gastrointestinal Tract in Relation to Autoimmune Disease, Allergy, and Atopy

Christina J. Valentine, MD, MS, RD
Assistant Professor Department of Pediatrics The University of Cincinnati Cincinnati, Ohio Neonatologist, Principal Investigator Division of Neonatology Perinatal and Pulmonary Biology Cincinnati Children’s Hospital Medical Center Cincinnati, Ohio
Human Milk Feeding of the High-Risk Neonate

W. Allan Walker, MD
Conrad Taff Professor of Nutrition and Pediatrics Department of Pediatrics Division of Nutrition Harvard Medical School Director, Mucosal Immunology Laboratory Pediatrics Massachusettes General Hospital Boston, Massachusettes
Development of the Intestinal Mucosal Barrier

James L. Wynn, MD
Assistant Professor of Pediatrics Pediatrics Duke University Durham, North Carolina
The Neonatal Gastrointestinal Tract as a Conduit to Systemic Inflammation and Developmental Delays

Christopher Young, MD
Neonatal Biochemical Nutrition and GI Development Laboratory Department of Pediatrics Division of Neonatology University of Florida Gainesville, Florida
Necrotizing Enterocolitis
Series Foreword

Richard A. Polin, MD

“Medicine is a science of uncertainty and an art of probability.”
—William Osler
Controversy is part of every day practice in the NICU. Good practitioners strive to incorporate the best evidence into clinical care. However, for much of what we do, the evidence is either inconclusive or does not exist. In those circumstances, we have come to rely on the teachings of experienced practitioners who have taught us the importance of clinical expertise. This series, “Neonatology Questions and Controversies,” provides clinical guidance by summarizing the best evidence and tempering those recommendations with the art of experience.
To quote David Sackett, one of the founders of evidence-based medicine:

Good doctors use both individual clinical expertise and the best available external evidence and neither alone is enough. Without clinical expertise, practice risks become tyrannized by evidence, for even excellent external evidence may be inapplicable to or inappropriate for an individual patient. Without current best evidence, practice risks become rapidly out of date to the detriment of patients.
This series focuses on the challenges faced by care providers who work in the NICU. When should we incorporate a new technology or therapy into every day practice, and will it have positive impact on morbidity or mortality? For example, is the new generation of ventilators better than older technologies such as CPAP, or do they merely offer more choices with uncertain value? Similarly, the use of probiotics to prevent necrotizing enterocolitis is supported by sound scientific principles (and some clinical studies). However, at what point should we incorporate them into every day practice given that the available preparations are not well characterized or proven safe? A more difficult and common question is when to use a new technology with uncertain value in a critically ill infant. As many clinicians have suggested, sometimes the best approach is to do nothing and “stand there.”
The “Questions and Controversies” series was developed to highlight the clinical problems of most concern to practitioners. The editors of each volume (Drs. Bancalari, Oh, Guignard, Baumgart, Kleinman, Seri, Ohls, Maheshwari, Neu, and Perlman) have done an extraordinary job in selecting topics of clinical importance to every day practice. When appropriate, less controversial topics have been eliminated and replaced by others thought to be of greater clinical importance. In total, there are 56 new chapters in the series. During the preparation of the “Hemodynamics and Cardiology” volume, Dr. Charles Kleinman died. Despite an illness that would have caused many to retire, Charlie worked until near the time of his death. He came to work each day, teaching students and young practitioners and offering his wisdom and expertise to families of infants with congenital heart disease. We are dedicating the second edition of the series to his memory. As with the first edition, I am indebted to the exceptional group of editors who chose the content and edited each of the volumes. I also wish to thank Lisa Barnes (content development specialist at Elsevier) and Judy Fletcher (publishing director at Elsevier), who provided incredible assistance in bringing this project to fruition.
Over the past several years, with improved survival of critically ill and very small preterm infants, neonatologists are focusing on nutrition of these infants as a means to prevent morbidities associated with intensive care, such as chronic lung disease and the complications of neurologic injuries. The intestine is being looked upon as more than a digestive absorptive organ, with the recognition that it has a very large surface area which serves as a barrier to potentially dangerous microbes and food antigens. When this breaks down, there is a huge potential for translocation of bacteria to the bloodstream, sepsis, inflammation, and accompanying short- and long-term complications.
The microbes that reside within the lumen of the intestine are increasingly being recognized as mediators of growth with the provision of nutrients that are products of their metabolism, as well as major mediators of inflammatory processes in the intestine. They play a major role in modulation of innate immunity as well as development of adaptive immunity. Alterations of the normal microbiota in the developing intestine of these infants (“dysbiosis”) are associated with diseases such as necrotizing enterocolitis (NEC).
New technologies are rapidly evolving that may help us assess inflammatory processes in the intestinal tract. Technologies that may assist the neonatologist to determine feeding readiness as well as propensity to develop diseases such as NEC and late onset sepsis are rapidly evolving. Methodologies to evaluate these as well as epigenetic mechanisms that lead to diseases such as metabolic syndrome are being developed and discussed.
In this revised edition, we continue to incorporate clinical neonatal gastroenterology and nutrition with up-to-date research. We hope that it will not only provide guidance for clinical care with clarification of some of the controversies related to nutrition, feeding, and neonatal intestinal disease but also stimulate new avenues of research that will be pertinent to optimizing the care of these infants and providing them the opportunity to reach their full genetic potential.

Josef Neu, MD
Section A
Basic Science of the Intestinal Tract
Chapter 1 Overview of Digestion and Absorption

Josef Neu, MD

• Protein Digestion and Absorption
• Carbohydrate Digestion and Absorption
• Lipid Digestion and Absorption
Along with its role as the largest and most active immune organ of the body, the intestine is involved in important endocrine and exocrine roles and also encompasses neural tissue equivalent to that of the entire spinal cord. The intestinal luminal microbiota interactions with the intestinal mucosa and submucosa are becoming increasingly recognized as critical in health and disease. In addition to these seemingly newfound functions of the gastrointestinal tract that will be discussed in subsequent chapters, the intestine’s role in digestion and absorption of nutrients remains of utmost importance in health. Its development during neonatal and early childhood periods needs to be understood to optimize nutrition during these highly critical windows of development. Here, basic physiology of some of the major aspects of macronutrient (protein, carbohydrate, and lipid) digestion and absorption during early life will be provided and related to developmental maturation and clinical strategies based on these principles.

Growth: There is a close interplay between overall size of the intestine and surface area. In the term infant, the length of the small intestine is about 200 cm, and the villous and microvillous architecture provides a huge surface area that is much larger than that of the skin. Half of the growth in length of the intestine occurs in the last trimester of gestation. 1
Digestion: Large molecular aggregates need to be processed by mechanical and chemical means starting in the mouth, stomach, and upper small intestine. The enzymatic and other chemical processes in luminal digestion involve interactions between gastric acid, lipases (lingual, gastric, pancreatic, and milk derived), salivary- and pancreatic-derived carbohydrases, pepsin, pancreatic-derived proteases, lipases, and bile.
Absorption: The intestinal epithelium is composed of a population of diverse cells whose functions differ along the aboral (or horizontal) as well as the crypt to villus (vertical) gradients. As ingested nutrients travel through the intestine, they are sequentially exposed to regions that have epithelia with very different absorptive characteristics; permeability, transporter, and enzymatic functions differ markedly along the proximal-distal portions of the intestine.
Processes for digestion and absorption of protein, carbohydrates, and lipids are described separately in this chapter. A brief general review of major physiologic processes for each of the macronutrients is first provided, then development of these processes during fetal and early postnatal life is described. Correlations of some of these principles to patient care will also be presented.

Protein Digestion and Absorption

The composition of proteins ingested by neonates largely reflects that in either mother’s milk or commercial formulas. Digestion of proteins begins in the acidic environment of the stomach and continues in the small intestine under the influence of pancreatic proteases and peptidases.
Dietary proteins in human infants are, with very few exceptions, not absorbed intact. Rather, they must first be digested into amino acids or dipeptides and tripeptides. Proteolytic enzymes are secreted into the lumen of the upper digestive tube from two primary sources: (1) the stomach secretes pepsinogen, which is converted to the active protease pepsin by the action of acid; and (2) the pancreas secretes a group of potent proteases, chief among them trypsin, chymotrypsin, and carboxypeptidases, which require activation by enterokinase. Through the action of these gastric and pancreatic proteases, dietary proteins are hydrolyzed within the lumen of the small intestine predominantly into medium and small peptides (oligopeptides).
These small peptides, primarily dipeptides and tripeptides, are absorbed into the small intestinal epithelial cell by cotransport with H + ions. 2, 3 Once inside the enterocyte, the vast bulk of absorbed dipeptides and tripeptides are hydrolyzed into single amino acids by cytoplasmic peptidases and exported from the cell into blood. Only a very small number of these small peptides enter blood intact.
The mechanism by which amino acids are absorbed by the epithelial cell is similar to that of monosaccharides. The luminal plasma membrane of the absorptive cell bears several sodium-dependent amino acid transporters—one each for acidic, basic, neutral, and amino acids. 4 These transporters bind amino acids only after binding sodium, after which a conformational change allows entry of sodium and the amino acid into the cytoplasm, followed by its reorientation back to the original form. Thus, absorption of amino acids is dependent on the electrochemical gradient of sodium across the epithelium. Further, absorption of amino acids, like that of monosaccharides, contributes to generating the osmotic gradient that drives water absorption. The basolateral membrane of the enterocyte contains additional transporters that export amino acids from the cell into blood. These are not dependent on sodium gradients.

Developmental Aspects of Protein Digestion and Absorption


Gastric Acidity
The first traces of gastric acidity appear in 4-month-old fetuses. 5 The human fetus has the potential to produce gastric acid and gastrin from the middle of the second trimester. Parietal cell activity is present in the body, antrum, and pyloric regions in the fetus from 13 to 28 weeks. 5 - 7 When comparing full-term and premature infants, hydrochloric acid secretion was found to be much lower in premature infants than in full-term infants. 8
Gastric acid secretion is limited in very-low-birthweight (VLBW) infants. However, both basal and pentagastrin-stimulated acid secretion doubles from the first to fourth week of postnatal life in preterm infants. 9 The actual pH of the stomach contents in infants is substantially influenced by food intake. The entry of milk into the infant’s stomach causes a sharp increase in the pH of the gastric contents and a slower return to lower pH values than in older children and adults. 10

Gastric Proteolytic Activity
The output of pepsin is low in the newborn infant and increases until the third postnatal month. The range of values found in the second and third postnatal months is less than the range of adult values. 11 In contrast, pepsin activity in biopsy specimens from the stomachs of infants and children did not change between the ages of 6 months and 15 years. 12 Formula feeding evokes an increase of pepsin activity in the stomach content of 3- to 4-week-old orogastrically fed premature infants. 13

Pancreatic Proteolytic Activity
The protease cascade in the small intestine is catalyzed by food-stimulated secretion of enterokinase from the upper small intestinal epithelium. Enterokinase catalyzes the conversion of pancreatic pro-proteases to active enzymes ( Table 1-1 ). Even though enterokinase is detectable at 24 weeks’ gestation, its concentration is relatively low and reaches only 25% of adult activity at term. 14 This theoretically can be limiting to protein digestion and may be responsible for an increased capability of larger antigens or microorganisms to pass into the intestine without breakdown by luminal enzymes.

Proteolytic enzymes contained in gastric juice
Requires acid environment of stomach to hydrolyze protein
Synthesized in the gastric chief cells as inactive pre-proenzymes (pepsinogen) Intestine and Pancreas Enterokinase—an intestinal brush border enzyme that activates pancreatic proteases and is stimulated by trypsinogen contained in pancreatic juice Pancreatic Endopeptidases

Trypsin: cleaves peptide bonds on the carboxyl side of basic amino acids (lysine and arginine)
Chymotrypsin: cleaves peptide bonds on the carboxyl side of aromatic amino acids (tryosine, phenylalanine and tryptophan)
Elastase: cleaves peptide bonds on the carboxyl side of aliphatic amino acids (alanine, leucine, glycine, valine, isoleucine) Pancreatic Exopeptidases

Carboxypeptidases A and B: zinc-containing metalloenzymes that remove single amino acids from the carboxyl-terminal ends of proteins and peptides
Carboxypeptidase A: polypeptides with free carboxyl groups are cleaved to lower peptides and aromatic amino acids
Carboxypeptidase B: polypeptides with free carboxyl groups are cleaved to lower peptides and dibasic amino acids
Pancreatic enzymes begin to form at about the third fetal month, 15 and pancreatic secretion starts at the beginning of the fifth month of gestation. Levels of trypsin concentration encountered during the first 2 years of life are reached by the age of 3 months. From birth onward, the concentration of chymotrypsin (after pancreozymin-secretin stimulation) increases about threefold and reaches adult levels in 3-year-old children. Serial measurement of fecal chymotrypsin concentrations in preterm infants (23 to 32 weeks’ gestation) during the first 4 weeks of life demonstrated values generally similar to those found in term infants. Premature infants fed soy-based formula for 1 month exhibited higher trypsin activity after cholecystokinin-pancreozymin stimulation than did those fed a milk-based formula. 16

For a very few days after birth, most mammalian neonates have the ability to absorb intact proteins. This ability, which is rapidly lost, is of importance because it allows the newborn animal to acquire passive immunity by absorbing immunoglobulins in colostral milk. The small intestine rapidly loses its capacity to absorb intact proteins—a process called closure; and consequently, animals that do not receive colostrum within the first few days after birth will likely die from opportunistic infections.
The ability of the gastrointestinal tract to exclude antigenically intact food proteins increases with gestational age, and gut closure occurs normally before birth in humans. 17 Using lactulose-to-mannitol ratios, preterm infants’ (26 to 36 weeks’ gestation) intestinal permeability was not related to gestational age or birthweight but was higher during the first 2 days of life than 3 to 6 days later. It is higher in preterm infants than in healthy term infants only if measured within 2 days of birth. This suggests rapid postnatal adaptation of the small intestine in preterm infants. 18

Proteolytic and Peptidase Activity
Beginning in the eighth week of gestation, villi are formed from the duodenum up to the ileum, and after week 9, differentiation of the crypts of Lieberkühn is observed. The activity of proteases is high (especially DPP IV) in the differentiating microvillous zone of primitive enterocytes. The gradient of apex-base activity of the villus is maximal on the apex of the villi. In one study, brush border and intracellular proteolytic enzyme activities were measured in fetuses (8 to 22 weeks’ gestation), children (7 months to 14 years of age), and adults. The peptidase activities in all three of the groups were comparable, suggesting that the small intestine of the term and preterm newborn should be able to efficiently digest peptides. 19

Clinical Correlations
Acid secretion limitations in premature infants should be kept in mind when considering the use of histamine-2 (H 2 ) blockers, which are widely prescribed in many neonatal intensive care units. Studies suggest that critically ill premature infants treated with H 2 blockers have a higher incidence of nosocomial sepsis and necrotizing enterocolitis. 20, 21 Although speculative, it is possible that with the already limited hydrogen ion production in the stomach of the premature infant, additional blockage further diminishes the acid barrier to microorganisms and allows for a higher load of bacteria in the more distal regions of the intestine.
In terms of protein absorption, the mechanisms for brush border hydrolysis are present early; dipeptides and tripeptides are absorbed faster than amino acids, and protein digestion and absorption rarely appear to be an issue in premature infants. 22
Despite the potential limitations of digesting and enzymatic capability in premature infants, data showing significant benefits using hydrolyzed protein fractions appear to offer only minimal advantage over whole protein formulas. 20, 23 Studies have yet to demonstrate a benefit of hydrolyzed protein formulas over human milk.
Hydrolyzed formulas are also extensively prescribed to prevent allergic and atopic disease. However, a recent Cochrane meta-analysis showed no evidence to support feeding with a hydrolysed formula for the prevention of allergy over exclusive breastfeeding. Furthermore, in high-risk infants who are unable to be completely breastfed, there is limited evidence that prolonged feeding with a hydrolyzed formula compared with a cow’s milk formula reduces infant and childhood allergy and infant cow’s milk allergy. 24

Carbohydrate Digestion and Absorption

Starches and complex carbohydrates must first be hydrolyzed to oligosaccharides by digestive processes in the mouth, stomach, and intestinal lumen. This is accomplished primarily through salivary and pancreatic amylases. Oligosaccharides must then be hydrolyzed at the epithelial brush border to monosaccharides before absorption, and the key catalysts in these processes are the brush border hydrolases, which include maltase, lactase, and sucrase. Dietary lactose, sucrose, and maltose come in contact with the surface of absorptive epithelial cells covering the villi where they engage with brush border hydrolases: maltase, sucrase, and lactase.

