Gut Flora, Nutrition, Immunity and Health

Gut Flora, Nutrition, Immunity and Health

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Gut Flora, Nutrition, Immunity and Health Edited by

Roy Fuller & Gabriela Perdigon

Blackwell Publishing

© 2003 by Blackwell Publishing Ltd Editorial Offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK Tel: +44 (0)1865 776868 108 Cowley Road, Oxford OX4 1JF, UK Tel: +44(0)1865 791100 Blackwell Publishing USA, 350 Main Street, Maiden, MA 02148-5020, USA Tel: +1 781 388 8250 Iowa State Press, a Blackwell Publishing Company, 2121 State Avenue, Ames, Iowa 50014-8300, USA

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ISBN 1-4051-0000-1 Library of Congress Cataloging-in-Publication Data Gut flora, nutrition, immunity, and health/edited by Roy Fuller & Gabriela Perdigon. p. cm. Includes bibliographical references and index. ISBN 1-4051-0000-1 1. Intestines - Microbiology. 2. Nutrition. 3. Functional foods. 4. Digestion. I. Fuller, R. II. Perdigon, G. (Gabriela) [DNLM: 1. Intestines-microbiology. 2. Nutrition. 3. Dietary Supplements. 4. Digestion — physiology.

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Contents Preface Acknowledgements 1

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The Intestinal Microflora G.W. Tannock From Petri dish to polyacrylamide gel Catalogues show diversity A phoenix arises Is it all the same in the end? The formative years Rules and regulations, but mind your language Food and the Large Intestine S. Macfarlane and G.T. Macfarlane The large intestine Interactions of the microflora with the host Effect of diet on the colonic microflora Diet and bacterial species composition in the large intestine Diet and bacterial metabolism Intestinal bacteria and vitamins Bacterial growth substrates in the large intestine Breakdown of complex carbohydrates by intestinal bacteria Protein breakdown by gut microorganisms Toxological implications of amino acid fermentation Effects of carbohydrate on amino acid fermentation Short-chain fatty acids Effect of diet on SCFA production In vitro studies on SCFA production SCFA and cell metabolism SCFA and colon cancer Lactate formation by gut microorganisms The Health Benefits of Probiotics and Prebiotics G.R. Gibson, R.A. Rastall and R. Fuller Summary Introduction

xi xii 1 1 7 7 12 14 15 24 24 26 26 26 28 30 31 32 35 36 38 39 40 40 41 41 41 52 52 52

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Probiotics Composition of probiotic preparations Tracking probiotics through the gut Prebiotics Oligosaccharides as prebiotics Lactulose Inulin and fructooligosaccharides Galactooligosaccharides Soybean oligosaccharides Lactosucrose Isomaltooligosaccharides Glucooligosaccharides Xylooligosaccharides Current status Persistence of the prebiotic effect to distal regions of the colon Anti-adhesive activities against pathogens and toxins Targeted prebiotics Attenuative properties Defined health outcomes of probiotics and prebiotics Improved tolerance to lactose Protection from gastroenteritis Coronary heart disease Colon cancer Vitamin synthesis Irritable bowel syndrome Improved digestion and gut function Immunomodulation Mineral bioavailability Conclusions

55 55 57 59 59 60 60 61 62 62 63 63 64 64 65 65 65 66 66 66 67 67 68 68 68 68 69 69 69

Intestinal Microflora and Metabolic Activity A. Perez Chaia and G. Oliver Dietary carbohydrates Bacterial fermentation SCFA production SCFA and electrolytes absorption Colonic metabolism Physiological consequences of SCFA absorption Probiotics and the intestinal metabolism of carbohydrates

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The Role of the Immune System C.M. Riera, M. Maccioni and C.E. Sotomayor Overview of the immune system Introduction Innate immune response

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77 79 82 84 87 87 89

99 99 100

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Early induced immune response Adaptative immune response B lymphocytes and the immunoglobulins T lymphocytes and the T cell receptor complex APCs and the MHC molecules Education of lymphocytes in the primary lymphoid organs Education of T lymphocytes in the thymus Education of B lymphocytes in the bone marrow The immune system functioning Peripheral lympoid tissues, a place where lymphocytes meet the antigen Lymphocyte traffic Activation of T cells Cell-mediated immune response: generation of armed effector T cells Generation of Thl and Th2 cells Cytotoxic T cells Humoral immune response Activation of B cells in the secondary lymphoid organs Kinetics of the immune response Endogenous regulation of the immune response Tolerance to T cell repertoire Tolerance to B cell repertoire Immunoregulation

103 104 104 109 110 115 115 117 119 119 120 121 122 122 124 125 125 130 130 131 132 133

Behaviour of the Immune System in Eating Disorders A. Marcos, E. Nova and S, Lopez-Varela Relationship between nutrition and the immune system The defence of the organism Immune system behaviour under malnutrition Effects of micronutrients on the immune system Antioxidant vitamins The immune system in eating disorders Eating disorders as a clear example of malnutrition Cytokine participation over the lack of infection in eating disorders The interrelationship between leptin and cytokines Conclusions

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Mucosal Immune System and Malnutrition M.E. Roux, N.H. Slobodianik, P. Gauffin Cano and G. Perdigon Mucosal immunity Introduction Organized mucosal lymphoid tissues Antigen uptake Role of epithelial, toll-like receptors and dentritic cells Tolerance to food

137 137 138 139 143 146 146 146 147 149 155 155 155 156 156 156 160

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Effect of nutritional factors on the microenvironment of mucosal immune system Nutrition and immunity Nutritional effects on immune mechanisms Essential amino acid deficiency and immune response Nutritional deficiencies and mucosal immune defence systems in animals Probiotics in malnutrition Introduction Protein malnutrition and immune response Effects of probiotic addition to a renutrition diet 8

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Immune Activation Versus Hyporesponsiveness and Tolerance in the Gut J. Chin and A. Mullbacher Introduction Innate and adaptative immunity Antigen and the T cell response Antigen-presenting cells in the GIT T cell function in the GIT - the CD4 population y5 T cells Turnover of T cells Intestinal epithelial cells Peyer's patches - suppression of T cell responsiveness Probiotic bacteria — a biological role as live or dead organisms Immune activation by probiotic bacteria feeding Probiosis — is it Gram-positive versus Gram-negative bacteria or is it live versus dead bacteria? Summary and perspective

160 161 161 162 163 166 166 167 168

178 178 179 180 181 181 183 184 184 185 187 187 192 195

Food Hypersensitivity and Allergic Diseases R.K. Chandra Introduction Immunological mechanisms Food allergens Clinical manifestations Identification of high risk infants for prevention Preventive measures Breast feeding Hydrolysed formulas Other measures Management of food allergy

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Nutritional and Microbial Modulation of Carcinogenesis R. Hughes and I. Rowland Introduction

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201 201 202 202 204 204 204 205 206 206

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Bacteria and cancer overview Bacterial metabolites Enzyme activities P-glucuronidase and [3-glycosidase Enzymic hydrolysis of plant flavonoids Nitroreductase and nitrate reductase Metabolism of heterocyclic amines Metabolism of sulphur-containing compounds Secondary bile acids Fecapentaenes Reduced exposure to toxic compounds Formation of toxic and protective agents during fermentation Carbohydrate fermentation Short chain fatty acids Products of colonic protein fermentation Probiotics, prebiotics and cancer

209 211 211 211 212 213 213 214 215 216 217 217 218 218 219 220

The Role of Nutrition in Immunity of the Aged S. Walrand, M.-P. Vasson and B. Lesourd Introduction Aging and immune function Innate immune system Age-related changes in skin and mucous barriers Age-related changes in phagocytic cells Age-related changes in natural killer cells Acquired immune system Age-related involution of thymic function Quantitative and qualitative changes in T cell subpopulations in the elderly Age-related changes in T cell function Changes in B cell number and function in the elderly Nutritional regulation of immune function of the aged Effect of protein-energy malnutrition on immune responses in aged people Alterations in polymorphonuclear functions during PEM in the elderly Alterations in monocyte/macrophage functions during PEM in the elderly Alterations in T-cell mediated immunity during PEM in the elderly Alterations in B-cell mediated immunity during PEM Immune response during nutritional recovery in undernourished elderly persons Influence of nutritional recovery on PMN function in old age Influence of nutritional recovery on T cell function in old age Influence of nutritional supplementation in healthy well-nourished elderly subjects Concluding remarks

237 237 238 238 238 238 243 244 244 244 246 247 248 248 248 250 250 252 252 252 253 255 258

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Conclusions G. Perdigon and R. Fuller

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Preface It is a self-evident truth that food is essential for the growth and survival of the consumer. However, there are foods which, as well as contributing essential nutrients, also contain additional components which improve disease resistance and general health status over and above that induced by ingestion of conventional foods. The importance of this concept is reflected in the recent development of the so-called functional foods. These can occur naturally or be produced by addition or by modification. They exemplify the relationship that exists between nutrition, gut flora, immunology and health. Some have the capacity to modify the composition or activity of the gut microflora. This, in turn, can modulate the immune system of the gut which has very significant consequences because it accounts for a large proportion of the total immune system of the body. These beneficial effects on the immune response may be manifest as an increase in activity which suppresses pathogens or they may be responsible for down-regulation of the response which will prevent allergic and inflammatory reactions. This book contains chapters on the basic principles of nutrition, gut microecology and immunology as well as chapters which discuss the way in which this knowledge may be used to explain the positive and negative effects of food consumption. There is information on the effects of conventional food and also on supplements designed specifically to target the gut microflora. These approaches either add viable microorganisms to the diet (probiotics) or include chemical agents, usually oligosaccharides, which stimulate specific groups of bacteria in the lower gut (prebiotics). These are artificial ways of modulating the gut flora, but we should also be aware of the fact that any ingested food has the potential to affect the gut flora in some way, either beneficially or adversely. We should be aiming to create an optimal composition of the gut flora which will maximize its effect on the host animal. These approaches are necessary because of the unnatural diet and lifestyle which we have acquired, especially in developed countries, resulting in gut flora changes. The balance between the gut flora and the host has become deranged and we should be striving to return to the natural symbiotic relationship that exists in wild animals. Food may also contain components which have a direct effect on the immune system and consequently on health by inducing allergic responses. This sort of response will be seen in only a small proportion of the individuals which encounter the antigen responsible and there is now some evidence that the priming of these particular subjects is dependent on the quality of their gut flora in early life. Some problems of later life may also be related to the composition of the gut flora. Certainly, it is well established that the gut microflora changes as we age. The changes associated with senescence, such as failing immunity, may reflect changes in the intestinal microflora induced by altered feeding habits. The following chapters in this book address

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these aspects of health. It is hoped that by presenting them together in one volume we can draw attention to relevant relationships and to the way in which they interact. Hopefully we will be able one day to recommend the ideal diet to ensure optimal health from the cradle to the grave. Alas it is not yet possible to do so but by presenting the available information we hope to stimulate discussion and improve our appreciation of the current state of our knowledge and perhaps become better acquainted with the potential offered by this approach. This book draws on the skills and knowledge of a wide variety of experts in the subjects covered in this book; we thank all of the authors for their excellent contributions. Roy Fuller Gabriela Perdigon

Acknowledgements The editors are extremely grateful to Dr Marta Medici for her important contribution to the preparation of the manuscript.

Editor's note

I would like to express my profound gratitude to Dr Roy Fuller for giving me the opportunity to share with him the editorship of this book. Without his invaluable help I would have found it impossible. Gabriela Perdigon

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G.W. Tannock Department of Microbiology, University of Otago, Dunedin, New Zealand, and Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada

From Petri dish to polyacrylamide gel Much of our knowledge of the composition of the gut microflora of humans has been derived from bacteriological studies that utilised the traditional techniques of culture, microscopy and the determination of the fermentative and other biochemical capabilities of bacterial isolates. This work required the use of apparatus such as anaerobic glove boxes, or test tubes flushed with an oxygen-free gas, to provide a suitably reduced environment that would enable extremely oxygen-sensitive bacteria to be cultivated. The numerically predominant members of the microflora are obligately anaerobic bacteria and it is estimated, by statistical extrapolation from the number of bacterial species that have actually been cultivated, that perhaps hundreds of species may be capable of inhabiting the human intestinal tract. Hence the logistics associated with the quantification, isolation and identification of obligately anaerobic bacteria are formidable. Selective bacteriological culture media have been essential for accurate analysis of the microflora because they enabled enumeration of specific bacterial populations to be made (Summanen et al., 1993). Unfortunately, few culture media used in the analysis of the microflora are absolutely selective and misinterpretations by the novice are easily made. Not all of the species comprising a population may be able to proliferate with equal ease on the selective medium. This introduces bias to the results. Total bacteria microscopic counts, utilising the 4',6-diamidino-2-phenylindole (DAPI) stain and computer imaging, has revealed average total bacterial cell counts in human faeces approaching 1 x 10 per gram (wet weight) (Tannock et al., 2000). Even state-of-the-art bacteriological methodologies still only permit about 40% of this bacterial community to be cultivated on non-selective agar medium in the laboratory (Tannock et al., 2000). Thus a large proportion of the bacterial cells seen in microscope smears have never been investigated. Although some of these cells may be non-viable, it is likely that many are viable but non-cultivable due to their fastidious requirements for anaerobiosis or, more likely, due to the complex nutritional interactions that can occur between the inhabitants of bacterial communities. These nutritional complexities may be difficult, if not impossible, to achieve in laboratory culture media. With respect to identification of bacterial species, one must admit that the success of

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phenotypic identification methods often relies on the experience of the laboratory worker and a dose of intuition. This is because there is considerable intraspecies variation with regard to biochemical properties. Additionally, phenotypic characteristics are readily influenced by the culture conditions under which the bacteria are maintained and tested. Nevertheless, without the pioneers of anaerobic bacteriology (Veillon, Prevot, Hungate, Holdeman and Moore, Sutter and Finegold and their respective colleagues) intestinal microflora research would have remained at a primitive standard of knowledge. (Holdeman & Moore, 1973; Finegold et al., 1974; Moore & Holdeman, 1974; Finegold & Sutter, 1978; Moore et al., 1978; Summanen et al., 1993). The results of Carl Woese's investigations revealed that small ribosomal subunit RNA (16S rRNA in the case of bacteria) contained regions of nucleotide base sequence that were highly conserved and that these were interspersed with hypervariable regions (V regions) (Woese, 1987). These hypervariable regions contained the signatures of phylogenetic groups and even species. Members of the gut microflora can be accurately identified by extraction of DNA from a pure culture of a bacterial isolate, polymerase chain reaction (PCR) amplification of the 16S rRNA gene using universal primers that target conserved bacterial sequences, and determination of its nucleotide base sequence. The whole gene (about 1500 bp) should be sequenced for utmost accuracy, but a sequence of 500 bp will provide useful information. The sequence can be compared to those available in gene databanks in order to determine the closest match and, hence, an identification of the bacterial isolate. There are currently about 16000 16S rRNA gene sequences stored by the ribosomal database project II, for example, although not all are of complete genes (http://rdp.cme.msu.edu). Doubtless there are intestinal species that are still not represented in gene databanks. 16S rRNA gene sequences can be used to construct phylogenetic trees of the members of the intestinal microflora. This contributes greatly to bacterial taxonomy. Molecular taxonomy provides a concrete basis for identification of bacterial species. A sequence of As, Ts, Gs and Cs is tangible and not influenced by the cultivation conditions to which the bacteria have been subjected. Comparison of 16S rDNA similarities can also be used in the analysis of bacterial communities (Raskin et al., 1997). Universal primers can be used in PCR to amplify 16S rDNA from bacterial cells in natural samples. The amplified 16S rDNA sequences are cloned, screened (some sequences will have been cloned more than once) and sequenced. Alignment of the sequences with those stored in databanks permits the recognition of which species were represented in the habitat, and detects those that cannot be cultivated by conventional bacteriological techniques. In a study of this type reported by the Dore group (Suau et al., 1999), three bacterial divisions represented 95% of the faecal microflora of a human subject: Bacteroides-Prevotella, Clostridium coccoides group and Clostridium leptum group. The diversity within these divisions is, however, vast (Tables 1.1-1.3) and a full inventory of the inhabitants of the human gut awaits the results of further nucleic acid-based studies. The Welling group at the University of Groningen, The Netherlands (Franks et al., 1998), has pioneered fluorescent in situ hybridisation (FISH) as a means of investigating the intestinal microflora. They have derived oligonucleotide probes, labelled with fluorescent dyes, that target 16S rRNA sequences. Although an array of hundreds of probes could be used to enumerate bacterial species in faecal samples, due to practical considerations, a smaller collection of probes that recognise large phylogenetic groups of bacteria have been

