Advances in Food and Nutrition Research Volume 37

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 37

ADVISORY BOARD

DOUGLAS ARCHER Washington. D.C.

JESSE F. GREGORY 111 Gainesville. Florida

SUSAN K. HARLANDER St. Paul, Minnesota

DARYL B. LUND New Brunswick, New Jersey

ROBERT MACRAE Hull, England

BARBARA 0. SCHNEEMAN Davis, Calfornia

STEVE L. TAYLOR Lincoln, Nebraska

ADVANCES IN

FOOD AND NUTRITION RESEARCH VOLUME 37

Edited by

JOHN E. KINSELLA College of Agricultural and Environmental Sciences University of California, Davis Davis, California

ACADEMIC PRESS, INC. A Division of Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, 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 photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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DEDICATION

This book is dedicated to the memory of John Edward Kinsella.

Born in Wexford, Ireland, John E. Kinsella was an internationally distinguished scientist and a dedicated administrator. He received his bachelor’s degree in natural and agricultural sciences from National University in Dublin in 1961, and his master’s degree in biology in 1965 and doctor’s degree in food chemistry in 1967 from Pennsylvania State University. Joining the faculty at Cornell University in 1967, he was named chair of the Department of Food Science in 1977, serving until 1985. He became associate director of Cornell’s Institute of Food Science in 1977, and director from 1980 to 1987. At Cornell, he was designated Liberty Hyde Bailey Professor of Food Chemistry, a named chair, in 1981 and General Foods Distinguished Professor of Food Science, an endowed chair, in 1984. He was a Fulbright Professor of Food Science and Nutrition at University College Cork, Ireland in 1984. In the spring of 1989, he was Campbell-Tyner Eminent Scholar at Florida State University. He came to the University of California, Davis in September 1990 as Dean of the College of Agricultural and Environmental Sciences. Dean Kinsella received numerous academic honors, including the Borden Award (1976) for research in biochemistry of mammary tissue and milk, the Babcock Hart Award (1987) for research in food science and nutrition, the Atwater Award (1988) in recognition of international nutrition and food science research, the Spencer Medal Award (1991) for outstanding research in food and agricultural chemistry, the Chang Award (1991) for research and advancement in lipid and flavor chemistry, and the Tanner Lectureship (1991) for scientific contributions to food chemistry. An authority on the biochemistry of dietary fatty acids, he was also the holder of eight patents. He served as an officer and consultant for many organizations, including the U.S. Department of V

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DEDICATION

Agriculture, World Bank, National Research Council’s Board on Agriculture, National Cancer Institute, and the Food and Nutrition Board of the National Academy of Science’s Institute of Medicine. At the University of California, Davis, Kinsella was instrumental in establishing the University of California FoodSafe Program, a consumer-oriented food safety education program. He was shepherding the College of Agricultural and Environmental Sciences through a bold reorganization aimed at guiding the College’s mission of teaching, research, and public service into the next century. John E. Kinsella died on May 2, 1993. He is survived by his wife Ruth Ann, his sons Sean and Kevin, his daughters Helen and Kathryn, and his granddaughter Hannah.

CONTENTS

CONTRIBUTORS TO VOLUME 37 ............................. PREFACE ................................................

xi xiii

Food. Diet. and Gastrointestinal Immune Function

James J . Pestka I . Introduction ....................................... I1. Overview of the Gastrointestinal Immune System ...... I11. Diseases Involving the Gastrointestinal Immune System ........................................... IV . Impact of Food Constituents and Contaminants on Gastrointestinal Immunity ........................... V . Modification of Gastrointestinal Immunity through Food and Diet .......................................... VI . Research Needs ................................... References ........................................

1 2

22 35 45 53 56

Effect of Consumption of Lactic Cultures on Human Health

Mary Ellen Sanders I. I1. I11. IV .

V.

VI . VII .

Introduction ....................................... General Physiology ................................. Health Targets ..................................... Safety Issues ...................................... Considerations for Strain Selection ................... Research Needs ................................... Conclusions ....................................... References ........................................

67 71 78 114 115 116 121 121 vii

...

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Defining the Role of Milkfat in Balanced Diets

Louise A . Berner

I . Introduction ....................................... I1 . Background Information-Dietary Fat Consumption and Composition ...................................... 111. Dietary Fat. Dairy Products. and Coronary Heart Disease ........................................... IV . Dietary Fat. Dairy Products. and Cancer Risk ......... V . Milkfat as Part of the Total Diet ...................... VI . Conclusions and Research Needs .................... References ........................................

131

133

153 208 224 236 239

Biochemistry of Cardiolipin: Sensitivity to Dietary Fatty Acids

Alvin Berger. J . Bruce German. and M . Eric Gershwin

I . Introduction ....................................... I1 . Discovery of Polyglycerophospholipids ............... 111. Abundance of Polyglycerophospholipids .............. IV . Pathways of PolyglycerophospholipidSynthesis ....... V . Intracellular Location of Polyglycerophospholipid Synthesis ......................................... VI . Conformation of Cardiolipin in Biomembranes ......... VII . Degradation of Polyglycerophospholipids ............. VIII . Oxidation of Cardiolipin ............................ IX . Association of Enzymes with Cardiolipin .............. X . Cardiolipin Acyl Composition ....................... XI . Influence of Diet and Other Factors on Cardiolipin Content ................................ XI1 . Possible Role of Cardiolipin in Resistance to Ethanol-Induced Membrane Disordering .............. XI11. Immunologic Activity of Cardiolipin .................. XIV . Chemical Synthesis of Acyl-Specific Cardiolipin Derivatives ........................................ xv . Chromatographic Separation of Cardiolipin ............ References ........................................

260 261 261 264 267 270 274 275 275 289 300 302 304 314 317 318

CONTENTS

ix

Diseases and Disorders of Muscle

A . M . Pearson and Ronald B . Young 1. I1 . I11. IV . V. VI .

INDEX

Introduction ....................................... Disorders and Diseases of Muscle .................... Disorders of Energy Metabolism ..................... Diseases of the Connective Tissues ................... Research Needs ................................... Summary ......................................... References ........................................

...................................................

340 341 374 387 405 409 410 425

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CONTRIBUTORS TO VOLUME 37

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Alvin Berger, Department of Food Science and Technology, University of California, Davis, Davis, California 95616 (259) Louise A. Berner, Nutrition Consultant, Sun Luis Obispo, California 93401 ( I 3 I ) J . Bruce German, Department of Food Science and Technology, University of California, Davis, Davis, California 95616 (259) M. Eric Gershwin, Division of RheumatologylAllergy and Clinical Immunology, University of California, Davis Medical School, Davis, California 95616 (259) A. M . Pearson, Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331 (339) James J . Pestka, Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824 (1)

Mary Ellen Sanders, Microbiology Consultant, Littleton, Colorado 80122 (67) Ronald B. Young, Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama 35899 (339)

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PREFACE

Although the role of foods in providing nutrients (and nonnutrients) to maintain metabolic functions for normal functions (i.e.. energy generation and growth and maintenance of tissue integrity) has been well elucidated, the role of food components in disease prevention, particularly as mediated by the immune system, is finally receiving deserved attention. In this regard, the importance of the gastrointestinal (GI) immune system in health and disease is being systematically clarified. Perturbation or dysfunction of the GI immune system is associated with diseases such as microbial infections, allergies, inflammatory states, autoimmunity, and neoplasms. Ongoing research in cellular and molecular immunology is providing a greater understanding of the interaction of dietary components with the GI immune system. More complete knowledge of dietary factors affecting the GI immune system may provide opportunities to produce foods that ameliorate disturbances and/or enhance GI immune system functions and overall health. In this volume, Pestka reviews the nature of the GI immune system and the complexity and varied functions of the mucosal immune system. Diseases involving the GI immune system, hyperactivity, allergenicity, food-sensitive enteropathies, inflammatory bowel disease, and immunosuppression of the GI system can be affected by dietary components. Nutritional therapies for intestinal diseases, immune-compromised subjects, and autoimmune disorders are now feasible, and the possibility of providing probiotic agents to colonize the gut with beneficial bacteria is of emerging interest. Sanders provides a comprehensive overview of the current information concerning the possible therapeutic benefits of lactic acid bacteria (lactobacilli), particularly those in cultured dairy foods. The author discusses the diversity and complexity of the gastrointestinal ecology and the difficulty of experimentation in this field. The value of food microbes in the treatment of gastric disturbances, ...

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PREFACE

lactose intolerances, diarrhea, and cancer suppression by reducing mutagens, possibly in cholesterol reduction, and by immune stimulation, is reviewed. Probiotics for human food are now being commercialized; however, standards for ensuring functional criteria are needed. The issue of the safety of cultured microbes and the possibility of transfer of genetic material warrants monitoring. The understanding of the role of dietary fat in atherogenesis, atherosclerosis, and thrombosis is still incomplete. Although some useful general guidelines have emerged concerning food fats, the effects of relative amounts and ratios of different fatty acids in different foods is still incomplete. Dietary fat is needed to provide energy, sufficient essential fatty acids (1-3 g/day), and fat-soluble vitamins. In this context it should be relatively easy to prescribe rather accurately the fatty acid needs for various ages and life stages, and the associated energy need patterns, rather than the current approach of advising what fats not to consume. It is time to discuss quantities of specific fatty acids and relate these to the optimum mixture of foods which should be consumed. In this regard, forbidding important foods which deliver essential nutrients (because they contain fats which if eaten in inordinate quantities exacerbate some common chronic diseases) is simplistic at best and may be predisposing certain subjects to the danger of other deficiencies. Thus, the tendency to advise against dairy foods because butterfat is conducive to atherosclerosis, when consumed as the predominant source of fat, may not be generally beneficial to all consumers. Dairy foods are a principal source of calcium, riboflavin, and several other nutrients. The chapter by Berner reviews the literature and addresses the questions of the role of dairy food milkfat in health and disease. The data indicate that dairy foods are desirable in a normal, prudent diet because they ensure a balanced intake of nutrients. Ongoing research to improve the fatty acid composition of dairy foods is outlined. Among the important roles of dietary fatty acids is their influence on composition of cellular and intracellular membranes. This, in turn, can affect several metabolic functions (e.g., receptormediated uptake; mediation of cellular signaling; regulation of membrane-bound enzymes), and in the case of mitochondria, this affects reactions related to energy generation. Cardiolipins are a unique class of acylpolyglycerolphosphatides that are particularly

PREFACE

xv

enriched in the outer membrane of cardiomuscular mitochondria where they may affect enzyme functions. Cardiolipins have an unusual fatty acyl composition that is more resistant to dietary fatty acids than other lipid classes. The role and possible functions of cardiolipins are reviewed; however, little is known about whether modification via diet affects function in animals, and data on humans are not available. Muscle tissue is a significant source of nutrients, and its integrity is important for production efficiency and food quality. The final chapter in this volume catalogs the diseases and disorders of muscle tissue, including sarcoplasmic proteins and connective tissue components. Some of these are related to nutritional disorders and nutrient imbalances. Advances in Food and Nutrition Research fills an important niche in providing a timely, comprehensive review of topics and subject matter that link food, nutrition, health promotion, disease prevention, and amelioration. Scholarly in-depth reviews are welcome. JOHN E. KINSELLA

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ADVANCES I N FOOD AND NUTRITION RESEARCH. VOL. 37

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION JAMES J. PESTKA Department of Food Science and Human Nutrition Michigan State University East Lansing. Michigan 48824

1. Introduction 11. Overview of the Gastrointestinal Immune System

111.

IV.

V.

VI.

A. Anatomy of the Gastrointestinal Tract B. Nonspecific Immune Mechanisms C. Specific Immune Mechanisms Diseases Involving the Gastrointestinal Immune System A. Inadequacy of Normal Function B. Hyperactivity C. Immunosuppression Impact of Food Constituents and Contaminants on Gastrointestinal Immunity A. Microbes and Microbial Products B. Food Allergens C. Chemicals in Food D. Nutritional Composition Modification of Gastrointestinal Immunity through Food and Diet A. Breast-Feeding 8. Detection and Avoidance of Food Antigens and Allergens C. Development of Hypoallergenic Foods D. Control of Microbial Flora E. Autoimmune Therapy by Oral Tolerance Induction F. Nutritional Therapies Research Needs References

1.

INTRODUCTION

The evolution of higher organisms was dependent on the concurrent development of defensive barriers that excluded the external environment from the increasingly complex internal milieu. Although epithelial struc1 Copyrighl 0 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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tures such as skin provided for innate defense, more specialized elements were required in the alimentary canal because this was the specific site of absorption from the external environment and because intestinal lumen contents included low and high molecular weight products of digested food, ingested microbes, and the natural commensal microflora. Thus, the intestine of higher organisms evolved as a partially penetrable filter that largely prevents entry of macromolecular material but allows passage of small nutrients. When deleterious components penetrate the barrier, resultant immune reactions must limit this penetration without compromising the function or integrity of the intestine. The importance and complexity of the gastrointestinal (GI) immune system are evidenced readily by serious effects that are manifested in its failure or dysregulation, for example, microbial infections, allergy, neoplasms, inflammatory diseases, and autoimmunity. Advances in cellular and molecular immunology have led to a greater understanding of the GI immune system and its interaction with diet. This information undoubtedly will lead to novel approaches to maintenance of its homeostasis through diet as well as through therapies for immunologically mediated diseases. In addition, regulatory concerns may arise over the foods eaten by certain sensitive individuals. Ultimately, this new information could afford opportunities for the development of novel foods that benefit general or select populations. The purposes of this chapter are (1) to provide an overview of the GI immune system in health and disease, (2) to relate its function to food composition and nutritional value, and (3) to discuss the current status of and future prospects for beneficial modification of this system through food and diet. Although emphasis is placed on the human GI immune system, generalities are drawn from well-developed animal models. II. OVERVIEW OF THE GASTROINTESTINAL IMMUNE SYSTEM

A. ANATOMY OF THE GASTROINTESTINAL TRACT Discussion of the GI immune system requires a general understanding of the anatomy of the digestive tract. The GI tract (Fig. I) is approximately 30 feet long and is composed of four layers including a mucus membrane lining, a highly vascular submucus coat, longitudinal muscle layers, and an additional oblique layer of muscle fibers (Burke, 1985). Food entering the mouth is disrupted mechanically by the teeth and mixed with lubricating salivary secretions. This material then travels via the pharynx and esophagus to the stomach. On entering the stomach, food is digested by gastric

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

3

FIG. I . Anatomy of the gastrointestinal tract.

juices that contain pepsin, lipases, and acids. The stomach absorbs little food because the food is not yet in diffusible form. Opening the pyloric valve allows stomach contents to enter the small intestine (Burke, 1985). This organ is approximately 20 feet long and has been subdivided into sections called the duodenum, jejunum, and ileum (Fig. 1). The inner mucosal surface has numerous circular folds that con-

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FIG. 2. Sectional views of the small intestinal lining. (A) Circular folds containing villi.

(B)Expanded view of multiple villi found on folds. (C) Expanded view of villi containing absorptive lymphatic and blood capillaries.

tain small protuberances called villi (Fig. 2). Villi effectively increase the total surface area almost 600-fold, thus enhancing the absorptive capacity of the small intestine. Villi contain blood capillaries that permit absorption of monosaccharides and amino acids as well as lymph capillaries that facilitate absorption of fatty acids and glycerol. Enzymes and hormones secreted by the liver and pancreas provide numerous accessory functions to the small intestine. Because of its length, surface area (equivalent to a singles tennis court), and digestive capacity, most absorption (>80%) occurs in the small intestine. Undigested food material enters the large intestine, travels to the colon, and finally exits the anus. Clearly, the large surface area, high nutrient content, and absorptive capacity of the alimentary canal make it particularly prone to penetration by microbial agents and food macromolecules. This challenge is met by an extraordinary array of nonspecific and specific immume mechanisms.

B. NONSPECIFIC IMMUNE MECHANISMS Nonspecific or “innate” immunity is the front line of host defense against microorganisms in the gut and other sites (reviewed by Walker,

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

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TABLE I MECHANISMS OF NONSPECIFIC IMMUNITY IN THE GASTROINTESTINAL TRACT

Mechanism Physical Secretory Microfloral Cellular

Examples Epithelial membranes, intestinal mobility Gastric acidity, bile acids, proteolytic enzymes, mucus Site occupation, bacteriocins, volatile fatty acids Macrophages, pol ymorphonuclear phagocytes

1983; Newby, 1984). These mechanisms might be considered constitutive properties of the normal host. The general types of nonspecific immunity in the gut are summarized in Table I. A fundamental mechanism used by a host to avoid microbial infection is performed by the epithelial barriers, which exclude most (299%)proteins in the intestinal tract (Newby, 1984). Constant renewal of epithelial cells as they move from the crypt insures that damaged villi do not remain in the intestine as foci for infection. Motility of the gut contents also keeps the small intestinal microfloraat low levels relative to the large intestinal microflora, which exist in a more static environment. Secretions provide another major form of nonspecific immunity in the GI tract. For example, the low pH of the stomach (c4.0) along with popsin facilitates destruction of pathogens, as well as of their toxins and immunogenic macromolecules. Bile acids and pancreatic secretions that contain proteases such as trypsin and carboxypeptidase also can function as protectants against microbial pathogens. Also, gastric and intestinal epithelia are covered by a moving layer of mucus that is shed continuously into the lumen. Mucus is a glycoprotein that consists of a long polypeptide chain surrounded by oligosaccharide units that protect it against proteolytic attack. In addition to functioning as a lubricant and as protection for the stomach and intestine from acidic pH, mucus provides a vehicle for antibacterial substances (secretory immunoglobulin A, lysozyme, lactoferrin) and prevents passage of large molecular weight materials into enterocytes. The intestinal microflora represent a stable ecosystem that diminish opportunities for microbial infection. By occupying binding sites on the enterocytes, decreasing gut pH, producing volatile fatty acids, elaborating

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bacteriocins, and increasing motility, these commensal microbes provide an important element of nonspecific defense (Newby, 1984). Microbial agents or antigens that do penetrate the epithelial barrier first may encounter mononuclear phagocytes (blood monocytes or tissue macrophages) and polymorphonuclear phagocytes (PMNs or granulocytes) which nonspecifically defend the systemic compartment. PMNs can cross over blood vessels and are a primary defense against infectious agents, whereas macrophages can be recruited to an inflamed site and can be activated subsequently to an enhanced killing state. Certain proteins in blood also can serve as reinforcing nonspecific defense mechanisms (Pestka and Witt, 1985). Interferon, formed by virus-infected cells, can inhibit replication of other unrelated viruses. Kinins constitute a group of peptides that, when activated, are involved in inflammation and blood clotting. Finally, the complement system, a series of proteins and enzymatic reactions, can bring about the lysis of an invading cell. The complement cascade is activated either nonspecifically by bacterial components (endotoxin, protein A) in the “properdin pathway” or specifically by attachment of an antibody to an invading cell in the “classical pathway.” The nonspecific mechanisms described here act synergistically to prevent entry of intestinal macromolecules or establishment and infection by enteric microorganisms. Thus, under normal conditions, relatively large numbers of a microorganism would be required to initiate infection. However, abilities such as attachment and motility can enhance the virulence of a microbe and contribute to the failure of innate defense. Nonspecific immunity also might be depressed by a variety of factors including decreased gastric acidity via antacid ingestion, diminished commensal microflora as a result of antibiotic administration, or damage to epithelial barriers. In such cases, the numbers of a pathogen such as Salmonella that are required for establishment of an infection would be diminished greatly. Intricate specific defense mechanisms therefore must have evolved to protect higher organisms under these circumstances. C. SPECIFIC IMMUNE MECHANISMS When a microbial invader overcomes nonspecific immune defenses, a mammalian host can activate a system that recognizes and then inactivates a foreign material or “antigen” that is then removed or destroyed (Pestka and Witt, 1985). Fundamental to this specific or “acquired” immune system are capacities (1) to recognize minute differences in the chemical structure of an antigen and (2) to “remember” these structures for long periods of time. Antigens are typically high molecular weight (>10,000) proteins or polysaccharides. Components of bacteria, fungi, and viruses

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such as cell wall, flagella, capsule, and toxins are excellent antigens and are multivalent, that is, have more than one antigenic determinant or recognition site. Abbas et af. (1991) suggested that specific responses could be divided into (1) a cognitive phase, (2) an activation phase, and (3) an effector phase (Fig. 3). The cognitive phase refers to the binding of foreign antigens to specific receptors on lymphocytes that are present prior to antigen stimulation. The activation phase refers to the series of events that is induced in lymphocytes as a consequence of specific antigen recognition. Activation events include proliferation, which leads to expansion of antigen-specific lymphocytes and differentiation from cognitive to effector functions. Activation requires antigen as well as “helper” or “accessory” signals from another cell for a complete signal. Finally, the effector phase represents the active functional manifestations of antigen recognition and activation. Several effector responses to an antigenic stimulus exist. First, one or more components of the specific immune system can be induced to bring about the removal of the antigen. Second, cooperative interactions can occur between specific and nonspecific immune mechanisms, thereby enhancing overall host defense. Third, an antigenic stimulus can induce

FIG. 3. Phases of the specific immune response. Based on Abbas et al. (1991).

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“tolerance,” a “specific” type of unresponsiveness. Thus, a host can recognize and tolerate self-antigens. Therefore, the ability of the immune system to develop a memory allows the host to prevent future reinfection by an invading organism as well as to avoid mounting a self-destructive immune response. I . lmmunocompetent Cell Types Many highly specialized cell types that facilitate varied and intricate specific humoral and cell-mediated immune reactions have been described. Leukocytes are the cells within the immune system that carry out these critical functions, which include resistance to infection, homeostasis of cell maturation, antibody production, and immune surveillance against nascent neoplastic cells (Dean and Murray, 1991). These specialized cells are derived from stem cells in the bone marrow that proliferate, differentiate, and mature into lymphocytes, granulocytes, macrophages, and other specialized cells during a process called hematopoiesis (Fig. 4). Many aspects of leukocyte development are regulated by cell-to-cell interactions and by cytokines. Cytokines, including interleukins, lymphokines, and monokines, are soluble protein factors that have specific effects on cell growth, differentiation, and maturation. Leukocyte subsets can be identified by cell differentiation (CD)antigens present on the cell surface using techniques such as immunofluorescenceand flow cytometry or using functional assays in cell culture. In most cases, the cell types involved in generalized systemic immunity (Table 11) also play key roles in gastrointestinal immunity. Lymphocytes carry out critical regulatory and effector activities in specific immunity. Lymphocytes that mature in the tissue equivalent of the avian bursa are termed B cells. B cells are responsible for humoral (antibody-mediated)immunity and carry immunoglobulins on their surface. Lymphocytes that mature in the thymus are known as T cells, which can have both effector and regulatory functions (Table 11). Effector T cells are involved in cell-mediated immune sequelae such as cytotoxicity and delayed-type hypersensitivity. Regulator T cells control the maturation of effector T and B cells via cognate (cell-to-cell)interactions and cytokines. The immune response thus can be enhanced or depressed through T helper (Th) or T suppressor (T,) cells, respectively. Regulator T cells also contribute to the development and maintenance of tolerance. Committed B and T cells undergo a second stage of differentiation on encountering antigen in “secondary” lymphoid organs such as spleen and gut-associated lymphoid tissue. Here, B and T cells segregate into specific areas where they are exposed to filtering blood and lymph, thus facilitating contact between lymphocytes and circulating antigens. This contact ulti-

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION ERY'THROCYTES 1

GRANULOCYTES: Neutrophlls Eosinophlls Basophlls

MEGAKARYOCYTES

1

MONOCYTES LYMPHOCYTE PROGENITORS

I

PLATELETS

\( MACROPHAGES

!

B ELL

THYMUS T CELL (Thymocyte)

PLA'SMA CELLS

T HELPER

T EFFECTOR

FIG. 4. Hematopoiesis and differentiation of lymphocyte progenitors.

TABLE I1 GENERAL FUNCTIONS OF LEUKOCYTES I N SPECIFIC IMMUNITY

Cell type Lymphocytes B cells T cells Accessory cells Macrophages Monocytes Killer cells Natural killer cells Mast cells

Functions Antigen presentation, antigen recognition, antibody secretion Antigen recognition, control maturation of R cells and T effector cells, cytotoxicity Phagocytosis, antigen presentation Phagocytosis, antigen presentation Antibody-mediated cellular cytotoxicity Tumor cell lysis IgE-mediated hypersensitivity

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mately results in the observed humoral and cell-mediated effector functions of specific immunity. The mechanisms of antigen uptake in the gut and subsequent immunologic events are among the most remarkable aspects of immunity in higher organisms. In addition to B and T cells, macrophages and monocytes derived from hematopoiesis can phagocytose infectious particles and function in antigen presentation. Mast cells can respond to various antigens and generate a hypersensitivity response. Additionally, mononuclear cells known as “killer” cells can bind to antibodies and facilitate tumor cell lysis. Other cell types with spontaneous cytolytic activity for neoplastic cells have been called natural killer (NK) cells.