Developmental Aspects of Carbohydrate Digestion and Absorption

Carbohydrate Digestion
There is no difference in amylase activity in preterm and term human milk. The isoamylase of preterm milk is of the salivary type, just as in term milk. There is no great variation in amylase activity during a feeding or from one feeding to another. 25, 26 This can survive the relatively mild acidity and the lower activity of pepsin in the stomach of the newborn infant. Amylase in saliva is present in lower concentrations in children than in adults. 27
Pancreatic amylase activity has been demonstrated in amniotic fluid and pancreatic tissue from 14- to 16-week-old fetuses. 28, 29 Although salivary amylase activity rapidly increases shortly after term birth, pancreatic amylase remains low until 3 months and does not reach adult levels until nearly 2 years of age. 30

Carbohydrate Absorption

Activity of sucrase and lactase is lower in young fetuses than in specimens from the small intestinal mucosa of adults. 14 Sucrase activity is present in the fetal colon and disappears before birth. 31 The presence of lactase in the fetal colon (13 to 20 weeks of age) has also been described previously. 14 Villa and coworkers 32 confirmed that intestinal lactase is low between 14 and 20 weeks of gestation and exhibits a relatively high level of activity at 37 weeks; amounts of lactase messenger RNA (mRNA) correlated with the enzymatic activity. It is interesting that lactase mRNA was not detectable in the colon of normal adult subjects, whereas it was detectable at low levels in fetal colon. 33 Sucrase-isomaltase in the human fetal intestine is present in a different form from that in adults, and it differs in degree of glycosylation (different electrophoretic mobility) and in the size of the polypeptide. 34 The difference in polypeptide length in fetuses and adults can be related to low activity of pancreatic proteases in the fetal intestinal lumen.

Studies suggest that colonic fermentation activity is adequate for colonic salvage of lactose even during the second week of life. Using a stable isotope method for serial assessment of lactose carbon assimilation, Kien and associates 35 demonstrated efficient absorption of lactose in premature infants (30 to 32 weeks’ gestation and 11 to 36 days of age). Despite that finding, a study in which 130 preterm infants fed standard preterm formula with and without lactase showed that lactase-treated infants grew faster over the first 10 days of life but similarly thereafter, suggesting that limitations in lactose absorption are short-lived in the preterm infant. 36

Absorption of Monosaccharides
Glucose absorption in infants is less efficient than in adults ( Table 1-2 ). Kinetics of glucose absorption are related to gestational age and appear to be affected by diet and exposure to glucocorticoids. 37 Other studies demonstrated that carrier-mediated monosaccharide absorption increases the first 2 postnatal weeks in infants born at 28 to 30 weeks’ gestation. 38

Glucose uptake is Na + dependent.
Fructose is absorbed through facilitated diffusion.
Galactose and glucose are actively transported.
1 SGLT1 is the transport protein responsible for Na + -dependent glucose transport.
2 Glut-2 transports glucose out of the cell into the portal circulation.

Clinical Correlations
Because pancreatic secretion is poorly developed in the first several months after birth, this mode of starch hydrolysis could serve as a limiting factor that leaves substantial undigested starch in the intestine. Many infant formulas, including those formulated for preterm infants, contain partially hydrolyzed starches. The more extensively the starch is hydrolyzed, the less reliance is placed on an immature digestive capability, but the greater the osmolality. Whether there is any advantage of these hydrolyzed starch formulas over those containing disaccharides or lactose has not been established.
A study in premature infants was designed to ascertain whether the timing of feeding initiation affected the development of intestinal lactase activity and whether there are clinical ramifications of lower lactase activity. 39 Early feeding increased intestinal lactase activity in preterm infants. Lactase activity is a marker of intestinal maturity and may influence clinical outcomes. Whether the effects of milk on lactase activity were due to the greater concentration of lactose in human milk compared with that in formula has not yet been determined. 39
The finding of low lactase activities in the intestine of fetuses has led to the notion that premature babies cannot tolerate lactose. 14 The presence of a high lactose concentration in human milk should not be a contraindication for its use in the VLBW infant. Microbial salvage pathways that convert nonabsorbed lactose to short-chain fatty acids that can be absorbed and utilized for energy production are functional in these infants ( Fig. 1-1 ). 35 Furthermore, feedings for VLBW infants rarely are initiated at levels intended to meet the infants’ entire nutritional requirements and usually are advanced slowly. The rationale for using a lactose-free formula instead of human milk or even a commercial lactose-containing formula is weak and theoretically may be harmful. Slow initiation of enteral feedings is unlikely to exceed the lactose hydrolytic and salvage capability of the small and large intestines.

Figure 1-1 Lactase deficiency, fermentation by microbes in the distal intestine and production of short-chain fatty acids (SCFA).

Lipid Digestion and Absorption

The bulk of dietary lipid is triglyceride, composed of a glycerol backbone with each carbon linked to a fatty acid through an ester moiety. Foodstuffs typically also contain phospholipids, cholesterol, and many minor lipids, including fat-soluble vitamins. In order for the triglyceride to be absorbed, two processes must occur ( Fig. 1-2 ):

• Large aggregates of dietary triglyceride, which are virtually insoluble in an aqueous environment, must be broken down physically and held in suspension—a process called micellar emulsification.
• Triglyceride molecules must be enzymatically digested through triglyceride hydrolysis to yield monoglyceride and fatty acids, both of which can efficiently diffuse or be transported into the enterocyte .

Figure 1-2 A, Bile acid emulsification of lipids. B, Lipase hydrolysis of triglyceride.
The key mediators in these two transformations are bile acids and lipases. Bile acids are also necessary to solubilize other lipids, including cholesterol.

Emulsification, Hydrolysis, and Micelle Formation
Bile acids promote lipid emulsification. Bile acids have both hydrophilic and hydrophobic domains (i.e., they are amphipathic). On exposure to a large aggregate of triglyceride, the hydrophobic portions of bile acids intercalate into the lipid, with the hydrophilic domains remaining at the surface. Such coating with bile acids aids in breakdown of large aggregates or droplets into smaller and smaller droplets. For a given volume of lipid, the smaller the droplet size, the greater the surface area, which provides greater surface area for interaction with lipase.
Hydrolysis of triglyceride into monoglyceride and free fatty acids is accomplished predominantly by pancreatic lipase. The activity of this enzyme is to clip the fatty acids at positions 1 and 3 of the triglyceride, leaving two free fatty acids and a 2-monoglyceride. As monoglycerides and fatty acids are liberated through the action of lipase, they retain their association with bile acids and complex with other lipids to form micelles, which are small aggregates (4 to 8 nm in diameter) of mixed lipids and bile acids suspended within the ingesta. Micelles, providing much greater lipid surface area than the original fat globule, allow for amplified interaction with the brush border of small intestinal enterocytes, where the monoglyceride and fatty acids are taken up into the epithelial cells.
The major products of lipid digestion—fatty acids and 2-monoglycerides—enter the enterocyte by simple diffusion across the plasma membrane. A considerable fraction of the fatty acids also enter the enterocyte through a specific fatty acid transporter protein in the membrane.
After entry into the cell, medium-chain triglycerides, which require only minimal emulsification by bile acids, undergo a relatively simple process of assimilation in which they do not undergo re-esterification and chylomicron formation, as the long-chain lipids do. Medium-chain triglycerides are taken directly into the portal venous system; chylomicrons formed from long-chain fats enter the lymphatics. In conditions that involve obstruction of the lymphatics, feeding formulas containing primarily medium-chain triglycerides rather than long-chain triglycerides are recommended.

Developmental Aspects of Lipid Digestion and Absorption

Bile Acids
Bile acids are critical to efficient fat digestion and absorption. These processes are limited in VLBW infants because the duodenal concentration of bile acids is low owing to lower synthesis and ileal reabsorption. 40 Lower micellar solubilization leads to inefficient cell-mucosal interaction and subsequently lower absorption of the molecules of the mucosal–cell surface interface. Long-chain fatty acids but not medium-chain fatty acids depend on bile acids for solubilization and, thus, are the most susceptible to inefficient absorption.

Bile Salt–Stimulated Lipase
Human milk contains esterolytic activity that is not detectable in bovine milk. 41 It has been shown that the digestion of long-chain triglycerides proceeded only in the presence of bile salts by an enzyme, later classified as bile salt–stimulated lipase (BSSL), which is present in human colostrum and in preterm and term milk. 42 - 44 It has been estimated that in milk produced during the first 2 weeks of lactation, 40% of triglycerides are hydrolyzed within 2 hours, and during later lactation, only 20% of triglycerides are hydrolyzed. 44, 45 This apparent decrease is caused by an increase in milk fat content during lactation, rather than a real change in absolute BSSL activity.
The significance of the presence of BSSL for the digestion of milk lipids is supported further by studies of low-birthweight preterm infants (3 to 6 weeks old) fed raw or heat-treated (pasteurized or boiled) human milk. Fat from the former was absorbed more (74%) than the latter two (54% and 46%, respectively). 46

Pancreatic Lipases
In adults, pancreatic juice contains two enzymes involved in triglyceride hydrolysis. The so-called pancreatic lipase is more active against insoluble, emulsified substrates than against soluble ones. The second lipase, also called pancreatic carboxylase esterase, is more active against micellar or soluble substrates than against insoluble, emulsified substrates. In contrast to the first lipase, it is strongly stimulated by bile salts. Colipase removes the inhibiting effect of bile salts on lipase. Studies usually do not differentiate between these lipases. Generally, lipases show the lowest values after birth. 47 The increase toward adult values occurs within the first 6 months of life, which is earlier than in the case of amylase. Premature and VLBW infants have lower values than do full-term neonates. 48 During the first week of life, lipase activity increases about fourfold in premature infants. 47
In healthy preterm infants between days 3 and 40 postnatally, this activity increased linearly (both in infants at gestational age 29 to 32 weeks and 33 to 36 weeks). 40 At 1 month of age, values reached 35% of values found in 2- to 6-week-old babies.

Clinical Correlations
Although it has been mentioned that there appear to be differences for long-chain versus medium-chain triglycerides in the need to for bile acids, studies have shown medium-chain triglycerides to be just as readily absorbed as long-chain triglycerides. 49 The mechanisms of this are speculated to reside in greater gastric lipolytic activity of the longer-chain lipids. This is supported by a Cochrane review that showed no differences in growth, necrotizing enterocolitis, or other morbidities in babies fed primarily medium- versus long-chain triglycerides. 50
Most essential fatty acids provided to neonates are derived from the ω-6 family (linoleic acid). This is because much of the lipid derived from formulas or intravenous lipid solutions is from vegetable oil, which is rich in the ω-6 but not the ω-3 fraction. The likelihood of health benefits to babies provided greater quantities of the ω-3 lipids than they are currently receiving requires additional study and is discussed in Chapter 12 .


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Chapter 2 Maturation of Motor Function in the Preterm Infant and Gastroesophageal Reflux

Anna Maria Hibbs, MD, MSCE

• Upper Gastrointestinal Motility and Physiology
• Diagnosis of Gastroesophageal Reflux and Gastroesophageal Reflux Disease
• Physiologic Gastroesophageal Reflux
• Gastroesophageal Reflux Disease Symptoms
• Gastroesophageal Reflux Disease Diagnostic Tests
• Gastroesophageal Reflux Disease Treatment
• Summary
Gastroesophageal reflux (GER) is defined as the retrograde passage of gastric contents into the esophagus. In term and preterm infants, GER is usually a benign physiologic process, but it meets the definition of gastroesophageal reflux disease (GERD) if it causes clinical symptoms or complications. 1, 2 A multitude of gastrointestinal, respiratory, and other symptoms have been attributed to GERD, including apnea, worsening of lung disease, irritability, feeding intolerance, failure to thrive, and stridor. However, determining whether reflux is the cause of symptoms in an infant can be challenging. The approach to an infant with suspected GERD is further complicated by the paucity of available medications demonstrated to be safe or effective in this population.