The Intestinal Microflora

Table 1.1

3

Cultivated bacterial species within the Bacteroides-Prevotella group

Porphyromonas salivosa Porphyromonas catoniae Porphyromonas gingivalis Porphyromonas cangingivalis Porphyromonas endodontalis Rikenella microfusus Cytophaga fermentans Bacteroides putredinis Bacteroides splanchnicus Bacteroides vulgatus Bacteroides fragilis Bacteroides stercoris Bacteroides uniformis Bacteroides eggerthii

Bacteroides caccae Bacteroides ovatus Bacteroides thetaiotaomicron Bacteroides forsythus Bacteroides distasonis Bacteroides merdae Prevotella heparinolytica Prevotella pallens Prevotella veroralis Prevotella denticola Prevotella oulora Prevotella oris Prevotella oralis Prevotella ruminicola

used (Table 1.4). It has been estimated that these probes currently detect about 90% of the members of the faecal microflora (Harmsen & Welling, 2002; Harmsen et al., 2002). Technical difficulties can influence the accuracy of the results. The oligonucleotide probes must reach their target sequence, which is inside the bacterial cell, by passing through the cell wall. This is more easily achieved with some bacterial species than with others (Welling et al., 1997). The method is best for enumeration of the numerically predominant members of the microflora, the lowest level of detection being 10 cells per gram (Welling et al., 1997).

Table 1.2

Cultivated bacterial species within the Clostridium coccoides group

Clostridium polysaccharolyticum Clostridium herbivorans Clostridium populeti Clostridium coccoides Clostridium nexile Clostridium oroticum Clostridium clostridiiforme Clostridium celerecrescens Clostridium xylanolyticum Clostridium symbiosum Clostridium aminovalericum Clostridium aminophilum Coprococcus eutactus Butyrivibrio crossotus Butyrivibrio fibrisolvens

Eubacterium xylanophilum Eubacterium ventriosum Eubacterium eligens Eubacterium formicigenerans Eubacterium contortum Eubacterium rectale Eubacterium hadrum Eubacterium halii Eubacterium ramulus Lachnospira pectinoschiza Ruminococcus obeum Ruminococcus hansenii Ruminococcus productus Ruminococcus torques Ruminococcus gnavus Roseburia cecicola

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Table 1.3

Cultivated bacterial species within the Clostridium leptum group

Fusobacterium prausnitzii Eubactehum siraeum Clostridium sporosphaeroides Clostridium leptum Ruminococcus flavefaciens

Ruminococcus bromii Eubacterium plautii Clostridium viride Eubacterium desmolans Termitobacter aceticus

DNA-RNA hybridisations provide a means of determining the proportions in which specific bacterial groups occur within the microflora. In this method, bacterial RNA is extracted from samples and dot-blotted to membranes. The membranes are probed with a collection of radioactively labelled oligonucleotides, each specific for a particular bacterial group. A probe (currently Bact338) that hybridises to a conserved rRNA sequence in the majority of bacterial cells is used as a reference against which the hybridisation results of the other probes are compared. This provides a means of calculating the proportions that the various populations form in the total bacterial community. In work carried out in France, six oligonucleotide probes detected, on average, 70% of the 16S rRNA hybridised by the universal probe in faecal extracts from 27 human subjects. Bacteroides-PrevotellaPorphyromonas accounted for 37% of the faecal microflora, Clostridium coccoides group for 16% and the Clostridium leptum group for 14%. Bifidobacteria, enterobacteria and LactobacillusStreptocoaus-Enterococcus each comprised only about 1% of the microflora (Sghir et a!., 2000).

Table 1.4 Bacterial groups quantified by DNA probes in the faeces of adult humans in Europe by Harmsen and Welling (2002)

Image Not Available

After Harmsen and Welling (2002).

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Oligonucleotide probes can only be derived if the microbial members of the ecosystem are known. Phylogenetic analysis of the community, such as that carried out by Suau et al. (Suau et al., 1999), or perusal of the 16S rDNA sequences derived from cultured bacterial species, are thus prerequisite to successful use of FISH or DNA-RNA hybridisations. Another molecular approach to monitoring the composition of complex communities overcomes this limitation. PCR coupled with temperature gradient gel electrophoresis (TGGE) has been shown by Zoetendal et al. (1998) to provide an excellent method for comparative monitoring of the faecal microflora. Since this pioneering work, TGGE has been superseded by denaturing gradient gel electrophoresis (DGGE). In PCR/DGGE, fragments of the 16S rRNA gene are amplified by PCR. One of the primers has a GC-rich 5' end (GC clamp). 16S fragments are amplified from the bacterial community in the sample and DGGE separates the 16S molecular species within the resulting mixture. The double-stranded 16S fragments migrate through a polyacrylamide gel containing a gradient of urea and formamide until they are partially denatured by the chemical conditions. The fragments do not completely denature because of the GC clamp, and migration is radically slowed when partial denaturation occurs. Because of the variation in the 16S sequences of different bacterial species, chemical stability is also different; therefore different 16S 'species' can be separated by this electrophoretic method. A profile of 16S sequences, thought to represent 90-99% of the bacterial community, is generated. Individual fragments of DNA can be cut from DGGE gels, further amplified and cloned, then sequenced. The sequence can be compared to those in gene databanks in order to obtain identification of the bacterium from which the 16S sequence originated. Depending on the length of the sequence, identification to at least bacterial phylogenetic group can be made (Muyzer & Smalla, 1998; Zoetendal et al., 1998). A sequence from the bacterial species or phylogenetic division identified in this way might be used in further studies as the target for FISH enumeration, or DNA-RNA hybridisation, of the specific bacterial population. PCR, wonderful molecular biological tool though it is, poses some problems: while culture bias is removed, another bias is introduced because polymerase chain reactions are known to amplify DNA sequences from mixed populations with differing efficiency (Reysenbach et al., 1992). Chimeric sequences can be derived during PCR where there is a mixture of template DNAs in the reaction mix (Kopczynski et al., 1994), and there can be heterogeneity with regard to 16S rRNA sequences within species, and even within a single bacterial cell (Nubel et al., 1996). Nevertheless, the depth and breadth of gut microflora research has been considerably improved through the use of nucleic acid-based methodologies. For the microbiologist faced with the identification of hundreds of bacterial isolates, PCR amplification and whole gene sequencing may not be regarded as essential. Indeed, some microbial ecologists may view modern studies of the faecal microflora as 'death by phylogeny'. PCR amplification of short sequences of 16S rDNA that are hypervariable can provide a rapid identification method when coupled with DGGE. The V2-V3 region of the 16S rRNA gene, for example, can be amplified from pure cultures of Lactobatillus isolates. The PCR amplicons, when examined by DGGE, allow the identification of many Lactobacillus species because their V2—V3 sequences have characteristic migration properties in the electrophoretic gel. This means that an identification ladder can be prepared from type cultures in order to identify isolates of intestinal lactobacilli (Walter et al., 2000). This approach has also been found to be appropriate for the identification of Bacteroides (Munro &

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Tannock, unpublished) and Bifidobacterium species (Satokari et al., 2001) and is doubtless applicable to many other bacterial genera. PCR primers that are group specific add a new dimension to this method of analysis (Walter et al., 2001; Requena et al., 2002). Hypervariable regions of the 16S rRNA gene also provide targets for the derivation of genus- and species-specific PCR primers. Numerous examples already exist in the scientific literature, but those useful in the identification of bifidobacteria can be cited here. Matsuki et al. (1999) have reported PCR primer sets that will differentiate between nine bifidobacterial species (B. catenulatum and B. pseudocatenulatum cannot be differentiated from each other). This is an outstanding accomplishment, considering the high degree of similarity in 16S rRNA gene sequences of the members of this genus (Leblond-Bourget et al., 1996). Targets for species-specific primers may be found elsewhere on the bacterial chromosome. PCR primers that anneal to conserved sequences in the 16S and 23S rRNA genes of lactobacilli, for example, allowed the amplification of DNA between these two genes (intergenic spacer region) (Tannock et al., 1999)- A high degree of hypervariability of the spacer region was observed and this permitted the derivation of primers that target these sequences and differentiate between Lactobacillus species. Eight primer sets that target the intergenic spacer region sequence for the identification of Lactobacillus species are currently available (Walter et al., 2000). The use of primer sets to identify bacterial species is logistically demanding and expensive. For example, consider the requirements to test each bifidobacterial isolate in nine separate PCR reactions to achieve identification. Probably the best use of species-specific primers is in the confirmation of the results obtained by identification methods such as PCR/ DGGE, and in the direct detection of species, without culture, in faecal or intestinal samples. The techniques described thus far are aimed at the analysis of bacterial communities or the identification of bacterial isolates at the level of genus or species. Bacterial species can be further divided into strains using molecular typing methods. These typing methods are generally based on restriction fragment length polymorphisms of bacterial DNA and require cultivation of the bacteria in the laboratory. Restriction endonuclease digests of DNA extracted from pure bacterial cultures are analysed by agarose gel electrophoresis. The resulting patterns of DNA fragments are characteristic of each strain. Ribotyping and pulsed field gel electrophoresis (PFGE) of DNA digests are examples of methods that have been used to differentiate between strains of intestinal bacteria (McCartney et al., 1996; Kimura et al., 1997). Such methodologies are extremely useful in tracking the fate of specific bacterial strains in the intestinal ecosystem, just as they are in studying the epidemiology of bacterial pathogens. An important observation resulting from the use of molecular typing concerns Lactobacillus—host relationships. Genetic fingerprinting of bacterial isolates was used to analyse the composition of the Lactobacillus populations present in the faeces of humans during a study aimed at measuring the impact of probiotic consumption on the composition of the faecal microflora. The composition of the faecal microflora of ten healthy subjects was monitored before (control period of six months), during (test period of six months) and after (post-test period of three months) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose of 1.6 x 109 lactobacilli). The composition of the Lactobacillus population of each subject was analysed by PFGE of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Consumption of the probiotic altered the composition of the Lactobacillus populations of the subjects, but to varying degrees. The presence of DR20 among the numerically predominant strains was

The Intestinal Microflora

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related to the presence or absence of a stable autochthonous population of lactobacilli during the control period. The probiotic strain did not predominate in samples collected from subjects with Lactobacillus populations of stable composition. The presence of lactobacilli capable of persisting long term (autochthonous strains) in the faeces of a subject appeared to preclude the establishment of DR20 (allochthonous) as the numerically dominant strain (Tannock et at., 2000). In a period of about 30 years, technological emphasis in the analysis of the intestinal microflora has progressed from 'culture-dependent' to 'culture-independent'. This reflects attitudes that have developed in microbial ecology in general. There is no doubt that sophisticated investigations of the intestinal microflora can be carried out by a combination of careful anaerobic culture of bacteria and the parallel use of nucleic acid-based methodologies.

Catalogues show diversity The expanding use of nucleic acid-based, culture-independent methods for the analysis of the composition of the gut microflora confirms the complexity of this bacterial collection. The results of molecular methods of analysis have somewhat altered our view of the predominant bacterial species that are present in the human gut. While Bacteroides-Prevotella continues to be a numerically predominant group, eubacteria/clostridia and fusobacteria attain more prominence as a result of the use of molecular analytical methods, whilst the importance of bifidobacteria in the adult microflora is lessened (compare Tables 1.4, 1.5 and 1.6 that provide observations from 2002, 1974 and 1995 respectively). Recent studies using DNA-based PCR, have revealed that each human has a bacterial community of unique composition in the faeces that provides a further 'fingerprint' in addition to that of the fingers and the genome. First observed during PCR/DGGE analysis of the faecal microflora, this uniqueness has been confirmed by FISH (Table 1.7). Such is the uniqueness of the faecal microflora, a definition of the 'human faecal microflora' now seems impossible unless expressed in very simple terms ('a collection of bacterial species in which obligate anaerobes predominate'). There may well be a core intestinal microflora that all humans share, but catalogues of bacteria arrayed in their phylogenetic divisions is unlikely to be particularly informative in this respect. It may be time to investigate the gut microflora, not in terms of phylogeny, but with regard to its functional aspects.

A phoenix arises Knowledge of the impact of the intestinal microflora on the biochemistry, physiology, immunology and disease resistance of the host animal has been derived almost exclusively from gnotobiological studies (Gordon & Pesti, 1971). Comparison of the characteristics of germfree animals (raised in the absence of microbial associates) and their conventional counterparts (presence of microbial associates) has demonstrated that the intestinal microflora as a whole has major impacts, referred to as the microflora-associated characteristics, on host attributes (Table 1.8).

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Table 1.5 The most prevalent bacterial species in the faeces of adults consuming a 'Japanese diet' or a 'Western' diet according to Finegold et al. (1974) Japanese diet

Western diet

Bacteroides fragilis subsp. thetaiotaomicron Bacteroides fragilis subsp. distasonis Peptostreptococcus sp. 1 Eubacterium rectale Bacteroides fragilis subsp. vulgatus Eubacterium sp. 4 Clostridium innocuum Eubacterium aerofaciens Eubacterium lentum Eubacterium sp. 20 Bacteroides fragilis subsp. ovatus Bacteroides fragilis subsp. fragilis Bacteroides subsp. other Peptostreptococcus productus Escherichia coli Bifidobacterium sp. 4 Clostridium sp. 14 Facultative streptococcus, other Streptococcus faecalis subsp. faecalis Streptococcus bovis

Bacteroides fragilis subsp. vulgatus Bacteroides fragilis subsp. other Bacteroides fragilis subsp. fragilis Bacteroides fragilis subsp. thetaiotaomicron Peptostreptococcus micros Bacillus sp. Bifidobacterium adolescentis D Eubacterium aerofaciens Bifidobacterium infantis, other Ruminococcus albus Bacteroides subsp. distasonis Peptostreptococcus intermedius Peptostreptococcus sp. 2 Peptostreptococcus productus Eubacterium lentum Facultative streptococcus, other Fusobacterium russii Bifidobacterium adolescentis A Bifidobacterium adolescentis C Bacteroides clostridiiformis subsp. clostridiiformis Peptococcus prevotii Bifidobacterium infantis subsp. liberorum Clostridium indolis Streptococcus faecium Bifidobacterium longum subsp. longum

Eubacterium sp. 24 Eubacterium sp. 32 Clostridium sp. 45 Peptococcus prevotii Bifidobacterium eriksonii After Finegold et al. (1974).