2 . Gut-Associated Lymphoid Tissue Differentiating generalized systemic immunity from mucosal immunity is useful (Fig. 5 ) . The systemic immune system includes all tissue involved in protecting the internal milieu from invading microorganisms. The mucosal immune system encompasses lymphoid tissue that borders the external environment of the gut lumen or other sites such as the bronchus and nasal regions. Although this classification is useful when analyzing diverse functions, many of the specific activities of lymphoid tissue in the systemic and mucosal compartments overlap; one compartment can impact the function of the other. Leukocytes are found at prominent locations in the gut-associated lymphoid tissue (GALT). GALT consists of extrinsic and intrinsic components (Mayrhofer, 1984). The extrinsic lymphoid tissue comprises the single mesenteric lymph node in rodents or multiple (>loo) lymph nodes found in the mesentery of humans and other animals. This extrinsic tissue includes sites that collect intestinal lymph and therefore interface between the gut mucosal and systemic immune compartments. The intrinsic component includes ( 1) organized follicles and groups of follicles (Peyer’s patches) in the small intestine, appendix, and large intestinal submucosa, (2) more diffuse lymphoid tissue, namely, lymphocytes, macrophages, and mast cells in the lamina propria, and (3) intraepithelial lymphocytes in the gut wall (Fig. 6). The intrinsic GALT components contain numerous subsets of immunocompetent cells that play a variety of roles. In humans, the pharynx is surrounded partially by tonsillar lymphoid aggregates that likely participate in immunity. Since the pharynx is shared by the alimentary tract and the respiratory system, inhaled particulate matter can be propelled into this area by ciliary action and swallowed, thus exposing the gut to inhaled antigens (Mayrhofer, 1984). Peyer’s patches have been studied thoroughly and found to contain a full complement of immune cells necessary for

FIG. 6. Gut-associated lymphoid tissue (GALT). Key elements of the intrinsic GALT include the Peyer's patches and lamina propria. Locations of B and T lymphocytes, macrophages (Ma), mast cells (MC), and intraepithelial lymphocytes are shown.

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induction of an immune response, that is, B, T, macrophage, and accessory cells. Lamina propria and intraepithelial lymphocytes apparently function primarily as effector cells. The intestinal lamina propria contains both immunoglobulin(1g)-secretingB cells (plasma cells) and highly specialized T cells that have the phenotype of memory T cells. When activated, these T cells can be characterized functionally as differentiated effector lymphocytes (Zeitz et al., 1990). The pattern of cytokines produced by lamina propria T cells and the responsiveness to certain cytokines also differ from those of other lymphocyte populations (James et al., 1990). Since T-cellderived cytokines are critical regulators for epithelial growth and differentiation as well as for connective tissue metabolism, lamina propria T cells might have additional significance in mucosal growth and transformation. The intraepithelial lymphocyte population has been subject to intense investigation and classified into several types based on differentiation markers and cytokine patterns. These lymphocytes have been suggested to contain regulatory and cytotoxic T cells (Kiyono et a / . , 1991; Lefrancois, 1991; Taunk et al., 1992). 3. Antigen Uptake in the Gut

The movement of high molecular weight antigens from the gut lumen to the blood circulation has been demonstrated experimentally in humans and animals on numerous occasions. Prior to uptake, the antigens must resist proteolytic activity in the lumen and penetrate the mucus/IgA layer to interact with the various absorptive cell types. Factors that disrupt mucosal barrier function and cause extensive uptake include immature gastrointestinalfunction, malnutrition,inflammation,and immunoglobulin deficiencies (Walker, 1987). Macromolecules may be taken up by the gut by at least two distinct mechanisms (Stokes, 1984). In the first, the intestinal epithelial cell can endocytose macromolecular aggregates and deliver these to the subepithelial space. In the second, antigens can be deliberately “sampled” by the Peyer’s patches. a . Uptake via the Intestinal Cell. Normally, epithelial cells are constantly proliferating in the crypt and migrating up the villus surface to the villus tip. Thus, cells of the crypt are undifferentiated whereas cells at the villus tip are differentiated with the capacity to absorb nutrients and provide a mucosal barrier. The microvillus and glycocalyx on the luminal surface are the key elements that facilitate this function. Walker (1985,1987) has reviewed extensively all aspects of antigen uptake by the small intestinal cell. Briefly, when luminal macromolecules are at suffi-

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cient concentrations to evade proteolysis or antibody neutralization and to penetrate the active glycocalyx compartment, they adsorb to the microvillus membrane (Fig. 7). Following evagination of the membrane, phagosomes arise from coalesced vacuoles and fuse with lysosomes to form the phagolysosomes, in which intracellular digestion occurs. Intracellular antigens that escape digestion migrate to the basal surface and are released into the interstitial space. Here, the macrophage functions as nonspecific defense to prevent movement of the antigens into the systemic compartment. Nevertheless, when present at high levels, macromolecular antigens may escape into the circulation and induce a series of immunologic sequelae. Thus, the fact that a small percentage of luminal antigens

Luminal Antigens

II

Phagosome formation I I

J!

0 t

Digestion

@

Exocytosis

L

Antigen and antigen fragments in ints titial space

FIG. 7. Antigen uptake by an epithelial cell. Luminal antigens at sufficient concentrations evade mucus and IgA barrier and interact with microvillous membrane of intestinal cell. Following adsorption/invagination,phagosomes are formed that fuse with lysosomes. Undigested antigen is released into the interstitial space following exocytosis. In some cases, these antigens can enter the systemic compartment and induce immunologic sequelae. Based on Walker (1987).

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JAMES J. PESTKA

can be detected in mesenteric lymph and portal blood is not surprising (Stokes, 1984). Husby (1988) has indicated that dietary antigens are taken up in amounts that are nutritionally insignificant but may be of immunologic importance. Local or systemic antibodies may retard the uptake but, in addition, actually may increase the uptake of unrelated antigens. In humans, the uptake of intact dietary antigen, free or in immune complexes, was reported in studies of healthy subjects and of patients with immune deficiency or allergy. For example, when the uptake of dietary antigen in healthy persons was investigated after a test meal using enzyme immunoassay and high performance liquid chromatography (HPLC) of serum samples, ovalbumin was taken up as intact antigen or as a high molecular weight immune complex constituent in all subjects. The antigen was measurable in serum up to 2 days after the meal. b. Uptake via the Peyer’s Patch. As an alternative to the process just described, antigens can be sampled by lymphoid nodules and groups of nodules known as Peyer’s patches (Fig. 6). These patches are the primary inductive sites for the gut mucosal immune response (Fig. 8). Size and

FIG. 8. Antigen uptake by the Peyer’s patch and lymphocyte homing to mucosal sites. Antigen (Ag) is sampled by M cells in dome epithelium and passed on to underlying lymphocytes. After Ag presentation, activated B and T cells proliferate, differentiate, and migrate via the mesenteric lymph node (MLN)and thoracic duct (TD)to general circulation. From here, they populate the lamina propria and other mucosal sites. Subsequent encounters with Ag result in differentiation of B cells to plasma cells that secrete immunoglobulin (Y), primarily IgA.

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15

numbers of follicles within Peyer’s patches and small intestinal distribution are both age and species dependent (reviewed by Owen and Ermak, 1990). For example, in rodents Peyer’s patches contain 2-1 1 follicles of uniform size and are found throughout the small intestine. In contrast, human Peyer’s patches within the duodenum are small and consist of few follicles but become larger more distally in the ileum; thus, they correspond to the presence of endogenous microflora. Up to 1000 follicles have been noted in the large terminal ileal Peyer’s patch. These large aggregates slow the movement of gut luminal contents, thereby increasing the potential for sampling of antigens. Peyer’s patches are dome shaped and have epithelium absent of villi that contains M cells and M-cell-associated lymphocytes (Owen and Ermak, 1990). M cells exhibit short microvilli and do not digest antigen. Thus, they facilitate delivery of intact antigen into the underlying lymphoid tissue. Since antigenically stimulated lymphoid follicles in the Peyer’s patches protrude into the gut lumen, they have an enhanced capacity for antigen sampling and thus act as sentinels. Macromolecular and particulate antigens-including viruses, bacteria, and small parasites-can be phagocytosed by M cells and passed into the Peyer’s patches. B cell zones that contain germinal centers exist beneath the Peyer’s patch dome. These locales are the sites of B cell proliferation, switching to an IgA+ phenotype, and maturation that is based on affinity for antigen. Near the B cell zones are T cell areas that contain regulatory and effector subsets. In addition to B and T cells, Peyer’s patches contain “accessory cells” (macrophages and dendritic cells) that are capable of presenting antigens. Both B and T cells from the Peyer’s patch are capable of “homing” to distal sites in the body and, thus, can mediate a variety of wide-ranging effects (Fig. 8). This full complement of immunocompetent cells in the Peyer’s patches specifically facilitates ( 1 ) humoral and (2) cell-mediated responses that are focused primarily in the gut, as well as (3) concurrent regulatory effects in the gut and systemic compartment that include oral tolerance. 4. Specific Humoral Responses in the Gut

Humoral immunity is mediated by highly specific proteins known as antibodies, which are secreted by plasma cells derived from B cells in response to antigens. Antibodies belong to the globulin class of serum glycoproteins and thus have been termed immunoglobulins (Igs). The general structure of an Ig consists of two heavy (H)and two light (L) polypeptide chains (Fig. 9) resulting in a total molecular weight in the range of 150,000 to 190,000 daltons for a basic monomer. Although interchain disulfide bonds exist, hydrophobic, electrostatic, and hydrogen bonding

16

JAMES J. PESTKA

r

ANTIGEN

‘ITES

1

FIG. 9. General structure of an immunoglobulin. An immunoglobulin (Ig) consists of heavy

(H)and light (L)chains. An Ig has two specific binding sites, each composed of the variable regions of the H and L chains. The F, region contains determinants for various functions such as placental transfer, complement fixation, and mast cell binding.

interactions are primarily responsible for maintaining the basic Ig structure. Amino terminal ends of both heavy and light chains form the variable regions of the Ig and specifically bind antigen. These variable regions form a binding site that can recognize an antigenic determinant (epitope) approximately 6-7 amino acids in size. Since two pairs of heavy and light chains form identical binding sites, an Ig is said to be bivalent. This ability to bind two antigens simultaneously is used in macroscopic immunoassays such as the precipitation and agglutination reactions. The carboxyl end of the Ig is called the F, region. This region determines the biological functions of the antibody. The five major classes or “isotypes” of Igs are based on heavy chain structure (Table 111). Of these isotypes, IgA is of predominant importance in local immunity in the gut. a. IgA. IgA is critical in immunity to antigens in the gut and other mucosal sites, and accounts for 60% of total daily antibody production in humans (Mestecky, 1988; McGhee et al., 1989,1990). IgA is found both in mucus secretions (secretory IgA) of the gut and as a circulating Ig. Multiple IgA subclasses exist in humans, apes, and rabbits, whereas other species, such as mice, exhibit a single class (Ken-, 1990; Russell et al., 1991). The two human subclasses, IgAl and IgAz , have different distributions in the mucosal (secretory IgA) and systemic (circulatory IgA) compartments. The molecular form (polymeric or monomeric) and subclass are dependent on ( 1 ) type of antigen, (2) duration of immune response, and (3) route of exposure. The molecular properties, cellular origin, and possi-

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TABLE 111 PROPERTIES OF MAJOR IMMUNOGLOBULIN CLASSES

Immunoglobulin class

Percentage of total circulating Ig

Structure

Characteristics

IgA

15

Monomer, polymers

sIgA

-

Dimer

IBE

0.05

Monomer

IgG

80

Monomer

IOM

5-10

Pen tamer

IgD

0.05

Monomer

Found in blood and lymph Found in secretions such as saliva. milk, mucus; protects mucous membranes Found in blood and lymph; attaches to mast cells and basophils; associated with allergies and chronic parasitic infections Found in blood and lymph; transfers across placenta; fixes complement; attaches to phagocytes Found in blood, lymph; fixes complement; attaches to phagocytes Found mainly on surface of B cells

ble physiologic role of the IgA immune system have been the subject of several reviews (Bogers et al., 1991; Russell el al., 1991). Secretory IgA (sIgA) occurs as a dimer and includes two additional peptides: a J (joining) chain and a secretory component that enhances its transfer and enteric survivability (Mestecky et al., 1991). Antigens in the gut are most likely to encounter sIgA before any other Ig. Peyer’s patches usually are considered inductive sites for the IgA response. Antigens sampled by the Peyer’s patches encounter antigen-presenting cells, regulatory T cells, and B cells. From this location, committed IgA plasma cell precursors migrate via the mesenteric lymph node and thoracic duct to blood, spleen, and liver, and return to the gut lamina propria or are localized at distant mucosal sites (Fig. 8) (Czerkinsky et al., 1987). On differentiation at the mucosal level,. the resultant plasma cells produce dimeric IgA that, by noncovalent association, become complexed with secretory component, a protein that is synthesized by glandular cells.

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JAMES J. PESTKA

Secretory component facilitates transfer of sIgA across the epithelial layer into the lumen. Primary roles that have been suggested for sIgA are antigen exclusion, inhibition of adherence of microorganisms, intracellular virus neutralization, and excretion of IgA immune complexes. A key feature of sIgA is its inability to induce mechanisms such as opsinization or complement activation, which potentially could be damaging to the intestine. Rather, IgA induces antigen clearance by taking advantage of the normal clearing activities of the gut (Newby, 1984). Interaction with nonspecific immune parameters enables sIgA to inhibit entry of soluble antigens and restrict epithelial colonization of bacteria and viruses. Although the function of serum IgA is less defined, one possible role is the noninflammatory neutralization of toxins, viruses, and enzymes (Mestecky, 1988; Kerr, 1990). B cell activation, switching, proliferation, and differentiation to IgA synthesis are regulated tightly in the mucosal and systemic immune compartments by several cell types and cytokines (McGhee et al., 1989). A key element in differentiation of B cells to IgA production is the type 2 T helper cell, which can secrete the cytokines IL4, ILS, and IL6 (McGhee et al., 1989,1990;Taguchi et al., 1990). IL5 and IL6 stimulate IgA production in mitogen-stimulated B cells, a process that is enhanced by the presence of IL4 (Bond et al., 1987; Coffman et al., 1987; Murray et al., 1987). Another cytokine, TGF-P 1 , is a product of several cell types including T cells that is capable of inducing switching of IgM' cells to IgA+, as well as having a number of other pleiotropic effects (Coffman et al., 1989; Chen and Li, 1990; Kim and Kagnoff, 1990a,b). Finally, orally administered antigens can induce proliferation of Peyer's patch T cells that bear an F, a receptor. These cells may collaborate selectively with IgA-committed B cells via a putative immunoglobulin binding factor, but this factor has not been characterized molecularly (McGhee et d., 1989,1990). 6 . IgE. IgE is another key immunoglobulin that is thought to be involved in responses to parasitic infections. Present only in small amounts in serum, IgE exists primarily in association with mast cells and basophils. Binding of antigen to mast-cell-associated IgE and subsequent cross-linking results in the release of histamine and other inflammatory mediators (Fig. 10). Barrett and Metcalfe (1988) have reviewed the relationship between IgE and mucosal mast cells extensively. Numerous mast cells can be found within and beneath the gut mucosa (Fig. 6). For example, the human intestine contains 20,000 mast cells per mm3 (Norris et al., 1963). Thus IgE may be carried into the gut mucosa by mast cells and facilitate degranulation with histamine release on interaction with a food or microbial antigen. When this occurs in association with epithelia, the

FOOD. DIET. AND GASTROINTESTINAL IMMUNE FUNCTION

19

A

,;y-y,, .*"

MASTCELLS

".

B

FIG. 10. Activation of mast cells via IgE crosslinking. (A) Multivalent antigen (Ag) specifically binds to IgE attached to mast cells via F, receptors. (B) Crosslinking by Ag initiates degranulation of mast cells with concurrent release of vasoactive amines.

volume and flow of secretion can increase as can flushing action that removes the offending agent from the intestine. This response also contributes to certain food allergies (discussed in Section IILB). c. Other lgs. Protective function in the gut analogous to that served by IgA also may be attributed to secretory IgM, because on occasion its mucosal synthesis is elevated, particularly in selective IgA deficiency. IgG is not considered a secretory immunoglobulin since its external translocation requires passive intercellular diffusion. By binding complement, IgG actually can cause increased mucosal permeability and tissue damage, and thus contribute to long-term immunopathology in gut mucosal lesions.

5.

Specific Cell-Mediated Responses in the Gut

Understanding mechanisms for cell-mediated immune responses in the gut has just begun. These processes are likely to involve the two major types of cell-mediated immune response found in systemic immunity, namely, cytotoxicity and delayed-type hypersensitivity (DTH) reactions. Cytotoxic T cells defend a host against living antigens such as virusinfected cells or intracellular pathogens (Fig. 1 IA). In such a response, a target cell bearing a surface antigen interacts directly with a cytotoxic T

JAMES J. PESTKA

20 A

- -

0 . target cell

CTL

cJ+

lysed target cell lysis of more target cells

CTL

B

nonspecific cell killing lymphokine release

t

TDTHCBIl macrophage attraction, immobilization, and activation

FIG. 11. Major types of cell-mediated specific immune responses. (A) Cytotoxicity: cytotoxic T cell specifically recognizes and lyses target cell. (B) Delayed hypersensitivity: T cell releases cytokines that facilitate killing of target.

cell, resulting ultimately in the lysis of the target cell. The killing is unidirectional, so a cytotoxic T cells can kill target cells repeatedly. In allergic contact hypersensitivity, low molecular weight chemicals (haptens) can conjugate to proteins in the host and elicit a reaction involving cytotoxic T cells. Intestinal intraepithelial lymphocytes may have a novel and important role in recognizing and destroying transformed epithelial cells and colon cancers (Taunk et al., 1992). Certain other cell-mediated hypersensitivity reactions are referred to as "delayed-type'' since the time required for onset of effects is long compared with the time required for immediate-type reactions (Fig. 11B). DTH reactions can occur systemically for any type of antigen, living or nonliving; the sole requirement is that the antigen be bound to the cell surface. Recognition of the antigen by a specific T cell receptor induces activation of a T cell and subsequent cytokine secretion. These cells may cause nonspecific cell killing; others act on macrophages by drawing them to the

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21

site of inflammation where they are immobilized and activated. Such macrophages have enhanced ability to kill and destroy pathogens that normally would survive macrophage ingestion. Once formed, these macrophages kill nonspecifically, but their activation occurs through antigenspecific cytotoxic T cells. Rejection of allografts is the rejection of tissue grafts between members of the same species that differ genetically. Certain genetically determined cell surface antigens, called major histocompatibility antigens, are responsible for initiating this response. In this case, a local DTH reaction involving cytotoxic T cells occurs that produces an intense inflammation at the graft site. 6 . Common Mucosal Immune System

Antigen stimulation in the gut has been suggested to result in IgA secretion at other mucosal sites such as salivary glands and genitourinary sites. As described earlier, on antigen presentation in the Peyer’s patches, B and T cells migrate via the mesenteric lymph node into the systemic circulation. In addition to entering the lamina propria of the intestine, these cells can migrate to the salivary glands, to the eye via lacrimal glands, and into milk via the mammary glands (Fig. 8). The capacity of antigen-activated IgA B cell blasts from the Peyer’s patches to migrate to multiple mucosal sites has led to the concept of a common mucosal immune system (McDermott and Bienenstock, 1979). Although largely demonstrated in experimental animals, evidence exists for a common mucosal immune system in humans, based on detection of gut antigen-specific IgA at anatomically remote sites. Further, antigenspecific IgA-producing cells can be found in blood after oral immunization and before their appearance in saliva and tears (Russell et al., 1991). The advantage of a common mucosal response probably relates to the mobilization of humoral and cellular immune elements to various sentinel sites (e.g., mouth, eye, genitourinary tract) that can prevent infection on subsequent reexposure to a mucosal pathogen. 7. Stimulation of Specific Immunity

Stimulating the specific immune response within the gut to protect against various microbial illnesses clearly, is desirable. However, achieving long-term memory when immunizing orally is difficult because these antigens to be degraded by acidic pH and proteolysis in the gut (Stokes, 1984). Oral immunization with live organisms rather than nonreplicating ones is generally more effective for induction of IgA responses, implying that colonization and/or replication in the GI tract is

22

JAMES J. PESTKA

required (McGhee et al., 1992). Further, particulate antigens function much more effectively than soluble ones. Thus, close contact with key components of the gut is required to induce a GI immune response. The immunogenic character of an orally presented antigen can be preserved by administration with large amounts of sodium bicarbonate or in protective capsules (McGhee er al., 1992). Several adjuvants also have been used to induce gut IgA responses, including ox bile, muramyl dipeptide, concanavalin A, peptidoglycan and vitamin A, streptomycin, lysozyme, polyvalent cations, and DEAE dextran (Ernst et al., 1988). Other oral antigen administration systems have employed cholera toxin, biodegradable microspheres, and selective delivery of antigens by recombinant bacteria (McGhee et al., 1992). 8. Oral Tolerance as a Specific Response Effects on systemic immunity from encountering antigen by the oral route can range from the active sensitization described earlier to oral tolerance (immunological unresponsiveness) (Strobel and Ferguson, 1985). This effect is dependent on timing, dose, and frequency of antigen administration. Systemic unresponsiveness often can be observed concurrently with an intestinal immune response (Challacombe and Tomasi, 1980; Richman et al., 1981; Mattingly, 1983). The coexistence of a diminished systemic response with an enhanced mucosal response is thought to be favorable, since it reduces the chances of unfavorable inflammatory events while still protecting the host against agents in the gut environment. Oral tolerance in mice apparently becomes impaired with age (Kawanashi et al., 1990). Stokes (1984) and Ernst er al. (1988) have summarized possible mechanisms and the potential significance of oral tolerance. Ill.

DISEASES INVOLVING THE GASTROINTESTINAL IMMUNE SYSTEM

The relationship between disease and the GI immune system is exceedingly complex (Fig. 12). For example, normal function may be overridden by a highly virulent microorganism or by a series of mutation events leading to a neoplasm. Under these circumstances, inflammatory responses in the gut actually might contribute to the overall symptoms of the disease. Also, hyperactivity might lead to inflammatory sequelae such as food allergies or enteropathies and, on a more speculative level, to inflammatory bowel diseases and various immune complex and auto-

FOOD. DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

23

NORMAL FUNCTION ADEQUATE

No disease

Increased sensitivity to microbial infections, allergies,

Specific and nonspecific gastrointestinal immune mechanisms

Microbial infection, neoplasm

inflammatory bowel disease, autoimmune disorders HYPERACTIVITY

FIG. 12. Complex relationships between disease and the GI immune system.

immune disorders. A third possibility is that generalized immunosuppression can bring about increased sensitivity to microbial infections, allergies, or neoplasms. These options are complicated further by the fact that, in all these cases, mutually excluding GI immunity and general systemic immunity is difficult.