Upper Gastrointestinal Motility and Physiology
An understanding of GER in infants must begin with the physiology of the upper gastrointestinal tract. Esophageal motor function is well-developed in infants as early as 26 weeks gestational age. 3, 4 Swallowing triggers coordinated esophageal peristalsis and lower esophageal sphincter (LES) relaxation, as it does in more mature patients. 3 However, the velocity of propagation is significantly faster in term than preterm infants. 5 Manometry has also documented that spontaneous esophageal activity unrelated to swallowing tends to take the form of incomplete or asynchronous waves; this type of nonperistaltic motor activity occurs more frequently in preterm infants than adults. 3
The LES, which blocks GER, is made up of intrinsic esophageal smooth muscle and diaphragmatic skeletal muscle. 6 Although premature infants were once thought to have impaired LES tone, several manometry studies have documented good LES tone, even in extremely low-birthweight infants. 3, 7, 8 In term and preterm infants, as in older patients, transient LES relaxations (TLESRs) unrelated to swallowing are the major mechanism allowing GER by abruptly dropping lower esophageal pressure below gastric pressure. 3, 8, 9 These TLESRs may occur several times per hour in preterm infants, although most TLESR events are not associated with GER. 9 Preterm infants with and without GERD experience a similar frequency of TLESRs, but infants with GERD have a higher percentage of acid GER events during TLESRs. 9 It has been hypothesized that straining or other reasons for increased intra-abdominal pressure may increase the likelihood of a GER event during a TLESR. Although LES relaxations also occur during normal swallowing, these are less often associated with GER events than isolated TLESR events. 9
In addition to the anatomic and physiologic factors described that increase the likelihood of the retrograde passage of gastric contents into the esophagus, infants ingest a much higher volume per kilogram of body weight, about 180 mL/kg per day, than older children and adults. 10 In the neonatal intensive care unit (NICU) population, preterm and term patients with nasogastric or orogastric feeding tubes may experience more reflux episodes owing to mechanical impairment of the competence of the LES. 11, 12
Gastric emptying is also an important factor in the passage of fluids through the upper gastrointestinal tract. One small study showed that between 25 and 30 weeks gestational age, gastric emptying time seems to be inversely and linearly correlated with gestational age at birth. This study also found that simultaneously decreasing the osmolality and increasing the volume of feeds accelerated gastric emptying, although changes in osmolality or volume alone did not have a significant effect. 13 Emptying also occurs faster with human milk feedings than with formula. Several small studies suggest that prebiotics, probiotics, and hydrolyzed formulas may speed gastric emptying time in formula-fed infants. 14 - 16 Fortification of human milk may slow gastric emptying time. 17 The clinical significance of these findings with regard to GER remains uncertain, however. Although it seems logical that slower gastric emptying would be associated with increased GER, a study of the relationship between gastric emptying and GER in preterm infants found no association. 18

Diagnosis of Gastroesophageal Reflux and Gastroesophageal Reflux Disease
Although infants have a propensity to experience frequent GER, most GER is physiologic and nonpathologic. GERD is defined as GER that causes complications. 1, 2 Unfortunately, in infants, particularly preterm infants, complications of GER are difficult to characterize. Clinicians disagree about which symptoms are caused by GER or GERD. 19 There is mixed evidence in the literature to support or refute most of the proposed complications of GER in infants, including apnea, 20 - 30 worsening of lung disease, 31 - 34 and failure to thrive. 35 An ongoing problem, particularly in the preterm population, is that many of the putative symptoms of GERD also frequently occur for other reasons. For instance, preterm infants without GERD also frequently experience apnea, lung disease, or feeding intolerance.

Physiologic Gastroesophageal Reflux
Nonpathologic GER occurs frequently in both preterm and term infants. Among 509 healthy asymptomatic infants aged 3 to 365 days monitored with an esophageal pH probe, the mean number of acid reflux episodes in 24 hours was 31.28, with a standard deviation of 20.68. 36 The reflux index, the percentage of time the esophageal pH was less than 4, ranged from less than 1 to 23, with the median and 95th percentile being 4 and 10, respectively. For this reason, a reflux index of 10 is often considered the threshold value for an abnormal study, but it must be remembered that none of the infants in this study were thought to suffer from symptomatic GERD, and clinical correlation with symptoms is required to make the diagnosis of GERD. Among the neonates in this study, the 95th percentile for the reflux index was as high as 13.
In a smaller study of 21 asymptomatic preterm neonates with a median postmenstrual age of 32 weeks, continuous combined esophageal pH and impedance monitoring detected refluxed fluid in the esophagus by impedance for a median of 0.73% (range, 0.3% to 1.22%) of the recording time, and acid exposure detected by pH monitoring for a median of 5.59% (range, 0.04% to 20.69%) of the recording time. When using combined pH and MII monitoring, detection of acid exposure may exceed volume exposure because the esophageal pH may remain depressed for a time after most of the bolus has been cleared, as well as for a variety of other technical reasons. 37 Norms for acid and nonacid reflux are less well defined in preterm than term infants owing to the practical and ethical barriers involved in placing esophageal pH probes in a large number of asymptomatic preterm infants. However, the data from this small study make it clear that GER events occur frequently in asymptomatic infants, and a wide range of reflux measurements may be seen in healthy preterm infants without GERD.
In a study of otherwise healthy infants seen in general pediatric practice, half of all parents reported at least daily regurgitation at 0 to 3 months of age. 38 The peak prevalence occurred at 4 months, with 67% reporting regurgitation, but thereafter declined rapidly. Thus, benign regurgitation was the norm in the first few months of life. Parents reported regurgitation to be a problem when it was associated with increased crying or fussiness, perceived pain, or back arching. The prevalence of regurgitation perceived as a problem peaked at 23% at 6 months but was down to 14% by 7 months. Most of these children did not receive treatment for GERD from their pediatrician, suggesting that a diagnosis of GERD was only made in a minority of these patients. Infants who did and did not experience frequent regurgitation between 6 and 12 months of age were subsequently followed a year later. 39 At this time, none of the parents described regurgitation as a current problem, and only one child experienced spitting at least daily. That child had not experienced frequent regurgitation at 6 to 12 months of age. Infants who had frequent spitting at 6 to 12 months of age did not experience more infections of the ear, sinuses, or upper respiratory tract, nor did they experience more wheezing. In general, this cohort demonstrates that in most infants regurgitation is a benign process that is outgrown. However, it was noted that in the 1-year follow-up assessment, parents of infants who had frequent regurgitation at 6 to 12 months were more likely to report prolonged meal times (8% versus 0%) and frustration about feeding their child (14% versus 4%), even though regurgitation symptoms were no longer present. It is not clear whether this represents a true difference in feeding behavior or parental perception in a group likely to be sensitized to feeding issues.

Gastroesophageal Reflux Disease Symptoms
Although the definition of GERD hinges on the presence of troublesome symptoms or complications, identifying whether symptoms are in fact caused by reflux can be challenging in infants. 1, 2 Symptoms frequently attributed to GERD in infants include regurgitation, Sandifer posturing, worsening of lung disease, food refusal or intolerance, apnea, bradycardia, crying or fussiness, and stridor. Regurgitation may be a symptom of GERD in infants but in itself is not a sufficiently sensitive and specific finding to make a diagnosis. 2 In addition, otherwise healthy infants without sequelae from their regurgitation, so-called happy spitters, do not require treatment. 1 Clustering regurgitation with other symptoms may increase the accuracy of diagnosis, as demonstrated by the I-GERQ-R infant reflux questionnaire. 2, 40 However, the validity of such questionnaires has not been established in the NICU population, which includes preterm infants and sick term neonates who have multiple competing causes for the symptoms frequently attributed to GERD.
Although GERD and bronchopulmonary dysplasia seem to be associated, the presence or direction of causality have not been determined. 2, 31 - 34 Patients with increased work of breathing may generate more negative intrathoracic pressures, thereby promoting the passage of gastric contents into the esophagus. Conversely, aspirated refluxate could injure the lungs. Finally, there may be no causal link in most patients, with immaturity and severity of illness predisposing to both conditions. In addition, part of the apparent association between BPD and GERD may be due to an increased index of suspicion for GERD in patients with BPD, leading to increased rates of diagnosis. 33
A similar issue exists for apnea in premature infants. Although in animal models esophageal stimulation may trigger airway protective reflexes, 41 there is insufficient evidence in human infants to confirm that reflux causes apnea. 2, 30 In addition, apnea may itself trigger reflux. 42, 43 Finally, it may be that immature infants are simply prone to both apnea and reflux, with no causal association. 44 In a cohort of infants referred for overnight esophageal and respiratory monitoring for suspicion of GERD as a cause of apnea, desaturation, or bradycardia, fewer than 3% of all cardiorespiratory events were preceded by a reflux event. 45 The infant with the highest percentage had 4 of 21 cardiorespiratory events preceded by GER. Conversely, 9.1% of reflux events were preceded by a cardiorespiratory event. This study shows that it is more common for a cardiorespiratory event to precede reflux than for reflux to precede a cardiorespiratory event. Cardiorespiratory events preceded by reflux were not more severe than those not preceded by reflux. Furthermore, even in this population referred for suspicion of GER-triggering cardiorespiratory events, only a small minority of cardiorespiratory events were in fact preceded by reflux. This suggests that even if all of these temporally related events were also causally related, and even if a treatment were completely efficacious at eliminating GERD, most cardiorespiratory events would not be eliminated by GERD treatment. However, data from small or moderately sized research cohorts cannot rule out the possibility that reflux can trigger most cardiorespiratory events in a small subset of patients. Because bedside recording of apnea events is known to be inaccurate, correlation of apnea with feeding or reflux events in a specific patient requires formal simultaneous respiratory and esophageal monitoring studies.
It is unclear what component of the refluxate triggers complications. Infants experience less acid GER than older children or adults, owing in large part to frequent buffering of gastric contents by milk. Although most GER events in infants are nonacid, 46, 47 at least some preterm infants are able to experience significant acid GER, often defined as an esophageal pH of less than 4 for more than 10% of the recording. 22, 36 Acid GER predominates in infants preprandially, and nonacid GER postprandially. 44, 46 However, it is not clear whether acidity is the mechanism by which reflux causes complications in infants. 2 The other characteristics of the refluxate that have been postulated to be associated with symptoms include the height of the bolus in the esophagus, the volume of the bolus, or the pressure exerted on the esophagus.

Gastroesophageal Reflux Disease Diagnostic Tests
Numerous tests exist to measure acid and nonacid GER in infants ( Table 2-1 ). Esophageal pH probes measure acid reflux, and esophageal multichannel intraluminal impedance measures the presence of fluid in the esophagus regardless of pH. Impedance and pH sensors can be combined in one esophageal probe to give the most information about the frequency and timing of both acid and nonacid GER. Many systems have the capacity to be run in conjunction with respiratory monitoring, or for a family member or health care provider to mark the timing of a clinical symptom, in order to attempt to temporally correlate symptoms and GER events. An upper gastrointestinal radiographic series is useful for assessing anatomic abnormalities that may contribute to or mimic GER but is a poor measure of the frequency or severity of GER because it only captures a brief window in time. A nuclear medicine scintigraphy study can identify postprandial reflux and aspiration and quantify gastric emptying time. There is no current gold standard diagnostic modality for GERD in infants. In part, this is because it is still not clear what component of reflux, such as its frequency, volume, acidity, or height, is most likely to cause complications in infants, and each test measures different parameters. A recent international consensus statement on GERD concluded that no single diagnostic test can prove or exclude extraesophageal presentations of GERD in pediatrics. 2 Furthermore, many NICU patients are too small for endoscopy to directly assess esophagitis, so esophageal symptoms can only be inferred from vague symptoms, such as food refusal or fussiness. Finally, because the diagnosis of GERD relies on the presence of clinical complications, no physiologic test that only characterizes the frequency or characteristics of GER events in a patient can by itself confirm the diagnosis of GERD.


Gastroesophageal Reflux Disease Treatment
Nonpharmacologic therapies for GERD include positioning, thickening feeds, and decreasing the volume while increasing the frequency of feeds. When milk protein allergy is thought to be mimicking or triggering GERD, changing to a more elemental formula may also be appropriate. In the run-in period for a randomized control trial of a pharmacotherapeutic intervention for GERD, the majority of infants seemed to improve over a 2-week period with such a multipronged conservative management strategy, although this effect simply could also be attributed to time and maturation. 48 Thickening feeds has been shown to decrease episodes of clinical vomiting, although it does not seem to decrease physiologic measures of GER. 49 Although typical positioning precautions for an infant with a diagnosis of GERD include elevating the head of the bed, there is not an advantage to supine upright versus supine flat positioning. 49 Prone positioning seems to be associated with fewer GER events than supine but is generally contraindicated owing to the increased risk for sudden infant death. 49, 50 Lateral positioning with the right side down results in more frequent reflux events than left lateral positioning, but it is not clear whether this results in more symptoms. 51
Medications for the treatment of GERD are among the most common drugs prescribed in the NICU. 52 - 54 In the United States, pharmacotherapy primarily consists of drugs to decrease gastric acidity, such as the histamine-2 (H 2 ) receptor antagonists and proton pump inhibitors (PPIs), and prokinetics, such as metoclopramide and erythromycin ( Table 2-2 ).

Because both GER and the symptoms commonly linked to GERD, such as feeding intolerance and apnea, change rapidly with time and maturation, valid studies of GERD in infants must account for this effect in their study design. A study that simply measures symptoms before and after a therapy is likely to find improvement related to maturational effects, whether or not the therapy was truly efficacious. In addition, although many studies have demonstrated physiologic changes in response to pharmacotherapy, the gold standard for the treatment of GERD must be improvement in the symptoms that define the disease. Several recent well-conducted studies accounting for maturational changes have raised further questions about the efficacy and safety of common GERD drugs. 55 - 57
Because of the difficulties in proving that a putative complication of GER is indeed caused by reflux, along with the questionable efficacy of available GERD medications, it must be remembered when treating an individual patient that a treatment failure may stem from either the application of drugs to symptoms not caused by GERD or a failure of pharmacotherapy to improve true GERD. Apparent treatment successes may result from either a true treatment effect or natural maturational changes in the GERD or symptoms misclassified as resulting from GERD ( Table 2-3 ). Pharmacotherapy should be stopped if symptoms fail to improve with therapy. If an improvement is seen, a trial off therapy in several weeks should be considered because maturational changes may have been the cause of the initial apparent response or may obviate the need for therapy in the near future.
Table 2-3 POSSIBLE ETIOLOGIES OF APPARENT IMPROVEMENT OR LACK OF IMPROVEMENT AFTER INITIATION OF GERD THERAPY *   Symptoms Correctly Attributed to GERD Symptoms Erroneously Attributed to GERD Improvement after initiation of therapy The therapeutic intervention was successful. The therapy is efficacious in treating GERD symptoms. or The therapeutic intervention was not successful owing to lack of efficacy of the therapy, but improvement in the symptoms due to maturation caused the apparent response to therapy. The therapeutic intervention was not successful because the symptoms were not triggered by GERD, but improvement in the symptoms due to maturation caused the apparent response to therapy. The therapy may or may not be efficacious in treating true GERD. No improvement after initiation of therapy The therapeutic intervention was not successful owing to lack of efficacy of the therapy. The therapeutic intervention was not successful because the symptoms were not triggered by GERD. The therapy may or may not be efficacious in treating true GERD.
* The severity of gastroesophageal reflux disease (GERD) and the symptoms frequently attributed to GERD, such as apnea, feeding difficulties, or lung disease, rapidly change with time and maturation in infants. Interpretation of a response or lack of response to therapy hinges on understanding that both GERD symptoms and causally unrelated symptoms may change with time, complicating the interpretation of an apparent response to therapy. In addition, many of the symptoms that have been proposed to be triggered by GERD have many other competing causes in preterm infants, and it is difficult to definitively determine whether they are caused by GERD.