The impact of specific bacteria, lactobacilli, on the biochemistry of the murine gut milieu has been investigated (Table 1.9), but gnotobiotic research has been sadly neglected during the last decade. This is probably because of the high cost of maintaining germfree animals and a lack of suitable bacteriological methodologies to advance the field in a manner relevant to medical research. It is only recently that the molecular mechanisms associated with intestinal microflora—host relationships have begun to be studied (Hooper et al., 2001). Made possible by the development of molecular biological tools, this research coincides with the complete genome sequencing of selected animal species and offers unprecedented opportunities to identify the molecular foundations of gut microflora—host relationships. The intimate contact between the bacterial community of the gut and the animal host is a notable feature of this relationship. Numerous bacterial cells are confined within a relatively

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9

Table 1.6 The most prevalent bacterial species in the faeces of adults consuming a 'Japanese diet' or a 'Western diet' according to Moore and Moore (1995) Rural native Japanese

North American Caucasians

Bacteroides vulgatus Eubacterium aerofaciens 1 Bacteroides FB Bifidobacterium adolescentis Eubacterium aerofaciens 2 Enterococcus faecium Fusobacterium prausnitzii Bacteroides thetaiotaomicron Bacteroides fragilis Bacteroides ovatus Bacteroides uniformis Lactobacillus salivarius Ruminococcus gnavus Bacteroides stercoris Streptococcus SO3 Bacteroides FO Ruminococcus bromii Peptostreptococcus DZ 2 Peptostreptococcus productus 2b Lactobacillus acidophilus 5

Eubacterium aerofaciens 1 Bacteroides vulgatus Eubacterium rectale 2 Ruminococcus bromii Fusobacterium prausnitzii Eubacterium rectale 1 Bifidobacterium longum Peptostreptococcus DZ Bifidobacterium adolescentis Eubacterium biforme Ruminococcus obeum Gemmiger formicilis Bacteroides stercoris Ruminococcus CE Eubacterium aerofaciens 2 Eubacterium eligens Peptostreptococcus productus 2b Bacteroides thetaiotaomicron Bacteroides caccae Coprococcus comes Peptostreptococcus productus 1b Peptostreptococcus productus 2c Bacteroides uniformis Bacteroides distasonis Butyrivibrio crossotus Eubacterium rectale 3h Escherichia coli Eubacterium rectale 3f Lactobacillus acidophilus Bacteroides uniformis 2 Bacteroides FO

After Moore and Moore (1995).

small, defined space and are separated from the sterile tissues of the host by an epithelium composed of a single layer of enterocytes. In the ileum of mice, and some other animal species, filamentous segmented bacteria related to the clostridia attach by one end to enterocytes, particularly in the vicinity of Peyer's patches (Klaasen et al., 1992). The method of attachment involves the insertion of the base of the filament into a murine enterocyte. Actin rearrangements within the enterocyte form a socket by which the filament becomes permanently attached to the mucosal surface (Jepson et al., 1993). The mechanism by which

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Table 1.7

Variation in the composition of the faecal microflora of 15 healthy adult humans % of total faecal microflora (n = 15)1

Bacterial group

1.5-96.3 0.4-44.6 0.1-23.0 6.2-69.0 0.5-38.5

Bacteroides-Prevotella Bifidobacterium Atopobium-Eggerthella-Colinsella Eubacterium rectale-Clostridium coccoides Clostridium leptum 1 Measurements made by FISH. Stebbings, Harmsen, Welling, Munro and Tannock, 2001, unpublished data.

Table 1.8 Comparison of selected biochemical properties of the intestinal tracts of germfree and conventional animals Property

Conventional1

Germfree1

Bile acid metabolism

Deconjugation, dehydrogenation and dehydroxylation

Absence of deconjugation dehydrogenation and dehydroxylation

Bilirubin metabolism

Deconjugation and reduction

Little deconjugation and absence of reduction

Cholesterol

Reduction to coprostanol

Absence of coprostanol

B-aspartylglycine

Absent

Present

Intestinal gases

Hydrogen, methane and carbon dioxide

Absence of hydrogen and methane. Less carbon dioxide

Short-chain fatty acids

Large amounts of several acids

Small amounts of a few acids

Tryptic activity

Little activity

High activity

Urease

Present

Absent

p-glucuronidase (pH 6.5)

Present

Absent

Extent of degradation of mucins

More

Less

Serum cholesterol concentration

Lower

Higher

1

Conventional = raised in association with a normal microflora; germfree = raised in the absence of demonstrable microbes.

The Intestinal Microflora

Table 1.9

11

The impact of lactobacilli on the biochemical characteristics of mice

Characteristic

Reference

Bile salt hydrolase activity in intestinal contents is due mainly to presence of lactobacilli

Tannock et al. (1989)

More unconjugated bile acids in intestinal contents when lactobacilli are present

Tannock et al. (1994)

Azoreductase activity in large bowel contents reduced when lactobacilli present

McConnell & Tannock (1991)

(3-glucuronidase activity reduced in large bowel contents of male mice when lactobacilli are present

McConnell & Tannock (1993b)

Enzyme activities associated with duodenal enterocytes unaffected by presence of lactobacilli

McConnell & Tannock (1993a)

Serum cholesterol concentrations unaffected by presence of lactobacilli in the gastrointestinal tract

Tannock & McConnell (1994)

the bacteria cause rearrangement of the enterocyte cytoskeleton is not known, but likely involves the passage of effector molecules from the bacterial cell to the enterocyte: much as occurs when membrane ruffling of enterocytes is induced by Salmonella typhimurium (Galan, 1996). The presence of filamentous segmented bacteria in the mouse gut results in the increased expression of a murine gene involved in the fucosylation of the asialo GM1 glycolipid associated with enterocytes (Umesaki et al., 1995). Curiously, a similar phenomenon has also been observed when ex-germfree mice were colonised with Bacteroides thetaiotaomicron (Bry et al., 1996). This bacterial species utilises L-fucose, salvaged from intestinal glycoconjugates, as an energy source. The induction of fucosylation of glycoconjugates in the bowel of mice was shown to be dependent on the presence of a critical concentration of Bacteroides cells (106 —107 CFU/ml) and on the ability of the bacteria to utilise L-fucose. A mutant strain, unable to utilise L-fucose, was less efficient at inducing fucosylation. The linkage between Lfucose utilisation and the signalling system that induces fucosylation of intestinal glycoconjugates is mediated by bacterial protein FucR (Hooper et al., 1999, 2000). It is proposed that the bacteroides, by influencing host biochemistry, ensure that L-fucose is constantly available as an energy source in a highly competitive environment. Bacteroides thetaiotaomicron affects the expression of other genes in the mucosal cells of the ileum of ex-germfree mice. Using high-density oligonucleotide-based DNA microarrays for the analysis of the host transcriptional responses, it was shown that these bacteria regulated the expression of a broad range of mouse genes (Table 1.10) that participate in diverse and fundamental physiological functions (Hooper et al., 2001). Defining the mechanisms by which the bacteria communicate with host cells will surely prove a fascinating area of research.

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Table 1.10 Examples of murine genes regulated by colonisation of the gut by Bacteroides thetaiotaomicron

Image Not Available

After Hooper et al. (2001).

Is it all the same in the end? Despite the diversity of human faecal microflora profiles generated by PCR/DGGE from bacterial DNA, the functioning of the bacterial community may be similar in all humans. It is clear from the studies of Freter (1988) that there is redundancy in the mechanisms by which the intestinal community is regulated. Further, although the concentration of shortchain fatty acids in the faeces of human subjects is highly variable when measured temporally, the proportions of the respective acids is roughly the same from individual to individual (Table 1.11). Thus the acetic to propionic acid ratio is within the range 2.5 to 5.0 (mean 3.4), and the propionic to butyric acid ratio 0.6 to 1.4 (mean 0.8). Therefore, regardless of

The Intestinal Microflora

Table 1.11

13

Concentrations of short-chain fatty acids in human faeces

Image Not Available

After Tannock et al. (2000).

bacterial species composition, the microflora as a whole may function in the same way, producing the same metabolic end-product profile. If the catalogue of bacterial species is markedly different from one individual to another, one can envisage that the antigenic diversity may be different from one subject to another, and that the impact of the microflora on the immune system will also differ. In this respect, further investigations using gnotobiotic animals will be necessary to advance knowledge of this host-microflora relationship. Nevertheless, since the innate defence mechanisms of the host relate more to the recognition of bacterial molecular patterns (Table 1.12) rather than antigenic specificity, the taxonomy of the intestinal inhabitants may be irrelevant (Medzhitov & Janeway, 2000; Akira et al., 2001; Krieg, 2001).

Table 1.12

Pattern-recognition receptors on effector cells of the innate immune system

Bacteria-associated molecular patterns

Pattern-recognition receptors

Lipopolysaccharide Peptidoglycan Flagellin Mannans DNA (unmethylated CpG dinucleotides)

Toll-like receptor 4 Toll-like receptor 2 Toll-like receptor 5 Mannan-binding lectin Toll-like receptor 9

14

Gut Flora, Nutrition, Immunity and Health

The formative years Comparison of the immunological characteristics of germfree (raised in the absence of microbes) and conventional (raised in the presence of microbes) animals has demonstrated that the presence of the normal microflora stimulates the reticuloendothelial tissues of the animal, particularly those tissues associated with the intestinal tract. Peyer's patches and mesenteric lymph nodes are more developed, the lamina propria of the intestinal wall contains more neutrophils and lymphocytes, and the serum contains a higher concentration of immunoglobulins in conventional animals compared to germfree (Gordon & Pesti, 1971). Other striking differences between germfree and conventional animals concern intestinal intraepithelial lymphocytes that in conventional mice are about equally divided into y5 + and y5+ subpopulations. In germfree mice, y5+ lymphocytes are predominant in the intestinal epithelium. The y5+ subpopulation quickly increases in size when ex-germfree mice acquire an intestinal microflora (conventionalisation) (Kawaguchi et al., 1993; Umesaki et al., 1993). The influence of particular bacterial groups on the development of the immune system during early life has not been investigated systematically. Studies with germfree mice, monoassociated with bacterial species, or injected with heat-killed bacteria have been reported. These studies, reviewed by McCracken and Gaskins (1999), are open to several criticisms, and firm conclusions about the significance of specific bacteria in 'programming' the immune system is lacking. A practical application of properly conducted studies of the impact of the gut microflora on the ontogeny of the immune system would be to understand the increasing incidence of atopic disorders, including asthma, of children in affluent countries (Burr et al., 1989; Burney et al., 1990; Bjorksten, 1997). Atopic disorders are characterised by dominant T helper 2 (Th2) mechanisms and the production of immunoglobulin E (IgE) to common environmental antigens. Asthma is an atopic disorder characterised by activation and recruitment of eosinophils to the lung resulting in chronic swelling and inflammation of the airways. The reasons for the increase in atopic disorders in developed countries are unknown, but several factors in addition to hereditary predisposition that may be of significance, all concerning microbial exposure in childhood, have recently been identified in retrospective studies: a decline in diseases such as tuberculosis, measles and influenza that characteristically stimulate a T helper 1 (Thl) type of immune response (Bjorksten, 1997; Romagnani, 1997; Martinez & Holt, 1999); immunisation with whole-cell pertussis vaccine which is a strong promoter of IgE synthesis with an effect on primary and secondary antibody responses to heterologous antigens (Pauwels et al., 1983); treatment with oral antimicrobial drugs during the first two years of life, especially multiple courses of antimicrobials (Farooqi & Hopkin, 1998). The mechanism by which antibiotics influence the programming and development of the immunological system most likely involves alterations to the collection of bacterial species inhabiting the intestinal tract. Treatment of young children with broad-spectrum, oral antibiotics might produce perturbations in the composition of the intestinal microflora such that bacteria important in promoting Thl mechanisms are depleted at a crucial age. These observations fit the 'Hygiene hypothesis' proposed by Strachan (1989) who demonstrated an inverse relationship between birth order in families and the prevalence of hayfever and suggested that this was due to an increased likelihood of infection in younger siblings

The Intestinal Microflora

15

due to transmission of pathogens from older children in the family. It is possible that declining family size, better personal cleanliness and improved living conditions, and widespread administration of antimicrobial agents to babies have altered the microbial exposure of families and have thus resulted in a higher prevalence of allergies. The composition in the human infant differs from that of the adult. Particularly noticeable are the higher numbers of facultative anaerobes (enterococci, enterobacteria) and bifidobacteria in relation to the total microflora when compared to that of adults (Tables 1.13—1.15). A general decline in the numbers of enterococci and enterobacteria in the faeces occurs as the intestinal microflora of infants matures (Tannock & Cook, 2002) and as shortchain fatty acids increase in quantity and diversity in the intestine. Bifidobacteria are predominant members of the infant intestinal microflora, forming up to 91% of the total bacterial community in breast-fed babies and up to 75% in formula-fed infants (Harmsen et al., 2000). Clearly, these groups of bacteria that predominate in the intestine during the formative years may be worthy of considerable attention in relation to the ontogeny of the immune system.

Rules and regulations, but mind your language The intestinal microflora is a well-regulated bacterial community in which homeostatic forces are strongly operative. The DGGE profile of the faecal community of adults, for example, is constant over long periods of time. Antibiotic administration by the oral route to children causes marked changes in bacterial community profiles (Fig. 1.1), but the composition of the microflora generally returns to pretreatment status by ten days after treatment has ceased. Although the factors that regulate the composition of the microflora are essentially unknown, it can be speculated that competition for nutrients that are present in growthlimiting amounts in the ecosystem is of major importance, just as it is in other environments (Alexander, 1971). Amensalism, in which the metabolic products of one bacterial group inhibit the growth of another bacterial group, is also likely to be of significance. This is apparent from study of the biological succession in the infant mouse gut where enterococcal and enterobacterial numbers are initially high, but rapidly decline at about the time of weaning. Once the mice begin to consume solid food at about 15 days of age, obligately anaerobic bacteria establish in the large bowel and the populations of enterococci and enterobacteria quickly drop to levels observed in adult mice (Lee & Gemmel, 1972). It is these types of autogenic factors that regulate the composition of the intestinal microflora and which negate attempts to modify the bacterial community permanently by the administration of 'probiotics' (Tannock et al., 2000). Recent research carried out by Bassler and colleagues (1999) has demonstrated that bacterial cells communicate with one another to the extent that they can sense the density of cells in their vicinity. Increasing density of cells leads to the induction of previously silent genes which are then expressed. Quorum sensing, or the control of gene expression in response to cell density, is used by both Gram-positive and Gram-negative bacteria to regulate a variety of physiological functions. In all cases, quorum sensing involves the production and detection of extracellular signalling molecules called autoinducers (Bassler,

16

Gut Flora, Nutrition, Immunity and Health

Table 1.13 The faecal microflora of infants of different ages consuming human milk or formula feed

Image Not Available

After Stark and Lee (1982).