A. INADEQUACY OF NORMAL FUNCTION 1 . Microbial Infection A multitude of bacteria, parasites, and viral agents cause gastroenteritis or penetrate the gut prior to eliciting systemic infection. The capacity to override the GI immune system is dependent on the numbers of ingested pathogen and on virulence factors such as toxigenicity, adherence, and invasiveness (Walker and Owen, 1990). Thorne (1986) outlined five levels of pathogenesis for bacterial diarrheal diseases: (1) bacteria produce toxin but do not adhere and multiply (e.g., Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum); ( 2 ) bacteria adhere and produce toxin (e.g., enterotoxigenic Escherichia coli, Vibrio cholerae); ( 3 ) bacteria adhere to the mucosa and destroy the brush border (e.g., enteropathogenic E. coli); (4) bacteria invade the mucosa and multiply intracellularly (e.g., Shigella spp.); and

24

JAMES J. PESTKA

(5) bacteria penetrate mucosa and spread to lamina propria and lymph nodes (e.g., Campylobacter, Yersinia). With each increasing level of action, the pathogen focus moves from the mucosal to the systemic compartment. Hence, the specific immune response must be escalated. For example, some types of Salmonella can move into the systemic compartment causing generalized illnesses such as enteric and typhoid fevers. This escalation concurrently can unleash destructive potential that will damage the host seriously. In addition to the aforementioned mechanisms of virulence, the antigen sampling process itself may become a major portal of entry for pathogens (Owen and Ermak, 1990). Wells et al. (1988) hypothesized that, in some instances, a motile phagocyte may ingest an intestinal bacterium, transport it to an extraintestinal site, fail to accomplish intracellular killing, and liberate the bacterium at the extraintestinal site. This hypothesis was based on the observation that intestinal bacteria that most readily translocate out of the intestinal tract are categorized as facultative intracellular pathogens. Also, intestinal particles without inherent motility (e.g., yeast, ferritin, starch) move out of the intestinal lumen within hours of their ingestion. Finally, the rate of translocation of intestinal bacteria can be altered with agents that modulate immune functions such as phagocytosis. Thus, systemic infection by translocating intestinal bacteria could be a result of the antigen-sampling process that evolved to regulate the immune response to intestinal antigens. A normal mucosal immune response occasionally might enhance viral infection. Sixbey and Yao (1992) have shown that, when bound to dimeric IgA, Epstein-Barr virus (EBV) can enter epithelial cells through secretory component-mediated IgA transport. This IgA-dependent infection of mucosal tissue may be a mechanism for the involvement of EBV in cases of human nasopharyngeal carcinoma. 2 . Intestinal Cancer Lower intestinal tract cancer is the second most frequent cause of cancer death in the United States, typically affecting persons over age 50. Diet is thought to be a major factor in the prevalence of this disease. Successful establishment of intestinal neoplasms suggests a failure in normal immune surveillance. Immune surveillance refers to the capacity of cell-mediated immune-effector cells (1) to recognize unique cell surface antigens that distinguish spontaneously arising tumors from normal cells and (2) to destroy the neoplasm (Dean and Murray, 1991). This concept is supported by the observation of increased neoplasia associated with primary immunodeficiency diseases and immunosuppressive

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

25

therapy. Immune surveillance may involve direct T cell killing, antibodydependent cellular cytotoxicity, or natural killer cell cytolysis. Some investigators question the validity of the concept of immune surveillance, since it largely has been demonstrated with a limited subset of cancers caused by oncogenic viruses (Abbas et al., 1991). Immunologic mechanisms in intestinal malignancy are reviewed in depth by Itzkowitz and Kim (1988). B. HYPERACTIVITY 1 . Adverse Reactions: Intolerance or Allergy

Adverse food reactions can be defined as clinically abnormal responses to a food or food additive. The concepts of “food allergy” and “food intolerance” frequently are misused and interchanged when discussing adverse reactions. Based on the criteria of the Committee on Adverse Reactions of the American Academy of Allergy and Immunology (Atkins and Metcalfe, 1984), a food intolerance is defined as an idiosyncratic, pharmacologic, metabolic, or toxic reaction in which the immune system does not participate. Known and alleged food intolerances are summarized in Table IV. These nonimmunologic effects have been discussed extensively by Anderson ( 1984); further discussion is beyond the scope of this chapter. Other poorly defined symptoms that have been attributed to foods include allergic tension fatigue syndrome, hyperactiv-

TABLE IV GENERAL CLASSES OF FOOD INTOLERANCE‘

Type of reaction Anaphylactoid Metabolic Pharmacologic Food intoxication Food additive

Examples Food-induced histamine release, histamine poisoning Lactose intolerance, hypoglycemia Caffeine Natural food toxins, bacterial and fungal toxins Salicylates, tartrazine, monosodium glutamate, sulfites

Based on Anderson (1984).

26

JAMES J . PESTKA

ity, schizophrenia, cerebral allergy, and environmental illness (Kettelhut and Metcalfe, 1987). A variety of controversial diagnostic tests has been used to verify these other syndromes. Metcalfe (1992) evaluated these conditions and concluded that (1) in most instances no clear relationship exists between these diagnoses and food ingestion, (2) the lack of association with food suggests other etiologies, and (3) well-designed clinical trials rather than controversial tests such as sublingual provocation, subcutaneous/intracutaneous provocation, and in vitro leukocytotoxic tests should be used to evaluate these syndromes. In contrast to food intolerances, food allergies or hypersensitivities are indicative of adverse reactions in which the gut and systemic immune systems play distinct roles (reviewed by Taylor, 1980; Atkins and Metcalfe, 1984; Ferguson, 1984; Stern and Walker 1985). Sampson and Metcalfe (1991) estimated that 1-2% of the general population may exhibit food allergies. Allergies can be classified pathogenically into reaginic (involving IgE) and nonreaginic classifications (Gel1 et al., 1975) (Table V). Sometimes these reactions are categorized as immediate or delayed-onset type. In such food-related hypersensitivities, the gut immune system may be an initial target, but hypersensitivity can be manifested further at the systemic level. Absolute separation into immediate and delayed hypersensitivities must be done with caution because the interval between ingestion and symptoms is dependent on quantity of food ingested, degree of hypersensitivity, threshold for complaints, and other factors that are not always related directly to the immune event taking place (Taylor, 1980). TABLE V CLASSIFICATION OF HYPERSENSITIVITIES

Classification Reaginic Type I: Immediate hypersensitivity Nonreaginic Type 11: Antibody mediated Type 111: Immune-complex mediated Type IV: T-cell mediated

Mechanisms Involves IgE-mediated release of vasoactive amines, arachidonic acid metabolites, and cytokines from mast cells IgM and IgG specific for tissue or cell surface antigens induces complement activation. leukocyte recruitment and activation Circulating immune complexes and IgM/lgG induce complement activation, leukocyte recruitment and activation Delayed hypersensitivity caused by T-cell activation; direct target cell lysis by cytotoxic T cells

FOOD. DIET. AND GASTROINTESTINAL IMMUNE FUNCTION

27

2 . Reaginic (Type I ) Hypersensitivities Key components of a Type I reaction to a food are a sensitizing antigen, specific IgE response, and IgE-binding mast cells or basophils. Examples of antigenic substances that have been identified in foods include cow's milk protein, egg, codfish, shrimp, peanut, and soybean. Although serum antibodies to ingested proteins can be detected early in life (Gunther et al., 19601, the capacity to produce problematic antigen-specific IgE responses apparently is determined genetically. IgE-secreting cells can be found in the gut lining and other mucosal sites as well as in the systemic compartment. Mast cells and basophils have receptors that bind the F, region of IgE (Barrett and Metcalfe. 1988). On cross-linking of these receptors through binding of multivalent antigen (allergen), the mast cell releases various inflammatory mediators including histamine (Fig. 10). Mast cell degranulation with subsequent exposure of adjacent tissues to mast cell mediators can cause local changes in vasopermeability, enhanced mucus production, muscle contraction, pain, and inflammatory cell recruitment. This local anaphylaxis also can bring about translocation of macromolecules across the intestinal barrier. Mast cells bound to IgE molecules that react with food antigens can be identified throughout the body, even in skin. Thus, food antigens entering the systemic compartment by mechanisms described earlier can initiate degranulation of mast cells at other sites in the body. Clinical manifestations of reaginic hypersensitivities may range from minor to more ominous effects that may be localized to the GI tract, to more distant sites, or to both places. The oral cavity often is the initial site of symptoms such as swelling and burning of the lips, mouth, and throat. Responses in the gut may result in abdominal swelling, nausea, cramping, vomiting, and diarrhea. Other sites of clinical signs include skin where itching, fluid accumulation, and eczema might be seen. Asthma and allergic rhinitis also can occur, particularly in children. Systemic anaphylaxis describes the simultaneous onset of a reaginic response at multiple organ sites that includes the effects just described coupled with sometimes fatal hypotension with shock (Sampson et al., 1991b). Systemic anaphylaxis can occur after minor symptoms have been observed in previous exposures to the offending food or may be manifested unexpectedly. Unequivocal verification of reaginic food allergy requires antigen identification, demonstration of a relationship between antigen exposure and adverse reaction, and determination of the immunologic mechanism involved (Atkins and Metcalfe, 1984). Hence, no single test can verify a

28

JAMES J. PESTKA

TABLE VI METHODS FOR DIAGNOSIS O F TYPE I FOOD ALLERGIES

Preparation of medical history In vivo skin test In vivo tests RAST ELISA Basophil histamine release Oral food challenge Elimination diets

food allergy. Various methods that can be used to diagnose reaginic food allergies are listed in Table VI. Initially, a careful medical and dietary history must be taken to exclude reactions that might be misinterpreted as food allergies. Such reactions include enzyme deficiencies, gastrointestinal disease, anatomical defects, chemical reactions, collagen vascular diseases, endocrine disorders, and psychological factors (Metcalfe, 1992). Initial evaluation can be followed up by immunologic testing and food challenge elimination trials. Immunologic testing consists of in uiuo and in uitro procedures that employ food extracts. For example, skin testing can be performed by applying aqueous food extracts to a scratch or puncture. A demonstrable weal compared with a control site can be used to support the possible involvement of suspect food antigens. In uitro tests such as the radioallergosorbent test (RAST) and enzyme-linked immunosorbent assay (ELISA) are useful in identifying antigen-specific IgE in patient serum (Atkins and Metcalfe, 1984). Basophil histamine release assays have been found to correlate with skin tests but are difficult and expensive to perform. Although other immunologic tests such as cytotoxic testing, provocative subcutaneous testing, and provocative sublingual testing employ the use of extracts, these tests are unproven (Metcalfe, 1992). Final verification that a particular dietary antigen causes an allergic response is dependent on observing the response to oral challenge, particularly when the association between certain foods and a clinical sign remains uncertain. Because of the threat of systemic anaphylaxis, oral food challenge always should be conducted under supervision of a physician. 3. Nonreaginic (Types 11, III, I V ) Hypersensitivities

Strong evidence is not available to suggest the potential for Type I1 food-mediated allergies. However, food-related Type I11 reactions have been noted that involve deposition of antigen-antibody complexes at a

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

29

reaction site, followed by complement fixation and cell-mediated tissue damage. Circulating immune complexes that contain food antigens have been noted (Cunningham-Rundles et al., 1979a,b; Pagnelli et al., 1979, 1980; Frick, 1982). Other experimental evidence includes translocation of cow’s milk antigen (Bock et al., 1983), elevated Ig-producing cells in cow’s milk allergy (Savilahti, 1973; Stern et al., 1982), and deposition of IgG, IgM, and complement (Matthews and Soothill, 1970; Shiner et al., 1975). The precise mechanisms of Type I11 hypersensitivities remain elusive. Based on transplantation studies, Strobel (1990), postulated that Type 111 reactions in gut mucosa lead to aberrant expression of histocompatibility antigens, infiltration of inflammatory cells, and an increase in intraepithelial lymphocytes. These changes are followed by an increase in crypt cell turnover and elongation of crypt cells. Type IV hypersensitivities are referred to as delayed-type hypersensitivities because of the time required for onset of effects. These reactions involve recognition of a cell-bound antigen by a T cell and subsequent cytotoxic action; reactions can occur between 6 and 25 hr after ingestion of food. Clinical manifestations of Type IV hypersensitivity involve inflammation of the GI tract with attendant symptoms (Atkins and Metcalfe, 1984). Elevation of intraepithelial lymphocytes, a group of cells including T cells or cells that share many phenotypic markers and functional activities with T cells, appear to be a common element in foodmediated Type IV reactions such as cow’s milk allergy (Asquith, 1970; Ashkenazi et al., 1980; Stern, 1982). Intraepithelial lymphocytes may function locally in Type IV responses by a mechanism as yet unknown. 4 . Food-Sensitive Enteropathies

Several diseases exist in which small intestinal pathology appears to be associated with a local immune response in the gut (Mowat, 1984). Clinical features of food-sensitive enteropathies in infants include diarrhea, malabsorption, and failure to thrive, all of which occur soon after the introduction of antigen in diet. Although sometimes defined in a descriptive fashion, these diseases very likely involve reaginic or nonreaginic mechanisms. Examples include celiac disease, cow’s milk protein intolerance, and soy enteropathy. Strobe1 (1990) has suggested that foodsensitive enteropathies result from a breakdown of oral tolerance, which allows a cell-mediated and/or IgE-mediated immune response to develop. Underlying reasons are multifactorial and encompass genetics, environment, maturity, infection, and autoimmune responses. a . Celiac Disease. Celiac disease is defined as a permanent condition of gluten intolerance that is associated with characteristic gluten-

30

JAMES J. PESTKA

sensitive changes in the small intestinal mucosa (Ferguson, 1984). This tissue characteristically returns to normal with a gluten-free diet but relapses on further challenge with gluten. Celiac disease involves interactions between genetic and immunologic factors and diet (Cole and Kagnoff, 1985). The condition typically begins in the first 2 years of life following exposure to gluten and is related to initial exposure to an offending food antigen (Mowat, 1984). Major histocompatibility complex genes apparently represent a major component contributing to disease susceptibility (Kagnoff, 1989). A viral protein also may play a role in the pathogenesis of celiac disease, perhaps via immunologic cross-reactivity between an antigenic determinant shared by the viral protein and (Y gliadins. When Husby (1988) studied uptake of ovalbumin and p-lactoglobulin in children with celiac disease, either on a gluten-free diet or after gluten challenge, and in controls with normal gut mucosa, both antigens were present in high molecular weight fractions of the sera, presumably as immune complexes. Levels of the antigens in serum did not differ among the children with celiac disease and the controls eating the gluten-free diet. However, in 4 of 5 celiac children, the uptake of both antigens was increased after gluten challenge, indicating the potential for increased antigen uptake in celiac disease. Several studies suggest that cellmediated reactions, particularly of intraepithelial lymphocytes, are of critical importance in celiac disease (Kagnoff, 1989; Russell et al., 1991; Troncone and Ferguson, 1991). Intestinal T cell-mediated reactions in experimental animals have shown pathologic features similar to those of celiac disease. This pathology includes changes in villus and crypt architecture, crypt hyperplasia, elevated numbers of intraepithelial lymphocytes, and increased intraepithelial lymphocyte mitosis. Since pathogenesis in celiac disease might be viewed as failure of the normal inhibition of immune responses to gluten in the gut, therapeutic control of these immunoregulatory mechanisms might provide an approach to treating this disease and other food protein-sensitive enteropathies.

b. Cow’sMilk Protein Intolerance. Cow’s milk protein intolerance (CMPI) is an enteropathy that includes syndromes resulting from intolerance of one or more cow’s milk proteins. The condition is estimated to affect between 1 and 3% of children (Mike and Asquith, 1987). CMPI occurs during the first 6 months of life but, unlike celiac disease, subsides in subsequent years. Symptoms include diarrhea, vomiting, weight loss, urticaria, wheezing, and eczema. Interestingly, breast-fed infants can be sensitized to cow’s milk that has been ingested by the mother (Gerrard and Shenassa, 1983). Mike and Asquith (1987) suggested that Type I, 111, and IV responses may be active in CMPI to varying extents

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

31

among children. In a model proposed by Jackson et a f . (1983), CMPI was related to increased permeability during the course of acute gastrointestinal infections. Type I reactions may occur on entry of unprocessed antigens, thereby enhancing entry of more antigens into the systemic compartment. Ensuing local and systemic reaginic and nonreaginic effects then might determine the extent of the illness. c . Soy Protein Enteropathy. Mucosal and systemic reactions to soy proteins have been identified in children and adults. Mechanistically, this response appears to involve a Type 111 reaction mediated by IgG antibodies (Mike and Asquith, 1987). Young animals fed soy protein develop very high titers of the IgG, a subclass that could form immune complexes, activate complement, and elicit a Type I11 hypersensitivity response (Barrett et al., 1978). Morphologic effects are consistent with this possibility and include villous atrophy and infiltration of the villi and lamina propria with mononuclear cells. Nevertheless, the immunologic mechanisms of soy protein enteropathy are still vague.

5 . Inflammatory Bowel Disease Inflammatory bowel disease is a classification that includes two similar clinical entities, ulcerative colitis and Crohn’s disease (regional enteritis). Ulcerative colitis is an inflammatory disease that begins in the rectum and proceeds upward (Burke, 1985). The condition usually affects young adults under age 30. Extensive sloughing of mucosal cells occurs in this disease, and involves ulcerations that can coalesce into the deep muscular layer of the colon. Crohn’s disease patients exhibit similar symptoms; the terminal ileum is affected particularly but the disease can be found in any segment of the intestinal tract. The pathogenic mechanisms of inflammatory bowel disease remain enigmatic and have been proposed to include autoimmunity, toxic environmental factors, and diet (Mike and Asquith, 1987). Immune sensitization to intestinal epithelial antigens is common in families with chronic inflammatory bowel disease. The high frequency of sensitization among asymptomatic relatives suggests that it predisposes individuals to gut tissue injury (Fiocchi et al., 1989). Although heightened humoral and cell-mediated immunologic manifestations clearly occur in the gut, whether these conditions actually cause the disease or are serious secondary effects that perpetuate the inflammatory process is not clear. With respect to humoral immune function, both numbers and functional characteristics of B and T cells vary with inflammatory bowel disease, but a definitive correlation has not been proven (Fiocchi, 1990).

32

JAMES J. PESTKA

Further, anticolon antibodies are detectable in ulcerative colitis, but similarly are found in other conditions. Altered immune regulation sometimes results in a disproportionately increased number of IgG-producing cells in the mucosa; this immune dysregulation may contribute to persistence of inflammatory bowel diseases (Brandztaeg et al., 1985). Vascular complement activation. also might be a process in active inflammatory bowel disease lesions, and presumably relates to the degree of inflammation and immune complex formation (Halstensen e? al., 1989). Circulating antigen-nonspecific suppressor T cells are found in the early stages of Crohn’s disease (James et al., 1987). These and other data suggest that the suppressor T cells are markers of an underlying and persistent antigen-specific immune response to an as yet unidentified antigen or set of antigens. James et al. (1987) have postulated that this underlying antigen-specific response is the result of a primary immunoregulatory abnormality involving an imbalance between the effects of antigen-specific helper and T suppressor cells that recognize a common antigen or antigens present in the mucosal environment. Critical areas of study in intestinal inflammatory diseases are identification of unique and nonunique lymphoid cell sets in the intestine and clarification of how these sets operate in the intestinal microenvironment (Elson et a/., 1986). The latter goal will require understanding the mechanisms by which these sets communicate with and regulate one another via cell surface molecules and soluble mediators. Cytokines that have been investigated in some detail or in a preliminary fashion include IL1, IL2, IL4, interferon y (IFNy), and colony-stimulatory factors (Fiocchi, 1989). For example, Kusugami et al. (1989) demonstrated that reactivity to IL2 distinguishes intestinal mononuclear cells from inflammatory bowel disease patients from those from controls. How intestinal immune homeostasis is maintained in normal individuals, so they do not have inflammatory disease despite the presence of endogenous stimulatory factors such as endotoxin that are inflammatory elsewhere in the body, is particularly unclear. The role of food and diet in inflammatory bowel diseases is also complicated (Frieri et al., 1990). Clarification of these processes perhaps will provide new perspectives on mechanisms involved in chronic intestinal inflammatory diseases and clarify the role of food in initiating or exacerbating these diseases. 6 . Other Immunologic Diseases: Immune Complex and Autoimmune Types

The gut immune system may play a pivotal regulatory role in other immune diseases that are mediated by the nonreaginic hypersensitivity

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

33

reactions described in Table V. These diseases result from aberrant, excessive, or uncontrolled immune reactions or via autoimmunity resulting from immune responses to self-antigens (Abbas et al., 1991). Some immunologic diseases involve antibodies that can (1) form immune complexes and deposit at various tissue sites (e.g., poststreptococcal glomerulonephritis, systemic lupus erythematosus) or (2) attach to self-antigens that are circulating or fixed, thus inducing an autoimmune response (e.g., Goodpastures’s syndrome, Grave’s disease). Others involve T-cellmediated injury (e.g., myasthenia gravis, viral myocarditis). Jonsson, Mountz, and Koopman (1990) have evaluated pathogenesis of autoimmunity relative to oral mucosal diseases. Although the role the gut immune system plays in most autoimmune and immune-complex diseases is vague, evidently this system is involved in a syndrome known as IgA nephropathy. IgA nephropathy is the most common form of glomerulonephritis that, in some cases, can lead to kidney failure (Schena, 1990). Apparently, this disorder is caused by mesangial deposition of IgA-containing immune complexes formed by polymeric IgA (pIgA) that is overproduced in response to antigens presented at mucosal surfaces (Emancipator and Lamm, 1989). To test whether antibodies to dietary antigens might be involved in the pathogenesis of IgA nephropathy, Nagy et al. (1988) measured IgG and IgA serum antibody activities to gluten, a gluten fraction called glyc-gli, a-lactalbumin, p-lactoglobulin, casein, and ovalbumin in patients with IgA nephropathy. The IgA activities to gluten antigens and a-lactalbumin were increased significantly in IgA nephropathy over levels seen in age-matched healthy controls. These researchers suggest that their results showed a relationship between the intestinal humoral immune system and IgA nephropathy, and indicated that antibodies to dietary antigens in some patients may be involved directly in the pathogenesis of IgA nephropathy.

C. IMMUNOSUPPRESSION The GI immune system can be depressed for a variety of reasons, which can impact a host’s capacity to defend against microbial pathogens in the gut and to control intake of protein antigens. The association between various immunodeficiency disorders and heightened incidence of gastrointestinal disease has been reviewed extensively by Ament ( 1984). For example, selective IgA deficiency is a type of congenital immunodeficiency that occurs in 1 of 400 persons. Absence of sIgA in the intestine allows uptake of protein antigens and creates a propensity for increased

34

JAMES J. PESTKA

Type I and 111 allergies (Cunningham-Rundles, 1991). Also, radiation and chemotherapy used in transplantation and cancer treatment also can contribute to depressed GI immune function (Cunningham-Rundles and O’Reilley, 1986). Acquired immunodeficiency syndrome (AIDS) affects all aspects of immunity because of a depressed T helper population. Because it is the largest lymphoid organ in the body, the GI tract is considered a potential reservoir for human immunodeficiency virus (HIV), the agent that causes AIDS. Indeed, most AIDS patients exhibit a variety of gastrointestinal symptoms including diarrhea. Initial depletion of CD4+ (T helper cells) has been postulated to result in impaired IgA+ B cell development, which reduces IgA secretion in the lamina propria (Smith et al., 1992). This change, coupled with impaired gastric acid secretion, results in increased bacterial colonization leading to recruitment of activated monocytes and macrophages that promote chronic inflammation, villus atrophy, and malabsorption. Progression of the HIV disease results in reduced function among cytotoxic T cells, mucosal macrophages, and monocytes. Immunosuppression and weight loss brought about by these events makes the AIDS patient increasingly susceptible to a variety of enteric pathogens that are considered rare in normal populations (Table VII).

TABLE VII ENTERIC PATHOGENS ASSOCIATED WITH

HIV INFECTION^ Group Viruses Bacteria

Fungi Protozoa

GenudSpecies Cytomegalovirus Herpes simplex virus Salmonella spp. Shigella flexneri Campylobacterjejuni Clostridium dificile Candida spp. Giardia lamblia Entamoeba histolytica Isospora belli Cryptosporidium spp. Microsporidium spp.

Based on Smith et al. (1992).

FOOD. DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

IV.

35

IMPACT OF FOOD CONSTITUENTS AND CONTAMINANTS ON GASTROINTESTINAL IMMUNITY

A. MICROBES AND MICROBIAL PRODUCTS Many food- and waterborne microbial agents can colonize the GI tract and induce gastroenteritis (Centers for Disease Control, 1990). These agents can be bacterial (Table VIII), viral (Table IX), or parasitic (Table XI. As discussed earlier, other foodborne microbial agents can evade the GI immune system and cause systemic disease. Endogenous components or secreted products of bacteria can cause a variety of toxic effects. Clearly, staphylococcal enterotoxin and botulinum toxin must survive ingestion and evade GI immunity in the gut prior to inducing their specific toxic effects. Bacterial products also can affect immune function directly. For example, lipopolysaccharide (LPS or endotoxin), a component of gram-negative bacteria, is a potent polyclonal activator of B cells and can induce cytokine release by monocytes. McGhee et al. (1989) proposed that gram-negative bacterial antigens and LPS actually contribute to the normal differentiation and development of Peyer’s patches. Staphylococcal enterotoxin has been deemed a “superantigen” because of its capacity to activate a large number of T cell clones (Marrack and Kappler, 1990). What the long-term effects on GI immunity of such T cell activation in the intestinal tract would be is not clear. Finally, cholera toxin is the most potent mucosal immunogen known (McGhee et al., 1992), and can act as an adjuvant when ingested simultaneously with other proteins to induce both mucosal and systemic Ig responses. Mycotoxins are low molecular weight secondary metabolites that are produced by various fungi and frequently are found in foods (Pestka and Casale, 1990). Because they resist digestion and processing, mycotoxins can enter into the intestinal tract; because of their size, they evade normal GI immune mechanisms of protection. Their toxic effects are variable and can include cancer, impaired reproduction, and gastroenteritis. The capacity of mycotoxins to impair a variety of normal immune functions has been reviewed by Pestka and Bondy (1990). In at least one case, the GI immune system is known to be affected specifically. In the mouse, vomitoxin (deoxynivalenol) can stimulate B cells polyclonally to produce hyperelevated levels of serum IgA, probably via a T-cell-related mechanism (Bondy and Pestka, 1991). The quality and quantity of this IgA results in increased IgA immune complex formation and accumulation in the kidney, with current hematuria (Dong et al., 1991). This experimental model bears many similarities to human IgA neDhroDathv.