Acid-Blocking Medications
H 2 receptor antagonists and PPIs decrease the acidity of gastric fluid and esophageal refluxate. They act on the H 2 receptors in acid-producing gastric parietal cells, decreasing acid production below normal fasting basal secretion rates as well as suppressing meal-associated acid production. Acid in the esophagus or airway is thought to trigger many of the proposed complications of reflux in NICU patients, such as food refusal, failure to thrive, and pharyngeal or vocal cord edema. Examples of H 2 receptor antagonists include ranitidine, cimetidine, and famotidine.
Few randomized clinical trials of H 2 receptor antagonists have assessed their impact on GERD symptoms in either neonates or premature infants. In a small but statistically significant crossover trial of combined ranitidine and metoclopramide in preterm infants with bradycardia attributed to GERD, infants experienced significantly more bradycardic events when receiving reflux medications than when receiving placebo. 56 This unexpected finding is biologically plausible; histamine receptors are present in the heart, and ranitidine has been implicated in causing bradyarrhythmias. 58 - 64 Because most cardiorespiratory events are not associated with GER, 45 the lack of effect found in this study could have been driven either by the misattribution of frequent bradycardia to GERD or by a lack of drug efficacy. Bradycardia is likely to have poor specificity for the identification of GERD given the multiple other triggers for bradycardia in premature infants, including apnea of prematurity and vagal stimulation, and most cardiorespiratory events are not preceded by reflux even among infants suspected of having GERD. 45 Notably, this crossover study of ranitidine and metoclopramide, which appropriately accounted for maturational changes, also demonstrated a clinically and statistically significant decrease in bradycardic events over a 2-week period in both the treatment and placebo groups. This finding underscores the importance of accounting for temporal changes in processes influenced by maturation, such as GER, apnea, and bradycardia.
In a randomized trial of H 2 receptor antagonists, very-low-birthweight infants were randomized to cimetidine or placebo. 65 The investigators hypothesized that cimetidine could decrease liver enzyme–mediated oxidative injury in the lung. Although this was not a study of GERD treatment, it is one of the few studies in which very-low-birthweight infants were randomized to an H 2 receptor antagonist early in life. Strikingly, it was stopped by the data safety monitoring committee for increased death and intraventricular hemorrhage in the treatment group. The mechanism of these apparent adverse effects is unknown. The increase in adverse events could have occurred by chance or could be a true adverse event related to cimetidine, which may or may not be generalizable to other H 2 receptor antagonists.
In a small double-blind study, infants aged 1 to 11 months were randomized to a higher or lower dose of famotidine, with a subsequent placebo-controlled withdrawal. 57 Infants receiving famotidine had less frequent emesis than those receiving placebo. Infants on the higher famotidine dose also had a decreased crying time and smaller volume of emesis. However, famotidine was associated with increased agitation and a head-rubbing behavior attributed to headache, raising some concerns about possible side effects in the general infant population.
PPIs irreversibly block the gastric hydrogen/potassium adenosine triphosphatase responsible for secreting hydrogen ions into the gastric lumen. Currently, no PPIs are labeled for use in patients younger than 1 year. Nevertheless, between 1999 and 2004, PPI prescriptions for infants increased exponentially, with the highest rates of use in infants younger than 4 months. 66 Common PPIs include omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, and rabeprazole.
Although PPIs have been shown to decrease gastric acidity in infants in physiologic studies, there is a paucity of masked randomized studies in infants that assess PPI impact on GERD symptoms and account for underlying maturational changes over time. In a study by Orenstein and colleagues, outpatient infants who had failed a run-in period of nonpharmacologic management were randomized to lansoprazole or placebo. 55 There was no difference in symptoms between the groups, with slightly more than half of the infants in each group experiencing improvement over the study period. However, a significant increase in serious adverse events in the lansoprazole group was seen; among these adverse events, a nonsignificant increase in lower respiratory tract infections was noted.
In addition to drug-specific side effects, such as leukopenia and thrombocytopenia with ranitidine, class effects resulting from the change in gastric pH may be seen with H 2 receptor antagonists and PPIs. For instance, increasing evidence suggests that gastric acidity may play an important role in host immune defense. In an observational study, use of H 2 receptor antagonists was associated with increased necrotizing enterocolitis. 67 In another cohort study, ranitidine use was associated with late-onset sepsis in NICU patients. 68 However, in these observational studies, confounding by indication or severity of illness cannot completely be excluded as the cause of this apparent association. Consistent with the findings in the observational studies, in one small interventional study, gastric acidification was shown to decrease necrotizing enterocolitis. 69 Higher rates of gastric colonization with bacteria or yeast have also been associated with ranitidine, but without a detectable increase in clinical infection. 70 In older patients, a possible association between acid suppression and lower respiratory tract infections, including ventilator-associated pneumonia, remains controversial in the literature. 71 - 79 Acid suppression has also been associated with Clostridium difficile infection in some adults. PPIs seem to carry a higher risk than H 2 receptor antagonists, presumably owing to more effective acid suppression. 80, 81 The relationship between PPI use and C. difficile colonization or infection has not been reported in infants.
Increasing gastric pH can theoretically also have nutritional consequences. Acid reduction may decrease calcium absorption as a result of decreased ionization of calcium in the stomach. The U.S. Food and Drug Administration (FDA) recently released a class labeling change for PPIs based on concerns that adults on high doses or prolonged courses of PPIs seem to experience more fractures. 82, 83 The impact of acid suppression by PPIs or H 2 receptor antagonists on bone health in either healthy neonates or preterm infants with osteopenia of prematurity is unknown. Vitamin B 12 absorption is also dependent on gastric acidity, but the impact of gastric acid suppression on B 12 status in infants has also not been described.

Drugs to promote gastrointestinal motility are thought to act by improving esophageal motility and LES tone. Prokinetics are also often used to shorten gastric emptying time, although a relationship between GER and delayed gastric emptying in infants has not been proved. 18
Metoclopramide and erythromycin are the primary prokinetics currently approved in the United States. Cisapride was removed from the market because of the risk for serious cardiac arrhythmias and QT prolongation. 84 Domperidone is not approved in the United States because of concerns about QT prolongation in neonates. 85
Metoclopramide is a dopamine receptor antagonist. The Cochrane systematic review of GERD therapies in children found both therapeutic benefit and increased adverse effects with metoclopramide treatment. 86 However, most of the improvements seen were in physiologic measures of GER and not in the symptoms of GERD. A subsequent systematic review of metoclopramide therapy for GERD in infants found insufficient evidence for either efficacy or safety in this population. 87 Published after these reviews, the previously described placebo-controlled crossover study of ranitidine and metoclopramide demonstrated a lack of efficacy and an increase in bradycardia in the treatment group, although this finding could be attributed to ranitidine and not metoclopramide. 56
Metoclopramide can cause neurologic sequelae because it crosses the blood-brain barrier and acts on central dopamine receptors. Possible neurologic complications of metoclopramide in infants include irritability, drowsiness, oculogyric crisis, dystonic reaction, and apnea. 87 In 2009, the FDA issued a warning about the risk for tardive dyskinesia with prolonged or high-dose metoclopramide use. 88 Tardive dyskinesia has no known treatment and consists of involuntary body movements, which may persist after the drug is stopped. It is unknown whether term or preterm infants are at greater or lesser risk for tardive dyskinesia than older patients.
Erythromycin is an analog of motilin, a hormone normally produced by duodenal and jejunal enterochromaffin cells that promotes gastrointestinal migrating motor complexes. 89 - 91 The prokinetics dose of erythromycin is typically lower than the antimicrobial dose, but a standard promotility dose has not been established in neonates or preterm infants. Infants older than 32 weeks gestational age may be better able than less mature infants to respond to stimulation of the motilin receptor. 92, 93
Most studies of erythromycin in preterm infants have focused on improving feeding intolerance and not specifically on GERD treatment. 92, 93 In a masked randomized trial of erythromycin to promote feeding tolerance in 24 preterm infants, GER was measured as a secondary endpoint. 94 Erythromycin did not decrease the time to reach full enteral feeds, and there were no changes in GER measured by pH probe. GERD symptoms were not reported in this study. A systematic review of erythromycin to promote feeding tolerance in premature infants concluded that erythromycin could promote the establishment of enteral feeding and was not associated with any adverse events. 95 However, the authors cautioned that since long-term adverse events had not been fully studied, erythromycin should be reserved for infants with severe dysmotility.
When used as an antibiotic, erythromycin may promote pyloric stenosis in infants. It is unknown whether a similar effect could occur with the lower doses and longer duration of therapy associated with use as a prokinetic, although pyloric stenosis was not reported in most of the current trials in preterm infants. 95 Chronic administration of erythromycin has the potential to impact gastrointestinal colonization, but the impact in the NICU population is unknown.
Erythromycin may increase serum levels of theophylline, digoxin, sildenafil, and some benzodiazepines and has been implicated in arrhythmias and QT prolongation when coadministered with cisapride. In addition, it also has a direct proarrhythmic effect due to prolongation of the QT interval. 96 In older patients, the risk for sudden death may be increased when erythromycin is used with other inhibitors of the same hepatic enzyme (CYP3A), such as cimetidine and methadone. 96

GER is common in term and preterm infants. The primary mechanism allowing reflux is TLESRs. Although the diagnosis of GERD requires the presence of complications resulting from reflux, ascertaining whether symptoms in a given patient are caused by GERD can be challenging. There is no gold standard diagnostic modality to diagnose GERD in the NICU population. Although esophageal impedance and pH measurements are the most commonly reported, linking measured GER with symptoms is still required to diagnose GERD. In the NICU population, few symptoms have been definitively shown to be caused by GERD, and most of the putative symptoms of GERD, such as feeding intolerance or apnea, have many possible etiologies. Furthermore, no pharmacologic interventions have been proved safe and effective in this population. Therefore, nonpharmacologic expectant management should be the mainstay of treatment for most infants.


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88 FDA, , 2009.
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Chapter 3 Development of Gastrointestinal Motility Reflexes

Sudarshan Rao Jadcherla, MD, FRCPI, DCH, AGAF, Carolyn Berseth, MD

• Embryologic Aspects of Motility Development
• Pharyngoesophageal Motility Reflexes in Human Neonates
• Gastrointestinal Motility Reflexes in Human Neonates
• Developmental Colonic Motility in Human Neonates
• Summary
• Implications and Controversies of Gut Motility
Gastrointestinal motility is very complex and is influenced by embryologic development and aberrations, vulnerable neurologic systems, maturational changes in central and enteric nervous systems, and rapidly changing anatomy and physiology during infancy. In the vulnerable high-risk infants in intensive care units, the influence of hypoxia, inflammation, sepsis, and other comorbidities complicates the feeding process and gastrointestinal transit. Despite the complexities, the simple physiologic functions of the neonatal foregut, midgut, and hindgut, respectively, are to facilitate the feeding process safely to steer the feedings away from the airway, gastrointestinal transit of luminal contents to modulate absorption and propulsion, and evacuation of excreta to maintain intestinal milieu homeostasis. These functions continue to advance through infant development, from fetus to adult. In this chapter, we review and summarize the developmental aspects of pharyngoesophageal motility, gastrointestinal motility, and colonic motility.

Embryologic Aspects of Motility Development
The airway and lung buds, pharynx, esophagus, stomach, and diaphragm are all derived from the primitive foregut and or its mesenchyme and share similar control systems. 1 - 4 By 4 weeks of embryologic life, tracheobronchial diverticulum appears at the ventral wall of the foregut, with left vagus being anterior and right vagus posterior in position. At this stage of development, the stomach is a fusiform tube with its dorsal side growth rate greater than its ventral side, creating greater and lesser curvatures. At 7 weeks of embryonic life, the stomach rotates 90 degrees clockwise, and the greater curvature is displaced to left. The left vagus innervates the stomach anteriorly, and the right vagus innervates the posterior aspect of stomach. At 10 weeks’ gestation, the esophagus and stomach are in proper position, with circular and longitudinal muscle layers and ganglion cells in place. By 11 weeks, swallowing ability develops; by 18 to 20 weeks, sucking movements appear; and by full term, the fetus can swallow and circulate nearly 500 mL of amniotic fluid. Thus, swallow-induced peristaltic activity begins in fetal life. 5, 6
Regulators of motility underlie excitatory and inhibitory neurons and form the basis for Starling’s law of the intestine ( Fig. 3-1 ) 7 in that luminal stimulation results in ascending contraction and descending relaxation, facilitating bolus transport. This sequential enteric reflex pattern results in the phenomenon of peristalsis. The mediators of these enteric reflexes are the excitatory neurons that underlie the parasympathetic vagal pathways supported by the dorsal motor nucleus (DMN) of the vagus and the inhibitory neurons that underlie the VIPergic or nitrergic pathways. 8, 9 At the myenteric plexus level, the cholinergic postganglionic excitatory neurotransmitter acetylcholine and the inhibitory transmitter vasoactive intestinal polypeptides (VIP) and nitric oxide (NO) are responsible for peristalsis.