1999). Well-known paradigms include intercellular signalling in the formation of fruiting bodies by myxobacteria, the production of luminescence by Vibrio fischeri when colonising the light organ of squid, nisin production by Lactococcus lactis, and signalling between Staphylococcus aureus cells mediated by peptides. Recent studies suggest that bacteria are multilingual. It appears that many bacteria possess a species-specific language as well as a species-non-specific language. These findings imply that bacteria can assess their own

The Intestinal Microflora

Table 1.14 Enterococcal populations in the faeces of ten adult humans

Image Not Available

After Tannock et al. (2000).

Table 1.15 Populations of lactose-fermenting enterobacteria in faecal samples from adult humans

Image Not Available

After Tannock et al. (2000).

17

18

Gut Flora, Nutrition, Immunity and Health

Fig. 1.1 PCR/DGGE profiles of the faecal microflora of a child treated with an antibiotic. Lane a, control sample collected prior to antibiotic administration; lane b, day 3 of amoxycillin treatment; lane c, day 9 post amoxycillin treatment. Note return of microflora profile in day 9 post-treatment sample to that existing prior to antibiotic administration.

population numbers and also the population density of other species of bacteria in the vicinity. Distinct responses to the intraspecies and interspecies signals allow a particular species of bacteria to properly modulate its behaviour depending on whether it makes up a majority or a minority of any given consortium (Schauder & Bassler, 2001). The interspecies language involves the autoinducer AI-2 whose synthesis depends on the LuxS protein. Homologues of LuxS are known to be produced by more than 30 species of bacteria, representing both Gram-negative and Gram-positive species. In contrast, Gram-negative communication is mediated by the LuxI/LuxR language. These bacteria produce and respond to secreted acylated-homoserine lactone autoinducers that accumulate in the external environment as the cells grow. In contrast, Gram-positive bacteria do not employ homoserine lactones and do not have the LuxI/LuxR signalling system. Instead, they secrete peptide signalling molecules that are recognised by two-component sensor proteins. Signal

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19

transduction results in the activation of target genes. Quorum sensing may be especially important to bacteria that form biofilms (Davies et al., 1998). Biofilms occur on certain epithelia lining the proximal regions of the gastrointestinal tract of some animal species. These biofilms are composed of lactobacilli which adhere to stratified, squamous, epithelia of the mouse and rat forestomach, the porcine pars oesophagea and the avian crop. Additionally, obligately anaerobic bacteria, such as Bacteroides and Clostridium species, inhabit the mucus layer associated with the large bowel epithelium of the rodent gut (Savage et al., 1968). Perhaps the gut microflora will provide a rich source of information concerning communication within microbial communities?

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genetic susceptibility, innocuous and/or microbial antigens and the immune system. Current Opinion in Immunology, 9, 773—5. Satokari, R.M., Vaughan, E.E., Akkermans, A.D.L., Saarela, M. & De Vos, W.M. (2001) Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology, 67, 504—13. Savage, D.C., Dubos, R. & Schaedler, R.W. (1968) The gastrointestinal epithelium and its autochthonous bacterial flora. Journal of Experimental Medicine, 127, 67—76. Schauder, S. & Bassler, B.L. (2001) The languages of bacteria. Genes and Development, 15, 1468-80. Sghir, A., Gramet, G., Suau, A., Rochet, V., Pochart, P. & Dore, J. (2000) Quantification of bacterial groups within the human fecal flora by oligonucleotide probe hybridization. Applied and Environmental Microbiology, 66, 2263—6. Strachan, D.P. (1989) Hay fever, hygiene, and household size. British Medical Journal, 299, 1259-60. Suau, A. et al. (1999) Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Applied and Environmental Microbiology, 65, 4799—807. Summanen, P., Baron, E.J., Citron, D.M., Strong, C, Wexler, H.M. & Finegold, S.M. (1993) Wadsworth Anaerobic Bacteriology Manual, 5th edn. Star Publishing Company, Belmont. Stark, P.L. & Lee, A. (1982) The microbial ecology of the large bowel of breast-fed and formula-fed infants during the first year of life.Journal of Medical Microbiology, 15, 189-203. Tannock, G.W. (1995) Normal Microflora. An introduction to microbes inhabiting the human body. Chapman and Hall, London. Tannock, G.W. & Cook, G. (2002) Enterococci as members of the intestinal microflora of humans. In: Enterococci: pathogenesis, molecular biology and antibiotic resistance (ed. M. Gilmore, P. Courvalin, D. Clewell, G. Dunny, B. Murray & L. Rice). American Society for Microbiology, Washington, D.C. Tannock, G.W. & McConnell, M.A. (1994) Lactobacilli inhabiting the digestive tract of mice do not influence serum cholesterol concentration. Microbial Ecology in Health and Disease, 7, 331-4. Tannock, G.W., Dashkevicz, M.P. & Feighner, S.D. (1989) Lactobacilli and bile salt hydrolase in the murine intestinal tract. Applied and Environmental Microbiology, 55, 1848-51. Tannock, G.W., Tangerman, A., Van Schaik, A. & McConnell, M.A. (1994) Deconjugation of bile acids by lactobacilli in the mouse small bowel. Applied and Environmental Microbiology, 60, 3419-20. Tannock, G.W., Tilsala-Timisjarvi, A., Rodtong, S., Ng, J., Munro, K. & Alatossava, T. (1999) Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yogurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons. Applied and Environmental Microbiology, 65, 4264—7. Tannock, G.W., Munro, K., Harmsen, H.J.M., Welling, G.W., Smart, J. & Gopal, P.K. (2000) Analysis of the fecal microflora of human subjects consuming a probiotic containing Lactobacillus rhamnosus DR20. Applied and Environmental Microbiology, 66, 2578—88.

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Umesaki, Y., Setomaya, S., Matsumoto, S. & Okada, Y. (1993) Expansion of the yS T cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germfree mice, and its independence from the thymus. Immunology, 79, 32—7. Umesaki, Y., Okada, Y., Matsumoto, S., Imaoka, A. & Setoyama, H. (1995) Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse. Microbiology and Immunology, 39, 555-62. Walter, J., Tannock, G.W., Tilsala-Timisjarvi, A. et al. (2000) Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Applied and Environmental Microbiology, 66, 297—303. Walter, J., Hertel, C, Tannock, G.W., Lis, C.M., Munro, K. & Hammes, W.P. (2001) Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human faeces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology, 67, 2578—85. Welling, G.W., Elfferich, P., Raangs, G.C., Wildeboer-Veloo, A.C., Jansen, GJ. & Degener, J.E. (1997) 16S ribosomal RNA-targeted oligonucleotide probes for monitoring of intestinal tract bacteria. Scandinavian Journal of Gastroenterology Supplement, 222, 17—19. Woese, C.R. (1987) Bacterial evolution. Microbiology Reviews, 51, 221-71. Zoetendal, E.G., Akkermans, A.D. & De Vos, W.M. (1998) Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and hostspecific communities of active bacteria. Applied and Environmental Microbiology, 64, 3854-9.

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S. Macfarlane & G.T. Macfarlane University of Dundee, MRC Microbiology and Gut Biology Group, Ninewells Hospital Medical School, Dundee DD1 9SY, UK

The large intestine In health, the large bowel is the principal area of permanent bacterial colonisation of the human gastrointestinal (GI) tract. That this is so is determined by the anatomy of the GI tract and by physiological and environmental factors in the upper gut, such as low pH, short retention time and the production of antimicrobial substances in the stomach (Xu et al., 2001), as well as the rapid rate of transit of digestive material through the small intestine (two to four hours). The movement of digestive substances slows markedly in the colon, where the average rate of transit is about 60—70 hours, although interindividual transit times vary considerably (Cummings et al., 1992). Long retention times and a good supply of nutrients facilitates the development of complex microbial communities in the large bowel. The colon is generally about one metre long in adults living in Western societies, with an internal surface area in the region of 1300 cm . It contains approximately 200 g of digestive material, most of which is water resident in bacterial cells (Banwell et al., 1981; Cummings et al., 1990, 1992). Average daily faecal output in the United Kingdom is somewhat over 100 g, and bacteria are the major component of stool, accounting for 40—45% of solids in individuals living on Western diets (Stephen & Cummings, 1980; Cabotage et al., 1990; Cummings et al., 1992). In addition to diet, the length of time digestive residues spend in the large intestine is an important determinant of bacterial metabolism. Long colonic transit times affect the digestion of carbohydrates and proteins, and through carbohydrate depletion and the formation of putrefactive products (see later), may be viewed as a potential risk factor in large bowel cancer. There is also a strong correlation between transit time and bacterial mass in the colon, where fast transits are associated with high stool weights and increased excretion of bacterial dry matter (Stephen et al., 1987). The inverse relationship between colonic transit time and stool weight is in large part due to intestinal bulking, which stimulates propulsion of bacterial cell mass and food residues through the bowel (Read & Eastwood, 1992). From anatomical, microbiological and environmental perspectives, the proximal bowel (caecum, ascending colon) and distal gut (descending colon, sigmoid/rectum) are quite different to each other. When digestive residues from the ileum enter the caecum, they

Food and the Large Intestine

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encounter a pool of partially-digested foodstuffs and bacteria, which rapidly begin to utilise simple carbon and nitrogen sources and initiate the breakdown of complex carbohydrates and proteins. Because of the production of fermentation acids, pH is reduced to about 5.5 or less in the proximal colon (Cummings et al., 1987), and due to relatively high levels of substrate availability, the caecum and ascending colon are sites of the most intense microbial activity, although bacterial cell population densities increase distally through the colon (Macfarlane et al., 1992b), as shown in Fig. 2.1.

Fig. 2.1 Viable bacterial counts (shaded bars) in large intestinal contents obtained from human sudden death victims, and SCFA production (open bars) from endogenous substrates in gut contents after 48 hours anaerobic incubation.

Wiggins and Cummings (1976) considered that the movement of digestive substances through the bowel had two distinct phases: time spent in the caecum and ascending colon, or 'mixing region', and time spent in storage of semi-solid faecal material in the distal bowel and rectum. On a daily basis, the caecum receives over 1 kg of material from the small gut, and as a consequence, caecal contents are fluid allowing this region of the bowel to serve as a mixing chamber. Due to water absorption, as faecal material moves through the bowel, the moisture content of intestinal contents decreases from 86% in the caecum to 77% in the distal colon (Cummings et al., 1990). Studies on human subjects using radio-opaque pellets have demonstrated that caecal residence accounts for about 30% of total colonic transit time, and that gut contents spend approximately 18 hours in the caecum (Wiggins & Cummings, 1976). As a result of bacterial substrate utilisation in the proximal large bowel, especially with regard to readily digestible carbohydrates, many bacterial carbon sources become depleted as digestive material moves through gut. This is known to influence the growth of some bacterial populations (Gibson et al., 1993), the types and amounts of fermentation products that are formed, as well as gut pH, which rises towards neutrality in the distal bowel (Cummings et al., 1987). This in turn affects physiological and biochemical processes

26

Gut Flora, Nutrition, Immunity and Health

in the microbiota, such as the binding of bile acids to food residues (Eastwood & Hamilton, 1968), hydrogen disposal (Macfarlane et al., 1992b; Gibson et al., 1993), peptide fermentation (Smith & Macfarlane, 1998), the activities of proteolytic and peptidolytic enzymes (Gibson et al., 1989; Macfarlane & Macfarlane, 1992, 1995), as well as enzymes involved in bile acid (Hylemon & Glass, 1983) and steroid metabolism (Bokkenheuser et al., 1977). Interactions of the microflora with the host The colonic microbiota interacts with the intestine in many ways, for example, bacterial cell mass stimulates peristaltic movement, thereby facilitating the passage of digestive residues through the bowel, while studies with germfree animals suggest that small intestinal uptake of sugars, amino acids, minerals and vitamins is more efficient than in animals with a conventional microflora (Abrams, 1983). This was related to greater longevity of enterocytes and the absence of bacterial toxins and inhibitory substances. Germfree animals also differ from normal animals in that they have thinner intestinal walls, fewer migration motor complexes, reduced sensitivity to peptides, higher gut trypsin activities and, in rodents, enlarged caecae (Norin & Midvedt, 2000). As will be discussed in more detail later, some bacterial fermentation products, such as xbutyrate, are metabolised and used as energy sources by the colonicmucosa. Intestinal bacteria also interact with several components of the host immune system, and are required for its normal development (Forestier et al., 2000; Helgeland & Brandtzaeg, 2000). Microorganisms growing in the large gut are also susceptible to variations in host physiology. Nutritional and emotional stress, together with stress resulting from disease, affect the composition of the intestinal microflora, particularly with respect to lactobacillus and enterobacterial populations (Tannock, 1995). This probably also has an effect on the metabolism of the microbiota. Holdeman et al. (1976) concluded that increased Bacteroides thetaiotaomicron in Skylab astronauts was due to anger stress. The mechanism was thought to be increased gastrin and adrenalin secretion which resulted in changes in the absorptive properties of the gut, blood flow and bowel motility.