TABLE VIII INFORMATION RELEVANT TO OUTBREAKS OF BACTERIAL GASTROENTERITIS"

Selected symptoms Causative agent

Patient age

Vomiting

Fever

Diarrhea

Incubation period

Duration of illness

Mode of transmission

All

Common

Rare

Usually not prominent

1-6 hours

10 days

Food, water, pets, fecal-oral

Occasional

Variable

12-72 hours 2-6 days

3-5 days

Enteropathogenic

Adults, infants, and children Infants

Enteroinvasive

Adults

Occasional

2-3 days

1-2 weeks

Food. water, PTP," fecal-oral Food, water, PTP, fecal-oral Food, water, FTP, fecal-oral

Bacillus cereus and Staphylococcus aureus Carnpylobacterjejuni

Escherichia coli Enterotoxigenic

Variable

Watery to profuse watery Variable Watery to profuse watery Common May be dysenteric

1-3 weeks

Enterohemorrhagic

'

Salmonella spp.

Shigella spp.

Yersinia enterocolitica

Vibrio cholerae

(10 years of age (So%), 15 months-75 years All groups, especially infants and young children All groups, especially 6 months-10 years All groups, especially older children and young adults All groups

Common

Rare or mild

Occasional

First watery. then grossly bloody

3-5days

7-10days (1-12 days)

Food, PTP, fecaloral

Common Loose, watery, occasionally bloody

8-48 hours

3-5 days

Food, water, fecaloral

Occasional

Common May be dysenteric

1-7 days

4-7 days

Food. watar, PTP, fecal-ord

Occasional

Common Mucoid, occasionally bloody

2-7 days

I day-3 weeks (average 9 days)

Food. water, PTP. pets, fecal-oral

Common

Variable

9-12 hours

3-4 days

Fecal-oral, food, water

From the Centers for Disease Control (1990). PTP, Person-to-person.

May be profuse and watery

00 W

TABLE IX INFORMATION RELEVANT TO OUTBREAKS OF VIRAL GASTROENTERITIS"

Selected symptoms" Causative agent

Patient age

Vomiting

Fever

Incubation period

Duration of illness

Mode of transmission

Young children and elderly people Infants, young children, and adults

Occasional

Occasional

1-4 days

Common for infants. variable for adults

Occasional

1-3 days

2-3 days. occasionally 1-14 days 1-3 days

Young children

Common

Common

7-8 days

8- 12 days

Older children and adults

Common

Rare or mild

18-48 hours

12-48 hours

Food. water, PTP,' air,(/fecal-oral

Rotavirus Group A

Infants and toddlers

Common

Common

1-3 days

5-7 days

Group B

Children and adults

Variable

Rare

3-7 days

Group C

Infants, children, and adults

Unknown

Unknown

56 hours (average) 24-48 hours

Water, PTP, food,'/ air." nosocomial. fecal-oral Water. PTP. fecal-oral

3-7 days

Fecal-oral

Astrovirus Calicivirus

Enteric adenovirus Nonvalk virus

From the Centers for Disease Control (1990).

" Diarrhea is common and is usually loose, watery, and nonbloody when associated with gastroenteritis. '-PTP, Person-to-person. Not confirmed.

Food. water. fecal-oral Food. water. nosocomial. fecaloral Nosocomial. fecal-oral

TABLE X INFORMATION RELEVANT TO OUTBREAKS OF PARASITIC GASTROENTERITIS"

Selected symptoms Causative agent

Patient age

Fever

Balantidium coli

Unknown

Cryprosporidiurn spp.

Children, adults with AIDS

Enrarnoeba histolytica

All groups, adults

Variable

Giardia lamblia

All groups, children

Rare

lsospora bclli

Adults with AIDS

Unknown

" From the Centers for Disease Control (1990).

' PTP, Person-to-person.

Diarrhea

Rare

Occasional mucus or blood Occasional Profuse, watery

Abdominal

Incubation period

Duration of illness

Mild to severe pain

Unknown

Unknown

Occasional cramping

Occasional mucus or blood Loose, pale, greasy stools

Colicky

Loose stools

Unknown

Cramps, bloating, flatulence

Mode of transmission

Food, water. fecal-oral 1-2 weeks 4 days-3 Food. water, weeks FTP," pets. fecal-oral 2-4 weeks Weeks to Food. water, months fecal-oral 5-25 days 1-2 weeks Food. water, to months fecal-oral and years 9-15 days 2-3 weeks Fecal-oral

40

JAMES J. PESTKA

TABLE XI SIMILARITIES BETWEEN VOMITOXIN-

INDUCED

IgA NEPHROPATHY A N D H U M A N IgA NEPHROPATHY

Increased serum IgA Increased po1ymeric:monomeric IgA Increased in uirro IgA production Increased IgA specific for dietary antigens Increased CD4:CD8 T-cell ratio Increased IgA-bearing cells Increased mesangial IgA Increased serum IgA complexes Hematuria Predilection for males Long-term persistence

the most common form of human glomerulonephritis (Table XI). Males particularly are prone to disease in the experimental model and in human disease and can be affected by as little as 2 ppm of vomitoxin in the diet (Greene et al., 1992). Since this level can be found in cereal-based food products (Abouzied et al., 1991),vomitoxin might be speculated to be an etiologic agent in human IgA nephropathy.

B. FOODALLERGENS Although foods contain a multitude of proteins, very few of these can trigger IgE-mediated food allergies. Typical food allergens are naturally occurring water-soluble proteins that are heat and acid stable and resist digestion. Taylor (1992) has noted that, with the exception of cow’s milk proteins and egg proteins, most allergenic proteins in foods are of plant or marine origin. Some common allergenic foods are listed in Table XII, TABLE XI1 C O M M ~ NALLERGENIC

FOODS^

Legumes, especially peanuts and soybeans Crustacea: shrimp, crab, lobster, crayfish Milk, including cows’ milk and goats’ milk Eggs from all avian species Tree nuts: almonds, walnuts, Brazil nuts, hazelnuts, etc. Fish: cod, haddock, salmon, trout, etc. Mollusks: clams, oysters, scallops, etc. Wheat Reprinted with permission from Taylor (1992).

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

41

Many food proteins can be perceived as “foreign” by a host and can stimulate both humoral and cellular immune responses. However, most immunogenic food proteins stimulate IgG rather IgE (Taylor, 1992). Thus, allergenicity does not correlate with immunogenicity. Production of IgE antibodies in food allergies apparently is associated with a heritable genetic predisposition (Aas, 1978). A s discussed earlier, binding of an allergen by specific IgE present on the surface of mast cells and basophils results in degranulation with release of histamine and other allergy mediators (Fig. 10). To induce this response, the allergen must be of sufficient size to “bridge” between two IgE molecules. The optimal molecular weight for an allergen is in the range of 10,000 to 70,000 daltons (Taylor et al., 1987). These size estimates are based on (1) capacity to be immunogenic, (2) intestinal permeability of the protein, and (3) the bridging requirement (Taylor, 1992). A second key characteristic of an allergen is the presence of multiple immunogenic sites (or epitopes) that facilitate bridging between two IgE molecules. These epitopes must be spaced at an appropriate distance on the protein molecule. Finally, a common feature of allergenic proteins is the capacity to resist the digestive process as well as heat and acid treatments (Taylor, 1992). Taylor (1992) has reviewed the chemistry and detection of specific food allergens extensively. He noted that progress in food allergen identification has been facilitated by protein separation techniques such as SDS-polyacrylamide gel electrophoresis and immunoblotting with serum from food-allergic individuals. Allergenic food proteins identified by such techniques are summarized in Table XIII. C. CHEMICALS IN FOOD When exogenous chemicals interact with lymphoid tissue, immune homeostasis might be disrupted and induce undesirable “immunotoxic” effects such as ( 1 ) immunosuppression, (2) uncontrolled proliferation, (3) impaired host resistance, (4) allergy, and (5) autoimmunity (Dean and Murray, 1991). Therefore, foodborne chemicals (not including macromolecules) have the potential to interact with the gastrointestinal immune system. Chemicals that are potentially immunotoxic in the gut might be found among natural components, additives, growth promoters, animal drugs, and various contaminants (Table XIV). However, most information relative to these chemicals often is based on systemic immune effects that follow injection at relatively high doses. Miller (1987) reviewed intolerance to food additives such as tartrazine, benzoates, food flavors, and colors and concluded that the mechanisms for production of adverse reactions have not been demonstrated to have

42

JAMES J. PESTKA

TABLE XI11 K N O W N FOOD ALLERGENSU

Source

Allergen

Cows' milk Egg white Egg yolk Peanut Soybean

P-Lactoglobulin. a-lactalbumin. caseins Ovomucoid ( g a l dI), ovalbumin (gal dII). conalbumin (gal dIII) Lipoprotein, livetin, apovitellenin I, apovitellenin V1 Peanut I. concanavalin A-reactive glycoprotein Kunitz trypsin inhibitor, P-conglycinin. glycinin, unidentified protein (20.000 daltons) Albumin protein (1800 daltons) Unidentified proteins ( 16.000-30.000 daltons) Unidentified protein (30,000 daltons) Papain Glutelin fraction, albumin proteins (14,000-16.000 daltons) Trypsin inhibitor Albumins and globulins Allergen M (gad c l ) , a-parvalbumin Antigen 1 (9000-20,000 daltons), antigen I1 (31.000-34.000 daltons), transfer ribonucleic acid

Green pea Potato Peach Papaya Rice Buckwheat Wheat Codfish Shrimp

" Reprinted with permission from Taylor (1992).

immunologic components that usually are associated with hypersensitivity reactions. Animal growth regulators that exist as residues in food theoretically could alter immune function. For example, estrogenic compounds such as diethylstilbestrol (DES) can depress T-cell-related functions including aspects of cell-mediated immunity and helper activity for antibody responses (Kalland er al., 1979; Luster er al., 1979,1980; Kalland, 1980).

TABLE XIV EXAMPLES OF POTENTIALLY IMMUNOTOXIC

INGESTED CHEMICALS ~~

~~

Type

Examples

Growth promoters Drugs Pesticides

Diethylstilbestrol Penicillin, ethanol Carbamates, organochlorines, organophosphates, organotins Polychlorinated biphenyls, polybrominated biphenyls

Contaminants

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

43

Host resistance to Lesreria, Trichinella, and transplantable tumors also is depressed in DES-exposed mice (Dean et al., 1980). Drugs entering foods as residues have the potential to induce an immunologic effect. For example, p-lactam antibiotics including penicillin are capable of inducing allergic responses (Ahlstedt et al., 1980). Although penicillin alone cannot elicit an immune response, biotransformation products that react with self proteins can induce antibody responses (Parker, 1982) or be carried by gastrointestinal contents, bacteria, or bacterial products (Dean and Murray, 1991). Notably, a higher frequency of allergy occurs after intramuscular penicillin administration than after oral administration (Ahlstedt et al., 1980; Van Arsdel, 1981). Ingestion of drugs of abuse also can alter immunologic function. Alcohol abuse in humans results in greater numbers and severity of infections (Tapper, 1980), impaired humoral immunity (Gluckman et al., 1977). and dysregulation of T cell function (Berenyi et al., 1975). Pesticide residues also might modulate immune function. Dean and Murray (1991) reviewed evidence that suggests certain pesticides or formulation contaminants can impair immunity in rodent models. These chemicals include four general classes: ( I ) carbamates, (2) organochlorines, (3) organophosphates, and (4) organotin compounds. Greatest risk was suggested to be posed by pesticides such as organochlorine that are stable in the environment. Pesticide contamination also presents a greater risk to the developing immune system than to the mature adult. Finally, inadvertent food contaminants can pose an immunotoxic risk. For example, contamination of rice with polychlorinated biphenyls (PCBs) in Japan increased susceptibility of exposed persons to respiratory infections (Shigamatsu et al., 1978). People exposed to PCBs in rice in China exhibited depressed serum IgM and IgA and altered T cell number and function (Chang et al., 1980; Lee and Chang, 1985). The accidental substitution of the flame retardant Firemaster BP-6 for a magnesium oxide food supplement in livestock feed resulted in widespread contamination of milk and dairy products with polybrominated biphenyls (PBBs) in Michigan during 1973. High levels of PBBs were found in serum and adipose tissue of Michigan residents (Bekeisi et al., 1978). Exposed persons exhibited a variety of immunologic abnormalities including depressed T cell numbers, depressed lymphocyte function, and increased Ig levels (NIEHS, 1983; Bekesi et al., 1987). Although substantial information is available that certain ingested chemicals have the potential to alter systemic immunity, a definite need exists to expand these findings to the GI immune system using models of oral exposure at realistic doses.

44

JAMES J. PESTKA

D. NUTRITIONAL COMPOSITION That the frequency and severity of GI infections increase in undernourished individuals is well known (Chandra and Wadhwa, 1989). Protein-energy malnutrition is a primary cause of immunodeficiency (Christou, 1990). Fatal outcomes in children with diseases such as mumps and measles can occur because of such generalized immunosuppression. Specific immunologic effects include thymic atrophy, decreased spleen weight, decreased T cell counts, and impaired production of cytokines, thymic hormones, and antibodies. Thus, both cellular and humoral immunity are affected. With respect to GI immunity, McMurray et al. (1977) showed that moderate malnutrition depressed secretory IgA levels. Chandra and Wadwha (1989) reviewed specific effects of malnutrition on gut immunity (Table XV), including depression of IgA-producing cells and fewer intraepithelial lymphocytes. Antibody responses after viral vaccine administration are reduced and natural killer cell activity decreases. Additionally, the number of bacteria that bind to epithelial cells is increased. Osogoe et al. (1991) examined the effects of refeeding subsequent to starvation on the plasma cell population in the lamina propria of the small intestinal villi in adult rats, using an immunohistochemical method to detect IgA, IgM, and IgG. Extensive hyperplasia of intestinal plasma cells could be induced by refeeding after starvation. A large majority of the proliferating intestinal plasma cells expressed IgA, whereas cells bearing IgM or IgG occurred in extremely small numbers in the lamina propria. The mechanism of this extensive plasma cell hyperplasia might involve enhanced transmission of antigenic macromolecules across the mucosal barrier. Also, the origin of the expanded plasma cell population was sugTABLE XV EFFECTS OF MALNUTRITION ON GASTROINTESTINAL IMMUNITY

Secretory component synthesis depressed Reduced numbers of IgA-producingcells Decreased total secretory IgA Decreased specific secretory IgA Reduced numbers of intraepithelial lymphocytes Impaired lymphocyte migration Increased bacterial adherence to epithelial cells L1

Based on Chandra and Wadhwa (1989).

FOOD, DIET, AND GASTROINTESTINAL IMMUNE FUNCTION

45

gested to be correlated with B cell precursors in the germinal centers of the Peyer’s patches, which apparently were more resistant to starvation than other lymphoid cells. Single nutrient effects on immunity also have been characterized and reviewed extensively (Chandra and Chandra, 1986; Delafuente, 1991 ;Latshaw, 1991) with respect to systemic immunity. These effects are likely to be equally important in gut immune function.

V.

MODIFICATION OF GASTROINTESTINAL IMMUNITY THROUGH FOOD AND DIET

A.

BREAST-FEEDING

Innate and specific defense mechanisms must be well developed for the epithelium to function as an impermeable barrier to proteins and their fragments. However, such defense mechanisms are not fully developed in the infant during the postpartum period, particularly when born prematurely (Walker, 1987). Newborn infants are especially susceptible to pathologic penetration by deleterious intestinal contents because of this delayed maturation of the mucosal barrier. Repercussions of this immaturity include enhanced susceptibility to infection, potential for hypersensitivity reactions, and formation of immune complexes. In some situations, these conditions can be fatal. Clinical conditions that have been associated with an immature mucosal barrier include allergy, necrotizing enterocolitis, dermatitis, malabsorption, sudden infant death syndrome, and toxigenic diarrhea in early childhood, as well as inflammatory bowel disease, nephritis, and autoimmunity in adulthood (Walker, 1985). Human milk is a natural means of passively assisting a sensitive neonate against the hazards of a deficient GI immune system. Ingestion of colostrum decreases antigen penetration (Udall et al., 1979). Intake of colostrum apparently enhances the maturation of mucosal epithelial cells and accelerates the development of an intact mucosal barrier (Heird and Hansen, 1977). Secretory IgA exists in milk and can prevent uptake of bacteria and luminal antigens (Walker, 1985). The capacity of colostrum to prevent cow’s milk allergy apparently is related to the IgA content (Savilahti et al., 1991). In addition to antibodies, human milk contains viable leukocytes as well as many other substances that impede bacterial colonization and prevent antigen infiltration. However, care must be taken by the nursing mother to minimize ingestion of allergenic foods that could be carried over into breast milk to sensitize an infant (Gerrard and Shenassa, 1983).

46

JAMES J. PESTKA

B. DETECTION AND AVOIDANCE OF FOOD ANTIGENS AND ALLERGENS Based on a survey of adults with allergies, Ericksson (1978) found that 24% of individuals asked believed that they experienced allergic symptoms after handling or ingesting food; 25% of parents interviewed believed that their children experienced adverse food reactions (Kayosaari, 1982; Bock, 1987). However, only one-third of these perceived food allergies could be verified by controlled clinical trials (Atkins et af., 1985). Because of the large number of people with food allergies (1-2% of the general population) and the even greater number with perceived food allergies, Metcalfe (1992) emphasized the critical need for proper classification of food-related diseases to facilitate development of rational approaches to and policies for dealing with suspected food allergies in the public and private sectors. Concurrent with this need is the need to label foods appropriately to alert the segment of the population that is susceptible to reaginic and nonreaginic hypersensitivities or various enteropathies when exposed to certain food proteins. Taylor (1992) highlighted certain processing and distribution errors that currently make avoiding certain food proteins through labeling problematic (Table XVI). He further suggested that, to avoid these oversights, food processors should establish quality assurance programs that are based on immunoassay procedures that are capable of detecting small amounts of food proteins in a complex matrix. Although clinicians use the sera of food-allergic patients to detect food (Porras et a / . , 1985; Yunginger et al., 1988; Keating et af., 1990; Gem et al., 1991), these sera are unlikely to become available for wide usage. A more practical approach would be to develop specific immunoassays for specific foods that would indicate allergenic potential, for example, the gluten assay described by Skerritt and Hill (1991).

TABLE XVI FACTORS AFFECTING ENTRY O F TRACE ALLERGEN A N D ANTIGEN LEVELS INTO A FOODa

Source of food ingredients unclear Inadequate cleaning of equipment Use of re-work Inadvertent/intentional addition of unlabeled ingredient Bulk distribution Based on Taylor (1992).

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C. DEVELOPMENT OF HYPOALLERGENIC FOODS Another goal for certain food products might be deliberate reduction of allergenic potential. Hypoallergenic foods are those that are completely incapable of inducing an allergic response in an individual (Taylor, 1992). Currently, direct challenge of an allergic person is the best means of assessing whether a food contains an active allergen. These approaches have been used for casein and whey hydrolysate formula (Walker-Smith et al., 1989; Merritt et al., 1990; Sampson et al., 1991a),rice (Yoshizawa and Arai, 1990), and edible oils from peanuts, soybeans, and sunflower seeds (Taylor et al., 1981; Bush et al., 1985; Halsey et al., 1986). Also, immunoassays may be used to evaluate the reduction of allergenicity in a food. For example, human serum IgE has been used to assess the allergenicity of peanut products (Nordlee, 1981),milk proteins (Pahud et al., 1985; Asselin et al., 19891, and rice proteins (Wantanabe et al., 1990). In the future, developing libraries of human monoclonal antibodies that react with key allergenic epitopes in foods may be possible to facilitate studies on the effect of processing on reduced allergenicity . Animal studies also may provide insight into the effect of processing on food allergens. Poulsen et al. (1990) compared intestinal anaphylactic reactions in sensitized mice challenged with untreated bovine milk and homogenized bovine milk. When given orally, homogenized milk resulted in IgE production in 10 of 14 mice, whereas untreated milk resulted in IgE production in only 1 of 12 mice. In contrast to untreated milk or saline, homogenized milk caused a large increase in the mass of the proximal gut segment of orally sensitized mice; only mice both sensitized and challenged orally with homogenized milk showed degranulation of mast cells in the intestinal wall. These observations suggest that homogenization of bovine milk may render the milk more aggressive in its ability to induce intestinal reactions. The study suggests that mice may be an attractive experimental animal model for mimicking the intestinal anaphylactic reactions of humans allergic to cow’s milk or other proteins. D. CONTROL OF MICROBIAL FLORA I . Reduction or Elimination of Microbial Pathogens For immunosuppressed populations, insuring that certain categories of food are pathogen-free, using appropriate processing techniques such as pasteurization or retorting, may be desirable. Appropriate packaging, labeling, and distribution must complement the development of such prod-

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ucts, and could be achieved best by implementing Hazard Analysis Critical Control Point procedures. 2. Ingestion of Probiotic Cultures

Based on theories on the beneficial effects of lactic acid bacteria on intestinal flora that were developed by Metchnikoff many years ago (Hughes and Hoover, 1991), interest in the effects of ingesting fermented milk products has been widespread. Probiotic agents have been defined as “a live microbial feed supplement which beneficially affects the host animal by improving its microbial balance” (Fuller, 1991). The underlying concept is that colonization of the gut by beneficial bacteria would impede attachment and entry of various pathogenic bacteria. Thus nonspecific immunity would be enhanced. Other benefits of probiotic cultures include improvement of lactose tolerance of milk products, anti-tumorigenic activity, reduction of serum cholesterol, and synthesis of B-complex vitamins (Gilliland, 1990). Lactobacillus spp. and Bifidobacterium spp. have been considered for use as probiotics in humans. Colonization of the gastrointestinal tract is thought to be host specific (Barrow et al., 1980; Tannock et al., 1982; Lin and Savage, 1984; Isolauri et al., 1991). Some researchers have reported that, on ingestion, only a small number of Lactobacillus strains survive at the terminal ileum (Bouhnik et al., 1992). However, Lidbeck et al. (1987) showed that fecal counts of Lactobacillus acidophilus increased after ingestion. Goldin et al. (1992) found that a newly isolated human strain of Lactobacillus (GG) was recoverable in the feces of human volunteers up to 7 days after ingestion and that the strain was resistant to the effects of ampicillin. Also, ingested lactobacilli have been suggested to alter fecal bacterial enzyme activities (Pedrosa et al., 1990). Bijidobacterium species are part of the major anaerobic microflora of the colon (Mitsuoka, 1984). Several fermented dairy products have been introduced commercially that may have probiotic effects (Colombel et al., 1987; Marteau et al., 1990; Hughes and Hoover, 1991). Using an intestinal perfusion technique in humans, Pochart et al. (1992) demonstrated the survival of bifidobacteria in the upper gastrointestinal tract. A streptomycin-resistant Bifdobacterium species was used to monitor survival and colonization of ingested bifidobacteria in humans (Bouhnik et al., 1992). The investigators concluded that exogenously administered Bifidobacterium spp. did not colonize the colon but that high concentrations of the exogenous bifidobacteria were compatible with metabolic probiotic activities.

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In evaluating the probiotic efficiency of an organism, knowing the effective therapeutic dose is critical (Saxelin et al., 1991).elements et al. (1983) detected batch to batch variation in probiotic efficiency of freeze-dried lactic acid bacteria products. To assess attachment capacity of some common dairy strains of bacteria, Elo et al. (1991) used a human colon carcinoma line. When compared with an adhesive strain of E. coli, Lactobacillus casei GG had potent attachment capacity whereas attachment by L . acidophilus, Lactobacillus bulgaricus, and various Bifidobacterium strains was weak or absent. Obviously, maintaining viability in any probiotic fermented dairy product that is marketed will be critical. Such maintenance will require intensive study on effects of processing, additives, and packaging in storage. Hughes and Hoover (1991) extensively reviewed research on the challenges and opportunities of commercial BiJidobacterium fermented milk products.