Figure 3-1 Schematic representation of the afferent and efferent components of the peristaltic reflex, Starling’s law of the intestine. When luminal stimulation occurs by mechanoreceptor, chemoreceptor, osmoreceptor, or tension receptor activation, there ensues a cascade of proximal afferent and distal efferent activation. This results in sequential proximal excitatory and distal inhibitory neurotransmission, resulting peristalsis to facilitate gastrointestinal transit. At the level of the esophagus, such sequences also facilitate aerodigestive protection.
Enteric nervous system–mediated contractile activity is prominent in function by full-term birth and is essential for propulsive activity. Variations in gut motility and peristaltic patterns occur in prematurely born neonates and are discussed in the latter part of this chapter. The earlier subtype of enteric neurons to develop is the nNOS neurons, and although there are some exceptions, NO-mediated transmission develops earlier and is more prominent during prenatal and postnatal development than in adults. At the tissue level, postreceptor modification mechanisms and excitation-contraction coupling mechanisms are distinctly different in the skeletal and smooth muscle components. 10, 11 These mechanisms were seen to develop postnatally and mature in their functional capabilities in developmental animal models. 12 - 14

Pharyngoesophageal Motility Reflexes in Human Neonates

Maturation of Esophageal Peristalsis and Upper Esophageal Sphincter and Lower Esophageal Sphincter Functions
Using micromanometry methods, pharyngeal, upper esophageal sphincter (UES), esophageal body, and lower esophageal sphincter (LES) functions have been characterized in neonates. 15 - 17 Remarkably, the resting UES tone increases with maturation and is dependent on the state of alertness and activity. The average resting UES pressure (mean ± SD) in preterm neonates at 33 weeks postmenstrual age (PMA) was 17 ± 7 mm Hg; in full-term neonates, it was 26 ± 14 mm Hg; and in adults, it was 53 ± 23 mm Hg. With growth and maturation, the muscle mass and therefore the tone and activity of the UES improve. Similarly, changes in LES length and tone have been observed with growth. 16 - 19 By determining the LES high-pressure zone in developing premature infants, investigators have determined changes in esophageal length during postnatal growth in premature and full-term infants. Specifically, the esophageal lengthening occurs in a linear fashion in neonates during growth. 18

Maturation of Basal and Adaptive Esophageal Motility
During the propagation of the pharyngeal phase of swallowing, the UES relaxes, and esophageal body waveforms propagate the bolus from proximal to distal end, which is accompanied by LES relaxation to allow the bolus to enter the stomach ( Fig. 3-2 ). This whole integrated sequence of reflexes constitutes primary peristalsis, which is swallow dependent. Evaluation of consecutive spontaneous solitary swallows during maturation (preterm at 33 weeks PMA vs. preterm at 36 weeks PMA) and growth (preterm-born and full term-born vs. adults) confirmed significant ( P < .05) differences in the basal UES resting pressure, UES relaxation parameters, proximal and distal esophageal body amplitude and duration, magnitude of esophageal waveform propagation, and segmental peristaltic velocity. Specifically, the characteristics of UES and primary esophageal peristalsis exist by 33 weeks PMA; however, they undergo further maturation and differentiation during the postnatal growth and are significantly different from those of adults. 15

Figure 3-2 Example of spontaneous primary esophageal peristalsis in a premature infant evoked on pharyngeal contraction, upper esophageal sphincter relaxation, forward propagation of esophageal body waveforms, and lower esophageal sphincter relaxation. Such sequences facilitate swallowing and esophageal clearance. Note the brief respiratory modification and deglutition apnea during the pharyngeal waveform, suggesting cross-communications between the pharynx and airway.
The esophagus is the frequent target for the anterograde bolus from the oropharynx, as in swallowing, and for the retrograde bolus from the stomach, as in gastroesophageal reflux. During either event, the bolus comes in close proximity to the airway, and evolving postnatal mechanisms facilitate pharyngeal and airway protection. For example, during primary esophageal peristalsis, there is a respiratory pause called deglutition apnea that occurs during the pharyngeal phase of swallow (see Fig. 3-2 ). This brief inhibition in respiration is due to a break in respiratory cycle (inspiratory or expiratory) and is a normal reflex. On the other hand, during esophageal provocation events, esophageal peristalsis occurs independent of pharyngeal swallowing, called secondary peristalsis ( Fig. 3-3 ). Although the nature and composition of bolus within the pharyngeal or esophageal lumen can vary, peristalsis remains the single most important function that must occur to favor luminal clearance away from the airway. This reflex peristaltic response and airway protection are the end result of the activation and interaction of receptors, afferents, brain stem mediation, efferents, muscles, and effectors. In premature infants, the mechanosensitive, chemosensitive, and osmosensitive stimulus can provoke the esophagus, and the resultant reflexes that protect the airway and digestive tract include secondary esophageal peristalsis and UES contractile reflexes. 20 - 22 These reflexes prevent the ascending spread of the bolus and favor descending propulsion to ensure esophageal clearance.

Figure 3-3 Example of swallow independent secondary esophageal peristalsis in a premature infant in response to a mid-esophageal infusion. Absence of pharyngeal waveform, presence of propagating esophageal body waveforms, upper esophageal sphincter contraction, lower esophageal sphincter relaxation, and complete esophageal propagation are also noted. Such sequences are evoked during esophageal provocations and contribute to esophageal and airway protection by facilitating clearance.
These reflexes advance during maturation in premature infants. Premature infants were studied twice at 33 weeks and 36 weeks mean PMA. The occurrence of secondary peristalsis was volume dependent, and the characteristics were different with advanced maturation. At 36 weeks PMA, completely propagated secondary peristalsis was greater with liquids than with air, proximal esophageal waveform duration signifying proximal esophageal clearance time was shorter for air and liquids, and the propagating velocity for liquids was faster. Additionally, as the premature infant grew older, the occurrence of secondary peristalsis increased significantly with increment in dose volumes of air or liquids. These findings are suggestive of the existence of vagovagal protective reflex mechanisms that facilitate esophageal clearance in healthy premature neonates and indicate that these mechanisms improve with growth.
Similar to the occurrence of secondary peristalsis, esophageal provocation can result in an increase in UES pressure (see Fig. 3-3 ). 23, 24 This reflex is the esophageal-UES contractile reflex, and is mediated by the vagus. We observed that the occurrence of UES contractile reflex was also volume dependent and that the characteristics improved with advanced maturation in healthy premature neonates. This reflex may provide protection to the aerodigestive tract, thus preventing the proximal extent of the refluxate, as in spontaneous gastroesophageal reflux events. Concurrently, the LES relaxes to facilitate bolus clearance. This is called the LES relaxation reflex response.

Gastrointestinal Motility Reflexes in Human Neonates
Although fetal peristalsis is recognized, local neural transmission and integration of peristalsis mature throughout fetal life and continue to develop during the first postnatal year. Peristalsis is mediated by gastric motility, which is mediated by stomach muscle contractions occurring at a rate of 3 to 5 times/minute, duodenal contractions at a rate of 9 to 11 times/minute, and midgut contractions at about 6 to 8 times/minute ( Figs. 3-4 and 3-5 ). 25, 26 These local contractions are coordinated throughout the length of the intestine by neural regulation modulated by the enteric nervous system, autonomic nervous system, and central nervous system. The interstitial cells of Cajal (ICCs) are specialized muscle cells located primarily in the duodenum and upper small intestine. 2, 27, 28 They also play a role in triggering coordinated contractions in the intestine. Finally, motor function can be modulated by gastrointestinal hormones and peptides, which may exert endocrine, paracrine, or neurocrine activity, resulting in inhibitory (e.g., peptide YY, nitrergic, VIP) or excitatory (e.g., cholinergic-muscarinic, cholecystokinin, substance P) modulation. All of the muscles and neural structures are present by 32 weeks’ gestation, although full neural and neuroendocrine integration is not achieved until late in infancy. 29, 30

Figure 3-4 Example of nonmigrating (A) and migrating (B) gastroduodenal motility in a human neonate in the fasting state. A, A representative manometric recording depicting nonmigrating activity in a term infant. Fasting motor activity recorded in the antrum is shown in the top line, activity in the antropyloric junction in the second line, and duodenum in the third and fourth lines. B, A representative manometric recording in the same infant and three duodenal leads. The arrow indicates the presence of migrating motor complex, a phenomenon mediated by motilin, serotoninergic system, or vagal parasympathetic system.
(Adapted from Jadcherla SR, Klee G, Berseth CL. Regulation of migrating motor complexes by motilin and pancreatic polypeptide in human infants. Pediatr Res 1997;42:365-369.)

Figure 3-5 Migrating motor complex results from stimulation of motilin receptors after enteral erythromycin. A, Example of motility recording in an infant at 26 weeks of gestation 30 minutes after the enteral erythromycin. No evidence of migrating motor activity is seen. B, Example of motility recording in an infant at 33 weeks of gestation 30 minutes after the administration of intragastric erythromycin. Phasic contractions appear in the antrum and are temporally coordinated with the occurrence of phasic activity in the three duodenal recording ports.
(Adapted from Jadcherla SR, Klee G, Berseth CL. Regulation of migrating motor complexes by motilin and pancreatic polypeptide in human infants. Pediatr Res 1997;42:365-369.)

Gastric Motor Functions
The gastric fundus accommodates ingested nutrients by receptive relaxation and is mediated largely by the vagal nerve. However, little is known about receptive relaxation in neonates and infants. In contrast to the fundus, the antrum has tonic and phasic activity and is responsible for the churning of nutrients with secretions to initiate early digestion and empty stomach contents into the duodenum. Contractile activity in the antrum is coordinated with that in the duodenum to promote emptying of contents into the upper small intestine. Hence, physical characteristics of nutrients trigger feedback to the antrum to hasten or slow emptying. Gastric emptying is not altered by feeding temperature or non-nutritive sucking. However, it is delayed during extreme stress, such as the presence of systemic illness. Calorically denser formula hastens gastric emptying. 31 The administration of drugs for clinical care, such as opioids or mydriatics, may also impair gastrointestinal function. 32 Interestingly, bolus feedings appear to delay gastric emptying in some preterm infants, presumably by rapid distention. 33 Infant massage improves feeding tolerance, and this has been shown to be mediated by stimulation of vagal activity. 34

Small Intestine Motor Functions
The intrinsic contractile rhythm of the stomach, duodenum, and small intestine is present as early as 24 weeks’ gestation. Full neural integration is inadequate at birth. Gastric emptying is slower in the preterm infant than the term infant, and overall intestinal transit is slower. Overall gut transit can vary from 7 to 14 days and depends on gestational maturation.

Maturation of Gastrointestinal Motility
The small intestine exhibits two basic patterns of motor activity: fasting response and fed response. During fed response, the muscle layers contract in a disorganized fashion, resulting in active, continuous mixing and churning of nutrients and secretions and producing chime. Fed response facilitates transport of nutrients distally to facilitate digestion and absorption. Although an adult-like fed response is seen in most term infants in response to bolus feeding, about half of preterm infants exhibit such a response. 35 In contrast, in fasting state (see Figs. 3-4 and 3-5 ), the gut contractility cycles in four phases starting with a state of quiescence (phase I). Progressing with time, solitary or groups of uncoordinated contractile waveform clusters occur at various levels of the gut, increasing in number and intensity (phase II). Subsequently, the contractile waveforms are sustained for 2 to 10 minutes and migrate sequentially distally down the length of the gut distally (phase III, or the migrating motor complex [MMC]). MMCs are responsible for about 50% of the forward movement of nutrients and are considered the “intestinal housekeeper.” This robust, well-organized pattern is replaced by randomly occurring contractile waveforms that terminate in the reappearance of quiescence (phase IV). The entire sequence of phases I through IV is called the interdigestive migrating motor complex (IMMC; see Fig. 3-5 ). The appearance of the MMC is also controlled by the ICC, which can be triggered by the hormone motilin. Plasma concentrations of the motilin cycle fluctuate in the adult, and the peak is associated with the occurrence of the MMC. Preterm infants exhibit fasting levels of motilin that are similar to those seen in adults, but motilin fails to cycle in the preterm infant. The initial amino acid configuration of the antibiotic erythromycin mimics that of the hormone motilin, and low doses of erythromycin trigger initiation of the MMC in preterm infants older than 32 weeks gestational age. 29, 36 Administration of erythromycin fails to trigger MMCs in infants younger than 32 weeks, suggesting that the motilin receptor cannot be activated by erythromycin or absent before 32 weeks’ gestation. Thus, the absence of the MMC in the very preterm infant appears to be the result of overall immaturity of the integration of motor pattern, absence of the motilin receptor, and absence of fluctuating levels of motilin. Contractile waveforms in the antrum and small intestine occur as single isolated events or in clusters. 37 The characteristics of these clustered contractions changes with increasing gestational age, culminating in the appearance of the MMC at about 34 to 36 weeks’ gestation. 38, 39
The method of feeding influences motor patterns during fasting as well as feeding. The provision of small early feedings, as opposed to no feeding or nonnutritive feedings (e.g., sterile water), accelerates the maturation of fasting motor patterns, 40 - 43 which in turn are associated with better feeding tolerance. Interestingly, small feedings (e.g., 20 mL/kg per day) induce maturation of motor patterns comparable to that induced by larger feedings ( Fig. 3-6 ). 43 This induction of maturation is likely neurally mediated because hormone release is not as robust in response to small feedings as for larger feedings. 42 In one animal model, it has been shown that the acceleration of maturation of motor patterns is associated with an increase in nitrergic neurons, 44 both of which may regulate motor activity. Additionally, feeding diluted formula slows the onset and intensity of feeding responses. 40, 45

Figure 3-6 Small intestinal motor activity in term infant (40 weeks of gestational age) during fasting and progressing through initiation of milk infusion. Presence of migrating motor complex is followed by a brief period of quiescence before feeding is initiated. Quiescence is replaced by persistent motor activity in all four duodenal recording channels shortly after feeding infusion is begun.
(Adapted from Berseth CL. Neonatal small intestinal motility: motor responses to feeding in term and preterm infants. J Pediatr 1990;117:777-782.)

Developmental Colonic Motility in Human Neonates
There is a significant lack of data on colonic motility in preterm human infants, and this is largely due to technical limitations, the need for invasive approach, and ethical concerns. Some evidence can be gleaned from animal studies that, in common with humans, intestinal contents are propagated through the bowel before birth. Colonic motility is quite distinct from small intestinal motility, and regionalization of contractions in different regions of the colon occurs. ICC-mediated, slow-wave activity causes colonic contractions when the depolarization is of sufficient amplitude. The internal anal sphincter, a specialized thickening of circular muscle, maintains a state of tonic contraction, thus maintaining continence in association with the external sphincter. Distention of the rectum, typically with feces, results in an enteric nervous system–dependent reflexive relaxation of the sphincter (rectoanal inhibitory reflex). 46 It is not surprising, then, that the passage of meconium is inversely related to gestational age at birth. 47 One might postulate that colonic distention results in neural feedback that inhibits motor function in the upper intestine. Indeed, the stimulation of delayed meconium passage is associated with better feeding tolerance. 48 The authors of recent studies suggest that effective colonic contractions do occur but that these are not mediated by the enteric nervous system. 49, 50

Postnatal maturation of the gastrointestinal motility reflexes are dependent on sensory and motor regulation of local and regional enteric reflexes integrated and modulated by the vagus nerve. These reflexes mature in evolution frequency, magnitude, response sensitivity, and associated responses with advanced postnatal maturation.

Implications and Controversies of Gut Motility in Neonatal Gastrointestinal Therapies

• Because the relative importance of different neurotransmitters to gastrointestinal contractile activity changes with infant growth and development, agents that modify composition of gut secretions or prokinetic effects successfully in adult gut will not necessarily have similar effects in infants and children.
• Erythromycin, as a prokinetic agent, may be effective in inducing migrating motor complexes in premature infants older than 33 weeks PMA.
• Enteral trophic nutrition is associated with acceleration of gut motility patterns.
• If feeding intolerance limits the ability to provide full feeding volumes to an infant, smaller feeding volumes may be just as capable of inducing maturation.
• An infant who is intolerant to bolus feedings may tolerate feedings that are given over 1 hour every 3 hours. An infant who has large gastric residuals may tolerate feedings better when longer intervals between feedings are provided.