Effect of diet on the colonic microflora Diet and bacterial species composition in the large intestine Until the introduction and widespread study of prebiotic oligosaccharides over the last decade or so, consistent changes in the effect of diet on the composition of intestinal bacterial populations have been hard to demonstrate (Hudson et al., 1981). However, early work investigating faecal bacteria in different human populations (Hill et al., 1971) showed that people in the United Kingdom and United States had considerably higher levels of anaerobes than Japanese, Indians or Ugandans, who had substantially more facultative anaerobes in their faeces. Other studies on the effects of a high-beef diet on ten human volunteers showed that animal protein consumption had little observed effect on the composition of the colonic microbiota (Hentges et al., 1977). The control diet, meatless and high-beef diets had 80, 82 and 179 g protein, respectively. A total of 84 bacterial species and subspecies were studied in

Food and the Large Intestine

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this investigation, and high interindividual variations in microbiota composition were found. The ratio of strict anaerobes to facultative anaerobes was reported to be 15 : 1 on the meatless diet, and 34 : 1 on the high-meat diet. When six human volunteers were given dietary fibre supplements comprising 5.4 grams per day wheat bran, mean stool weight increased from 103 ± 40 g to 226 ± 90 g, while total anaerobe counts were reported to increase significantly, particularly clostridia (Fuchs et al., 1976). This was also found in Japanese consuming high-fibre diets (Finegold et al., 1974). In another feeding study in which 10 g/day gum arabic were given to human volunteers, the proportion of faecal bacteria able to ferment the polysaccharides increased from 6% to 50%, which was attributed to adaptation of the microflora to the new substrate (Wyatt et al., 1986). The gum arabic degraders were identified as bifidobacteria and bacteroides. At the end of the feeding period, the microbiota returned to its original state. Other investigations have indicated that severe long-term changes in food intake, such as converting to a vegetarian diet, can have significant effects on the composition of the faecal microflora in rheumatoid arthritis patients (Peltonen et al., 1997), though these conclusions were made on the basis of changes in bacterial cellular fatty acid profiles in faeces, and it is not known which bacterial populations were affected. Other studies have demonstrated that inulin changes the species composition of the large intestinal microbiota, through reducing numbers of putrefactive microorganisms and increasing bifidobacterial populations (Gibson et al., 1995). In this study, bifidobacteria increased from 8% to 20% of the total anaerobe count. There are now many examples demonstrating the effects dietary oligosaccharides (prebiotics) have on the composition and structure of intestinal bacterial populations (Hayakawa et al., 1990; Gibson & Robertfroid, 1995; Buddington et al., 1996). In a recent investigation, the faecal microflora was found to be markedly changed in patients receiving total enteral or parenteral nutrition (Schneider et al., 2000). This study showed that marked reductions in anaerobes and facultative anaerobes occurred in subjects receiving parenteral nutrition, with many Gram-positive (e.g. eubacteria, bifidobacteria, peptostreptococci) and Gram-negative species (Veillonella, Bacteroides, Prevotella) disappearing entirely, whereas total enteral nutrition reduced faecal counts of anaerobic species, and increased numbers of facultative anaerobes (Enterococcus, Citrobacter, Acinetobacter). These observations were explicable on the basis that total parenteral nutrition deprived bacteria growing in the intestine of dietary substrates, thereby precipitating changes in microbiota composition, whereas the increased numbers of facultative species in enterally-fed patients was linked to diarrhoea. As is well known, some of the most striking effects of diet on bacterial communities in the large gut can be seen in the microbiotas of breast- and formula-fed infants. Although there are inconsistencies in the literature with respect to the composition of these developing bacterial communities, this may be a reflection of interindividual, cultural, environmental and methodological differences distinct to individual studies. Most investigations have indicated that strong differences in microbiota composition exist between these two groups of children, and that bifidobacteria, in particular, are the predominant organisms in babies receiving formula-based diets (Beerens et al., 1980; Stark & Lee, 1981). This can be seen in Fig. 2.2, which summarises results from two studies. In the first investigation by Benno et al. (1984), which comprised 35 breast-fed and 35 formula-fed

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Image Not Available

Fig. 2.2 Bacterial populations in breast- and bottle-fed infants, showing differences in bifidobacterial species composition between these two groups. Results are adapted from Benno et al. (1984) and Beerens et al. (1980). children, bifidobacteria were detected in high numbers in both groups, but enterobacteria, enterococci, anaerobic Gram-positive cocci, eubacteria, bacteroides and lactobacilli all occurred in considerably lower numbers in children being breast fed. In the second study, by Beerens et al. (1980), in which 81 bifidobacterial isolates were acquired from 12 faecal samples in breast-fed children, and 153 isolates were obtained from 39 stool samples taken from bottle-fed babies, B. bifidum were found to predominate in latter group, due to the presence of a range of growth-enhancing factors in human milk. Neither infant formulas nor cows' milk contain these substances, and consequently, they did not promote the growth of B. bifidum, although B. infantis and B. longum were found to be able to grow on these foods. In Japan, B. breve and B. infantis were reported to be the numerically important bifidobacteria in infant stools (Mitsuoka, 1989), however, B. pseudocatenulatum, B. adolescentis, B. longum and B. parabifidum were observed to be present in considerable numbers in breast-fed babies in Europe (Kleessen et al., 1995). One reason why breast-fed children have intestinal bifidobacterial populations distinct to those in bottle-fed children is that many different microbiologically active substances are known to be present in human milk such as glycoproteins and glycolipids, fucose, neuraminic acid, lactose, N-acetylglucosamine, a variety of oligosaccharides based on lactose (Miller et al., 1994), as well as bifidogenic nucleotides (Gil & Rueda, 2000). These compounds are not assimilated to a significant extent in the infant small intestine, which has led to the suggestion that they are selectively utilised as carbon and energy sources by bifidobacteria in the large bowel (Brand Miller et al., 1995). Diet and bacterial metabolism While it has been difficult historically to show significant changes in intestinal bacterial communities through modifying diet, a great many studies through the years have

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demonstrated that the types and amounts of food that we eat profoundly affects bacterial metabolism in the large intestine. This can be detected by measuring faecal bacterial enzyme activities, or specific products of bacterial metabolism, such as mutagens, secondary bile acids and steroid metabolites in faeces, or by following urinary excretion of bacterial fermentation products, such as phenolic and indolic metabolites. Thus, the inclusion of 15 g fibre/day to a low-fibre diet in 15 volunteers resulted in increased (35%) bacterial [3-glucuronidase synthesis, and increased (11%) excretion of acid steroids in faeces, while neutral steroids were reduced by 9% (Ross & Leklem, 1981). In a comparison of volunteers fed high- and low-meat diets, shifting from high to low meat significantly reduced faecal [3-glucuronidase (Reddy et al., 1974), which is an enzyme involved in the synthesis of mutagenic substances in the bowel. Similarly, individuals consuming a normal Western diet were found to have higher levels of this enzyme in their faeces, together with other enzymes involved in the formation of genotoxic compounds (7-[3dehydroxylase, nitroreductase) than vegetarians (Goldin et al., 1980). In contrast, more recent work feeding large doses of inulin (20—40 g/day) to elderly people with constipation showed that faecal levels of (3-glucuronidase and (3-glucosidase were largely unaffected, even by such large doses of fermentable carbohydrate, while there was no major changes in faecal short-chain fatty acids (SCFA) or lactate (Kleessen et al., 1997). The work of Christl et al. (1992) is summarised in Fig. 2.3. These workers showed in a study with six methanogenic volunteers, that adding 15 mmol sodium sulphate per day to the diet strongly inhibited breath methane excretion in three of the subjects, while stimulating the activities of dissimilatory sulphate-reducing bacteria (SRB) in faeces. SRB compete with methanogens for the mutual electron donor hydrogen in the gut, and after sulphate

Fig. 2.3 Effect of feeding 15 mmol sodium sulphate per day on breath methane excretion and the activities of faecal sulphate-reducing bacteria in human subjects.

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Gut Flora, Nutrition, Immunity and Health

feeding was stopped, these individuals began to revert to their original methanogenic status, as SRB lost the ability to compete for hydrogen in the absence of inorganic terminal electron acceptor (sulphate). The three volunteers who converted from being methanogenic on the high sulphate diet naturally harboured low, but detectable levels of SRB (ca. Log10 3.3 per gram) in their faeces, while those who did not respond to sulphate feeding had no detectable SRB at any time. The significance of this investigation is that it showed that even small changes in diet could result in profound ecological disturbances in the colonic microbiota. Moreover, because the principal metabolic product of SRB is sulphide, which is highly toxic to human colonocytes, stimulating the growth of SRB in the bowel could have long-term health implications for the host. Urinary sulphate results from oxidation of methionine and cysteine by the host, and intestinal bacteria are not involved in these processes. However, measurements of faecal sulphide and urinary sulphate levels in humans have demonstrated that the breakdown of dietary protein and bacterial fermentation of sulphur-containing amino acids by colonic bacteria is an important source of sulphide generation in the large bowel (Magee et al., 2000). Although faecal sulphide concentrations do not directly correspond to those in the large gut, this feeding trial, with five healthy subjects, showed that both faecal sulphide and urinary sulphate excretion were significantly related to meat intake (see Fig. 2.4), and that sulphurcontaining amino acids in meat were metabolised by both the host and the microbiota. Intestinal bacteria and vitamins Amongst their many activities, colonic microorganisms are involved in vitamin metabolism in several ways. They can directly synthesise vitamins that become available to the host, act

Fig. 2.4 Effect of increasing meat protein on bacterial production of sulphide (squares) in the large bowel, and urinary excretion of sulphate (circles).

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on foodstuffs to release vitamins, and actively assimilate, degrade or inhibit their uptake from the bowel. In rats, intestinal bacteria have been shown to be a significant source of vitamin B under some nutritional circumstances (Fridericia et al., 1927). Similarly, more than half of human vitamin K requirements are supplied by bacteria growing in the large bowel, through synthesis of menaquinones with vitamin K-like activity by anaerobic species, such as the bacteroides (Ramotar et al., 1984; Fernandez & Collins, 1987). Adults are generally resistant to deficiency in this vitamin (Suttie, 1985), however, haemorrhagic disease, resulting from vitamin K deficiency occurs occasionally in bottle-fed babies (von Kries et al., 1988). Human intestinal bifidobacteria are known to excrete water-soluble vitamins (Yasui et al., 1992). Marked species and strain differences occur, but high levels of folate, thiamine and nicotinic acid are known to be formed by B. bifidum and B. infantis strains, in comparison to B. breve and B. longum, while these vitamins are not produced by B. adolescentis to any significant degree. Vitamin B12 and pyridoxine are also synthesised by B. bifidum, B. breve, B. longum, B. infantis and B. adolescentis, but they do not appear to be excreted by these organisms (Deguchi et al., 1985).

Bacterial growth substrates in the large intestine Large communities of protein degrading and amino acid fermenting species exist in the colon, however, in numerical and metabolic terms, saccharolytic bacteria predominate in the microbiota (Finegold et al., 1974; Moore & Holdeman, 1974; Holdeman et al., 1977). The principal substrates available to gut microorganisms include endogenously produced mucopolysaccharides, such as salivary, bronchial, gastric hepatic, biliary, small and large intestinal mucins. Carbohydrate residues in the oligosaccharide side chains may account for as much as 85% of the mass of some mucin molecules (Smith & Podolski, 1986), which consist of varying amounts of fucose, galactose, N-acetyl glucosamine, N-acetyl galactosamine and neuraminic acid (Hoskins & Boulding, 1981). Tissue mucopolysaccharides such as hyaluronic acid and chondroitin sulphate, which contain glucuronic acid, N-acetyl glucosamine, glucuronic acid and N-acetyl galactosamine (Jeanloz, 1970) occur in association with desquamated epithelial cells. Pancreatic endopeptidases and other hydrolases and dietary carbohydrates, proteins and peptides that escape breakdown in the small bowel (Macfarlane & Cummings, 1991) are also digested and recycled by the microbiota. Ileostomy patients have provided useful information on the types and amounts of food that escape digestion in the upper GI tract. Dietary substrates are more important in quantitative terms than those of endogenous origin, and their utilisation by the microbiota is illustrated in Fig. 2.5. Substrates of dietary origin principally comprise plant storage polymers such as starches (Englyst & Cummings, 1987) and inulin (Brighenti et al., 1996), together with structural polysaccharides found in the plant cell wall (Englyst et al., 1988, 1989), gums and mucilages that provide protection against insects and microorganisms (Read & Eastwood, 1992), as well as non-digestible oligosaccharides (NDO) such as raffinose, galactooligosaccharides, stachyose and fructooligosaccharides (Wiggins, 1984; Ito et al., 1990; Gibson et al. 1995; Rumney & Rowland, 1995). It is difficult to determine the amounts of endogenous

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Gut Flora, Nutrition, Immunity and Health

Fig. 2.5 Scheme showing the significance of dietary carbohydrates and proteins to bacterial growth in the large intestine. carbohydrate broken down by bacteria in the large gut, and estimates of dietary substrate availability vary considerably, however, it is believed that about 8—18 grams of non-starch polysaccharides (NSP) (Bingham et al., 1990) and between 8 and 40 g starch (Englyst & Cummings, 1987) may enter the colon on a daily basis. The principal polysaccharide growth substrates available for intestinal bacteria and their metabolic fates in the gut are illustrated in Fig. 2.6. Starches and plant cell wall polysaccharides (dietary fibre) are quantitatively the most important sources of carbon and energy in the microbiota, as shown in the results of an ileostomy study by Andersson et al. (1996) in Fig. 2.7. Dietary fibre includes celluloses and non-cellulosic polysaccharides, such as pectins, hemicelluloses, mucilages and gums. These polymers principally consist of glucose, galactose, arabinose, xylose and galacturonic acids (Cummings & Branch, 1986; Englyst et al., 1987).

Breakdown of complex carbohydrates by intestinal bacteria Fermentation in the large bowel liberates large amounts of energy, estimated to be the equivalent of between 15 and 40 g carbohydrates and proteins (Andersson et al., 1996). Many factors affect the breakdown of complex polymerised carbohydrates in the large gut, including, cooking (Collings et al., 1981; Selvendran, 1985), their chemical composition (Englyst et al., 1987), their rheological properties in intestinal contents, which are related to molecular size (Morris, 1992), food particle size (Brodribb & Groves, 1978; Heller et al., 1980), the solubility of the substrate (Englyst et al., 1987) and its occurrence in complexes with non-digestible plant materials such as lignins (van Soest, 1975; Southgate et al., 1976). Colonic transit time also affects the way some recalcitrant polysaccharides such as cellulose and certain hemicelluloses are broken down, with long transit times propitiating the process (Hummel et al., 1943; Stephen et al., 1987). The significance of transit time on fermentation is also shown in in vitro modelling studies with faecal bacteria in Table 2.1.

1 S o Q. CD

Q. r-+ I? CD

Fig. 2.6

Metabolic fates of starches, inulin and dietary fibre (non-starch polysaccharides) in the human large intestine.

£ du CD <-*•

CD (/)

rH-

CD

CO CO

34

Gut Flora, Nutrition, Immunity and Health

Fig. 2.7 Recovery of different dietary carbohydrates in a feeding study with human ileostomists. The volunteers were fed a low-starch basal diet with either low (open bars) or high (closed bars) levels of dietary fibre. Table 2.1 Effect of system retention time on conversion of complex carbohydrates into SCFA by faecal bacteria grown in a three-stage model simulating the large intestine1 Retention time (h)

27.1 66.7 1

SCFA formed (g/L)

Carbohydrate utilised (g/L)

Apparent conversion

9.2 6.7

14.6 14.9

63.2 44.8

Results are from Macfarlane et al. (1998b).