3 . Immunostimulatory Properties of Ingested Bacteria Intestinal microflora have a key role in the development of a normal immune response. For example, Bartizal et al. (1984) examined the effects of diet (chemically defined as opposed to natural ingredient), age, and microbial flora on the tumoricidal activity of NK cells from the spleens of mice. Germ-free mice raised on a chemically defined diet had significantly greater NK cell activity than their germ-free or “clean-conventional” (i.e., barrier-maintained) counterparts that were raised on a sterilized naturalingredient diet. NK activity of germ-free mice was increased dramatically after their alimentary tract was colonized with a complex intestinal flora. Conventional mice raised under clean (barrier) conditions had significantly lower NK cell activity than non-barrier-maintained mice. Changing germ-free mice from a chemically defined diet to a sterile natural-ingredient diet did not enhance NK cell activity. No significant differences in NK activity were evident among mice of different ages. These authors suggested that diet and microbial flora can modulate the NK cell activity of mice. Several investigators have hypothesized that organisms present in fermented dairy products may have immunostimulatory properties. Perdigon et al. (1986) reported that feeding of L. casei and L . acidophilus enhances peritoneal macrophage function and splenic lymphocyte activation in mice. Injection of mice with heat-killed L. casei LC99018 similarly was found to activate macrophage functions associated with microbicidal activity (Saito et al., 1987) and to augment host resistance to Listeria mono-

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cytogenes (Nomoto et a/., 19851, Pseudomonas aeruginosa (Miake, 19851, and transplanted tumors (Matsuzaki et a/., 1985). Antitumor activity also has been described for Lactobacillus strains by Friend and Shahani (1984) and for Streptococcus thermophilus by Kaklij et al. (1990). Lactic cultures stimulate cytokine production in some experimental models (De Simone et al., 1986; Yokukura et al., 1986; Nanno et a/., 1988).

E. AUTOIMMUNE THERAPY BY ORAL TOLERANCE INDUCTION Autoimmune diseases such as rheumatoid arthritis and multiple sclerosis appear to be initiated when systemic cell-mediated and humoral immune defenses target the body’s own tissue. Treatment of these diseases in humans typically involves use of immunosuppressive drugs that make the patient more susceptible to infection. Other approaches under study involve blocking the disease with monoclonal antibodies or synthetic peptides (Marx, 1991). As discussed earlier, oral tolerance involves suppression of systemic cellular and humoral immune responses by feeding a protein antigen. Several intriguing animal studies have been reported that show that simple ingestion of protein antigens that induce the aberrant anti-self responses actually can suppress autoimmunity via oral tolerance. For example, one animal model for multiple sclerosis involves injection of rats or guinea pigs with myelin basic protein, a component of the nerve fiber membranous sheath. This injection induces a systemic immune response directed specifically against the myelin sheath. Feeding of the myelin basic protein can suppress the onset of the experimental disease as well as ameliorate an ongoing response (Khoury et a/., 1990; Brod et al., 1991; Miller et a/., 1991; Whitacre et al., 1991). The mechanism for this protective response may involve the generation of T suppressor cells or the specific deletion of autoimmune T effector cells (Marx, 1991; Edgington, 1992). Deliberate induction of oral tolerance also has been tested in the inhibition of autoimmune effects in other models. Oral administration of insulin suppresses disease in a model for the autoimmune form of diabetes associated with insulitis (Zhang e f al., 1991). Nussenblatt et a/. (1990) demonstrated that oral administration of certain retinal antigens suppresses the eye inflammation associated with experimental autoimmune uveitis. Similarly, collagen-induced arthritis (a model for rheumatoid arthritis) can be suppressed by ingestion of collagen (Zhang et al., 1990).

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Suppression of autoimmunity by deliberate induction of oral tolerance might constitute a low technology and low cost alternative to the treatment of these diseases. Currently, human clinical trials are underway to test the efficacy of antigen feeding on multiple sclerosis, rheumatoid arthritis, and uveitis (Marx, 1991). An obvious research question that arises from these studies relates to the role antigens in normal foods have in protecting against or promoting certain autoimmune diseases.

F. NUTRITIONAL THERAPIES I.

Intestinal Diseases

Patients who have diseases of the small or large intestine, for example, ulcerative colitis, Crohn’s disease, and intestinal cancer, typically have impaired function of the digestive tract. The dysfunction impairs normal nutrient ingestion or absorption and predisposes the patient to malnutrition. Ultimately, this condition diminishes capacity of the intestine to respond appropriately to microbial and antigenic challenge in an effective and coordinated fashion. Diehl et al. (1983) reviewed approaches for intravenous hyperalimentation to diminish these effects. The general approach for preventing malnutrition requires the development of a profile of an individual patient’s nutritional state using anthropometric, biochemical, and immunologic tests to assess the body’s fat stores as well as somatic and visceral protein mass and immunocompetence. Ultimate success of parenteral nutrition support in intestinal diseases depends on proper placement of the catheter, maintenance of an aseptic environment, and surveillance of the patient’s metabolic status (Grant, 1980). Specific adjunctive nutritional therapies involving the enteral route also might be designed to bolster the immune system in certain instances of intestinal disease (Alexander and Peck, 1990) and cancer (Lowell et al., 1990).

2. AIDS-Related and Other Immunosuppressed Conditions A close relationship exists between adequate nutrition, gastrointestinal immune function, and long-term survival of AIDS patients (Simon et al., 1991). Crocker (1989) indicated that AIDS-related gastrointestinal disease is very common, and presents a challenge to all nutritional support clinicians because of long-term problems such as related weight loss, diarrhea, and malabsorption. The course of AIDS-related gastrointestinal disease is

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complicated by factors such as decreased food intake (resulting from fatigue and malaise), increased metabolic demand and nutritional requirements, and identifiable gastrointestinal pathology. Typically, chronic gastrointestinal dysfunction is caused by recurring opportunistic pathogens that are recalcitrant to chemotherapy. Chlebowski et al. (1989) investigated the clinical course of 71 patients with AIDS to determine relationships among nutritional status, gastrointestinal symptoms, and survival. Weight loss was observed in 98%, hypoalbuminemia was present in 83%, and gastrointestinal symptoms were present that included pharyngitis (54%), diarrhea (42%), nausea (23%), dysphagia (21%), and anorexia (18%). The degree of body weight loss and serum albumin level were associated strongly with survival. The authors concluded that (1) nutritional status may represent a major determinant of survival in AIDS and (2) the rate of albumin decrease may predict survival of individual patients with AIDS. Since even clinically stable AIDS patients have been diagnosed as chronically malnourished, patient care and long-term management of them ultimately must focus on fluid and electrolyte balance, nutritional support, and symptom control (Crocker, 1989). These persons are prone to rapid nutritional deterioration during disease exacerbations. Unfortunately, because of the eventual mortality associated with AIDS, physicians hesitate to prescribe aggressive nutritional support, particularly parenteral nutrition. The use of nutritional support as adjunctive therapy early in the course of disease also will be an issue with respect to the use of agents such as AZT, which are prescribed on a frequent basis for persons with AIDS. Although improving nutrition has not been shown to reverse any of the cellular manifestations of AIDS, nutritional support via counseling, food programs, or intervention with enteral or parenteral nutrition appears to improve strength and endurance, and enhance the overall quality of patient life. 3 . Immune Complex and Autoimmune Disorders

Skoldstam and Magnusson (1991) found that otherwise healthy and well-nourished patients with rheumatoid arthritis show significant clinical improvement when practicing prolonged fasting for 7 to 10 days. However, the improvement is reversible and is lost when eating is resumed. Although of little therapeutic value, the anti-inflammatory effect of short-term fasting might provide insight into some nutritional approaches to dealing with autoimmune diseases.

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RESEARCH NEEDS

Elucidation of the cellular and molecular basis of gut immune function will facilitate development of novel approaches for its homeostatic maintenance through diet as well as of novel food products that benefit general or select populations. Key basic research areas are identification of unique and nonunique leukocytes in the intestine, as well as clarification of how these cells function at the lurninal interface in the presence of food constituents and varied microflora. Such clarification requires an understanding of the mechanisms by which these sets of cells communicate with and regulate one another via cell surface molecules, cytokines, and neuroendocrine mediators. With increased understanding of GI immune function should come new methods of immunization against pathogenic microorganisms. A novel immunogenic approach also has been described for protection against intestinal carcinogens (Keren et al., 1986; Silbart and Keren, 1989),which involves conjugation of the carcinogen 2-acetylaminofluorine to cholera toxin and direct immunization of the intestine. Sensitive and accurate measures of immune function in the gastrointestinal tract are needed to elucidate more fully the effects of food constituents and contaminants. For example, experimental rodent models perhaps can be assessed best by monitoring leukocyte profiles by multiparameter flow cytometry. Specific leukocyte function can be measured by determining cytokine production in specific gut lymphoid tissue via immunoassay or the polymerase chain reaction technique. Results in experimental animal models then must be validated in humans using appropriate in v i m approaches with isolated human lymphocytes (Wood et al., 1992). One promising approach that may make possible the testing of hypotheses related to food and gut immunity involves the repopulation of immunodeficient mice with human lymphocytes (Gardner and Luciw, 1989). Additional information is needed on the molecular basis of the survival, uptake, and antigenicity of macromolecules in the intestinal tract. These questions will become even more pressing with the introduction of plant, animal, and microbial foods that have been modified by recombinant DNA techniques. One fundamental question is how multifactorial nonspecific immunity inhibits or favors survival and uptake of specific proteins. Another question relates to biotechnology-derived food products. For example, developing a transgenic cow that releases human Igs in its milk is theoretically possible (Lo et al., 1991). What effect would these human Igs have on adults and developing infants that ingest such milk? Another problem that has not been addressed adequately is the potential for sur-

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viva1 and uptake of DNA in the intestine. In preliminary work, we have demonstrated the rapid translocation of an orally presented plasmid into the blood of a mouse (L. Rasooly and J. Pestka, unpublished observations). Because of the large number of perceived food allergies, proper classification of food-related diseases and ingredient labeling are critical. Food processors need quality assurance programs that are based on sensitive immunoassay procedures capable of detecting small amounts of suspect food proteins in a complex matrix. Over the long term, the food industry must develop and identify foods that are devoid of or reduced in allergenic potential. Currently, direct challenge of sensitive persons and immunoassay with human IgE have been used to verify absence of allergenicity in a variety of food products. Also, immunoassays may be used in evaluating the reduction of allergenicity in a food. In the future, developing libraries of human monoclonal antibodies that react with key allergenic epitopes in foods may be possible, and may facilitate studies on the effect of processing on reduced allergenicity. Kessler et al. (1992) identified a regulatory framework for U.S. Food and Drug Administration evaluation of the safety of foods developed from biotechnology. High on this list is the potential of a donor food protein to be allergenic. One example would be the introduction of a peanut allergen into corn, thus making the new variety of corn allergenic to people allergic to peanuts. The authors suggested that employing recombinant DNA techniques to determine whether an allergenic determinant has been transferred from the donor to the new variety would be valuable. Since relatively few proteins are capable of eliciting an IgE response, elucidation of the structural requirements for allergenicity is both needed and feasible. As noted earlier, interaction of exogenous chemicals with lymphoid tissue may induce undesirable immunotoxic effects such as immunosuppression, uncontrolled proliferation, impaired host resistance, allergy, and autoimmunity. Chemicals that are potentially immunotoxic in the gut might be found among natural components, microbial products (including mycotoxins), additives, growth promoters, animal drugs, and various contaminants. Most information relative to immunotoxicity involves injection followed by assessment of systemic immunity. However, foodborne chemicals are most likely to have their greatest effect on GI immune function before they are absorbed and metabolized. Thus, studies on the effects of ingestion of chemicals at realistic levels on gut immunity are most useful. The potential for dysregulation of GI immunity by a foodborne chemical has been highlighted by the discovery that dietary

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exposure to vomitoxin increases serum IgA and induces IgA nephropathy in a rodent model (Dong et al., 1991). Progress has occurred in evaluating probiotic and immunologic effects of certain commensal and dairy strains of Lactobacillus and Bijidobacterium.A major problem in interpreting some experimental data is that the assays of immunologic function use leukocytes of systemic origin (e.g., peritoneal macrophages, blood lymphocytes, or spleen lymphocytes). Also, in some cases, cultures were injected into the animals. The specific effects of ingestion of lactic cultures on leukocytes found in the GI immune system must be elucidated. Additional research is needed on the role food antigens play in protecting against o r promoting certain autoimmune diseases (Marx, 1991). For example, can regular ingestion of a food that contains collagen reduce the severity of rheumatoid arthritis? Such a notion could lead to specific dietary recommendations for autoimmune individuals as well as to “designer’’ foods that enhance the ability of such individuals to cope with autoimmune diseases. Antigens might occur naturally in a food, might be added to the food, or might be recombinant proteins expressed in appropriate microbes, plants, or animals that are ingested normally. Using this final approach must be done cautiously since correct dosing for oral tolerance induction is critical to triggering the protective effect; overexposure actually may diminish the desired effect (Edgington, 1992). The specific effects of malnutrition and overnutrition on gastrointestinal immune function need to be investigated more fully. Hopefully, new dietary approaches can be used to maximize gut immunity in the face of a growing population that exhibits immunosuppression (including the elderly and AIDS patients) and chronic intestinal disease. In conclusion, the last two decades of immunologic research have expanded our understanding of gastrointestinal immunity greatly and have suggested possible routes for enhancing gut immune function. Although some study has been done of the interaction between food and GI immune function, the gap between basic immunology and the food and nutritional sciences is widening. This difference exacerbated by a general tendency in the popular literature and press to promote panaceas for stimulating immunity via special diets or foods. Equally disturbing is the potential for alarmism relative to adverse effects of food constituents, as exemplified by the promotion of pseudotests for immune function. With respect to gastrointestinal immunity, only through application of the basic research on topics described in this chapter can food and nutritional scientists offer

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rational and reliable approaches to enhancing the health of general and select populations via food and diet. ACKNOWLEDGMENTS This work was supported by Public Health Service Grant ES-03358, the National Kidney Foundation of Michigan, and the Michigan State University Agricultural Experiment Station. We acknowledge the kind assistance of Mary Rosner in manuscript preparation and Chris Oberg for illustrations.

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Cunningham-Rundles, C., Brandeis, W. E., and Good, R. H. (1979a). Bovine antigens and the formation of circulating immune complexes in selective immunoglobulin A deficiency. J. Clin. Invest. 64, 272-279. Cunningham-Rundles, C., Brandeis, W. E., Safai, B., O’Reilly, R., Day, N. K.. and Good. R. A. (1979b). Selective IgA deficiency and circulating immune complexes containing bovine proteins in a child with chronic graft vs host disease. Am. J. Med. 67,883-889. Czerkinsky, C., Prince, S. J., Michalek, S., Jackson, S., Russell, M. W., Moldoveau. Z., McGhee, J. R., and Mestecky, J. (1987). IgA antibody producing cells in peripheral blood after antigen ingestion: Evidence for a common mucosal immune system in humans. Proc. Natl. Acad. Sci. 84,2449-2453. Dean, J. H. and Murray, M. J. (1991). Toxic responses to the immune system. In “Casarett and Doull’s Toxicology” (M. 0. Amdur, J. Doull, and C. D. Klassen, eds.). pp. 282333. Pergamon Press, New York. Dean, J. H., Luster, M. I., Boorman, G. A., Luebke, R. W., and Lauer. L. D. (1980). The effect of adult exposure to diethylstilbestrol in the mouse: Alterations in tumor susceptibility and host resistance parameters. J. Reticuloendorhel. Soc. 28, 571-583. Delafuente, J. C. (1991). Nutrients and immune responses. Rheum. Dis. Clin. North A m . 17 (2), 203-212.

De Simone, C., Bianchi, B., Salvadori, R., Negri, R., Ferrazzi, M., Baldinelli, L., and Vesely, R. (1986). The adjuvant effect of yogurt on production of gamma interferon by Con A-stimulate$ human peripheral blood lymphocytes. Nutr. Reports Int. 33,419-433. Diehl, J. T., Steiger, E., and Hooley, R. (1983). The role of intravenous hyperalimentation in intestinal disease. Surg. Clin. North Am. 63, 11-26.

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Walker, R. I., and Owen, R. L. (1990). Intestinal barriers to bacteria and their toxins. Annu. Rev. Med. 41,393-400. Walker, W. A. (1983). Gastrointestinal transport of macromolecules in the pathogenesis of food allergy. Ann. Allergy 51,240-245. Walker, W. A. (1985). Absorption of protein and protein fragments in the developing intestine: Role in immunologic/allergic reactions. Pediatrics 75, 167-171. Walker, W. A. (1987). Pathophysiology of intestinal uptake and absorption ofantigens in food allergy. Ann. Allergy 59,7-16. Walker-Smith, J. A., Digeon, B., and Phillips, A. D. (1989). Evaluation of a casein and a whey hydrolysate for treatment of cow’s milk sensitive enteropathy. Eur. J . Pediatrics 149,68-7 I . Watanabe, M., Miyakawa, J., Ikezawa, Z., Suzuki, Y.. Hirao. T., Yoshizawa. T.. and Arai. S. (1990). Production of hypoallergenic rice by enzymatic decomposition of constituent proteins. J. Food Sci. 55,781-783. Wells, C . L., Maddaus, M. A., and Simmons, R. L. (1988). Proposed mechanisms for the translocation of intestinal bacteria. Reu. Infect. Dis. 10,958-979. Whitacre, C. C., Gienapp, I. E., Orosz, C. G., and Bitar, D. M. (1991). Oral tolerance in experimental autoimmune encephalomyelitis. 111. J . Immunol. 147 (7), 2155-2163. Wood, S. C., Karras, J. G., and Holsapple, M. P. (1992). Integration of the human lymphocyte into immunotoxicological investigations. Fund. Appl. Toxicol. 18,450-459. Yokokura, T., Nomoto, K., Shimizu, T., and Nomoto, K. (1986). Enhancement of hematopoietic response of mice by subcutaneous administration of Lactobacillus casei. Infecr. Imrnun. 52,156-160. Yoshizawa, T., and Arai, S. (1990). Production of hypoallergenic rice by enzymatic decomposition of constituent proteins. J. Food Sci. 55,781-783. Yunginger, J. W.. Sweeney, K. G., Sturner. W. Q., Giannandrea, L. A., Teigland. J. D.. Bray, M., Benson, P. A., York, J. A., Biedrzycki. L., Squillace, D. L.. and Helm, R. M. (1988). Fatal food-induced anaphylaxis. J. Am. Med. Assn. 260, 1450-1452. Zeitz, M., Schieferdecker, H. L., James, S. P., andRiecken, E. 0. (1990). Special functional features of T-lymphocyte subpopulations in the effector compartment of the intestinal mucosa and their relation to mucosal transformation. Digestion 46, 280-289. Zhang, Z. Y., Lee, C. S., Lider, O., and Weiner, H. L. (1990). Suppression of adjuvant arthritis in Lewis rats by oral administration of type I1 collagen. J . Immunol. 145 (8), 2489-2493.

Zhang, Z. J., Davidson, L., Eisenbarth, G., and Weiner, H. L. (1991). Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc. Natl. Acad. Sci. U.S.A. 88 (22), 10252-10256.

ADVANCES IN FOOD AND NUTRITION RESEARCH. VOL. 31

EFFECT OF CONSUMPTION OF LACTIC CULTURES ON HUMAN HEALTH MARY E L L E N SANDERS Microbiology Consultant Littleton. Colorado 80122

I. Introduction 11. General Physiology A. Gastrointestinal Ecology B. Fecal Recovery C. Adherence of Probiotic Cultures in the Human Intestine D. Production of Antimicrobials 111. Health Targets A. Lactose Digestion B. Diarrhea C. Cholesterol Reduction D. Cancer Suppression E. Immune System Stimulation F. Constipation G. Vaginitis IV. Safety Issues V. Considerations for Strain Selection VI. Research Needs VII. Conclusions References

I. INTRODUCTION

The opinions of experts on the role of lactic acid bacteria in promoting health in humans are diverse. Some are very optimistic; some are guarded. Consider these examples: Yogurt . . . is indicated as the best diet for balancing and preserving the biological functions of the gut. All these biological effects of yogurt are of great importance for the 67 Copyright 8 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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normalization of the autochthonous flora and the consequent increase of the body’s natural defense against infectious diseases and putrefactive and fermentative processes. (Bianchi-Salvadori, 1986)

.

[Lactic acid bacteria] impart nutritional and therapeutic benefits to the consumer. . The antimicrobial substances produced by these bacteria control the proliferation of undesired pathogens. . . . Their anticholesteremic properties assist in lowering serum cholesterol. It has been suggested that the tumor suppression trait of these microbes reduces the incidence of colon cancer. (Fernandes et al., 1987) There appears to be much potential for the use of bifidobacteria with other beneficial organisms as dietary adjuncts in cultured dairy products. Research has shown that in some cases bifidobacteriacan be used to control enteric infections, lower serum cholesterol levels, improve infant formulas, and make milk products more nutritious and more easily digestible by lactose-intolerant people. (Laroia and Martin, 1990) The therapeutic value of [Lacrobacillus acidophilus] has since been established for many diseases and disorders of the digestive tract. (Danielson et al., 1989) Consumption of cultured dairy products containing viable [lactic acid bacteria] alters favorably the gastrointestinal microecology, conferring protection to the host. (Fernandes and Shahani, 1989)

. . . It becomes . . . more and more important that we consume products containing the special lactobacilli, bifidobacteria, and yogurt cultures in foods on a daily basis. Those individuals who do so will help keep their immune systems activated to combat the influx of undesirable organisms and possible pathogens into their gastrointestinal system via the food chain. (Sellars, 1989) These generalized conclusions are remarkable because even the most basic of premises on which these conclusions are based-including the likelihood of intestinal implantation of dietary cultures, the role of diet on intestinal microflora, and the role of intestinal flora on human health-are still matters of intense scientific debate. Consider the guarded opinions of the researcher about substantiated health benefits, that are observed more commonly in the literature: The mechanisms of the beneficial effects of bifidobacteria are still insufficiently known, and further research and development are needed. (Kurmann and Rasic, 1991)

. . . An abundance of research results to support [health] claims of the current products have (sic) not been provided. (Kim, 1988) Many health promoting benefits . . . are purported to be gained by eating these fermented foods. Yet these claims are not fully substantiated by research and many conflicting opinions exist over what actual benefit is derived from eating these foods. (Truslow, 1986)

.