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Chapter 4 Development of the Intestinal Mucosal Barrier

Camilia R. Martin, MD, MS, Ricardo A. Caicedo, MD, W. Allan Walker, MD

• Nonspecific Mucosal Defenses
• Cell-Antigen Interactions: Innate Intestinal Mucosal Immunity
• Cell-Cell Interactions: Tight Junction Structure and Composition
• Altered Intestinal Permeability in Neonatal and Later Diseases
• Summary
The development of the gastrointestinal tract begins in early embryogenesis primarily facilitated by trophic factors present in amniotic fluid. Immediately after delivery, dietary and environmental factors and antigens interact with the intestinal tract, modulating its development in the postnatal period and ultimately influencing intestinal function and structural integrity. Intestinal development continues throughout early childhood and will eventually serve as the largest defense barrier and a critical component in the development of the innate immune system. 1, 2
The intestinal barrier includes multiple layers of defense that are elegantly coordinated and tightly regulated. These layers of defense include (1) nonspecific mucosal defenses, (2) specific cell-antigen interactions, and (3) specific cell-cell interactions forming the basis of tight junctions that serve to separate the luminal contents containing a myriad of microorganisms and food antigens from effector immune cells in the lamina propria and the internal milieu of the body. Abnormal gastrointestinal development or a breakdown in any of these barrier defenses can lead to pathologic stimulation of the mucosal immune system and thus to an imbalance between immune tolerance and inflammatory responsiveness, which results in an inappropriate response to antigenic challenges, increasing the host’s vulnerability to diseases of chronic, unregulated inflammation and dysregulated immunity. Specifically, breakdown of the barrier has been implicated in the pathogenesis of acute illnesses such as bacterial translocation, leading to sepsis and multiorgan system failure 3 as well as diseases that originate during infancy but manifest later in life, including inflammatory bowel disease, celiac disease, and extraintestinal disorders such as type 1 diabetes (T1D) and atopy.
This chapter provides an overview of intestinal barrier defenses but primarily focuses on the cell-cell interactions that form the structural and functional basis for one of the most important component of the intestinal barrier, the intestinal epithelial tight junction (TJ). How breakdown of this structure (leading to “leaky gut”) plays a role in the pathogenesis of neonatal as well as pediatric diseases is reviewed. In addition, we point to areas wherein a better basic understanding of this structure might lead to prevention or treatment of neonatal pathology related to leaky gut using nutritional or other immunomodulatory factors.

Nonspecific Mucosal Defenses
Specialized intestinal epithelial cells, as well as immune cells residing in the lamina propria of the gut, all contribute to provide local nonspecific barrier defenses that work together to protect the gut from colonization and translocation of potentially injurious pathogenic bacteria and antigens. Examples of nonspecific host defenses include digestive enzymes to eradicate ingested pathogens and destroy antigens, regular peristalsis to prevent bacterial stasis and rapidly eliminate antigen-antibody complexes, and polymeric immunoglobulin A (IgA), which is produced by plasma cells residing in the lamina propria and serves to bind luminal antigens reducing the likelihood of antigen penetration. 4
One critical component of the nonspecific host defenses is the intestinal mucus layer. Intestinal mucus is a complex matrix of water, electrolytes, mucins, immunoglobulins (sIgA), glycolipids, and albumin. 5 This protective layer traps bacteria, preventing direct epithelial binding by microorganisms and facilitating removal of bacteria. Mucins, a major component of the mucus layer, are produced by goblet cells. Although more than 20 mucin genes have been identified, MUC2 is the predominant mucin produced. Goblet cells continuously produce mucin to maintain a constant mucous layer; however, goblet cells also increase mucin production when exposed to specific factors such as hormones (e.g., histamine and serotonin produced by mast cells), 5 inflammatory mediators, and microbial-derived factors such as lipopolysaccharides, flagellin A, and lipoteichoic acids. 6 Goblet cells also produce proteins that help stabilize and repair the mucus layer. These proteins include intestinal trefoil factor and resistin-like molecule-β (RELM-β). 6 RELM-β also provides positive feedback to the goblet cell to increase mucin production.

Cell-Antigen Interactions: Innate Intestinal Mucosal Immunity
Paneth cells produce antimicrobial peptides (AMPs), although other intestinal epithelial cells may also have this ability, 7 in response to lipopolysaccharide (LPS) and other pathogenic antigens. Antimicrobial AMPs are conservatively preserved proteins of the innate immune system that have broad antimicrobial activity. They are currently categorized into two main families: defensins (α and β) and cathelicidins. AMPs exert their antimicrobial effects by creating pores in the organism’s cell membrane, promoting anion influx and eventual killing of the organism. 8 Other bactericidal compounds found in Paneth cells include lysozyme and phospholipase A 2 type IIA. 9 Refer to Chapter 6 for further description of the innate immunity of the intestines.

Cell-Cell Interactions: Tight Junction Structure and Composition
Close cell-cell adherence and interactions form the basis of the intestinal defense barrier separating the inner milieu of the body from the potentially harsh environment of the intestinal lumen. The intestinal barrier is maintained by regulation and maintenance of two pathways: the transcellular pathway and the paracellular pathway. 10 The plasma membrane of the intestinal epithelial cell serves as barrier to most hydrophilic solutes, but the interepithelial paracellular space is partially sealed and forms an intact epithelial barrier. Intestinal epithelial cells adhere to each other through junctional complexes, which are located at the lateral membranes. These junctional complexes serve as trafficking police by not allowing passage of macromolecules but allowing for essential transfer of fluids, electrolytes, and small peptides. The interepithelial junction comprises three major components that have occlusive properties: TJs, adherens junctions (AJs), and desmosomes ( Fig. 4-1 ).

Figure 4-1 Tight junction proteins.
The TJ, sometimes also referred to as the zonula occludens (ZO), represents the major barrier within the paracellular pathway between intestinal epithelial cells. 11 TJs appear as close cell-cell contacts by electron photomicrograph ( Fig. 4-2 ), and the contacts correspond to continuous rows of transmembrane protein particles by freeze fracture electron microscopy. The TJ complex consists of integral proteins or “gatekeepers,” plaque proteins that anchor the complex to the actin cytoskeleton, and cytosolic and nuclear proteins that regulate transcription (and therefore paracellular solute permeability, cell proliferation, cell polarity, and tumor suppression) ( Table 4-1 ). This degree of complexity of the TJ complex correlates with its barrier function. 12

Figure 4-2 Schematic of tight junction function, with freeze-fracture replica.
(From Sawada N, Murata M, Kikuchi K, et al. Tight junctions and human diseases. Med Electron Microsc 2003;36:147-156.)
Table 4-1 TIGHT JUNCTION COMPLEX TJ Components Function Examples Integral proteins Modulate permeability Occludin, claudins, JAM-1 Plaque proteins Anchor the complex Zonula occludens proteins (ZO-1, ZO-2, ZO-3), transmembrane-associated guanylyl kinase inverted proteins (MAGI-1, MAGI-2, MAGI-3), multiple PDZ domain proteins, etc. Cytosolic and nuclear proteins Coordinate paracellular solute permeability, cell proliferation, cell polarity, and tumor suppression Regulatory proteins, tumor suppressors, transcriptional and post-transcriptional factors
Multiple TJ integral proteins have been identified (see Fig. 4-1 ); occludin and members of the claudins family, a group of at least 20 tissue-specific proteins, are the major sealing proteins. 12 The claudins, a family of integral TJ proteins, form ion-selective pores within the TJ strands, whereas occludin and junctional adhesion molecule (JAM) may have an adhesive or signal-transducing function as they interact with various cytosolic complexes. 13 A third JAM also appears to play a role but is not as well delineated. 11 Occludin was once thought to be the major protein contributing to TJ function. However, studies of occludin gene deletion mice demonstrated that they do not lose their intercellular structural morphology, and the barrier function of the intestine is not affected when examined electrophysiologically, despite growth failure and other phenotypic abnormalities. 14 In the intestinal epithelium, claudin-1 may directly associate with occludin laterally in the membrane within the same cell but not intercellularly. 11 The combination of these two proteins functioning together performs the major gatekeeper or barrier function of the tight junction. These sealing proteins, both transmembrane proteins, interact with cytoplasmic plaques that consist of different types of cytosolic proteins that function as adaptors between the TJ proteins and actin and myosin contractile elements within the cell. Acting together, they open and close the paracellular junctions. 15
This dynamic and complex network of proteins interacts but is also influenced by external factors. For example, permeability may be altered by pathologic insults or by other factors such as zonulin, a protein that appears to increase permeability across TJs and has been implicated in the pathogenesis of celiac disease and type 1 diabetes. 1, 16 In addition, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide have all been shown to cause barrier dysfunction, whereas toll-like receptor-2 (TLR2)-activated protein kinase C isoforms influence arrangement of ZO-1 junction proteins, increasing barrier integrity. Optimization of TJs is also mediated by the short-chain fatty acid, butyrate, and the amino acid glutamine. 1, 5

Tight Junction Function
The transcellular and the paracellular pathways regulate which substances cross the epithelial membranes. The transcellular pathway allows molecules to enter from the luminal side of the enterocytes by endocytosis and exit on the serosal side. This section focuses on the paracellular pathway, which is controlled primarily by the TJs, dynamic structures that readily adapt to a variety of developmental, physiologic, and pathologic circumstances. At the molecular level, TJs assume several major functions, two of which are briefly mentioned here: the “barrier” and “fence” functions (see Fig. 4-2 ).
Barrier function refers primarily to the ability to selectively allow particles and solutes to pass through the intercellular space. This can be measured using transepithelial electric resistance (TER) of a monolayer of cells in culture or by placing particles of different size or electrical charge on one side of the membrane and measuring the appearance on the other side. The latter measurements can be done in vitro or in vivo. The relationship between the number of TJ strands and TER is not a linear but a logarithmic one. 17
The fence function of the TJ maintains polarity of the cell. Heteropolymers of transmembrane proteins (primarily occludin, claudins, and JAM) make up TJ strands, which encircle the top of epithelium to delineate the border between the apical and basolateral membranes. There are major differences in the composition of lipids and proteins that constitute the apical and basolateral surfaces of intestinal epithelial cells, and the TJ impedes the lateral diffusion of lipids and proteins between the apical and basolateral membranes, 18 maintaining cell polarity. In other words, the TJ prevents intermixing of molecules in the apical membrane with those in the lateral membrane.
TJs can adjust their integrity in response to physiologic demands by adjusting their degree of phosphorylation. Sodium-glucose cotransport induces phosphorylation of myosin light chains in actin-myosin microfilaments surrounding the TJ, which contract and open the junctions, leading to increased permeability. 19

Factors Affecting the Tight Junction that Are Relevant to Neonatology

Intestinal Maturity and Timing of Intestinal Closure
Pathology of the immature intestine, particularly necrotizing enterocolitis (NEC; discussed at length later), has been linked with barrier dysfunction. The levels of enteropathogen overgrowth reported in preterm neonates with NEC suggest an increased transmucosal passage of bacteria. 20 In an early study, infants born before 34 weeks’ gestation had greater intestinal permeability to lactulose than more mature babies, whereas those of 34 to 37 weeks’ gestation achieved a “mature” intestinal permeability to lactulose within 4 days of starting oral feeds. 21 In neonates born before 28 weeks’ gestation, intestinal permeability at day 7 was higher, and carrier-mediated monosaccharide absorption at day 14 was lower, compared with neonates of 28 weeks’ gestation. The barrier function of the intestinal epithelium transiently decreases during the first week after birth in preterm neonates who are not enterally fed. 22 However, a recent study concluded that in infants of 26 to 36 weeks’ gestation, gut permeability is not related to gestational age or birthweight but is higher during the first 2 days of life than 3 to 6 days later.

In malnourished hospitalized patients, a significant increase in intestinal permeability is seen in association with phenotypic and molecular evidence of activation of lamina propria mononuclear cells and enterocytes. 23 Undernutrition leads to villous atrophy, with rapid recovery of mucosal barrier function when the intestine is renourished. This is also the case when there is a lack of enteral nutrition. Despite being a beneficial therapy in many clinical settings, particularly the neonatal intensive care unit (NICU), total parenteral nutrition (TPN) is the deprivation of enteral nutrition and is associated with intestinal changes in structure and function. Animal studies have demonstrated TPN-induced increases in bacterial translocation and intestinal permeability. In rats receiving TPN, loss of mucosal barrier function to both Escherichia coli and the permeability marker phenol red was greater than in enterally fed rats. 24 TPN administration in newborn piglets led to mucosal atrophy, increased paracellular permeability, and decreased intraepithelial lymphocytes (IELs), but bacterial translocation was not different between the two groups. 25 Intestinal permeability was significantly reduced in TPN-dependent rats receiving an enteral low-residue diet. 26 Enteral administration of lipid-rich nutrition enhanced barrier function and reduced the local intestinal inflammatory response in a rat model later experiencing induced shock mediated by activation of cholecystokinin receptors and subsequent inhibition of cytokine release. This same group showed similar results after induced shock as well, supporting the role of nutrition in intestinal barrier function for both preventative and treatment phases of intestinal injury. 27
In critically ill adult patients, TPN failed to improve urinary lactulose-to-mannitol ratios (indicators of paracellular permeability), whereas enteral nutrition led to recovery of barrier function. 28 In a study of preterm infants, early enteral feeding was associated with a reduction in gut permeability at 10 days of age, whereas feeding of human milk (vs. formula) was associated with decreased permeability at 28 days, and continuous versus bolus feeding did not affect permeability. 29 Because of feeding intolerance and the fear of NEC, very-low-birthweight (VLBW) neonates typically undergo a period of “luminal starvation,” during which time they receive very little food through the gastrointestinal tract. The parenteral route supplies most of their nutrition. Long-term TPN has been associated with breakdown of the mucosal barrier and places VLBW infants at very high risk for mucosal atrophy–related pathology. 30

Individual Nutrients
VLBW infants and other highly stressed patients are frequently deprived of glutamine, a conditionally essential amino acid that has been demonstrated to improve intestinal barrier function. 31 Glutamine plays a central role in numerous metabolic processes. In Caco-2 cells, a cell culture line widely used as a model of mature enterocytes, glutamine helps recovery from stress-induced increased permeability. 32 Animal models and human patients have been shown to benefit from enteral or parenteral glutamine supplementation, which attenuates mucosal atrophy and decreases bacterial translocation, sepsis, and even mortality. 33
Both the ω-3 fatty acids eicosapentaenoic acid (EPA, C20) and γ-linolenic acid (C18) are known to be anti-inflammatory, and both have been show to upregulate TJ function by increasing occludin in human vascular endothelial cell lines. 34 However, EPA enhanced fluorescein sulfonic acid permeability and lowered TER in Caco-2 cells. 35 The reasons for differences in permeability depending on cell type are not understood.
Butyrate, a short-chain fatty acid, also is able to enhance restoration of mucosal barrier function after thermal and detergent injury to the rat distal colon in vitro. 36 In Caco-2 cells, butyrate increases TER in a concentration-dependent manner. The mechanism may be through promotion of TJ protein synthesis or through activation of lipoxygenase by histone acetylation of DNA. 37 In HT29 colonocytes, butyrate significantly reduces paracellular permeability, probably through activation of peroxisome proliferator–activated receptor-γ. 38

Physiologic Stress
Premature infants, by circumstance, are weaned early from their mothers before development is complete. In porcine models, early weaning is associated with activation of intestinal mast cells (MCs), corticotrophin-releasing factor (CRF) and its downstream pathways. 39 Activation of both of these components leads to intestinal barrier dysfunction although the mechanisms are still incompletely understood.
In severely stressed, critically ill patients, intestinal permeability is closely related to the presence of mucosal ischemia. 40 Intestinal leakiness is triggered by a set of changes such as oxidative stress with increased production of nitric oxide and other reactive oxygen species, release of proinflammatory cytokines, reduction of intramucosal pH, and hypoxia. Potent oxidants damage cellular DNA and promote the peroxidation of lipid membranes and the downregulation of the expression of several key TJ proteins of the ileum and colon. 41 Critically ill neonates are subject to multiple physiologic stressors, including feeding restriction, ischemic injury of the intestinal mucosa, hemorrhagic shock, and systemic inflammatory response syndrome (SIRS; discussed later). All these conditions are linked to excess production of oxidative free radicals.