In these experiments, which were carried out using a three-stage continuous culture model of the colon, the conversion of a mixture of complex polysaccharides to SCFA was studied at two different system retention (transit) times. The results showed that the efficiency of polysaccharide fermentation was inversely related to bacterial growth rate, which was attributed to increased bacterial maintenance energy requirements at low growth rates. Microbiological factors are also important determinants of polysaccharide degradation, since bacteria capable of synthesising depolymerising enzymes must be present in the gut in sufficient numbers for the process to occur to any great extent. Interestingly, relatively few intestinal bacteria are able to digest complex plant cell wall polysaccharides on their own, and most of those that do so belong to the genera Bacteroides and Bifidobacterium (Salyers & Leedle, 1983; Degnan, 1992; Macfarlane et al., 1995). Polysaccharide fermentation is a highly desirable process in the large bowel and it is intimately linked to colonic physiology. The concerted activities of these polymer degrading species, together with other cross-feeding saccharolytic and hydrogenotrophic organisms in

Food and the Large Intestine

35

the large bowel, give rise to a wide variety of fermentation products. The vast majority of these substances are absorbed and undergo a variety of metabolic fates in the body (see later). Few investigations have looked at the way individual bacteria and defined groups of intestinal microorganisms utilise polysaccharides, and except in broad terms, little is known of the processes in which the breakdown of these substances supports such a complex microbiota. However, in vitro modelling studies on polysaccharide digestion by Bacteroides thetaiotaomicron and Bifidobacterium adolescentis, in arabinogalactan-limited chemostats, showed that growth rate and pH affected the way the polymer was digested (Degnan & Macfarlane, 1995). The Bacteroides outcompeted Bif. adolescentis at all specific growth rates at culture pH values between 5.0 and 6.0. However, at pH 6.5, the Bifidobacterium predominated in co-cultures at dilution rates above 0.24 per hour. Arabinogalactan depolymerising enzymes (p-galactosidase, (3-arabinofuranosidase) were catabolite repressed in B. thetaiotaomicron at high growth rates, but were constitutive and growth rate-associated in the Bifidobacterium. Measurements of residual carbohydrate showed that B. thetaiotaomicron extensively digested the galactose backbone of the polysaccharide, and to a lesser degree, the arabinose sidechains. Despite this, arabinose monomers and short-chain oligosaccharides accumulated in the bacteroides cultures, showing that carbohydrate transport was a relatively inefficient process in this organism. Bifidobacterium adolescentis utilised considerably less arabinogalactan than the Bacteroides, suggesting that the Bifidobacterium was better adapted to growing on oligosaccharides. Although bacterial competition for substrate was the most important ecological process observed in these studies, the experiments highlighted the existence of synergistic interactions between the two species that were growth-rate dependent. Protein breakdown by gut microorganisms Analysis of digestive materials in different regions of the large bowel shows that large amounts of soluble protein and peptides are present throughout the gut (see Fig. 2.8). Approximately 13 g dietary and endogenously produced proteins enter the large intestine every day in ileal effluent (Wrong, 1988), but mucus and other secretions, together with a constant turnover of colonic epithelial cells also make a contribution to the pool of organic N-containing compounds in the colon (Cummings et al., 1989). Intestinal bacteria produce a variety of proteolytic and peptidolytic enzymes (Macfarlane & Macfarlane, 1995) that hydrolyse these complex macromolecules to peptides and amino acids, which can then be assimilated by the organisms. Relatively minor variations are observed in the compositions of faecal amino acid pools in healthy adults living on uncontrolled Western diets (Wilson et al., 1968; Owens & Padovan, 1975), and the generation of free amino acids from proteins in the large intestine (see Fig. 2.8) probably has little nutritional significance to the host, due to bacterial competition for the substrate, and the fact that the colon lacks absorptive machinery needed for their uptake (Wrong, 1988). Studies on amino acid metabolism in the gut show that disappearance of these substances can result from either direct assimilation by bacteria, deamination, decarboxylation, or by their binding to bacteria and host epithelial cells. Adibi and Mercer (1973) and Padovan et al. (1975) found low concentrations (< 10 mM) of free amino acids in faecal material, which had escaped absorption in ileal fluids. Since the

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Gut Flora, Nutrition, Immunity and Health

Image Not Available

Fig. 2.8 gut.

Physiological effects of protein breakdown and amino fermentation in the large

majority (80—85%) of nitrogen in the ileum occurs as proteins and peptides (Summerskill & Wolpert, 1970), one of the most important aspects of their metabolism in the colon is peptidolytic digestion. This is supported by the fact that intestinal anaerobes prefer nitrogen in the form of peptides or ammonia, rather than as free amino acids (Pittman et al., 1967). Toxological implications of amino acid fermentation A number of acidic metabolites result from protein breakdown, including SCFA, branched chain fatty acids (BCFA) and a range of other non-volatile organic acids (Macfarlane & Gibson, 1995). Other products of dissimilatory amino acid metabolism include carbon dioxide, molecular hydrogen, ammonia, amines, indoles and phenols (Smith & Macfarlane, 1996a, b). Many of these products are physiologically active in host tissues, with ammonia, phenols, indoles and amines being of particular toxicological significance. Protein breakdown and amino fermentation in the colon are increased if there are low levels of fermentable carbohydrates available (see below) and by long intestinal transit times (Cummings et al., 1979), and as shown in Fig. 2.8, this can have a number of undesirable consequences for the host. Thus, ammonia is known to alter the morphology and metabolism of cells lining the colonic mucosa, as well as increasing DNA synthesis and affecting their lifespan (Visek et al., 1978). Furthermore, high concentrations of ammonia in the large bowel may select for neoplastic proliferation (Clausen & Mortensen, 1992; Matsui et al., 1995). In patients with liver disease, ammonia formed by bacteria in the large gut contributes to the onset of portal systemic encephalopathy, or hepatic coma (Weber et al., 1987). Valerate has also been implicated in the causation of hepatic coma, when patients with cirrhosis have raised blood and cerebrospinal fluid levels of this fatty acid (Vince, 1986). The formation of BCFA by intestinal bacteria is not usually associated with toxicity, however, in children with isovaleric acidaemia, the onset of confusion and stupor is accompanied by a marked rise in this metabolite (Tanaka et al., 1976).

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Image Not Available

Fig. 2.9 Measurements of pH, proteins, peptides, SCFA and other bacterial metabolites associated with protein breakdown in human intestinal contents. Results are taken from Macfarlane et al. (1992a, b), Macfarlane et al. (1998a) and Smith and Macfarlane (1996a, b; 1998). Amines, phenols and indoles have been implicated in schizophrenia, migraine and the onset of hypertensive symptoms (Dalgliesh et al., 1958; Anon, 1968; Boulton et al., 1970; Zuccato et al., 1993). Phenolic and indolic compounds are produced from the aromatic amino acids tyrosine, phenylalanine and tryptophan. They are also thought to act as cocarcinogens (Dunning et al., 1950; Bryan, 1971; Zuccato et al., 1993), and have been related to hyperactivity in children, which is associated with elevated faecal levels ofp-cresol (Adams et al., 1985). Amine production by intestinal bacteria mainly results from the decarboxylation of amino acids, but these metabolites are also formed in transmethylation and N-dealkylation reactions, and by the degradation of polyamines. Increased amine production by intestinal bacteria is seen in diarrhoea in pigs, where putrescine and cadaverine excretion is particularly high (Porter & Kenworthy, 1969), and in humans, where children with gastroenteritis had significantly higher concentrations of tyramine and phenylethylamine in their faeces (Murray

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et al., 1986), though this probably resulted from the intestinal upset rather than being the cause. Amines are pharmacologically active in many ways, since some, including tyramine, histamine, cadaverine and putrescine, act as pressor or depressor substances, and may act as stimulators of gastric secretion or as vasodilators (Drasar & Hill, 1974), while secondary amines are precursors of N-nitroso compounds (Shephard et al., 1987), which are potent carcinogens. Effects of carbohydrate on amino acid fermentation In vitro fermentation experiments with faecal bacteria (see Table 2.2) studying the effects of pH and carbohydrate availability on peptide fermentation, demonstrated that two environmental characteristics of the proximal colon (low pH, high carbohydrate (starch) availability), reduced the rate of ammonia production. Starch was also shown to reduce the formation of BCFA from peptides by approximately 37%, however, low pH was found to be a more important determinant affecting formation of these metabolites (Smith & Macfarlane, 1998). In contrast, previous work (Smith & Macfarlane, 1996a, b) indicated that while low pH also reduced the formation of amines and phenolic compounds, carbohydrate was more effective.

Table 2.2 Effects of pH and carbohydrate availability on products of dissimilatory amino acid metabolism in faecal bacteria1

Image Not Available

1

Results are from in vitro fermentation studies with peptides as the source of organic N (Smith & Macfarlane, 1996a, b; 1998). Values in parentheses show per cent inhibition of fermentation product accumulation.

These observations show that by increasing bacterial requirements for organic Ncontaining compounds for use in biosynthetic reactions, and through fermentation acid production, carbohydrate availability plays a major role in regulating dissimilatory metabolism of peptides in the colon. While the ultimate nutritional determinant affecting the formation of phenols and amines in the large bowel is availability of organic nitrogencontaining compounds (proteins, peptides, amino acids), these fermentation studies indicate that these toxic metabolites can be reduced by increasing the supply of fermentable carbohydrate to the distal colon, where the levels of putrefactive products are greatest (Fig. 2.9). Consequently, a reduction of putrefactive processes in the large bowel may be readily achievable through relatively simple changes to diet.

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Short-chain fatty acids SCFA are the principal products of carbohydrate and protein fermentation in the large bowel (Macfarlane & Gibson, 1995). The vast majority of SCFA (> 95%) formed by intestinal bacteria are absorbed and metabolised by the host (Cummings, 1995). This allows the salvage of energy from food that is not digested in the upper GI tract, and can account for up to 9% of the hosts energy requirements (Hume, 1995). SCFA have a wide range of physiological functions in the body, including colonocyte metabolism (Roediger, 1980), cell growth and differentiation (deFazio et al., 1992), epithelial cell transport (del Castillo et al., 1994), metabolism of lipids and carbohydrates in the liver (Demigne & Remesy, 1994), intestinal motility (Cherbut et al., 1996), as well as energy generation in muscle, kidney, heart and brain (Macfarlane & Cummings, 1991). SCFA have also been shown to inhibit phagocytic cell function (Eftimiadi et al., 1990; Tonetti et al., 1991). Acetate is the major SCFA formed by gut microorganisms, followed by propionate and butyrate, which are usually present in broadly similar amounts. SCFA concentrations are higher in the proximal colon, where the acidity of gut contents (Fig. 2.9) reflects the high levels of fermentable carbohydrate that are available to bacteria in this region of the gut. SCFA concentrations in the bowel range between 100 and 180 mmol/kg gut contents, with acetate, propionate and butyrate ratios being in the region of 60: 20 : 20. Studies using intestinal material obtained from human sudden death victims (Cummings et al., 1987) found that acetate, propionate and butyrate ratios were similar in the proximal and distal large intestine, which was surprising because nutritional and environmental conditions are very different in these regions of the gut. However, subsequent studies demonstrated that spot measurements of SCFA levels in colonic contents only provide an indication of the equilibrium that exists between SCFA formation and colonic absorption of these metabolites, and that bacterial fermentation product formation varied quantitatively and qualitatively in different parts of the colon, particularly in relation to butyrate and products of amino acid fermentation (Macfarlane et al., 1995). Dissimilatory amino acid metabolism produces a wide range of SCFA, including the BCFA isobutyrate, isovalerate and 2-methylbutyrate, which are respectively formed from the branched chain amino acids valine, leucine and isoleucine. BCFA are therefore good indicators that amino acid fermentation is occurring. In vitro fermentation experiments have demonstrated that SCFA are the principal products of protein digestion in the gut (Macfarlane et al., 1986). Approximately 30% of protein is converted to SCFA, with BCFA production being dependent on the amino acid composition of the protein substrate. Fermentation studies with faecal bacteria demonstrated that BCFA constituted 16% of all SCFA produced from bovine serum albumin and 21% of SCFA generated when casein was the substrate (Macfarlane & Allison, 1986). Studies with intestinal material obtained from human sudden death victims showed that BCFA in the proximal and distal colons amounted to 3.4% and 7.5% of total SCFA (Macfarlane et al., 1992b). These measurements established that protein breakdown and amino acid fermentation accounted for about 17% of all SCFA in the lumen of the proximal large bowel, and 38% of SCFA produced in the distal colon.

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Effect of diet on SCFA production While the composition of the diet might intuitively be expected to be an important factor that affects SCFA formation in the large intestine, it has been difficult to demonstrate in human feeding studies. This is because the vast majority of bacterial fermentation products are taken up by the colon, with the result that faecal measurements of SCFA tell us little about fermentation processes occurring in the large bowel (Cummings & Macfarlane, 1991). This has proved to be the case in a number of investigations on the effects of dietary changes on SCFA production, where the majority of human studies show little or no effect (Fleming, 1992). However, faecal butyrate excretion was shown to increase after giving resistant starch to healthy volunteers (Munster et al., 1994), although this was not observed to occur in a subsequent study using this substrate (Cummings et a/., 1996). In vitro studies on SCFA production While it is difficult to investigate the stoichiometry of SCFA formation in human feeding experiments, this can be readily achieved in vitro. SCFA production from polymerised carbohydrates has been found to be dependent on many factors, including the numbers and types of hydrolytic and saccharolytic bacterial populations in the colonic microbiota, as well as the amount of substrate available and its chemical composition. This is demonstrated in Table 2.3, which shows the results of in vitro fermentation studies with faecal bacteria and a variety of different plant structural and storage polysaccharides. Table 2.3 Short-chain fatty acid production from complex carbohydrates in in vitro fermentation experiments1

Image Not Available

1

From Cummings (1995).

These results show that not only do individual polysaccharide substrates yield varying amounts of SCFA, but that the types of fermentation products are different. Interestingly, large amounts of acetate were produced from pectin, which mainly consists of relatively oxidised galacturonic acid residues, whereas starch was the best source of butyrate. The significance of starch as a precursor of this important SCFA has also been shown in a number of other studies (Macfarlane & Englyst, 1986; Englyst et al., 1987).

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SCFA and cell metabolism Although colonocytes can utilise glucose and glutamine as energy sources, epithelial cells lining the colon obtain up to 70% of their energy from SCFA (Roediger, 1980, 1989). Butyrate is the preferred fuel for the mucosa, but propionate and acetate are also metabolised. Colonocyte utilisation of butyrate in vivo can be demonstrated by comparing SCFA in portal and arterial blood, and in colonic contents. In human sudden death victims, butyrate molar ratios declined from 21% in the gut lumen, to 8% in portal blood, showing that about 65% of the butyrate was cleared by the mucosa (Cummings et al., 1987). The fall to 8% in portal blood indicates a clearance of 65% of the butyrate by the mucosa. Since acetate and propionate are also utilised by colonic epithelial cells, 65% is a minimum estimate of butyrate uptake. Observations of the trophic effects that SCFA have on the colonic epithelium have led to the suggestion these metabolites might facilitate tumourigenesis (Freeman, 1986; Jacobs and Lupton, 1986), however, studies with colonic biopsies indicate that this is unlikely to be the case (Scheppach, 1991). SCFA and colon cancer Butyrate has been shown to arrest cell growth early in Gl and induce cell differentiation, while stimulating cytoskeletal organisation and alterations in gene expression (Prasad & Sinha, 1976; Kruh, 1980; Fregeau et al., 1992; Hague et al., 1995). The arrest of cell growth by butyrate is associated with differentiation, and this occurs in many human cell lines. Butyrate modulates the expression of many different genes and differentiation in tumour cells and is linked to changes in their cytoskeletal architecture and adhesion properties (Wilson & Weiser, 1992). One of the most important mechanisms of butyrate action is on histone acetylation (Kruh et al., 1995), where inhibition of this process enables hyperacetylation of histones to occur, which facilitates access of DNA repair enzymes.