. . The belief that the consumption of lactobacillus-containing (sic) preparations promotes health is based largely on anecdotal information. The scientific literature is

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distressingly sparse concerning controlled, scientifically valid investigations of fermented milk products and their influence on health. (Tannock, 1990) Some evidence has accumulated to show the antimicrobial and anticholesteremic activities of these microorganisms [bifidobacteria]. The evidence is scant, however, and the therapeutic value of this important group of bacteria has not been conclusively established. (Modler et a / . , 1990) Colonization of the gut by bifidobacteria taken orally has not been clearly demonstrated. Recent evidence . . . has suggested that colonization can take place only when a human or animal subject has been fed his (sic) own strain of bifidobacteria; thus, inclusion of generic strains of bifidobacteria in dairy products may have no therapeutic value. (Modler et al., 1990) According to present-day reports on antitumor activity of fermented milks, it is not justified to claim such an effect. (Driessen and de Boer, 1989)

. . . Given the variation in life-styles and genotypes among the human population, measures aimed at stimulating the activities of certain members of the normal flora of the gastrointestinal tract by the use o f . . . dietary additives are unlikely to be practical or worthwhile. . . . The successful implantation of bacterial strains which are antagonistic to pathogens or potential pathogens in groups of adult humans is likely to be extremely difficult. (Tannock, 1984) Unfortunately, there is not good scientific evidence that the ingestion of milk fermented by lactobacilli is any more beneficial to health than simply ingesting plain, pasteurized milk. For every article in the scientific literature that claims beneficial results from the ingestion of fermented milk, another article will provide evidence to the contrary. Most of the reported studies have not been adequately controlled, statistical analysis of the results is rarely made, and the conclusions are largely speculative. (Tannock, 1984)

The controversial nature of this field demands care in summarizing even peer-reviewed research papers. Many published studies fall far short of providing direct evidence for a healthful effect of lactic cultures on human health. Many papers cited as proof focus on animal models, in uitro experiments, or insufficiently controlled human studies with few subjects, or pay little attention to substantive rather than statistical significance. Lactic acid bacteria have been used to promote health for hundreds of years. Perhaps their most obvious effect is the inhibition of pathogen growth in traditionally fermented food systems, accomplished primarily by the acidogenic nature of these cultures. These bacteria subsequently may exert .positive benefits after consumption as residents of or while travelling through the gastrointestinal tract. These traits generally are attributed to lactic acid bacteria of the intestinal tract, primarily Lactobacillus acidophilus and Bijidobacterium, but also may include other lactobacilli, Streptococcus salivarius thermophilus, and Enterococcus. Stated healthful effects of lactic cultures include improved absorbability

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of nutrients, alleviation of lactose intolerance symptoms, metabolism of some drugs, reduction of serum cholesterol, reduction of enzymes implicated in carcinogen production, improvement of intestinal motility, stimulation of the immune system, reduction of tumor incidence, creation of an antagonistic environment for intestinal pathogens by production of inhibitors, blocking adhesion sites from pathogens, inactivating enterotoxins, alleviating constipation, and relieving vaginitis. This impressive array of potential benefits from these organisms has led to the marketing of many products for the stated or implied health benefits derived from the viable cultures contained within them. These products include nonfermented milk, fermented milks, yogurts, dried cultures, soft drinks, and candy. Although many products are formulated to use these intestinal lactic acid bacteria as supplements, international interest also exists in the production of milk products fermented with intestinal lactics (Gurr, 1984; Kurmann, 1988). This type of product is unique because the probiotic culture must be functionally fermentative and impart good flavor to the product in addition to promoting health. In practical terms, developing high populations of desirable microbes in products in which their growth was encouraged would be easier. However, problems with acid sensitivity could reduce populations during storage (Misra and Kuila, I99 1a,b) . In addition to original research papers, several comprehensive reviews have been published that discuss this field in a general fashion (Sandine et al., 1972; Shahani and Chandan, 1979; Shahani and Ayebo, 1980; Rasic, 1983; Friend and Shahani, 1984; Gurr, 1984; Lemonnier, 1984; Fernandes et al., 1987; Bourlioux and Pochart, 1988; Kim, 1988; de Simone et al., 1989; Fuller, 1989,1991;Goldin, 1989,1990;Gorbach, 1989,1990;Kroger et al., 1989; Sellars, 1989,1991; Alm, 1991; Renner, 19911, or focus specifically on lactose intolerance (Savaiano and Levitt, 1987; Savaiano and Kotz, 19881, gastrointestinal microbiology (Gorbach, 1971 ; Brown, 1977; Savage, 1977,1983a,b;Tannock, 1983,1984,1990;Bianchi-Salvadori, 19861, antimicrobial activities (Fernandes and Shahani, 1989), anticarcinogenic properties (Fernandes and Shahani, 1990), vaginal health (Redondo-Lopez et al., 19901, bifidobacteria (Poupard et al., 1973; Rasic and Kurmann, 1983; Bezkorovainy and Miller-Catchpole, 1989; Mitsuoka, 1989; Reuter, 1989; Laroia and Martin, 1990; Mitsuoka, 1990; Modler et al., 1990; Sandine, 1990; Hughes and Hoover, 1991; Kurmann and Rasic, 1991; Misra and Kuila, 1991a), L. acidophilus (Sandine, 1979,1990; Fonden, 1989; Gilliland, 1989), and strain selection for dietary adjuncts (Gilliland, 1979; Klaenhammer, 1982). These reviews should be consulted for additional perspective on this field.

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II. GENERAL PHYSIOLOGY

A.

GASTROINTESTINAL ECOLOGY

Although the human body is composed of loi3 eukaryotic cells, an estimated prokaryotic cells colonize the body surfaces and gastrointestinal tract (Savage, 1977). Therefore, only 10%of the cells in the human body are eukaryotic. The influence of this sizable prokaryotic population is undoubtedly significant, but exactly how these microbes influence health is the subject of much debate. Studies with germ-free animals have shown that colonization is not necessary for survival. However, germ-free animals are much more likely to succumb to infection than their colonized counterparts (Collins and Carter, 1978), suggesting that the normal gastrointestinal flora (without dietary supplementation) play a significant role in interfering with the establishment of intestinal pathogens (Tannock, 1984). The distribution of gastrointestinal microbes occurs horizontally, from the center of the lumen to the depths of the crypts, and vertically, from the esophagus to the anus (Savage, 1977). The specific occurrence of microbes depends on a variety of factors including age, health state, use of antimicrobial drugs, and, perhaps to some extent, diet (Hentges, 1980). The influence of diet on the normal gastrointestinal flora is still uncertain, prompting Tannock (1983) to state that his treatise on the effect of diet and stress on gastrointestinal microbiota was “of a speculative nature.” Summaries of studies on the flora of the gastrointestinal tract can be found in several reviews (Gorbach, 1971,1986; Brown, 1977; Savage, 1977). These surveys are consistent in reporting the relatively low level of bifidobacteria (6-1 1%) and even fewer lactobacilli (0-1.3%) in the feces of healthy human adults. The exception to this condition is the dominant presence of bifidobacteria in breast-fed infants (Stark and Lee, 1982). When attempting to design intestinal lactic cultures that will exert a positive effect on human health, it is reasonable to consider the extent of influence that would be expected to be exerted by microbes that may be minor components of the gastrointestinal population. This question is even more pertinent in light of the widely held belief that a dietary culture, even one possessing in uitro adhering capabilities, is highly unlikely to displace any bacterial strain that colonizes a healthy human intestinal tract (Savage, 1977). This theoretical criticism of dietary supplementation with cultures perhaps is addressed best empirically. Human gastrointestinal microbiology is notably a difficult field to study because of limits on direct experimentation (Savage, 1983a) and the difficult physiological requirements of

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the intestinal microbes. As indicated by Tannock (1984), “All investigators approaching the study of the normal flora of the gastrointestinal tract must sooner or later be horrified by the complexity of an ecosystem that contains about 500 species of bacteria, most of which are technically difficult to work with under laboratory conditions.” Analysis of fecal flora is a common technique used to evaluate the effect of feeding lactic cultures, but can never be a good index of the true character of the gastrointestinal microbial ecosystem (Savage, 1977). Savage (1983a) reviewed techniques for measuring intestinal colonization and concluded that no technique could be relied on to give acceptable data on microbial colonization of humans. Savage (1977) challenged the value of population studies and suggested that the biochemical activities of the gastrointestinal microbiota may be more significant than their numbers. Perhaps even low populations of microbes can exert significant biochemical influences in vivo.

B. FECAL RECOVERY Although of questionable value, studies abound that examine the microbial content of human feces after feeding with probiotic cultures. Among these studies are those of Lidbeck et al. (1987), who attempted to determine the effect of L. acidophilus supplementation on the flora of the intestinal tract by conducting fecal analyses. The effect of feeding on the microbial content of saliva also was determined. The results showed that feeding 500 ml/day of milk fermented by L. acidophilus (Arla Acidofilus) had little effect on the microbial content of saliva, but had a detectable effect on microbial content of feces. The major effect of L . acidophilus feeding was a decrease in Escherichia coli levels in 6 of 10 subjects and an increase in lactobacilli in feces. After supplementation was stopped, the flora returned to preexperimental levels. Lidbeck et al. (1988) also studied the effect of feeding L . acidophilus NCDO 1748 fermented milk to 10 healthy volunteers after oral administration of the antibiotics enoxacin (active against gram-negative aerobic bacteria) and clindamycin (active against anaerobic bacteria). Microbial analysis was conducted on feces throughout antibiotic treatment and Lactobacillus supplementation. The authors concluded that “although there was a partial restoration of the intestinal microflora due to the reestablishment of lactobacilli and enterococci, L. acidophilus administration could not accelerate the normalization of most other strongly suppressed microorganisms in the intestine.” Additionally, Lidbeck et al. (1991) studied the effect of consumption of fermented acidophilus milk on

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dietary intake, fecal flora, and aqueous fecal bile acids in 14 colon cancer patients. Only minor microbiological changes occurred. Goldin et al. ( 1992) followed the excretion of Lactobacillus GG in feces 3 and 7 days after subjects consumed GG. GG was administered as a yogurt for 7 days (daily dose: 3.6 x 10" bacteria), as a concentrate for 28 days (daily dose 4 x 10" bacteria), or as a whey drink for 35 days (daily dose 1.6 x 10" bacteria). Strain GG increased 4-6 log cycles during GG feeding in almost all subjects. Levels of recovered GG fell after feeding stopped, but these lactobacilli were still present at 104/gfeces after 7 days. This type of experiment has been done with bifidobacteria as well. Bouhnik et al. (1992) fed a strain of Bijidobacterium, tagged with streptomycin and rifampicin resistance markers, via a fermented milk product to 8 healthy volunteers, 3 times daily for 8 days. This strain increased to levels of over 108/gfeces, but gradually disappeared after ingestion ceased. The authors concluded that Bijidobacterium fed to humans does not colonize the colon, but populations could reach levels high enough to exert metabolic probiotic effects. Kageyama et al. (1984) studied the effect of bifidobacteria on fecal flora of leukemia patients. Fecal flora were identified from 56 patients (32 males, 24 females, aged 20-60) with six different types of leukemia. Patients consumed 200 ml Morinaga Bifidus milk daily for 3 months. The milk contained about 107/mleach of Bijidobacterium and L. acidophilus. (Note that the title of the paper in question and the discussion throughout implied that only Bijidobacterium was administered, when in fact equal quantities of both microbes were consumed.) This study presented data that showed differences in fecal flora between patients consuming the Morinaga milk product and patients not consuming the milk. All subjects were undergoing antileukemic drug therapy. Tabulated data showed a higher level of E. coli, Bacteroides, and Veillonella during the administration of antileukemic drugs without Bijidobacterium, but unclear data presentation left a lack of understanding of what composed the reported numbers. Data on other minor intestinal bacteria were presented also. In this case, 10 control cases were used, but whether these controls were leukemic patients was unclear. Among the 56 cases undergoing drug therapy, some variation in minor fecal constituents was noted also. A legitimate decline in fecal levels of Candida was observed in patients administered the milk product. Marginal differences in urine indican and blood endotoxin occurred in patients treated with antileukemic drugs and bifidobacteria. In summary, this study did show an effect of administration of Morinaga Bifidus milk containing both L. acidophilus and Bifidobacterium on fecal content of leukemic patients receiving drug therapy. The extent of this effect could not be determined fully since no statistical analyses were done and since controls

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for some of the experiments were unclear. Further, no effort was made to follow the numbers of infections in these patients so true clinical effects could be determined. Clearly an effect on fecal populations was observed; how this change affected the clinical state of the patients was left unexplored. Numerous similar studies performed over the years have proven that certain lactic cultures can survive passage through the intestinal tract, whereas others cannot. Important factors include resistance to physiological levels of intestinal bile acids and feeding of the culture under conditions (usually in conjunction with food) that help neutralize stomach acid at or above pH 4. After consumption of a bacterium, recovery from the feces is certainly not evidence of implantation, even if recovery persists for a period after consumption has stopped, nor is recovery an indication that the probiotic culture is likely to impart a desirable influence. The administered microbes may reside exclusively in the large intestine, where many important benefits cannot be realized. Continued fecal recovery of these microbes after feeding has stopped simply may be because of residence time in the large intestine that exceeds microbial generation time, and may have little to do with colonization. C. ADHERENCE OF PROBIOTIC CULTURES IN THE HUMAN INTESTINE Adherence refers to the ability to stick to solid surfaces. Lactic cultures might be expected to have a greater impact on human health if they were capable of adhering to portions of the epithelial surfaces of the human intestine. Adherence differs from colonization because the latter implies the ability to adhere and also replicate. Mechanisms of adherence are diverse and complicated. However, retention of bacteria in the human intestine can result from specific adherence to epithelial cells, from nonspecific adherence to other intestinal bacteria, or from entanglement in mucus. Direct studies on adherence in humans are invasive and therefore essentially impossible to do. However, the contents of the human ileum, colon, and feces have been examined microbiologically. The presence of microbes in these intestinal or fecal contents is not proof that colonization has occurred, although absence suggests that colonization has not occurred (assuming suitable microbiological techniques). The body of scientific research on adherence deals with animal models, so their significance to humans could be questioned. As a comment on in uitro studies of adherence to human fetal intestinal cells, Tannock (1990) writes, “These observations presumably have little significance, since association of lactobacilli with intestinal epithelia in vivo has not been demonstrated.” Still, efforts continue toward identifying in uitro models that may mimic the in

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uiuo human environment. Experimentation with Caco-2 polarized human intestinal epithelial cells in culture (Chauviere et al., 1991) and other improved cell lines hopes to establish such a model. Confirmation of results from these in uitro systems with clinical data is necessary before these results would be considered valid for humans. The intestines of different species vary in chemical composition and nutrient availability (Tannock, 1990). Therefore, the observation of host specificity of adherence (Wesney and ,Tannock, 1979; Tannock et al., 1982; Lin and Savage, 1984) is not surprising. This host specificity can be achieved by specific adhesins and receptors on bacterial and host cells and/or by nutritional or physiological adaptation to different cell types or gut environments. Conway and Kjelleberg (1989) identified an extracellular protein from Lactobacillus fermentum that mediates host-specific L . fermentum adhesion and patented a process that uses this protein to promote adhesion (Conway and Kjelleberg, 1988). Sat0 et al. (1982), using mucosal epithelial cells from pig embryo ileum on glass slides, concluded that a Bifdobacterium strain required its polysaccharide fraction to adhere. The significance of this study is completely dependent on how well this in uitro model using pig intestinal cells represents a human system. Conway et al. (1987) studied the survival of lactic acid bacteria in the human stomach and their adhesion to intestinal cells as a means to identify cultures suitable as dietary adjuncts. Cultures were screened for survival in gastric juice aspirated from healthy humans (21-42 years old). Also, a Lactobacillus preparation was intubated directly into stomachs of volunteers, with and without skim milk. At 20-min intervals, gastric fluid was removed and bacterial survival was recorded. Lactobacillus acidophilus strains ADH and N2 were used. Strain-dependent survival in gastric juice occurred, but the stomach presented a challenge for culture survival. These results support the need to consume these microbes with food or in an encapsulated form. Goldin et al. (1992) reported that GG survived ex uiuo in human gastric juice at pH 3.0. Below this pH, survival was poor, suggesting that ingestion of cultures with food to raise the pH is important for gastric survival. These data are consistent with the results of Conway et al. (1987). Clements et al. (1983)verified survival of ingested lactobacilli from LactinexTM and Infloran Berna'" by sampling jejunal fluid in uiuo. Conway et al. (1987) studied adhesion using radioactive cells. Strain ADH was more resistant than strain N2 to low pH and gastric juice exposure in uitro and in uiuo. The cell wall of ADH was also resistant to lysis and bound significantly better to human ileal cells than did other strains. All strains (two L . acidophilus, one L . delbrueckii ssp. bulgaricus, and one S . saliuarius thermophilus) bound to pig ileal cells, but in the same strain-specific pattern seen with human cells. The addition of 1% skim milk increased the binding of ADH to human ileal cells by a factor of

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3. Since the survivability of cultures in phosphate-saline buffer mimicked results obtained with human gastricjuice, the authors recommended phosphate buffer for the selection of resistant strains. The impact of these results is minimized by the fact that only two L. acidophilus strains were evaluated. Further, the data from the adhesion assay has questionable physiological significance because of its in uitro nature, although an in uiuo source of the intestinal cells was used. The test determining survival in human gastric juice may be a useful screen for dietary adjuncts in the future. However, results should be generated with more than two strains to determine if phosphate-saline buffer is a reasonable substitute for gastric juice in general. Other authors have speculated on the role of carbohydrates in adherence of lactobacilli (Brooker and Fuller, 1975). Costerton et al. (1978) noted that extracellular glycocalyx is frequently expendable for bacteria that are removed from their natural habitat to be cultivated as pure cultures in the laboratory. Meaningful research requires preserving intestinal strains in their physiologically active state. The best method for such preservation is a subject for future research. As difficult as obtaining reliable information on adherence in human systems is, adherence is commonly believed to be required for a probiotic culture to exert most positive effects. Otherwise, residence time of the culture is believed to be too short. Clearly the ability to adhere is required for long-term implantation in the gut. However, even for adhering microbes, displacement of the established flora of the healthy adult intestine is difficult (Tannock, 1990), except with microorganisms that have an adaptive mechanism that directs displacement of other microbes or after a specific change in the physiological state of the person. Collectively, these facts suggest that adherence cannot be insured by simply feeding microbes that possess the potential to adhere. However, since the intestinal flora of an adult is not necessarily constant because of the influence of certain dietary, physiological, and symptomatic or asymptomatic disease states, chances may exist for alteration of the intestinal flora throughout the lifetime. Adherence may result if continued consumption of a suitable strain occurs during one of these opportunistic times. The newly adhering strain may be displaced, however, once the physiological condition returns to normal. Also, transient microbes may exert positive effects without adhering, which appears to be the case with yogurt bacteria that aid lactose digestion. Studies on fecal recovery of microbial dietary supplements show recovery of the fed microbe for days or even weeks after feeding has stopped. Although not a permanent condition, these studies suggest that residence times for these microbes may be sufficient to exert an effect. One could speculate that, if continuously consumed, transient populations of

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the dietary culture could possibly lower intestinal pH, produce antimicrobial compounds, neutralize toxins, utilize nutrients, and conduct other biochemical activities that could influence gastrointestinal ecology. D. PRODUCTION OF ANTIMICROBIALS The ability of lactic cultures to flourish at the expense of many other bacteria has been observed in food systems and is widely suspected to occur in the human intestine. In food systems, lactic acid bacteria create an environment that is inhospitable to many bacteria, including pathogens, by lowering the pH, decreasing the redox potential, and producing antimicrobial compounds including organic acids, hydrogen peroxide, and proteinaceous bacteriocins. In uiuo, the mechanisms and extent of competitive exclusion are much less clear. One aspect of lactic culture biochemistry that has attracted much research interest is the production of bacteriocins (Klaenhammer, 1988). The in uiuo significance of these compounds is not clear. Much of the literature published on bacteriocins is presented from the perspective of use of these substances or the producing microbes in food systems, not in humans (Babel, 1977; Gibbs, 1987). Characteristics of the bacteriocins required for these two different activities would be vastly different. However, claims continue to be made for the general antipathogenic effects of bacteriocin-producing lactic cultures in uiuo, although wellcharacterized bacteriocins purified from lactobacilli have been shown repeatedly to have a narrow spectrum of activity against closely related species and do not inhibit common gram-negative pathogens (Klaenhammer, 1988). Still, selection on the basis of bacteriocin-like activity is advised regularly as a screen for cultures that may be used for probiotic applications (Gilliland and Walker, 1990). Silva et a / . (1987) studied a low molecular weight protease (I 1000) and heat stable compound concentrated from Lactobacillus GG. This compound inhibited all 54 strains of 7 genera (E. coli, Streptococcus, Pseudomonas, Salmonella, Bacillus fragilis, Clostridium, and BiJdobacterium) tested. These properties are not consistent with those of a bacteriocin and seem more similar to those of an organic acid. When identical concentrations of pure lactic and acetic acids present in the GG extract were tested, no zones of inhibition were present. The authors concluded that the compound produced by GG must be distinct from lactic or acetic acid. Unfortunately, the organic acids were not tested in combination although they were present together in the GG extract. The authors did not eliminate the possibility of synergism with multiple organic acids or with other possible extract components. The involvement of lactic, acetic, or other organic acids in inhibition activity has not been clarified. The molecular weight,

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the spectrum of activity, and the physical stability of the compound all suggest the role of an organic acid. This antibacterial activity for a Lactobacillus strain is an observation of questionable significance. In the years since the publication, no follow-up on further characterization of the compound has been made. Meghrous et al. (1990)examined 13 strains of bifidobacteriafor bacteriocin production. One producer was found with a spectrum of activity that was narrow and limited to closely related gram-positive bacteria. Inhibition of clostridia in laboratory assays and in food systems by nisin, an antibiotic produced by Lactococcus lacris subsp. lactis, suggests the possibility that this compound may be effective against an intestinal pathogen such as Clostridium difficile.However, since L . lactis does not survive in the intestine, natural nisin-producing strains are not likely to be useful therapeutically. Further, nisin is degraded readily by proteolytic enzymes present during food digestion. Possibly, the genes that encode this bacteriocin could be transferred into bacteria that are likely to survive the stomach and intestinal environments, and possibly even implant in the intestine. However, no evidence exists that this bacteriocin is effective in an animal system. In addition to nisin, other bacteriocins from lactic cultures have demonstrated in uitro activity against pathogens, including Listeria (Bhunia et a f . , 1988; Harris et a!., 1989; Spelhaug and Harlander, 1989; Ahn and Stiles, 1990), Campylobacter (Lyon and Glatz, 1991), and Vibrio (Lyon and Glatz, 1991). In general, however, in uitro evidence suggests that bacteriocins of lactic acid bacteria are primarily effective against other lactic acid bacteria. The presence of some in uitro activity against some intestinal pathogens provides encouraging evidence that certain cultures may possess useful antipathogenic bacteriocin activity in uiuo. However, the effect of these bacteriocin-producing lactobacilli on other gastrointestinal microbes has never been studied in uiuo. The genetics of bacteriocin production in lactobacilli has progressed enough that comparative clinical studies of strains that are isogenic except for bacteriocin production genes could and should be conducted to determine the usefulness of this phenotype. Bacteriocin production by probiotic strains could have a negative effect by inhibiting or displacing native “desired” lactobacilli, not pathogens, in the gastrointestinal ecosystem. Ill.

HEALTH TARGETS

Research on the effects of consumption of lactic cultures on human health has focused on a few specific health targets, including lactose maldigestion, diarrhea, reduction of serum cholesterol, cancer, immune

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system stimulation, constipation, and vaginitis. This research is performed mostly from an empirical perspective, with little attention to culture selection, mechanism of effect, or degree of benefit that could be expected by the consuming public. Discussions of these specific health targets follow. A.

LACTOSE DIGESTION

Reviews on lactose intolerance have been prepared by Savaiano and Levitt (1987), Scrimshaw and Murray (1988), and Savaiano and Kotz (1988). The presence of lactose maldigestion in the majority of the human population (worldwide levels of about 70%) is well documented (Scrimshaw and Murray, 1988). Lactose-intolerant people may avoid milk and other dairy foods because of intolerance symptoms and therefore may consume suboptimal levels of calcium. This response is unnecessary and unfortunate (Gallagher et al., 1974;Gilliland and Kim, 1984),and provides excellent justification for the development of milk products that are digested more easily by lactase-deficient people. Note that some people may have milk protein allergies, which differ from lactase deficiency, that may prevent them from consuming milk products. Although lactose maldigestion may be prevalent worldwide, the symptomatic expression of lactose intolerance is less so. Most lactase-deficient people can consume one glass of milk per day asymptomatically (Savaiano and Kotz, 1988). Further, 85% of those individuals reporting discomfort with the consumption of milk and milk products characterize their symptoms as mild or state that they would not be deterred from drinking milk as a result (Scrimshaw and Murray, 1988). However, a greater percentage of people believe they are lactose intolerant than can be substantiated clinically. For example, when assessing individuals who claim to be intolerant of milk, 64% were shown to be lactose digesters (Rosado et al., 1987). Also, the incidence of symptoms in subjects fed lactose-free placebos also could reach 35% (Savaiano and Levitt, 1987). Therefore, lactose intolerance has a substantial psychological component. Dietary practices also can influence the expression of lactose intolerance. That a given quantity of lactose is tolerated more easily when consumed in milk or dairy products than when consumed in water is commonly accepted (Scrimshaw and Murray, 1988), although some dairy products are tolerated better than others. Yogurt, which may contain almost as much as or more lactose than milk, is tolerated better than milk. Some research has suggested that starter culture lactase may digest the lactose once the yogurt is consumed (Lin et al., 1991). Also, lactasedeficient people have been shown to be able to adapt to lactose by regular consumption of lactose-containing foods (Scrimshaw and Murray, 1988). The mechanism of this adaptation is not clear although, in animal studies,

80

MARY ELLEN SANDERS

three- to sixfold increases in the fecal microbial levels of j3-galactosidase occur. A change in the enzymatic activities of the intestinal bacteria or microbial populations toward greater ability to digest lactose may result in more tolerance to lactose. People clinically identified as lactose intolerant based on a standard lactose test may not show symptoms in day-to-day life since the quantity of lactose ingested at any one time in a normal diet is usually much less than that ingested in the lactose-tolerance test (Scrimshaw and Murray, 1988). These facts demonstrate that lactose deficiency exists as a multidimensional issue for humans. However, nonallergic people can consume at least some dairy foods, providing dietary options for good nutrition and much needed calcium. The choices and quantity of dairy foods that are available to lactoseintolerant individuals could be increased with proper formulation of culture-containing milk products. Although Lin et al. (1991) found that laboratory-preparedacidophilus milk containing lo8cfu/ml (counts almost two log cycles greater than those of commercially prepared acidophilus milk) showed only marginal effects on breath hydrogen values, similar milks prepared with yogurt cultures ( L . delbrueckii bulgaricus and S . salivarius thermophilus) elicited significantly lower breath hydrogen values in subjects. Of three L. acidophilus strains tested, one strain did show efficacy in alleviating symptoms, although yogurt cultures were better. These results clearly show that milk containing the proper cultures can be tolerated more readily by lactose-intolerant people. Improved lactose tolerance appeared to be due to in uiuo autodigestion of lactose by microbial lactase. The survival of intracellular microbial j3-galactosidase appears to be due to two factors (Savaiano and Levitt, 1987): the protective encasement of the enzyme by the microbial cell and the effect of the excellent buffering capacity of ingested dairy products on stomach pH. These conditions provide a mechanism for the microbial lactase to reach the intestine in an active form to digest the lactose. The results of Lin et al. (1991) also demonstrate the importance of strain selection, since certain microbes have greater efficacy in this role than others. In this regard, criteria for the selection of strains for their effectiveness in relieving lactose intolerance symptoms may be very different from and, in some cases, contradictory to those for selection for other proposed health benefits. For example, bile tolerance, a trait considered important for intestinal survival of probiotic cultures, may protect against bileinduced permeabilization of the microbe in the intestine, preventing intracellular microbial lactase from coming into contact with and digesting lactose. In fact, L. acidophilus NCFM, selected partly for its bile resistance for the Sweet Acidophilus'" product, performed worst in trials conducted by Lin et al. (1991).