Enteric Microbes
Interaction between intestinal epithelia and enteric microorganisms involves a complex and dynamic “cross-talk.” Commensal microbes maintain appropriate mucosal immune responses to both injury and pathogenic microbes. Enteric pathogens can disrupt the TJ of epithelial cells through a number of different virulence factors. Enteropathogenic E. coli (EPEC) dissociates occludin from the TJ and promotes phosphorylation of the 20-kDa myosin light chain (MLC20), inducing cytoskeletal contraction and a large permeability increase. 42 In a mouse model, EPEC significantly decreases barrier function in the ileum and colon through redistribution of occluding. 3 Similarly, enterohemorrhagic E. coli (EHEC)-infected mice demonstrated redistribution of the tight junction protein occludin as well as claudin-3. In addition, the expression of claudin-2 was increased correlating with increased intestinal permeability. 43 The protozoan Giardia lamblia similarly uses myosin light chain kinase (MLCK) to disrupt TJ function. 44 Clostridium difficile toxins cause disorganization of apical and basal filamentous (F-) actin and dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane in cultured intestinal epithelial cells. 45 Rotavirus, the most common cause of infantile gastroenteritis worldwide, and the opportunistic protozoan Cryptosporidium parvum have both been shown to induce a rapid increase in gut permeability in the acute phase, with recovery of barrier function within 20 days. 46
In polarized epithelial cells, rotavirus results in a paracellular leak and F-actin alteration. 47 The NSP4 protein of rotavirus induces decreased TER, redistributes F-actin, and prevents lateral targeting of the TJ-associated ZO-1 protein. 48 Vibrio cholerae , a diarrheagenic microbe, elaborates zonula occludens toxin (ZOT), which induces a redistribution of the F-actin cytoskeleton, correlating with increased mucosal permeability. 49 Although microbial interaction with intestinal epithelial cells may result in disruption of the tight junction intestinal barrier as described, microbial interaction may also enhance intestinal barrier function, specifically through TLR2 activation. Activation of TLR2 has been shown to increase phosphorylation of protein kinase Cα and protein kinase Cδ, resulting in reorganization of ZO-1. 50 Although TLR2 signaling appears important to strengthen barrier function during infection or injury, it does not appear important in maintaining barrier function homeostasis. 50

Pharmacologic Stress
One of the most commonly used drugs in the NICU, the nonsteroidal anti-inflammatory agent indomethacin, is primarily used for medical closure of the ductus arteriosus. Several studies have associated its use with gastrointestinal perforations. Furthermore, the prostaglandin pathway, which is inhibited by indomethacin, has been linked with a major mechanism (the PI3 kinase pathway) related to control of TJ proteins. Prostaglandins stimulate recovery of decreased paracellular resistance caused by calcium depletion through a mechanism involving transepithelial osmotic gradients and PI3 kinase–dependent restoration of TJ protein distribution. 46 The short- and long-term effects of indomethacin on intestinal barrier function are yet to be determined.

Cytokines and chemokines are soluble factors that mediate the inflammatory response by stimulating or activating lymphocytes and other immune cells. Many of the factors presented previously that alter intestinal barrier structure and function do so through activation of cytokines and chemokines. As discussed earlier, it is not unusual for premature infants to go through prolonged periods of lack of enteral substrate and parenteral nutrition dependency. Rapid villous atrophy ensues, affecting barrier function. Some of the altered levels in mediators that appear to play a role in the loss of intestinal barrier function in this clinical scenario include an increase in IFN-γ and a decrease in interleukin-10 (IL-10). 51 Coupled with these changes in cytokine levels is a reduction in the expression of proteins that compose the TJs. IFN-γ and TNF-α are found in high concentrations in intestinal mucosa affected with inflammatory bowel disease (discussed later). These cytokines increase permeability of enterocyte culture monolayers (decrease the TER), also through activation of MLCK. 52
The role of eicosanoids in inflammatory bowel disease is well established. 53 Eicosanoids are downstream inflammatory signaling proteins activated by arachidonic acid. Such mediators, prostaglandins and leukotrienes, further activate pathways, leading to the redistribution of occludin and other TJ proteins.

Altered Intestinal Permeability in Neonatal and Later Diseases

Prematurity, Necrotizing Enterocolitis, and Systemic Inflammatory Response Syndrome
The premature infant is faced with multiple potential threats altering the development of the intestinal barrier and mucosal immunity ( Table 4-2 ). 54 Developmental immaturity of the intestinal tract disrupts the natural innate defenses offered by nonspecific physical and chemical barriers. Inadequate production of digestive enzymes, mucus, and immunoglobulins and decreased intestinal peristalsis leave the premature infant vulnerable to pathogenic antigenic stimuli. In addition, common medication exposures (antibiotics, H 2 blockers, vasoconstrictors, sedatives, and paralytics) further decrease these inherent defenses by altering natural acidity and reducing peristalsis. Inadequate response by enterocytes is further compromised by prolonged absence of enteral feedings, leading to mucosal and villous atrophy. 55 Each of these components—developmental immaturity, intestinal-altering medications, and induced mucosal atrophy—results in altered intestinal barrier function and immunity and hence in a reduced ability for the neonate to eliminate pathogenic organisms, allowing for epithelial adherence and bacterial translocation.
Table 4-2 NEONATAL INTENSIVE CARE UNIT EXPOSURES AND POTENTIAL CONSEQUENCES ON INTESTINAL BARRIER DEFENSE AND BACTERIAL COLONIZATION IN PREMATURE INFANTS   Exposure Potential Consequences Nonspecific barrier defense Prematurity ↓ Immunoglobulin levels     ↓ Production of digestive enzymes     ↓ Production of mucus     Dysfunctional peristalsis   Delayed feeding Villous atrophy     ↓ Production of digestive enzymes     ↓ Production of mucus     ↓ Peristalsis   Medications     H 2 blockers ↓ Gastric acidity   Vasopressors and indocin ↑ Risk for intestinal ischemia and enterocyte injury   Sedatives and paralytic agents ↓ Peristalsis Bacterial colonization Prematurity Accentuated inflammatory response     Abnormal glycosylation pattern   Delayed feedings Delay in bacterial colonization   Broad-spectrum antibiotics Prolonged sterilization of gut     Delayed colonization of beneficial, commensal bacteria     Preferred bacterial colonization of pathogenic bacteria   Formula feeding and hospitalization Preferred bacterial colonization of pathogenic bacteria
From Martin CR, Walker WA. Intestinal immune defences and the inflammatory response in necrotising enterocolitis. Semin Fetal Neonatal Med 2006;11:369-377.

Necrotizing Enterocolitis
NEC is a devastating gastrointestinal disease predominantly observed in premature infants; its incidence varies inversely with gestational age and is estimated to be 0.3 to 2.4 cases per 1000 births, but it is 4% to 13% in neonates whose birth weight is less than 1500 g. 56 Of 2500 annual cases reported in the United States, 20% to 60% would require surgery, and the mortality rate remains high (20% to 28%). 57 The pathogenesis is incompletely understood and is likely multifactorial. As described previously, the preterm infant possesses and experiences multiple perturbations to postnatal intestinal and immune development, all of which likely work in concert to increase the vulnerability of the preterm infant to NEC. Predisposing factors in premature infants include incomplete innervation and poor motility of the premature gastrointestinal tract, stasis and bacterial overgrowth, low levels of protective mucus and secretory IgA, decreased regenerative mucosal capabilities, increased intestinal permeability, and exposure to medications that alter gastrointestinal acidity and bacterial colonization, such as prolonged exposure to H 2 blockers. 58, 59
Animal models and clinical observations suggest that NEC requires at least three factors to develop: mucosal injury, formula feeding, and the presence of bacteria. 60 The common pathway leads to an inflammatory response and ischemia-necrosis.
Intestinal bacteria and their toxic products may translocate across a compromised epithelial barrier, activating mucosal immune responses. Gram-negative rods ( E. coli, Klebsiella, Enterobacter, and Pseudomonas ) are common pathogens associated with NEC.
Figure 4-3 shows some of the measures that might be used to prevent NEC. The safest and most efficacious practices in the prevention of NEC include maternal breastfeeding, judicious advancement of enteral feedings, and careful infection control measures. Maternal breast milk may benefit the mucosal integrity by adding protective factors such as immunoglobulin, lysozyme, lactoferrin, macrophages, lymphocytes, neutrophils, gut maturation factors, glutamine, platelet-activating factor, acetylhydrolase, and prostaglandin E 1 (a splanchnic vasodilator). Initiation of trophic feeds in VLBW infants and avoidance of high osmolar solutions reduced the incidence of NEC. 61, 62

Figure 4-3 Measures for prevention of necrotizing enterocolitis.
To avoid unbalanced inflammatory responses in the gastrointestinal tract, it is critical to maintain the commensal microbiota, thus avoiding colonization with pathogenic microorganisms. Use of probiotics, live microorganisms introduced to the gut for their commensal beneficial immunoregulatory properties, may protect against NEC by different mechanisms, 63 including immune modulation, downregulation of inflammatory responses, regulation of intestinal permeability, increased mucin production, secretion of antimicrobial substances, inhibition of pathogen mucosal adherence, and stimulation of IgA production.

Systemic Inflammatory Response Syndrome
Severely stressed neonates with sepsis, trauma, or NEC are predisposed to the development of SIRS, which may lead to multiorgan dysfunction. SIRS may be regarded as the uncontrolled systemic expression of proinflammatory cytokines. A leaky gut can promote bacterial translocation, a phenomenon in which live bacteria or their products cross the intestinal barrier and can be retrieved in lymphoid tissues, liver, spleen, or blood. Data in animal models clearly support its existence, and it is likely to also occur in humans, but this has been difficult to ascertain in well-designed studies. 64 Gram-negative pathogens trigger SIRS through secretion of LPS, which in rats causes marked TJ morphologic alteration, MLCK phosphorylation, intestinal cytokine release, and bacterial translocation. 65
Intensive care unit patients who developed SIRS had persistently abnormal intestinal permeability tests during their critical illness and a significantly delayed improvement in permeability tests compared with the non-SIRS cohort. 66 However, several clinical studies have demonstrated that bacteria isolated from patients with systemic infections are often of the same strain as bacteria predominant in the fecal flora. The gram-negative bacteria present in the intestine often are the agents responsible for infectious complications in high-risk hospitalized patients, and the presumably translocated enteric bacteria are sometimes recovered from the mesenteric lymph nodes of these patients. 67 The value of enteral feeding should not be underestimated in critically ill patients. Early enteral feeding can improve intestinal permeability and is associated with a decrease in multiorgan dysfunction syndrome. 68
In addition to neonatal necrotizing enterocolitis, the leaky gut phenomenon has been implicated in the pathogenesis of a variety of diseases that present later in childhood, many of them chronic. The association between barrier breakdown and disease was first recognized in the critical care setting, in patients with multiorgan dysfunction. 69 A compromised intestinal epithelium, due to the combined effects of a genetic defect and stress or trauma, allows translocation of bacteria and antigens to the lamina propria, triggering an inappropriate or pathogenic immune response. 1 The resultant disorder may be limited to the gastrointestinal tract, as with infectious enteritis, but often will have extraintestinal manifestations, as in celiac disease and atopic conditions. In many cases, barrier dysfunction is postulated as a cause of disease, but there is evidence suggesting that it may also, or even only, be a result of the disease process. Some of the more common conditions associated with altered intestinal permeability are discussed individually here.