Lactate formation by gut microorganisms In the large intestine, lactate principally serves as an electron sink in carbohydrate fermentation. Unlike SCFA, the production of this organic acid is not directly coupled to energy generation, but is used to oxidise and recycle reduced pyridine nucleotides to maintain redox balance in fermentation reactions. These cofactors are generated in the initial stages of substrate catabolism, during glycolysis or in the pentose phosphate pathway, and must be continually reoxidised for fermentation reactions to continue. Lactate is formed by many colonic microorganisms, where both D- and L-enantiomers are produced. However, in the large intestine, relatively few organisms make D-lactate, and they mainly include species belonging to the Bacteroides fragilis group and the genus Lactobacillus (Holdeman et al., 1977). Measurements of lactate in material taken directly from the large intestine have demonstrated that it is mainly produced in the caecum and ascending colon (Macfarlane et al., 1992b). However, lactate concentrations are usually kept low in gut contents through the activities of cross-feeding bacterial populations such as desulfovibrios, bacteroides, propionibacteria, clostridia and enterobacteria, and because, like SCFA, lactate is efficiently absorbed by the colon.

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Fig. 2.10 Lactate is a major fermentation product of starch breakdown by intestinal bacteria in vivo, but not dietary fibre degradation, as shown by fermentation acid concentrations in human peripheral blood.

While D-lactate is continually being formed in the colon, it is normally a minor component of the lactate pool. This enantiomer is rapidly taken up from the large bowel and metabolised in the liver and kidneys (McNeil, 1982), but if there is an abnormal colonic fermentation in which high levels of carbohydrates are being broken down, D-lactate concentrations can become toxic, reaching levels as high as 20 mM in blood (Uribarri et al., 1998). Symptoms include ataxia, slurred speech, weakness, drowsiness and nausea. Faecal bacteriology usually shows a predominance of lactobacilli and bifidobacteria (Stolberg et al., 1982), while gut pH is usually below 5.0 (Caldarini et al., 1996), which probably selects for aciduric D-lactate producing species.

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Holdeman, L.V., Cato, E.P. & Moore, W.E.C. (eds) (1977) Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic and State University, Blacksburg. Hoskins, L.C. & Boulding, E.T. (1981) Mucin degradation in human colonic ecosystems. Journal of Clinical Investigation 67, 163—72. Hudson, M.J., Borrielo, S.P. & Hill, MJ. (1981) Elemental diets and the bacterial flora of the gastrointestinal tract. In: Elemental Diets (ed. R.I. Russell), pp. 105-25. CRC Press, Boca Raton. Hume, I.D. (1995) Flow dynamics of digesta and colonic fermentation. In: Physiological and Clinical Aspects of Short Chain Fatty Acid Metabolism (ed. J.H. Cummings, J.L. Rombeau & T. Sakata), pp. 119-32. Cambridge University Press, Cambridge. Hummel, F.C., Shepherd, M.L. & Macy, I.G. (1943) Disappearance of cellulose and hemicellulose from the digestive tract of children. Journal of Nutrition 25, 59-70. Hylemon, P.B. & Glass, T.L. (1983) Biotransformation of bile acids and cholesterol by the intestinal microflora. In: Human Intestinal Microflora in Health and Disease (ed. D.J. Hentges), pp. 189-213. Academic Press, New York. Ito, M., Deguchi, Y., Miyamori, A., Matsumoto, K. & Kikuch, H. (1990) Effects of administration of galactooligosaccharides on the human faecal microflora, stool weight and abdominal sensation. Microbial Ecology in Health and Disease 3, 285-92. Jacobs, L.R. & Lupton, J.R. (1986) Relationship between colonic luminal pH, cell proliferation and colon carcinogenesis in 1,2-dimethylhydrazine-treated rats fed high fiber diets. Cancer Research 46, 1727-34. Jeanloz, R.W. (1970) Mucopolysaccharides of higher animals. In: The Carbohydrates (ed. W. Pigman, D. Horton & A. Herp), pp. 589-625. Academic Press, New York. Kleessen, B., Bunke, H., Tovar, K., Noack, J. & Sawatzki, G. (1995) Influence of two infant formulas and human milk on the development of the faecal flora in newborn infants. Acta Paediatrica 84, 1347-56. Kleessen, B., Sykura, B., Zunft, H.-J. & Blaut, M. (1997) Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. American Journal of Clinical Nutrition 65, 1397—1402. Kruh, J. (1980) Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Molecular and Cellular Biochemistry 42, 65—82. Kruh, J., Defer, N. & Tichonicky, L. (1995) Effects of butyrate on cell proliferation and gene expression. In: Physiological and Clinical Aspects of Short Chain Fatty Acids (ed. J.H. Cummings, J.L. Rombeau & T. Sakata), pp. 275-88. Cambridge University Press, Cambridge. Macfarlane, G.T. & Allison, C. (1986) Utilization of protein by human gut bacteria. FEMS Microbiology Ecology 38, 19—24. Macfarlane, G.T. & Cummings, J.H. (1991) The colonic flora, fermentation and large bowel digestive function. In: The Large Intestine: Physiology, Pathophysiology and Disease (ed. S.F. Phillips, J.H. Pemberton & R.G. Shorter), pp. 51-92. Raven Press, New York. Macfarlane, G.T. & Englyst, H.N. (1986) Starch utilization by the human large intestinal microflora. Journal of Applied Bacteriology 60, 195—201. Macfarlane, G.T. & Gibson, G.R. (1995) Microbiological aspects of short chain fatty acid production in the large bowel. In: Physiological and Clinical Aspects of Short Chain Fatty Acid Metabolism (ed. J.H. Cummings, J.L. Rombeau & T. Sakata), pp. 87—105. Cambridge University Press, Cambridge.

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Macfarlane, G.T. & Macfarlane, S. (1992) Physiological and nutritional factors affecting the synthesis and secretion of extracellular metalloproteases by Clostridium bifermentans NCTC 2914. Applied and Environmental Microbiology 58, 1195-1200. Macfarlane, S. & Macfarlane, G.T. (1995) Proteolysis and amino acid fermentation. In: Human Colonic Bacteria: Role in Nutrition, Physiology and Pathology (ed. G.R. Gibson & G.T. Macfarlane), pp. 75-100. CRC Press, Boca Raton. Macfarlane, G.T., Cummings, J.H. & Allison, C. (1986) Protein degradation by human intestinal bacteria. Journal of General Microbiology 132, 1647—56. Macfarlane, G.T., Gibson, G.R., Beatty, E.R. & Cummings, J.H. (1992a) Estimation of short chain fatty acid production from protein by human intestinal bacteria, based on branched chain fatty acid measurements. FEMS Microbiology Ecology 101, 81—8. Macfarlane, G.T., Gibson, G.R. & Cummings, J.H. (1992b) Comparison of fermentation reactions in different regions of the colon. Jour nal of Applied Bacteriology 72, 57-64. Macfarlane, G.T., Gibson, G.R. & Macfarlane, S. (1994) Short chain fatty acid and lactate production by human intestinal bacteria grown in batch and continuous culture. In: Short Chain Fatty Acids (ed. HJ. Binder, J.H. Cummings & K. Soergel), pp. 44-60. Kluwer Academic Publishers, London. Macfarlane, G.T., Macfarlane, S. & Gibson, G.R. (1995) Co-culture of Biftdobacterium adolescentis and Bacteroides thetaiotaomicron in arabinogalactan-limited chemostats: Effects of dilution rate and pH. Anaerobe 1, 275—81. Macfarlane, G.T., Macfarlane, S., & Gibson, G.R. (1998a) Use of a three-stage compound continuous culture system to investigate bacterial growth and metabolism in the human colonic microbiota. Microbial Ecology 35, 180—87. Macfarlane, S., Quigley, M.E., Hopkins, M.J., Newton, D.F. & Macfarlane, G.T. (1998b). Effect of retention time on polysaccharide degradation by mixed populations of human colonic bacteria studied under multi-substrate limiting conditions in a three-stage compound continuous culture system. FEMS Microbiology Ecology 26, 231—43. Magee, E.A., Richardson, C.J., Hughes, R. & Cummings, J.H. (2000) Contribution of dietary protein to sulfide production in the large intestine: an in vitro and a controlled feeding study in humans. American Journal of Clinical Nutrition 72, 1488—94. Matsui, T., Matsukawa, Y., Sakai, T., Nakamura, K., Aoike, A. & Kawai, K. (1995) Effect of ammonia on cell-cycle progression of human gastric cancer cells. European Journal of Gastroenterology and Hepatology 7, S79-S81. McNeil, N.I. (1982) The absorption of lactate from the human large intestine. In: Colon and Nutrition (ed. H. Kasper & H. Goebell), pp. 141-3. MTP Press, Boston. Miller, J.B., Bull, S., Miller, J. & McVeagh, P. (1994) The oligosaccharide composition of human milk: Temporal and individual variations in monosaccharide components. Journal of Pediatric Gastroenterology and Nutrition 19, 371—6. Mitsuoka, T. (1989) Taxonomy and ecology of the indigenous intestinal bacteria. In: Recent Advances in Microbial Ecology (ed. T. Hattori, Y. Ishida, Y. Maruyama, R.Y. Morita & A. Uchida), pp. 493-8. Japan Scientific Societies Press, Tokyo. Moore, W.E.C. & Holdeman, L.V. (1974) Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Applied Microbiology 27, 961—79Morris, E.R. (1992) Physico-chemical properties of food polysaccharides. In: Dietary Fibre — a Component ofFood(ed. T.F. Schweizer & C.A. Edwards), pp. 41-56. Springer-Verlag, Berlin.

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Munster, I.P., Tangerman, A. & Nagengast, P.M. (1994) The effect of resistant starch on colonic fermentation, bile acid metabolism and mucosal proliferation. Digestive Diseases and Sciences 39, 834-42. Murray, K.E., Adams, R.F., Earl, J. & Shaw, KJ. (1986) Studies of the free amines of infants with gastroenteritis and of healthy infants. Gut 27, 1173—80. Norin, E. & Midtvedt, T. (2000) Interactions of bacteria with the host. Microbial Ecology in Health and Disease Supplement 2, 186—93. Owens, C.W.I. & Padovan, W. (1975) Quantitative method for estimating fecal amino acids. Clinical Chemistry 21, 1437-40. Padovan, W., Owens, C.W. & Ferguson, R. (1975) Creatinine and amino acid profiles of ileal and fecal fluids. Clinical Science and Molecular Medicine 49, 27P. Peltonen, R., Nenonen, M., Helve, T., Hanninen, O., Toivanen, P. & Eerola, E. (1997) Faecal microbial flora and disease activity in rheumatoid arthritis during a vegan diet. British Journal of Rheumatology 36, 64—8. Pittman, K.A., Lakshmanan, S. & Bryant, M.P. (1967) Oligopeptide uptake by Bacteroides ruminicola. Journal of Bacteriology 93, 1499—1508. Porter, P. & Kenworthy, R. (1969) A study of intestinal and urinary amines in pigs in relation to weaning. Research in Veterinary Science 10, 440—47. Prasad, K.N. & Sinha, P.K. (1976) Effect of sodium butyrate on mammalian cells in culture: a review. In Vitro 12, 125-32. Ramotar, K., Conly, J.M., Chubb, H. & Louie, T.J. (1984) Production of menaquinones by intestinal anaerobes. Journal of Infectious Diseases 150, 213-18. Read, N.W. & Eastwood, M.A. (1992) Gastro-intestinal physiology and function. In: Dietary Fibre — a Component of Food (ed. T.F. Sweitzer & C.A. Edwards), pp. 103—17. Springer-Verlag, Berlin. Reddy, B.S., Weisburger, J.H. & Wynder, E.L. (1974) Fecal bacterial (3-glucuronidase: control by diet. Science 183, 416-17. Roediger, W.E.W. (1980) Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa of man. Gut 21, 793—8. Roediger, W.E.W. (1989) Short chain fatty acids as metabolic regulators of ion absorption in the colon. Acta Veterinaria Scandinavica 86, 116—25. Ross, J.K. & Leklem, J.E. (1981) The effect of dietary citrus pectin on the excretion of human fecal neutral and acid steroids and the activity of 7-OC-dehydroxylase and p-glucuronidase. American Journal of Clinical Nutrition 34, 2068—77. Rumney, C. & Rowland, I.R. (1995) Non-digestible oligosaccharides - potential anti-cancer agents? British Nutrition Foundation Nutrition Bulletin 20, 194-203. Salyers, A.A. & Leedle, J.A.Z. (1983) Carbohydrate metabolism in the human colon. In: Human Intestinal Microflora in Health and Disease (ed. DJ. Hentges), pp. 129—46. Academic Press, New York. Selvendran, R.R. (1985) Developments in the chemistry and biochemistry of pectin and hemicellulosic polymers. Journat of Cell Science Supplement 2, 51—88. Shephard, S.E., Schlatter, C. & Lutz, W.K. (1987) N-Nitrosocompounds: relevance to human cancer. In: IARC Scientific Publications No. 57 (ed. H. Barrels, I.K. O'Neill & R.S. Herman), pp. 328-32. IARC Scientific Publications, Lyons. Scheppach, W.M. (1991) Short chain fatty acids are a trophic factor for the human colonic

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von Kries, R, Gobel, U., Shearer, MJ. & McCarthy, P.T. (1988) Vitamin K deficiency in breast-fed infants. Journal of Pediatrics 107, 650—51. Vince, A.J. (1986) Metabolism of ammonia, urea, and amino acids, and their significance in liver disease. In: Microbial Metabolism in the Digestive Tract (ed. MJ. Hill), pp. 83—105. CRC Press, Boca Raton. Visek, W.J., Clintron, S.K. & Truex, C.R. (1978) Nutritional and experimental carcinogenesis. Cornell Veterinarian 68, 3-39Weber, F.L., Banwell, J.G., Fresard, K.M. & Cummings, J.H. (1987) Nitrogen in fecal bacteria, fiber and soluble fractions of patients with cirrhosis: effects of lactulose and lactulose plus neomycin. Journal of Laboratory and Clinical Medicine 110, 259—63. Wiggins, H.S. (1984) Nutritional value of sugars and related compounds undigested in the gut. Proceedings of the Nutrition Society 43, 69-75. Wiggins, H.S. & Cummings, J.H. (1976) Evidence for the mixing of residue in the human gut. Gut 17, 1007-11. Wilson, D.R., Ing, T.S., Metcalfe-Gibson, A. & Wrong, O.M. (1968) In vivo dialysis of faeces as a method of stool analysis. III. The effect of intestinal antibiotics. Clinical Science 34, 211-21. Wilson, J.R. & Weiser, M.M. (1992) Colonic cancer cell (HT29) adhesion to laminin is altered by differentiation: adhesion may involve galactosyltransferase. Experimental Cell Research 201, 330-34. Wrong, O.M. (1988) Bacterial metabolism of protein and endogenous nitrogen compounds. In: Role of the Gut Flora in Toxicity and Cancer (ed. I.R. Rowland), pp. 227-62. Academic Press, New York. Wyatt, G.M., Bayliss, C.E. & Holcroft J.D. (1986) A change in human faecal flora in response to inclusion of gum arabic in the diet. British Journal of Nutrition 55, 261—6. Xu, J., Xu, X. & Verstraete, W. (2001) The bactericidal effect and chemical reactions of acidified nitrite under conditions simulating the stomach, journal of Applied Microbiology 90, 523-9. Yasui, H., Nagaoka, N., Mike, A., Hayakawa, K. & Ohwaki, M. (1992) Detection of Bifidobacterium strains that induce large quantities of IgA. Microbial Ecology in Health and Disease 5, 155-62. Zuccato, E., Venturi, M., Leo, G.D. et al. (1993) Role of bile acids and metabolic activity of colonic bacteria after cholecystectomy. Digestive Disease Science 38, 514—19.