81

LACTIC CULTURES AND HUMAN HEALTH

Martini et al. (l991a) published a paper describing the clinical effects of different species of lactic acid bacteria on lactose intolerance. In the first study, 7 lactase-deficient subjects were fed the following 5 meals in a random blind format: ( 1 ) yogurt made with undefined Yoplait starters; (2) yogurt made with S . salivarius thermophilus strain 3641 and L. delbrueckii bulgaricus strain 11842; ( 3 ) yogurt made with S . salivarius thermophilus strain 3641 and L. delbrueckii bulgaricus strain 880; (4) yogurt made with S . s. thermophilus strain TS2B and L. delbrueckii bulgaricus strain 11842; and (5) whole milk supplemented with 2% nonfat dry milk. Each meal contained 18 g lactose. Peak hydrogen excretion was about threefold higher with milk than with any of the yogurts. Fivefold more hydrogen was excreted over an 8-hr period after consumption of the milk than after consumption of any of the yogurts. No significant differences in any of the four yogurt patterns was observed, although a wide range of total (per g product) and specific (per mg protein) p-galactosidase levels was exhibited (Table I), implying that even the yogurt with the lowest 0-galactosidase activity contained sufficient enzyme for efficacy. A second study in this paper attempted to differentiate between the contributions of different strains to the alleviation of lactose-intolerance symptoms. In this experiment, 12 lactase-deficient subjects were fed the following meals: ( 1 ) low-fat milk; (2) yogurt made with L. delbrueckii bulgaricus and S . salivarius thermophilus; ( 3 ) milk fermented with only S . salivarius thermophilus; (4) milk fermented only with L . delbrueckii bulgaricus; (5) milk fermented with Bijidobacterium bijidum; and (6) milk fermented with L. acidophilus. Each meal contained 15 g lactose, but bacterial concentrations in the different products varied. Breath hydrogen was produced in decreasing levels from the following products: ( I ) milk; TABLE I

P-GALACTOSIDASE LEVELS OF FOUR YOGURT PRODUCTS FED To LACTOSEDEFICIENT SUBJECTS'

Yogurt starters

Total P-galactosidase activityh

Specific P-galactosidase activity"

Yoplait ST 3641 + LB I1842 ST 3641 + LB 880 ST TSZB + LB 11842

7.00 2.30 3.42 4.96

5.03 3.70 4.03 6.68

Reprinted with permission from Martini et a/. (1991a). Activity is pmol ONPG hydrolyzed/min/g product. ' Activity is pmol ONP released/min/mg protein.

82

MARY ELLEN SANDERS

(2) milk fermented with Bijidobacterium; (3) milk fermented with L. acidophilus; (4) milk fermented with S . salivarius thermophilus; ( 5 ) milk fermented with L. delbrueckii bulgaricus; and (6) milk fermented with both L. delbrueckii bulgaricus and S. salivarius thermophilus. The difference in breath hydrogen excretion among subjects consuming milk and those consuming milk fermented with both yogurt strains was ninefold. This study was the first published attempt to differentiate among the effects of different lactic cultures on lactose digestion. These results support the conclusions that, for given lactose loads, the presence of specific cultures enhances lactose digestion. Yogurt bacteria, alone or in combination, appeared to be the preferred bacteria for this application. The products containing L. acidophilus and Bijidobacterium were less effective, although populations of L. acidophilus were lower in the final product. This study suggests the importance of strain selection for a lactose-intolerance application. Martini et al. (1991b) determined the effect on lactose digestion of meal consumption and extra lactose consumption as well as yogurt consumption. In the first study, 12 lactase-deficient subjects were fed whole milk, commercial yogurt, a standard breakfast meal, or the same meal plus yogurt. Consumption of the meal with the yogurt did not affect peak breath hydrogen production, but did delay it I hr. In the second study, 10 lactasedeficient subjects were fed yogurt (10 g lactose), yogurt with added lactose (0-20 g), or milk (20 g lactose). The P-galactosidase activity contained in yogurt did not aid the digestion of lactose added in the form of additional milk or crystalline lactose. This study confirms that the lactose in yogurt is better tolerated than the lactose in milk. Yogurt will reduce lactose rnaldigestion, with or without concomitant meal consumption, but Pgalactosidase from yogurt cannot assist in the digestion of additional lactose consumed simultaneously. de Vrese et al. (1992) studied the effect of p-galactosidase-producing microbes contained in kefir fed to minipigs. Since, unlike yogurt, kefir does not contain free galactose (because of the presence of galactoseconsuming P-galactosidase-negative yeast), in vivo lactase activity was assayed by determining blood plasma levels of galactose (rather than breath hydrogen). This approach offers confirmation of the breath hydrogen method as a means to assay microbial lactase delivery. Two groups of five 64-pound pigs were given I liter kefir containing 100 mmol lactose per 1iter.and either fresh (70 U lactase activity) or heated (0.1 U lactase activity) kefir grains. Plasma was analyzed for galactose immediately before and up to 7 hr after feeding. The largest differences in galactose concentrations were measured 30- 180 min after feeding; pigs fed unheated kefir grains showed 30% higher plasma galactose levels 90 min after feeding. This study showed, in a minipig model, that microbial P-galactosidase

LACTIC CULTURES AND HUMAN HEALTH

83

significantly affects plasma galactose levels. Since the kefir contained no free galactose, the rise in plasma galactose was from increased intestinal lactose hydrolysis. Even greater differences in galactose levels might have been expected had baseline lactase levels been lower, as in lactose-intolerant individuals. This study did not differentiate between pgalactosidase activities of the mixed Lactobacilfuslyeast kefir culture, although the yeast p-galactosidase activity was a minor component. Although the study was conducted in an animal model, the results presented in this paper provide supporting evidence that consumption of dairy products containing p-galactosidase-containing microbes can decrease the physiological effects of lactose consumption by lactose maldigesters. Lactobacillus acidophilus cultures were effective in decreasing breath hydrogen levels of lactase-deficient subjects in a study by Kim and Gilliland (1983), in contrast to results of Lin et a f . (1991), McDonough et al. (1987), Payne et al. (1981), Welsh et a f . (1981), and Newcomer et al. (1983). The Kim and Gilliland (1983) study was criticized because of the short (3-hr) time of delay prior to sampling breath hydrogen. Sampling times of up to 8 hr are recommended (Savaiano and Levitt, 1987). Savaiano (personal communication) suggests that microbiological diversity of the culture called NCFM may account for some discrepancies between results from different laboratories. McDonough et al. (1987) did report an enhanced decrease of breath hydrogen when Sweet Acidophilus'" milk was sonicated prior to feeding, presumably predisposing the microbes to release lactase intestinally, Payne et al. (1981) reported a failure of commercially prepared Sweet Acidophilus'" milk to decrease breath hydrogen or to alleviate gastrointestinal symptoms. This study was criticized because the authors failed to analyze the microbiological characteristics of the milk prior to use. Since viable counts of lactobacilli in commercial unfermented acidophilus milk are frequently below 107/ml, perhaps the cell levels were lower than needed for efficient alleviation of symptoms. Savaiano and Kotz (1988) speculated that the failure of unfermented acidophilus milks to aid lactose digestion was the result of the use of frozen concentrate starter cultures. The concentration, freezing, and storage conditions may inactivate lactase. Combined with relatively low levels of culture in commercial milks, these processing steps may render the products ineffective against lactose maldigestion. Also, the bile resistance of commercial strains protects the culture from lysis once consumed, creating a barrier to lactase release. The contribution of bifidobacteria to the alleviation of lactose intolerance is currently speculative, since few publications are available on this topic. Roy et al. (1992) described the a-and p-galactosidase activities of the large colony type of Bijidobacterium infantis strain ATCC 27920. Using X-gal staining of polyacrylamide gels of cell-free extracts, three

84

MARY ELLEN SANDERS

bands of j3-galactosidase and one band of a-galactosidase activity were found. The study further characterizes the pH, temperature, and cation concentration optima of the enzymes, as well as their kinetic characteristics. The optimum pH (6.0-7.0) and temperature requirements (40") of the enzymes were consistent with human intestinal characteristics. The presence of these enzymes suggests that B. infantis may aid in the digestion of complex carbohydrates and may help alleviate lactose-intolerance symptoms in humans. The presence of some clinically useful j3-galactosidase activity in a Bijidobacterium strain is confirmed by Martini et al. ( 1991a). Clearly, from this and other work, certain cultures used in fermented dairy products can promote tolerance in lactose-intolerant people to lactose in milk. Mixed results have been obtained with consumption of Sweet Acidophilus'" milk, but the overwhelming evidence suggests that this commercial product is not very effective at decreasing breath hydrogen. The current commercial formulation of Sweet Acidophilus'" milk is not ideally suited to alleviate lactose-intolerance symptoms, but newer products produced with L. acidophilus and bifidobacteria blends of cultures have not been tested publicly. An unfermented culture-containing milk could be formulated to increase lactose digestion if the cultures used for this product contained suitably selected strains.

B. DIARRHEA Many studies have been conducted over the years to determine the effect of live lactic cultures on limiting the course of diarrheal diseases. Studies vary with culture used, dose used, target group (healthy vs diseased, pediatric vs adult), cause of diarrhea (viral or bacterial), and experimental format. Some experiments yielded positive results, some negative. Some conclusions appeared overextended; some were made carefully. All negative results could be discounted by criticizing culture selection or levels. Some positive results could be discounted because of poorly conducted experiments or marginal significance of results. However, a few well-done experiments suggest that, in carefully defined subpopulations, bacterial therapy can limit the course of diarrheal diseases. Additional research should be conducted on well-defined and properly selected cultures and their effect on the course of diarrheal diseases. The following discussion supports these conclusions. Colombel et al. (1987) conducted a double-blind placebo-controlled study in 10 healthy erythromycin-treated adults. Subjects were fed yogurt containing BiJdobacterium longum or a placebo yogurt in conjunction with erythromycin over two 3-day periods. Fecal weight, stool frequency,

85

LACTIC CULTURES AND HUMAN HEALTH

abdominal complaints, and presence of fecal clostridial spores were measured on days l and 3 of the test periods. Fecal weight (145 g/day vs 208 g/day), stool frequency (1.2/day vs 1.9/day), and abdominal complaints (1 person complaining vs 6) were reduced when patients consumed culture-containing yogurt but not the placebo (Table 11). Also, clostridial spores dropped significantly in subjects consuming bifid-yogurt. Spores were present in 7 of 15 subjects taking the placebo yogurt and in only 1 subject consuming bifid-yogurt. The authors concluded that bifid-yogurts could reduce antibiotic-induced alterations of intestinal flora, resulting in a decrease of gastrointestinal disorders. This study was conducted with a limited group (only 10 subjects) and should be considered only a pilot experiment, but the experimental design warrants acceptance of the results as valid. Siitonen et al. (19%) also studied diarrhea in healthy human volunteers treated with erythromycin. Of 16 subjects, 8 received yogurt made with strain GG and 8 received pasteurized yogurt. No significant difference was found in levels of total fecal lactobacilli or GG after 7 days of treatment, although 125 g yogurt containing GG was administered twice daily. Although no significant difference in Lacrobacillus counts was found, the duration of diarrhea was significantly shorter in the GG yogurt group (2 days) than in the placebo yogurt group (8 days). Authors report a lower incidence of stomach pain (23% in the GG group vs 39% in the placebo TABLE I1 EFFECT O F CONSUMPTION OF BIFIDOBACTERIUM LONGUM ON ERYTHROMYCIN-

INDUCED GASTROINTESTINAL EFFECTS I N

10 HEALTHY SUBJECTS'

Placebo

Stool weight (g/day) Stool number (/day) Abdominal discomfort Clostridial spores present

Yogurt

One day before erythromycin

Three days after erythromycin

One day before erythromycin

Three days after erythromycin

I lO(21)

208 (29)

132 ( 1 1 )

145 (16)

1 .O (0.15)

1.9 (0.35)

1.2 (0.13)

1.2 (0.13)

-

6

-

1

8

7

8

1

" Reprinted with permission from Colombel ef al. (1987).

86

MARY ELLEN SANDERS

group), diffuse abdominal pain, and nausea (data not shown), but statistical evaluation of these data was not provided. Clements et al. (1983) tested the ability of consumed commercial dried preparations of lactobacilli to prevent enterotoxigenic E . coli (ETEC)induced diarrhea in 23 healthy adults. Double-blind studies were conducted with two lots of Lactinex'" ( I .2 and 9.2 x lo8 viable lactobacilli per I oz package). No significant differences were found between the Lactinex'" and placebo groups with respect to ETEC attack rate, incubation period, duration of diarrhea, volume and number of diarrheal stools, and stool culture results. However, diarrhea resulting from treatment of the ETEC infection with neomycin was reduced in frequency and severity with one, but not a second, lot of Lactinex'". The authors concluded that LactinexTh'played no role in prevention or treatment of diarrhea caused by ETEC. The effect of Lactinex'T on neomycin-associated diarrhea was significant, but results were less than convincing because of the lot-to-lot variability of the culture. Since ETEC infection was induced in volunteers, results did not depend on a low frequency of symptom development in subjects. These results, however, do not support any prophylactic effect on ETEC-associated diarrhea. An earlier study by the same group (Clements et al., 1981) on LactinexTh'with 48 human volunteers showed a similar lack of efficacy. A study by Newcomer et al. (1983) to determine the effect of unfermented acidophilus milk on symptoms included 61 patients with irritable bowel syndrome with lactase sufficiency, 18 patients with lactase deficiency, and 10 healthy controls. The culture used in this study was NCFM, the acidophilus culture used to make Sweet Acidophilus'" milk. Milk contained at least 4 x lo6 cfu/ml and was consumed at a level of 720 ml/day for the irritable bowel group and between 0.25 and 4.5 8-oz glasses per day for the lactase-deficient group. The subjects were given milk randomly with or without added culture for a 10-wk period consisting of 2-wk periods in which milk was alternated with no milk. Cumulative indices were obtained for symptoms including abdominal paidcramps, diarrhea, bloating/gas, gurgling, and constipation based on subjective analysis by the subjects. No statistically significant difference was found between symptoms reported by the groups, for both lactase deficiency and irritable bowel syndrome subjects. This work can be criticized for the low levels of Lactobacillus used. However, the choice of bacterial levels can be defended easily since it was based on concentrations present in commercially available Sweet Acidophilus'" milk. Irritable bowel subjects consumed milk with each meal. This study clearly showed that no benefit for this type of diarrheal disease was obtained from Lactobacillus strain NCFM.

LACTIC CULTURES AND HUMAN HEALTH

87

The four papers discussed next present experiments that were conducted with pediatric cases. Pearce and Hamilton (1974) studied the effect of feeding a dried S . salivarius thermophilus (50-60%), L . acidophilus (35-45%), and L . delbrueckii bulgaricus (5%) preparation or a placebo to 94 children aged 3 years or younger who were hospitalized over a 10-monthperiod with acute-onset diarrhea. Each dose of culture contained at least lo8 viable bacteria in 400 mg (culture prepared by FermalacRougier Laboratories, Montreal). From 3 to 8 daily doses were administered, depending on the child's weight. The study was a double-blind design. No significant difference was found between placebo and treatment groups in the number and size of stool or the duration of diarrhea. The results did not differ when the data from short- and long-term cases were analyzed separately or together. This study is well controlled, with a large number of subjects, but shows no effect of this lactic culture treatment on alleviating diarrheal symptoms. Negative results could be attributed to strain selection, since no mention of the strain characteristics was made. Hotta et al. (1987) ran an uncontrolled study of 15 children under the age of 7 with intractable diarrhea (diarrhea that had an undetermined microbial cause, intractable clinical course, and high mortality) for at least 7 days prior to treatment. All children had various underlying diseases and all had been treated with various antibiotics (thought to cause the diarrhea). Clostridium difJicile was not found to be linked to these cases of diarrhea. Treatment included oral administration of (1) Bijidobacterium breve ( 109/g); ( 2 ) B . breve and Lactobacillus casei (each at 109/g); and/or (3) bifid-yogurt (MILMIL) containing B . breve ( 10'o/lOO ml), B . bijidum ( 10'o/lOOml), and L . acidophilus ( 109/100ml). Culture was administered at 3 g per day and yogurt at 60-600 ml per day. Of the patients, 10 received B . breve alone (BBG-01), 1 received the combination of B . breve, B . bijidum, and L . acidophilus in a yogurt product (MILMIL), 3 received L . casei and B . breve (BLG-B), and 1 received yogurt and the L . casei/B. breve combination culture. The B . breve strain was isolated from healthy breast-fed infants. No comment was made on the origin of the B . bijidum strain. Duration of treatment was not mentioned. In an average of all subjects, the duration of diarrhea prior to culture administration was 5-70 days (mean of 25.3 days); after treatment, duration was 3-14 days (mean of 7.0 days) (Table 111). Studies of fecal flora during treatment and recovery showed that fecal flora had stabilized in most, but not all, patients and contained large levels of bifidobacteria after diarrheal symptoms stopped. The aut h m attribute patient recovery to the bifidobacteria contained in the oral preparations and recommend viable bacterial preparations for diarrhea resulting from antibiotic therapy. This study was quite extensive in analy-

88

MARY ELLEN SANDERS

TABLE 111 EFFECT OF BlFlDO A N D ACIDOPHILUS ON DURATION OF DIARRHEA I N PATIENTS TREATED WITH VARIOUS ANTIBIOTICS"

Patient

Bacterio-therapyb

Duration of diarrhea before treatment (days)

Duration of diarrhea after treatment (days)

7 30 35

7 14 7 7 8 7 6 3 7 4 10 4 10 7 4

~

1

2 3 4 5 6 7 8 9 10 II 12 13 14 15

BLG-B BLG-B + MILMIL MILMIL BBG-01 BLG-B BBG-0 1 BBG-01 BBG-OI BBG-01 BBG-01 BLG-B BBG-01 BBG-01 BBG-01 BBG-OI

5 35 25 I1 10

25 9 70 7 30 40 40

Reprinted with permission from Hotta et al. (1987). For abbreviations. see text.

sis of patient physiological state, fecal flora, and diarrheal symptoms. Unfortunately, the lack of controls prevents a confident conclusion about the cause of the cure. However, the results are sufficient to warrant additional studies with adequate control groups and a greater number of subjects. Isolauri et al. (1991) studied 71 children between 4 and 45 months of age with acute viral (mostly rotavirus) diarrhea (less than 7 days of duration) and 8 control children with no gastrointestinal symptoms for the effect of Lactobacillus GG on shortening the course of diarrheal symptoms. Subjects were divided randomly into three groups to receive different dietary treatments. Group 1 (24 subjects) received strain GG-fermented milk, 125 g with cfu twice daily; Group 2 (23 subjects) received strain GG as a freeze dried powder with cfu twice daily; Group 3 (24 subjects) received fermented and pasteurized yogurt, 125 g twice daily (placebo). Each diet was given for 5 days. Oral rehydration of patients was conducted prior to nutritional therapy. All feeding products were obtained from Valio Dairies (Finland). The mean duration of diarrhea was reduced (95% confi-

LACTIC CULTURES AND HUMAN HEALTH

89

dence limit) in the groups receiving strain GG as a powder or a fermented product (1.4 days) compared with those receiving the placebo (2.4 days). Results were somewhat more pronounced when data from only patients with confirmed rotavirus infection were analyzed. This study showed that the duration of diarrhea could be reduced by feeding Lactobacillus GG with normal diet immediately after rehydration. This study was well controlled and conclusions seem warranted. Microbial treatments for pediatric diarrhea have not been limited to lactobacilli and bifidobacteria. Bellomo et al. ( 1980) treated pediatric diarrhea of various causes (104 patients from 1 month to 9 years old) with capsules of lyophilized Enterococcus faecium strain SF68 (3.8 x lo7 total cells; 53 treated) or L. acidophilus (5 x lo8),L. delbrueckii bulgaricus (5 X lo8), and L. lactis (4 X lo9) (51 treated). Patients were divided into two groups and were treated with one of the bacterial preparations. No untreated control patients were used. Treatment was for 3-10 days (treatment continued several days after recovery) with 1-2 capsules given per day, depending on the age of the child. Since most diarrheal disorders are self-limiting, all patients recovered within 4 days of treatment. However, in the group receiving the Enterococcus preparation, 62% recovered completely after 2 days of treatment, whereas only 35% recovered in the Lacrobacillus-treated group. These results suggest that Enterococcus strain SF68 may be useful in the treatment of miscellaneous diarrhea in children, and certainly was more effective than the Lactobacilluscontaining preparation used in this study. Unfortunately untreated illnesses and the varied treatment times make this study less than ideal. The data indicate at best a decrease of diarrhea duration of 1-2 days. Beck and Necheles (1961) ran an uncontrolled study of the effect of dried L. acidophilus (BacidTM)on 59 patients with various abdominal symptoms (constipation, antibiotic diarrhea, pancreatitis, diverticulitis, mucus colitis, ulcerative colitis, and colostomy-associated diarrhea). The authors concluded that, in most cases, a rapid improvement of symptoms occurred. Since no controls were run and since the target illnesses were a broad range of different diarrheal diseases, this conclusion is difficult to accept. This study is discussed because it is referenced frequently in support of the efficacy of Lactobacillus treatment for diarrheal diseases, and constitutes one source of unsubstantiated claims. Gorbach et al. (1987) treated 5 patients with relapsing C. difJicile colitis with 10'' (daily dose) viable Lactobacillus GG in skim milk for 7-10 days. Patients experienced 2-5 relapses over a 2- to I0-month period prior to treatment. After Lactobacillus treatment, no relapses occurred for 4 months to 4 years (Table IV). The authors stated that their experience supports the efficacy of treatment of C. difficile relapsing colitis

90

MARY ELLEN SANDERS

TABLE IV TREATMENT O F RELAPSING CLOSTRIDIUM DIFFICILE COLITIS WITH

LACTOBACILLUS

GG"

Patient (agekex)

Relapses (number)

Duration of illness (months)

Toxin titer before

A (35/M) B (93/M) C (241F) D (73/F)

2 3 3 4

2 4 3 6

111250 111250 111250 111250

E (5O/M)

5

10

Positive (no titer)

Toxin titer after 0 0 0 1/10 (delayed) 0

Follow-up with no relapses 16 mo 1 Yr 4 Yr 4 mo

4 Yr

Reprinted with permission from Gorbach et al. (1987).

with Lactobacillus GG. However, this study was not a controlled study and was done with only 5 patients. At best, this study suggests that further research may be warranted. Pozo-Olano et al. (1978) studied 50 travelers to Mexico for the effect of four daily tablets containing 3-6 X lo8 lactobacilli/tablet on diarrhea incidence and duration. The identification of the species, strain, or origin of the lactobacilli was not included in the publication. Subjects were divided randomly into two groups-one receiving the Lactobacillus preparation and the other receiving a placebo. The incidence of diarrhea in the experimental group was low (16 cases overall) and of short duration (most cases lasted only 2 days). No difference was detected between the two groups in incidence and, since no prolonged diarrhea occurred, no results could be generated on the effect of preparations on duration. Salminen et al. (1988) conducted a study on 21 gynecological cancer patients to determine whether oral administration of live L. acidophilus strain NCDO 1748 could alleviate intestinal side-effects of internal and external irradiation of the pelvic area. The test group received 150 ml of a fermented lactase-treated milk product containing 2 x lo9 /ml lactobacilli and 6.5% lactulose (added to support intestinal growth of the Lactobacillus strain). The control group received nothing. This experimental design did not control for effects of the fermented milk rather than the culture. Also, the lack of double-blind format brings into question the subjective reporting by both groups of their symptoms. Diet and antidiarrheal drugs were not controlled in the patients, but nutritional counseling was given to all subjects. The incidence of diarrhea was smaller in the yogurt group (10 reported incidences) than in the control group (34