Inflammatory Bowel Disease
Chronic inflammatory bowel disease (IBD) has a worldwide prevalence of more than 1 million and a rising incidence, estimated at 50,000 new cases yearly. The incidence of both IBD phenotypes (Crohn’s disease and ulcerative colitis [UC]) is also increasing in the pediatric population. An estimated 25% to 30% of IBD patients have the onset of symptoms or are diagnosed before the age of 20 years. 70 This early onset implies a higher lifetime morbidity burden; in the United States, the health care costs of IBD are estimated to exceed $1 billion annually. 71
The pathogenetic mechanisms are as yet undefined but are likely to involve a combination of genetic, environmental, and immunologic factors. One such innate immune factor, intestinal barrier function, has been associated with the development of IBD. Intestinal permeability is increased in humans with both UC 72 and Crohn’s disease. 73 Noninvasive permeability measurements such as urinary 51 Cr-labeled EDTA or lactulose-to-mannitol excretion ratio have been shown to correlate with disease activity. 74 Abnormal sugar probe tests have predicted clinical relapse in Crohn’s disease as much as 1 year in advance. 75 Increased permeability has also been documented in asymptomatic first-degree relatives of IBD patients, suggesting a genetic predisposition. 76 A very recent study in healthy first-degree relatives of Crohn’s disease patients found a significant association between abnormal lactulose-to-mannitol excretion ratios and a NOD2/CARD15 mutation that may predict more aggressive or complicated Crohn’s disease. 77
In both active and inactive IBD, interepithelial TJ structure is altered. Colonic mucosa from patients with UC and Crohn’s disease revealed dramatic, global downregulation of the key TJ transmembrane protein occludin in regions of active neutrophilic infiltration and in quiescent areas in the biopsy samples. 78 Occludin and zonula occludens proteins are dislocated from the apical to basolateral surface of epithelia from inactively inflamed Crohn’s mucosa. 79 Translating these findings to barrier function, Soderholm and colleagues demonstrated that in similar Crohn’s mucosa, luminal stimuli produce ultrastructural dilatations in TJ and induce a rapid increase in permeability to 51 Cr-EDTA. 80 This impaired intestinal barrier function is believed to allow the passage of luminal antigens to the lamina propria, where they stimulate an immune response, which when dysregulated leads to altered bacterial microbes and chronic inflammation. 81 Activated macrophages secrete proinflammatory mediators, which act on the TJ to perpetuate the permeability defect. Two such cytokines, central to the cascade leading to IBD, are TNF-α and IFN-γ. TNF-α disrupts TJ assembly and increases endosomal uptake of luminal antigens in ileal Crohn’s disease, whereas IFN-γ induces endocytosis of the major TJ proteins claudin-1 and occludin. 12 In synergy, they activate MLCK, leading to cytoskeletal changes that open the TJ, promoting a vicious cycle of increased leakiness and abnormal immune stimulation. 82
Clinical diagnosis of IBD relies primarily on histologic and radiographic findings. Intestinal permeability markers have been proposed as screening tests for IBD, with utility in active small bowel Crohn’s disease, but not in other phenotypes. In patients with Crohn’s disease in clinical remission, an increased intestinal permeability can predict those at significant risk for relapse in the next few months, with less than 20% of those with normal barrier function relapsing over the ensuing 6 months. 83
Treatment of IBD is evolving beyond immunosuppression and surgery. Enteral nutrition therapy with elemental diets has been shown to produce remission of active Crohn’s disease and to restore intestinal permeability. 84 Targeted molecular therapy of IBD with infliximab, a monoclonal antibody directed against TNF-α, has resulted in significant clinical improvement and documented mucosal healing in both pediatric and adult patients. It curtails inflammation by inducing apoptosis of effector T lymphocytes, downregulating IFN-γ production, and restoring mucosal architecture. 75 Crohn’s disease patients treated with infliximab had significant decreases in permeability measured by 51 Cr-EDTA excretion, accompanied by decreased disease activity indices. 85 Along the same lines, the barrier disruption may be corrected by inhibiting MLCK or using the anti-inflammatory cytokine IL-10 to counter IFN-γ, making these attractive targets for future therapies. 75

Celiac Disease
Also known as sprue or gluten-sensitive enteropathy, celiac disease (CD) is thought to be among the most common genetically determined conditions to affect humans, with a prevalence rate of 0.5% to 1% of the general population. 86 It occurs in individuals with a genetic predisposition, which is strongly linked to the human leukocyte antigen (HLA)-DQ2 and -DQ8 genotypes. Classically a disease of early childhood, it is now recognized as a common condition (up to 1 : 100 in some regions) with a wide spectrum of clinical manifestations that could be diagnosed at any age. 87
CD has become the model of intestinally mediated autoimmune diseases that result from a breach in the intestinal barrier. Structural changes in small intestinal epithelial cell TJ associated with increased ionic permeability have been observed in biopsy specimens from children with active CD. 88 The pathophysiology of CD is triggered by immune-mediated intestinal inflammation induced by gliadin (a protein component of certain grains, including wheat, barley, and rye) upregulating the release of zonulin, a reversible mediator of TJ disassembly, through MyD88. 89 Zonulin subsequently induces actin polymerization, followed by cytoskeletal changes, leading to “opening” of TJ and allowing for intraepithelial passage of gliadin into the lamina propria, where it is deamidated by tissue transglutaminase (tTG), producing antigenic epitopes. 90 These bind to HLA receptors on the surface of antigen-presenting cells and are presented to specific T lymphocytes, leading to an abnormal adaptive immune response that involves the release of cytokines and zonulin, perpetuating the epithelial injury. 1 In addition, gliadin itself was found to have a direct effect on TJ in Caco-2 cell cultures, reorganizing actin filaments and altering expression of occludin, claudin-3, and claudin-4. 91
CD is widely underdiagnosed, owing to under-recognition of its protean manifestations and, until recently, low yield and limited availability of screening tests. Improved methods have conferred sensitivity of 91%, specificity of 97%, and positive predictive value of 97% on measurement of IgA directed against tTG. 92 The gold standard for diagnosis is small bowel biopsy demonstrating villous atrophy and intraepithelial lymphocytic infiltration. The utility of intestinal permeability tests for screening a general population for CD has been studied, but these tests are not being widely used. 93 The only treatment for CD with documented efficacy is a strict gluten-free diet. Treatment is recommended because of associated long-term outcomes, including increased risk for intestinal malignancy, recalcitrant anemia, osteoporosis, liver injury, and neurologic deficits. Within 2 months of starting a gluten-free diet, small intestinal barrier function, as measured by sugar probe permeability tests, recovers, and this precedes histologic and morphometric recovery. 94 Furthermore, a gluten-free diet reverses gliadin-induced downregulation of the TJ component ZO-1. 95 A recent prospective cohort study of CD patients treated with strict gluten avoidance demonstrated that permeability normalized in 87% after 1 year of treatment and that permeability testing correlates better with trace gluten ingestion than serologic testing. 96 Adherence to a strict gluten-free diet is difficult; the searches for an alternative, more palatable treatment for CD will probably target molecules involved in altered permeability.

Type 1 Diabetes
The defining features of T1D—insulin deficiency and hyperglycemia—result from an immune-mediated destruction of insulin-secreting β cells in the pancreatic islets. 97 The interplay between impaired barrier function and a dysregulated proinflammatory response to dietary antigens has been implicated in the pathogenesis of T1D. 16, 98 As with Crohn’s disease, individuals with T1D have impaired intestinal barrier function and increased intestinal permeability demonstrated by studies using mannitol and lactulose. 99 In addition, T1D patients without CD had structural alterations such as aberrant microvilli and thickened TJs in their intestinal epithelia, as seen by transmission electron microscopy, and these correlated with abnormal sugar probe tests. 100
In T1D animal models (the diabetes-prone BB [BBDP] rat), an increase in intestinal permeability as demonstrated by an increased lactulose-to-mannitol excretion ratio is evident before the onset of disease. 101 This abnormality of the rodent gut barrier is associated with low expression of the major TJ protein claudin-1, confirming the correlation between structure and function in this disease process as well. 102 Oral administration of a zonulin inhibitor to BBDP rats blocks autoantibody formation and zonulin-mediated intestinal permeability increase, reducing the incidence of diabetes. 103
As in CD and the BBDP rat, zonulin may play a central role in mediating this passage of nonself antigens through the gut epithelium. Sapone and colleagues reported on a large subgroup of T1D patients in whom serum zonulin levels were elevated in correlation with increased lactulose-to-mannitol excretion ratios. 104 This zonulin upregulation was found in individuals at genetic risk for T1D and preceded the onset of overt T1D. Paralleling CD, the loss of intestinal barrier function preceding T1D may permit a switch from tolerance to immunity to nonself antigens that continuously cross the intestinal mucosa. 105 The high prevalence (up to 10%) of CD in the T1D patient population and shared HLA haplotype associations between the two diseases suggest a common mechanism of underlying intestinal barrier dysfunction. 106 In addition to a role in T1D pathogenesis, increased intestinal permeability leads to higher variation in postprandial blood glucose levels, complicating metabolic control. 107

Atopic Disease
The incidence of atopic diseases has dramatically increased over the past several decades in developed countries. More than 50 million Americans suffer from atopic diseases, generally referred to as allergy. Allergies are the sixth leading cause of chronic disease in the United States, costing the health care system $18 billion annually. Environmental and genetic factors play a role in susceptibility to allergy. The potential mechanism for this phenomenon has been coined the hygiene hypothesis. 108, 109 The hygiene hypothesis of atopic disease suggests that environmental changes in the industrialized world have led to reduced microbial contact at an early age and thus in the growing epidemic of atopic eczema, allergic rhinoconjunctivitis, and asthma. 110 In support of the importance of the intestinal ecosystem are the different colonization patterns observed in atopic versus healthy individuals 16, 111 and the potential benefits of probiotic supplementation. 112
The commensal microflora in the gut is essential for antigenic stimulation that leads to the Th1-type response of the gut-associated lymphoid tissue (GALT) and protects against the development of intestinal inflammation. The absence of commensal bacteria in animal models (e.g., germ-free mouse model) produces Th2-type hyperresponsiveness and lack of tolerance to orally administered antigens ( Fig. 4-4 ). 113

Figure 4-4 Diagram of the Th1-Th2 paradigm. Ig, immunoglobulin; Th, helper T cell; T naïve, naïve T cell.
Murine models implicate signaling through TLR4 as a mechanism by which the luminal flora can influence the response to food antigens. Antibiotic administration during the neonatal period reduced and altered gut wild-type and TLR4-deficient microflora, inducing an allergic phenotype. When commensal flora is allowed to repopulate (during the newborn period), the allergen-specific IgE and Th2 cytokine responses are reduced in antibiotic-treated mice. 114
Gastrointestinal disorders have been reported in children with atopic eczema in several studies. Caffarelli and coworkers showed that gastrointestinal symptoms (such as diarrhea, vomiting, and regurgitation) are more common among patients with eczema than in healthy controls. The frequency of symptoms correlated with extent of the disease. 115
Some microbial products have been shown to be efficacious for the treatment or prevention of allergy in both experimental models and clinical trials. New research on probiotics suggests that certain types of bacteria, especially lactobacilli, can ameliorate allergic inflammation. A prospective study of 159 children with a family history of atopy showed that increased intakes of retinol, calcium, and zinc and perinatal administration of probiotics reduced the risk for developing eczema. 116
In a double-blinded, placebo-controlled study of 62 mother-infant pairs, administration of probiotics to the pregnant and lactating mother increased the anti-inflammatory transforming growth factor-β2 in the breast milk. 117 The risk for developing atopic eczema during the first 2 years of life in infants whose mothers received probiotics was significantly reduced in comparison with that in infants whose mothers received placebo (15% and 47%, respectively; relative risk, 0.32 [95% confidence interval, 0.12 to 0.85]; P < .01). The infants most likely to benefit from maternal probiotic supplementation were those with an elevated cord blood IgE concentration. Administering probiotics during pregnancy and breastfeeding may enhance the immunoprotective potential of breastfeeding and provide protection against atopic dermatitis during the first 2 years of life.
Other gastrointestinal defects observed in atopic individuals include a deficiency of IgA and increased intestinal permeability. Mucosal permeability modulation and timing of antigen exposure are promising research areas in prevention of atopic diseases.

Autistic spectrum disorder (ASD) is a pervasive developmental disorder, with a complex clinical diagnosis as defined by the American Psychiatric Association, 118 and without known biomarkers or clear etiology. There have been many anecdotal reports of increased nonspecific gastrointestinal symptoms (such as abdominal pain, diarrhea, bloating, food aversion) in ASD patients. Figure 4-5 illustrates how gastrointestinal dysfunction may predispose to autism. Mucosal inflammatory responses to dietary proteins could be an explanation for a subset of ASD patients with gastrointestinal complaints. Nonallergic food hypersensitivity (adverse reaction) to dietary proteins is mostly mediated by cellular immunity and in small proportion by immunoglobulin E. Jyonouchi and associates showed that peripheral blood monocytes in patients with ASD and gastrointestinal symptoms have elevated production of TNF-α and IL-12 in response to cow’s milk protein. 119

Figure 4-5 Potential role of the gastrointestinal tract in autism.
There is growing evidence that children with ASD have an immune dysregulation with proinflammatory imbalance. Jyonouchi and associates also found that children with ASD have exaggerated innate immune responses most evident in TNF-α production (dominant Th2 pattern). 120 Cytokines have widespread systemic effects, including the central nervous system, and may be factors contributing to changes in mood and sleep patterns in ASD patients. Between 30% and 70% of ASD patients have circulating antibrain autoantibodies such as antibodies to neuron-axon filament proteins, 121 cerebellar neurofilaments, myelin basic protein, caudate nucleus, 122 serotonin receptor, brain endothelial cells, 123 and brain tissue. This evidence suggests autoimmunity as an important factor in ASD, but it does not clarify whether this is an etiologic factor or a secondary phenomenon. 124
Abnormal intestinal permeability has been reported in 43% of ASD children without clinical or laboratory findings consistent with a known intestinal disorder. 125
Dietary proteins such as gluten and casein are digested by intestinal peptidases in the lumen of the small intestine. The resulting short-chain peptides (e.g., gliadomorphin and casomorphin) are very similar to endorphins and are called exorphins . 126 These exorphins are neuroactive and may interfere directly with the function of the central nervous system if they enter the body. 127, 128 Exorphins have a high and physiologically significant affinity with the binding sites of endogenous opioid receptors. 129 Intravenous infusion of the exorphin B casomorphin-7 (exorphin) activates rat brain cells. 130 The neonate is more likely to experience this effect of exorphins because of the more permeable gastrointestinal tract and immature nervous system.
There are reports that gluten-free and casein-free diets improve behavior in ASD patients. 131 The improvement but not elimination of the neurobehavioral symptoms raises the possibility that elevated exorphins cause permanent damage to the infant brain. There is evidence of a possible association between gut function, immune dysregulation, and ASD, but the present data are insufficient to prove causality.
A better understanding of the role of gastrointestinal mucosal permeability may provide future answers about the pathology and treatment of ASD.

Other Diseases
A role for or an association with gut barrier breakdown and dysfunction has been reported in a wide variety of clinical conditions, some of which affect infants and children. A reversible impairment in gut permeability, associated with local immune cell and enterocyte activation, occurs in patients with obstructive cholestasis, paralleling observations in a rat model. 132 Other examples of diseases in which gut leakiness has been observed include juvenile rheumatoid arthritis, systemic lupus erythematosus, acute pancreatitis, and chronic renal failure. 133

The gastrointestinal system is a complex yet coordinated system of nonspecific barriers and defenses. Barrier function, especially the role of interepithelial TJs, is integral to overall gastrointestinal function, protecting the gut from invasion by pathogenic microbial agents or antigens and subsequent inflammation and tissue injury. Barrier dysfunction, or increased intestinal permeability, may be a pathogenetic mechanism underlying diseases such as NEC and SIRS in the neonate, as well as autoimmune and inflammatory conditions that present later in infancy or childhood. Developmental, environmental, and medical exposures early in life can affect TJs and barrier function, potentially leading to lifelong consequences. More studies are needed to further elucidate the mechanisms by which the intestinal barrier integrity is breached. Knowledge gained from these studies will help inform medical care practices to enhance intestinal barrier function, optimizing mucosal immunity and preventing the development of intestinal-mediated disease.


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