LACTIC CULTURES A N D HUMAN HEALTH

91

reported incidences), but no difference was observed in vomiting, nausea, abdominal pain, loss of appetite, or weight loss. The yogurt group reported more flatulence than the control group, likely caused by the lactulose in the yogurt. Although the study lacked some desirable controls, the results were sufficiently significant to warrant further research on this population subgroup. Of 14 studies reviewed that tested the effectiveness of a bacterial preparation (Lactobacillus, bifidobacteria, or enterococci) on alleviating diarrheal symptoms, results of 5 were negative, of 6 were questionably positive (suspect due to experimental set-up or data analysis), and of 3 were positive. In this last group, the major effect was on decreasing diarrheal duration or incidence. Preliminary evidence on the effectiveness of limiting C. difficife,rotavirus, and erythromycin-induced diarrhea suggests that these conditions may be worthy subjects for further study. Cfostridium d$$cile colitis can be especially persistent and difficult to treat (George, 1980), so alternative therapies could be useful. Overall, the positive results on limiting diarrheal diseases with lactic cultures are encouraging. Additional research will be necessary to clarify and define the parameters for effective bacterial therapy. How probiotic microbes diffuse diarrheal illness is not known. One proposed mechanism is competitive colonization. Competitive colonization occurs when one intestinal microbe interferes with the colonization of another. However, documentation of this phenomenon by observing lactic cultures displacing pathogens or preventing pathogen adherence has been difficult experimentally. In uitro tissue culture studies and animal model studies have attempted to shed some light on the mechanism of lactic culture interference with intestinal pathogens. Chauviere et al. (1991) studied the competitive exclusion of ETEC by heat-killed lactobacilli in a tissue culture of human enterocyte-like Caco2 cells. Heat-killed L. acidophifus LB (a human fecal isolate) cells were combined with E. cofi cells and added to the tissue culture plates. After incubation for 3 hr, the monolayers were washed and adhering E. coli were quantitated. High concentrations of lactobacilli ( lo9 /ml) were needed to induce a 75% inhibition of ETEC adherence. Adherence limitation was proportional to Lactobacillus concentration. Yamakazi et a f . (1985) challenged germ-free mice monoassociated with B. longum with 10'' viable cells/mouse of E. cofi or endotoxin. Monoassociated mice showed greater survival than germ-free mice. When observed 18 hr after challenge with E. coli, only 4 of 11 germ-free mice survived, whereas all 10 mice monoassociated with Bijidobacterium survived. Although these results are unambiguous, their significance is less clear. Whether the effect is caused by a direct protective action of the

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Bifdobacterium or by immune system stimulation that accompanies intestinal colonization is not known. The significance of these results for fully colonized humans is not known either. Tomoda et al. (1988) studied a group of patients undergoing drug therapy for leukemia who showed a higher level of Candida in their feces than did normal controls. Two groups were generated from 49 patients with > lo5 Candidalg feces. In these groups, 28 individuals were fed Bifdobacterium for 3 months as dried bacteria or in milk; the other patients were not treated. Of the 21 untreated patients, l l developed diseases due to Candida; the same result occurred for 12 of 28 treated patients. In the Bifdobacterium-treated group, half the patients showed a decrease in Candida levels to < 104/g feces; these patients showed a significantly decreased frequency of Candida infection. The analysis of these data is questionable, however, because bacterial counts were expressed only as < 104/gor > 105/g.Whether the group of patients whose count fell below 104/gsimply had lower Candida counts initially was not clear. The authors chose an arbitrary cut-off point on which to base their interpretations. If the Bifdobacterium-treated patients whose Candida counts dropped to less than 104/g were compared with the nontreated group with similar counts, the Bifdobacterium-treated patients had a higher incidence of Candida infections (14% vs 6%). No data were presented on untreated patients with initial counts above lO’lg that spontaneously fell to lower levels. If, in fact, a strict correlation between population of Candida and Candida infection is known, then more comprehensive data are needed to analyze exactly how Bijidobacterium affects Candida counts. C. CHOLESTEROL REDUCTION The use of certain lactic acid bacteria to assimilate cholesterol in uitro (Gilliland ef al., 1985; Lin et al., 1989; Gilliland and Walker, 1990), or to lower blood cholesterol in uiuo (Danielson et al., 1989) has been studied. The proposed benefit is based on the opinion that decreasing blood cholesterol lowers the risk of heart disease in humans. The importance of monitoring dietary cholesterol for the general public, although widely believed, has been challenged by some individuals in the medical community, primarily for overextension of results obtained from a minor fraction of the population to the general public (Ahrens, 1985). Therefore, the issue of lactic cultures and their effect on cholesterol has two facets: (1) the ability of lactic cultures to reduce effectively and substantively the amount of cholesterol absorbed by consumers and (2) the effect this cholesterol reduction may have on the health of the consumer.

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Discussion of the second point is beyond the scope of this chapter. However, since cultures could be marketed based on the widely held belief that decreasing dietary cholesterol is healthful, the remaining discussion will focus on the scientific evidence available on the effect of lactic cultures on cholesterol. The following studies were conducted in human subjects. Jaspers et al. (1984) studied the effect of consuming 681 g of three nonfat yogurts on total cholesterol, high-density lipoprotein (HDL) cholesterol, lowdensity lipoprotein (LDL) cholesterol, serum lipoproteins, and serum triglycerides of 10 healthy male subjects. Yogurts were differentiated by cultures used for fermentation: two unidentified yogurt cultures from Chr. Hansen’s Lab and a blend of strains (from K. Shahani, University of Nebraska) L. delbrueckii bulgaricus 202 and S . salivarius thermophilus EBC. These investigators also measured the levels of uric, orotic, and hydroxymethylglutaric acid in the three yogurts to address claims by previous investigators (Richardson, 1978) that these acids may serve as hypocholesterolemic agents in milk. Although some significant differences in total cholesterol, serum lipoproteins, LDL cholesterol, and HDL cholesterol were observed at certain points during the study, no significant change persisted throughout the course of the study, suggesting that any hypocholesterolemic effect of yogurt consumption is transient. In addition, no significant effect on serum triglycerides occurred. The authors also determined that concentrations of uric, orotic, and hydroxymethylglutaric acids in the yogurts were insufficient to affect cholesterol levels. Although this reference is cited frequently in support of a hypocholesterolemic effect of yogurt, the transient nature of the effect suggests that the effect is marginal. Lin et al. (1989) tested the effect of LactinexTM,a tablet containing L. acidophilus strain ATCC 4962 and L. delbrueckii bulgaricus strain ATCC 33409, on serum lipoprotein concentrations in 334 human subjects. The daily dose in the treatment group was approximately 8 X lo6 viable lactobacilli. Analysis was done of total cholesterol, LDL, HDL, and very low density lipoprotein (VLDL), and triglycerides. Several factors in the experimental design suggest that the results of this experiment are meaningful: the double-blind design, the large number of subjects, the lack of variability in assay results, and the inclusion of all subjects in both treatment and control groups. Results failed to show any effect of culture consumption on reduction of serum lipoprotein levels. Hepner et al. (1979) conducted two 12-wk studies on 54 healthy subjects with cholesterol levels higher than 200 mg/100 ml (17 and 34 individuals in studies I and 11, respectively) to evaluate the effect of 2% milk, yogurt, and pasteurized yogurt on serum cholesterol, triglycerides, and

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diet. Commercially available Dannon yogurt at a rate of three 240-ml cups per day was used. Bacterial strains were not identified. Subjects were encouraged to maintain their regular diet throughout the course of the experiment. Dietary logs indicated that calorie, protein, and carbohydrate intakes increased and cholesterol, fat, and fiber intakes were not changed. In Study I, the 12-wk experiment was divided into three 4-wk feeding trials, consisting of sequential 4-wk periods of yogurt feeding, control feeding, and milk feeding in one group, and of milk feeding, control feeding, and yogurt feeding in a second group. No group was used during the study to control for variations in sampling or cholesterol determination. When yogurt feeding came first, a significant drop (99% confidence limit) in serum cholesterol occurred after the first week. The levels remained constant through the end of the 4-wk period. This result is consistent with a 5-7% drop in serum cholesterol in the first week seen by Rossouw et a / . (1981), although the first week was used to establish baseline levels of serum cholesterol and no milk product was fed until the second week. The data imply that the effect observed by Hepner et al. (1979) may not be caused by yogurt but by spontaneous modification of dietary intake induced by participation in the study and keeping dietary records (Rossouw et a/., 1981). During the subsequent control period, serum cholesterol rose; cholesterol leveled off during the milk feeding (a nonstatistically significant increase of serum cholesterol was observed during the milk feeding period). When milk feeding came first, no significant difference was detected in serum cholesterol levels at the end of the milk feeding period or the control period. However, serum cholesterol levels fell significantly (99% confidence) during yogurt feeding. Study I1 was designed to differentiate between effects of eating yogurt, pasteurized yogurt, 2% milk, and a normal unsupplemented diet. Subjects were fed the dairy supplements constantly. Pasteurization of the yogurt did not affect the hypocholesterolemic effect in Study 11, indicating that viable starter cultures did not mediate any effect. Although a significant difference was detected between cholesterol levels of subjects eating the control diet those of subjects eating the diet supplemented with the nonpasteurized yogurt, problems with the control group make this result suspect. The serum cholesterol levels of the control group were significantly higher after 4 and 6 weeks (95% confidence) than at the beginning of the study. Further, the control group was used only for half the duration of the experiment (6 weeks). N o significant effect on serum triglycerides was detected in either study. The authors conclude from these studies that yogurt and, to a

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lesser degree, milk exert a hypocholesterolemic effect. The investigators did not address problems with the control group for Study I1 in which a significant rise in cholesterol levels was observed. Since statistical comparison of the data was done with the control group, the unexplained elevated serum cholesterol levels increased the difference between control and experimental groups, with the resulting emergence of “statistically significant” data. The decrease in serum cholesterol levels observed with yogurt feeding in Study I reflected a drop from 202 mg/dl to 191 mg/dl, a result of questionable importance. Although statistically significant data were reported in this publication, the authors do not address the substantive significance of this finding. The reduced cholesterol level persisted for 6 weeks, in contrast to the results of another study that found the hypocholesterolemic effect to be temporary (Jaspers et al., 1984). Issues of proper control of experiments suggest the need for confirmation of results found in this study. Further, since cultures did not mediate the effects, this study is of marginal significance to marketing of culture-containing milks. This study is used frequently, however, as a reference to substantiate a hypocholesterolemic effect of yogurt. Rossouw et al. (1981) studied the effect of consuming 2 liters per day of skim milk, yogurt, or whole milk on serum total cholesterol, triglycerides, and HDL and LDL cholesterol in 32 adolescent boys. Statistical differences in serum total cholesterol correlated strongly with the differences in total dietary fat and cholesterol intake in the whole milkand yogurt-modified diets, and could not be attributed to a hypocholesterolemic “milk factor.” Some effect was observed with skim milk, however, so the authors do not dismiss the possibility of a hypocholesterolernic factor in skim milk. All measured parameters returned to baseline levels within the follow-up week of unsupplemented diet, with the exception of the serum total cholesterol of the skim milk group. The duration of this effect was not determined. Clearly, no hypocholesterolemic factor was identified in the culture-containing milk product. Further, this experiment was conducted with adolescent boys with n o evidence of hypercholesterolemia. Since dietary control of serum total cholesterol is a concern only in the minority of individuals with the genetic inability to regulate serum cholesterol effectively, the significance of this study can be questioned. Thompson et al. (1982) tested the effect of consuming 1 liter of low fat (2%), whole, skim, or Sweet AcidophilusTMmilk, yogurt, or buttermilk on total, LDL, and HDL cholesterol in 68 healthy volunteers with normal or low blood lipids. Dietary records on non-milk foods consumed by

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subjects were not kept. This study showed no effect of consuming these milk products on blood lipids, indicating neither a hypocholesterolemic nor a hypercholesterolemic effect of nonfat or whole milk products. Bazzare et al. (1983) found a positive effect of yogurt feeding and calcium supplementation in 16 female subjects (but not in 5 male subjects) on decreasing total cholesterol levels and increasing HDL: total cholesterol ratios. Renner (1991) stated that Lactobacillus GG was shown to decrease cholesterol in healthy human volunteers, but no retrievable reference was provided. Gilliland and Walker (1990) proposed a strategy to select an L . acidophilus culture that would produce a hypocholesterolemic effect in humans. These researchers advised that L . acidophilus strains be selected based on human origin, the in uitro ability to “assimilate” cholesterol, growth in the presence of bile, and bacteriocin production. Experiments designed to differentiate among cholesterol assimilation abilities of different L . acidophilus strains showed that the measurement of this ability was time dependent and therefore somewhat arbitrary, depending on the time chosen for assay. Experimentation also has been done in animal systems. In rabbits, Kiyosawa et al. (1984) found a total aorta cholesterol-lowering effect of both skim milk and yogurt, implying that the milk, not the cultures, exerted some effect. Gilliland et al. (1985) studied some strains of L . acidophilus isolated from pig feces. When grown in MRS broth with oxgall and pleuropneumonia-like organism (PPLO) serum fraction as the cholesterol source, the cholesterol was reduced in the broth and increased in the bacterial cells. The effect was dependent on the presence of oxgall. One strain each, designated cholesterol assimilating and cholesterol nonassimilating, was used in a feeding trial of 18 5-wepk-old piglets. All pigs were fed a cholesterol-containing diet (1.8 g/kg body weight) during the experimental period. In addition, the pigs were divided into three groups, fed 50 ml 10% nonfat milk solids or nonfat milk solids plus 5 x 10’O of one of the two L . acidophilus strains. Blood samples were taken and analyzed for total serum cholesterol. After 10 days, the control group and the group fed the nonassimilating strain showed elevated but not statistically different total cholesterol levels (74.44 and 73.48 mg/dl, respectively). The group fed the cholesterol-assimilating strain showed significantly lower total cholesterol levels (62.29 mg/dl) (Table V). Danielson et al. (1989) isolated three strains of L. acidophilus from porcine feces. All three were able to reduce water-soluble cholesterol (polyoxyethanyl cholesteryl sebacate) from MRS broth with 0.2 or 0.4% oxgall by 30-80% in 24 hr. Since strain LA16 was considered best at in uitro cholesterol assimilation and other screenines. this strain was used

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TABLE V I N F L U E N C E OF FEEDING LACTOBACILLUS ACIDOPHILUS CELLS ON SERUM CHOLESTEROL LEVELS I N PIGS ON A

HIGH-CHOLESTEROL

DIET".^

Group

Day 0

Day 5

Day 10

Control P47" RP32"

52.23 ( I .88)c' 55.58 (4.70)c' 52.84 (3.00)c1

69.10 (3.91)CD' 72.01 (3.02)c2 61.48 (3.30)D'

74.44 (4.64)C* 73.48 (4.68)c2 62.29 (4.91)D'

" Reprinted with permission from Gilliland e t a / . (1985). Each value represents the mean cholesterol (mg/dl) from six pigs; numbers in parentheses represent the standard deviation. Values in the same column followed by different superscript letters are significantly different (P38 en% as fat and almost 16 en% as SFAs, beef was the single food category providing the highest proportion of SFAs. However, in a group eating less than 30 en% as fat and less than 10 en% as SFAs, pizza contributed the highest proportion of SFAs (McPherson et al., 1990). Much or most of the SFAs in the pizza were likely to be from cheese. Dairy foods may provide a larger proportion of total and saturated fat in low-fat diets than in higher fat diets. The estimates of dairy product contribution to SFAs by Krebs-Smith et al. (1990, 1992) and Witschi et al. (1990) exceed those made by Block et al. (1985a). Possible explanations are that Krebs-Smith et al. and Witschi et al. studied particular population subgroups rather than a cross section of the entire population, or that Block et al. failed to include dairy products present as ingredients in food mixtures in their milk group contributions. Block et al. (1985b) assessed food sources of several vitamins and minerals for adults participating in NHANES 11. These investigators found that milks, cheeses, and frozen desserts contributed approximately 30% of riboflavin, 17% of potassium, and 55% of calcium intakes (Block et al., 1985b). Krebs-Smith et at'. did not report how food groups contributed to intake of vitamins and minerals by women participating in the 1985 CSFII study, but did note that whole milk beverages contrib-

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uted 7.8% of SFA intake and 16% of calcium intakes (Krebs-Smith et al., 1992). Pennington and Young (1991) examined mineral intakes as part of the Total Diet Study by the Food and Drug Administration (FDA) and found that dairy products provided the following for adults: 42-46% of calcium, 18-23% of phosphorus, 13-15% of potassium; 10-13% of magnesium; and 10-12% of zinc intakes. For adolescents and children, dairy foods contributed much greater percentages of these nutrients. As part of the Bogalusa Heart Study, Nicklas ef al. (1992) examined 24-hr dietary recall data collected in 1973-1982 for 871 I0-year-olds. The percentage of energy from fat in the children’s diets ranged from about 18% to more than 60%. The sample was divided into four categories according to en% from fat (40%). As expected, total fat, SFA, and energy intakes were greater in the high-fat than in the low-fat groups. Although dairy foods made a larger contribution to the percentage of total fat consumed by the lowest fat intake group, children with the highest fat intakes consumed more dairy products (on average, 96 calories more from dairy products per day). Meat consumption was dramatically different among the groups: meat contributed more fat (8.1 vs 40.5 g/day) and more energy (129 vs 530 kcallday) to diets as fat increased from 40 en%. Although the children with diets containing deoxyCL (uncharged CL with 1’,3’-propanediolgroup instead of 1’,3’glycerol) = 0-benzyl CL (uncharged CL with 0-benzyl attached to C-2’) > derivatives with only one phosphodiester group.

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These authors did not set out to describe the binding affinity of different molecular species of CL to aCL. However, they found equal binding of the disodium salts of tetrapalmitoyl CL and beef heart CL. These molecules have a dramatically different acyl composition since bovine and ox heart CL contain only 1% 16: 0 and 7 2 4 7 % 18 : 2n-6 (Avanti Polar Lipids Catalogue; Gray, 1964). At least in their crude assay, acyl composition was unimportant. Brown et al. (1989) reported that, of 45 lupus patients that were LA positive, 6 reacted only to human cadaver heart tissue CL as the antigen and not to bovine CL. Of these patients, 88% had antibodies to human CL, whereas only 75% had antibodies to bovine CL. Hence, fatty acyl composition of the antigen may affect binding to aCL. Unfortunately, the authors did not examine the fatty acyl composition of the tissues used and did not quantify oxidation of the fatty acids of the human cadaver heart CL. Since the composition of CL varies widely with diet, one cannot estimate which fatty acid may be responsible for differences between the two CL sources. Gharavi et al. (1987) used quantitative isotype specific ELISA to determine the distribution of immunoglobulin isotypes and phospholipid specificities of aCL in 40 patients with one or more of the following aPL associated clinical complications: thrombosis, fetal loss, and thrombocytopenia. Of these 40 patients, 12 had IgG, IgM, and IgA aCL; 10 patients had IgG and IgM, 5 patients had IgG and IgA, and 3 patients had IgM and IgA aCL. No statistical association was found between any single isotype or any group of isotypes and thrombosis, fetal loss, or thrombocytopenia. The presence of IgG aCL in 36 of the 40 patients suggests that this isotype may be most important in determining clinical complications, but 4 patients without IgG aCL also appeared susceptible to thrombosis, fetal loss, and thrombocytopenia. IgG, IgM, and IgA aCL bound the negatively charged PL, PS, and PI, but not the zwitterionic PC. No significant difference between binding to CL and binding to other negatively charged phospholipids was found, suggesting that the specificity of these antibodies is for negatively charged phospholipids in general rather than for CL in particular. Staub et al. (1989) determined that most patients with LA activity have coincident antibodies to a group of negatively charged phospholipids. These researchers suggested that LA and aCL tests detect antibodies with overlapping specificities. Some discordance between the two assays has been described, however. One patient presenting with severe thrombotic disease (recurrent deep vein thrombosis, pulmonary embolism, inferior venocaval obstruction, myocardial infarction, and digital gangrene) showed strong LA activity. An ELISA showed no binding to the negatively charged phospholipids CL, PS, and phosphatidic acid, but binding

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to zwitterionic PE was demonstrated. Inhibition studies and affinity purification confirmed this finding. Interestingly, the serum did not bind to the KCT reagent when used as antigen in an ELISA. The pathogenic significance of anti-PE antibodies and their relationship to LA remains to be clarified. Further studies of the occurrence of anti-PE antibodies in patients with LA activity who have negative aCL tests are suggested. E. MOLECULAR NATURE OF THE REACTIVE EPITOPE In SLE, antibodies are produced predominantly to native dsDNA, in which case the reactive epitope is believed to be the helical structure of the molecule; to denatured ssDNA, in which case the reactive epitope is believed to be the individual bases; or to both molecules, in which case the reactive epitope is believed to be the sugar-phosphate group common to both ss and dsDNA (Cohen et al., 1971; Bourdage and Voss, 1988). In general, the immunochemical cross-reaction between DNA and CL leads most investigators to speculate that the arrays of phosphodiester groups on the nucleic acid backbone and in phospholipid micelles are the reactive epitopes (Schwartz, 1988; Voss, 1988). In fact, both molecules contain phosphodiester-linked phosphate groups that are separated by three carbon atoms. Ben et al. (1988) investigated the structural features of the interaction between DNA and anti-DNA in competition experiments with low molecular weight synthetic compounds. Two correctly spaced chemical components, a substituted aromatic ring system and a negatively charged acidic residue, were found to be required for the binding of most antiDNA antibodies to their respective antigens. These chemical elements are combined in the structure of several anionic dyes, including some certified food colors. The dyes were found to compete efficiently for lupus DNA. Therefore, this family of compounds may serve as a basis for the development of a new approach to drug therapy in SLE. Rauch et al. (1984) injected BALB/c mice intraperitoneally with a solution of the monoclonal autoantibody H102, which binds both DNA and CL suspended in Staphylococcus aureus (Cowan strain) cultured fluid, incubated with a liposomal preparation of CL, and emulsified with Freund’s adjuvant or saline. No adjuvant was used for subsequent immunizations. Mice injected with CL without the adjuvant showed low DNA binding and no significant CL binding. Mice given only the adjuvant without CL showed little or no binding to CL and DNA. However, mice given CL and the adjuvant produced both anti-DNA (antibodies) and aCL. This result suggests that CL and DNA share an epitope that is both antigenic and immunogenic, but whether the anti-DNA antibodies of

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SLE really originate from this kind of “antigenic mimicry” remains unknown (Schwartz, 1988). In competitive assays, large excesses of CL were needed to compete for single and double stranded serum anti-DNA antibodies, suggesting that only a minor population of anti-DNA antibodies binds to both DNA and phospholipid (Edberg and Taylor, 1986; Eilat et al., 1986). In competitive radioimmunoassays (RIAs), the ability of several phospholipid micelles to compete with denatured DNA for hybridoma autoantibody binding was as follows: CL=phosphatidate=denatured DNA >> PG; PS, PC, and PE failed to inhibit the reaction at the highest level tested, which was 832x more than that required for 50% inhibition by CL (Lafer et al., 1981). For other hybridoma antibodies, denatured DNA was up to 80 times more effective as a competitor than CL. For a base-specific autoantibody with a marked preference for a guanine- or hypoxanthine-containing determinant, no level of any phospholipid inhibited denatured DNA binding. Phosphatidate and PG may react as well as CL because both have a series of repeating phosphate groups on a micellar surface. In contrast, when PC, PE, and PS are presented as a micelle, the positively charged groups of these molecules exposed to the aqueous solvent may interfere with the binding of the phosphate groups with the antibody. These observations also support the notion that the reactive epitope is the phosphodiester linkage. In this experiment, phospholipid micelles were prepared by drying the lipids under NZand slowly adding saline citrate. Rauch and co-workers (Janoff and Rauch, 1986; Rauch et al., 1986) tested the structural specificity of different polymorphic forms of PE for structural specificity against 1 1 isolated hybridoma anticoagulants. The polymorphic forms of PE used were (1) dipalmitoyl PE, which forms a bilayer at 37°C; (2) dioleoyl PE; (3) monooleoyl PE at pH