The Health Benefits of Probiotics and Prebiotics

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G.R. Gibson,1 R.A. Rastall1 & R. Fuller2 1

Food Microbial Sciences Unit, School of Food Biosciences, University of Reading, Reading RG6 6BZ, UK, and2Russet House, Ryeish Green, Reading RG7 1ES, UK

Summary The rapid increase in the cost of pharmaceutical preparations and the rise in resistance, due to overuse of antibiotics has led to a search for alternative ways of influencing the composition of the gut microflora. Attention has turned towards food materials that may offer improved health benefits. So-called 'functional' foods have a currently high profile and many new products exist or are being developed. Early approaches involved vitamin and mineral supplementation to the diet. However, recent developments have focused on gut functionality which is a realistic target for enhanced food ingredients. Using diet to prophylactically manage the gut flora is both user friendly and attractive to the consumer. One popular approach is to include live microbes in the diet (probiotics). Another is to use non-viable food components (carbohydrates) that are preferentially utilised by certain components of the gut flora. Such prebiotics serve to induce beneficial effects by stimulating probiotic bacteria already present in the gut.

Introduction The bacterial microbiota in the human large intestine is thought to comprise 95% of the total cells in the body, representing 1012 cells/g dry weight faeces. Through the activities of the resident microflora, the colon plays a major role in host nutrition and health (Salminen et al., 1998). The gastrointestinal tract is sterile at birth and bacterial colonisation begins during the delivery process (from the maternal faecal or vaginal flora and/or the environment). The first bacteria to colonise the colon are facultatively anaerobic strains such as Escherichia coli and streptococci. These initial colonisers metabolise any traces of oxygen in the gut thereby

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reducing the environment into one of strong anaerobic conditions. The type of bacteria that subsequently colonise the gut depends largely on the feeding habits of the infant. Breast-fed infants develop a different gut flora to those on formula feeds. The breast-fed infant has bifidobacteria as the numerically predominant genus, whereas formula feeds give rise to a more complex, adult-like gut flora containing bifidobacteria but with no one genus showing a particular predominance (Stark & Lee, 1982; Benno et al., 1984). There is also evidence which shows that different species of bifidobacteria colonise the guts of breast-fed and formula-fed babies (Beerens et al., 1980). A major reason for this difference may be that breast milk contains a 'bifidus' factor (glycoprotein containing glucose, galactose, fructose and N-acetyl glucosamine) which stimulates the growth of specific bifidobacteria at the expense of other species (Yuhara et al., 1983; Yoshioka et al., 1991). Breast-fed infants generally have fewer gastrointestinal problems than their formula-fed counterparts and this may be attributed to the powerful anti-pathogen effects exerted by the bifidobacteria. The final phase of microflora acquisition occurs at weaning when a complex microflora develops. The resident gut microbiota ferments substances that cannot be digested by the host in the small gut, these substances include resistant starch, non-digestible carbohydrates and mucins (Table 3.1). The two main types of fermentation that are carried out in the gut are saccharolytic and proteolytic (Table 3.2). Saccharolytic fermentation is more favourable than a proteolytic fermentation due to the byproducts that are produced. The main end-products of saccharolytic fermentation are the short-chain fatty acids, acetate, propionate and butyrate. Acetate is metabolised in systemic areas like muscle tissues; similarly propionate is transported to the liver where it may interfere with cholesterol synthesis. Butyrate is an important source of energy for the colonocytes and has antitumour properties (Kruh et al., 1995). The end-products of proteolytic fermentation on the other hand, include nitrogenous metabolites (such as phenolic compounds, amines and ammonia), some of which are carcinogens. The proximal gut (right side) is essentially a site of saccharolytic fermentation, whereas the more distal (left side) sees more proteolytic fermentation (Macfarlane et al., 1992). This may be one reason why many gastrointestinal disorders (including ulcerative colitis and colon cancer) occur distally. An aim of prebiotics is to increase the amount of carbohydrate that reaches the colon. This would increase the amount of saccharolytic fermentation throughout the gut and reduce proteolysis. It is therefore apparent that activities in the gut microbiota can exert both positive and negative influences on health. This has led to the development of dietary ingredients that can fortify the former at the expense of the latter. Dietary modulation of the gut flora has been carried out for many years with the use of probiotics. Probiotics are live microbial feed additions that beneficially affect the host by improving its intestinal microbial balance (Fuller, 1989, 1992, 1997; Fuller & Gibson, 1997). Metchnikoff (1907) first described the action of probiotics. Probiotics are now widely available in most supermarkets and health food outlets, often in the form of fermented milk products but also as tablets, powders or capsules. The microorganisms after ingestion enter the gut and persist long enough for beneficial effects to be manifested. Prebiotics aim to stimulate certain indigenous bacteria in the gut rather than introduce viable bacteria as is the case with probiotics (Gibson & Roberfroid, 1995, 1999). The diet

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Table 3.1

Predominant products of carbohydrate metabolism in the human colon

End-product

Bacterial group involved

Metabolic fate

Acetate

Bacteroides, bifidobacteria, eubacteria, lactobacilli, clostridia, ruminococci, peptococci, veillonella, peptostreptococci, propionibacteria, fusobacteria, butyrivibrio

Metabolised in muscle, kidney, heart and brain

Propionate

Bacteroides, propionibacteria, veillonella

Cleared by the liver, possible glucogenic precursor, suppresses cholesterol synthesis

Butyrate

Clostridia, fusobacteria, butyrivibrio, eubacteria, peptostreptococci

Metabolised by the colonic epithelium, regulator of cell growth and differentiation

Ethanol, succinate, lactate, pyruvate

Bacteroides, bifidobacteria, lactobacilli, eubacteria, peptostreptococci, clostridia, ruminococci, actinomycetes, enterococci, fusobacteria

Absorbed, electron sink products, further fermented to SCFA

Hydrogen

Clostridia, ruminococci, fusobacteria

Partially excreted in breath, metabolised by hydrogenotrophic bacteria

contains non-digestible carbohydrates that are selectively fermented by indigenous beneficial bacteria. Lactic acid producing bacteria, especially bifidobacteria and lactobacilli, are the prime candidates targeted by prebiotics. Any compound that reaches the colon intact is a potential prebiotic. However, much of the interest in the development of new prebiotics is aimed at the non-digestible oligosaccharides such as fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides and soyaoligosaccharides, and the disaccharide lactulose. For a food ingredient to be classified as a prebiotic, it must meet the following criteria: (1) not be hydrolysed or absorbed in the upper part of the gastrointestinal tract; (2) be a selective substrate for one or a limited number of potentially beneficial bacteria commensal to the colon, e.g. bifidobacteria and lactobacilli, which are stimulated to grow; and (3) be able, as a consequence, to alter the colonic microflora towards a potentially more healthy composition and/or activity (Gibson & Roberfroid, 1995).

Health Benefits of Probiotics and Prebiotics

Table 3.2

55

Type of substrates available for bacterial growth in the human large intestine

Substrate Resistant starch Non-starch polysaccharides Unabsorbed sugars Oligosaccharides Chitins, amino sugars, synthetic carbohydrates, food additives Dietary protein Mucins Bacterial recycling Sloughed epithelial cells

Estimated quantity (g/day) 8-40 8-18 2-10 2-8 ? 3-9 ? ? ?

Probiotics It is clear that diet plays an important role in the maintenance and improvement of human health through the provision of growth substrates for the microbiota. To generalise, it is possible to categorise the gut microbiota components on the basis of whether they exert potentially pathogenic or health-promoting aspects (Gibson & Roberfroid, 1995; Naidu et al., 1999). Lactic acid producing genera such as the bifidobacteria or lactobacilli have along standing 'health image'. As such, attempts to stimulate microorganisms that carry out the latter could give many benefits. Composition ofprobiotic preparations The beneficial effects of probiotics have been widely reported (Fuller, 1992, 1997). Probiotic preparations and fermented milks containing viable cultures perceived as beneficial (e.g. lactobacilli, bifidobacteria) are used to establish populations in the gastrointestinal tract. To be effective, probiotics must be capable of being prepared in a viable manner and on a large scale (e.g. for industrial purposes), and during use and under storage the probiotic should remain viable and stable, be able to survive in the intestinal ecosystem and the host animal should gain beneficially from harbouring the probiotic. Clearly, the organisms should not have any adverse effects on the consumer. The organisms currently being used in probiotic preparations are either ones which have had a long use in food products without showing any side effects or they belong to that group of organisms which the Food and Drug Administration of the USA categorise as GRAS (generally regarded as safe), i.e. not known to have any pathogenic potential. For any potential benefits ofprobiotic use to become manifest, the micoorganisms must reach their target area in high enough numbers, should be resistant to gastric secretions and be relatively stable in bile acids present in the small gut. One added advantage would be good viability in the product and an ability to adhere to intestinal cells for increased persistence (Alander et al., 1999). Probiotic use in humans has a long history of use; the first reports on the ingestion of

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'soured milks' by Nomads appeared over two thousand years ago. Ellie Metchnikoff wrote a book called the Prolongation of Life about 100 years ago describing experiments with pure cultures. He believed that the complex microbial population in the colon was adversely affecting the host through so-called 'autointoxication'. However, in the early 1900s Metchnikoff (1907) observed longevity in Bulgarians and suggested that this may be linked to their gut flora and specifically to an elevated intake of milks with lactic acid bacteria in them (these would now be called probiotics). As such, he abandoned his practice of surgical removal of the colon, and began modification of the activity of the colonic microflora by the ingestion of soured milks. This was the beginning of the probiotic story. A Gram-positive rod, which he called the Bulgarian bacillus and later Bacillus bulgaricus, is probably the organism later known as Lactobacillus bulgaricus. This is now called L. delbrueckii subsp. bulgaricus, which together with Streptococcus thermophilus is responsible for the fermentation of milk to form traditional yoghurt. Over the years, many species of microorganism have been used as probiotics (Mitsuoka, 1990; Bengmark, 1996). They consist mainly of lactic acid bacteria (lactobacilli, streptococci, enterococci, lactococci, bifidobacteria) but also Bacillus spp. and fungi such as Saccharomyces spp. and Aspergillus spp. The commonest probiotics belong to the genera Lactobacillus (e.g. L. cam, L. acidophilus, L. rhamnosus, L. johnsonii, L. reuteri) and Bifidobacterium (e.g. B. bifidum, B. longum, B. breve). Three important observations stimulated the development of probiotics for human use. These all indicated that gut contents contained bacterial species that were 'protective'. The findings were: (1) (2) (3)

Germfree animals are more susceptible to infection than conventional counterparts i.e. those with bacteria in their gut. Oral antibiotics increased the susceptibility of animals to infection, implying that the resident microbiota normally exerted 'colonisation resistance'. Administration of faecal enemas could control antibiotic-associated diarrhoea normally caused by Clostridium difficile.

It is estimated that well over 10 million people in Europe regularly (each day) consume probiotics. The European market value is in excess of one billion euros per annum. Many different products exist and new developments are continuing at a rapid pace. Probiotics are marketed as health or functional foods, and claims are made for health benefits resulting from changes induced in the gut flora. There is a need for consumers to be provided with an independent assessment of physiological, microbial and safety aspects of these live microbial products and their ability to improve health. The approach of using diet to prophylactically manage the gut flora is both user friendly and attractive to the consumer. Currently the benefits being claimed are: improved resistance to infections, irritable bowel syndrome, chronic gut disorder (inflammatory bowel disease, colon cancer), lactose intolerance, coronary heart disease, recurrent vaginal thrush, skin problems, food allergy and enhancement of mineral bioavailability (De Vuyst & Vandamme, 1994; Collins & Gibson, 1999; Gibson & Angus, 2000; Shanahan, 2000; Hamilton-Miller, 2001; Madsen et al., 2001; Reid et al., 2001). Not all probiotics will be able to achieve these aims. For probiotics to exert any beneficial properties they must have robust survival properties in the gut, which is their first point of contact (Tannock, 1999). Moreover, they should not adversely affect immune up-regulation, produce toxins, disrupt colonocyte

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function or have the ability to transfer antibiotic resistance to the normal gut microflora (Sperti, 1971; Parker, 1974; Havennar & Huis in't Veld, 1992; Alander et al., 1997, 1999). A recent formal definition of probiotics was agreed by a working party of European scientists and is given as 'a live microbial feed supplement that is beneficial to health' (Salminen et al., 1998). This emphasised the importance of definitive improvements in health as well as the possibility that probiotics could have effects outside the gut, e.g. vagina, skin, mouth. The market progression in recent years has also been rapid and there are now many examples of foods that contain probiotics. These include: Live yoghurts: also called bio, active, bifidus. Manufacturers like Nestle, St Ivel, Danone, Onken, Vifit, introduced probiotic bacteria into yoghurt. Similarly the supermarkets also have their own versions. These bioyoghurts differ from conventional yoghurts in that other strains are added, or used in manufacture, in addition to the standard starter species (i.e. Lactobatillus delbreuckii subsp. bulgaricus, Streptococcus thermophilus}. Fermented dairy drinks: these liquid products contain high microbial numbers (up to 109 per mL). Examples are Yakult (Lactobacillus casei Shirota), Danone's Actimel (Lactobacillus cam Immunitass) and Nestle's LClGo (Lactobacillus johnsonii). Freeze-dried supplements: freeze drying of microbial cultures allows extremely high numbers to be incorporated into capsules and tablets. Examples in the UK are Multibionta which contains Lactobacillus acidophilus, Bifidobacterium bifidum and Bifidobacterium longum, Nature's Sunshine which contains several lactic acid bacteria and Protexin which contains a Streptococcus, two bifidobacteria and four lactobacilli. Other products exist, some of which include yeasts such as Saccharomyces cerevisiae and S. boulardii. Cheese: an example of a probiotic cheese is Inner Gut (Anchor). Squeezable yoghurt: available in tubes, e.g. Yo Squeez for children. Fromage Frais: some products contain live bifidobacteria and/or lactobacilli, e.g. Yoplait. Fruit juice: a widely used form is Proviva which contains Lactobacillus plantarum 299V.

Tracking probiotics through the gut Hamilton-Miller et al. (1999) examined bioyoghurts and other probiotic preparations for accuracy of their label descriptions. None of the bioyoghurts stated the viable count. Of the 37 other preparations tested, the majority gave an indication of the viable numbers present. Only about half the bioyoghurts accurately described the organisms in the product while the remainder used vague terms like 'special' or 'selected' to describe the flora. Of the 37 other products only 16 accurately fulfilled the label claims with respect to species present. Because there is variation between strains of the same species, it is important not only to identify the species but also to state which strain is being used. Four cases where this is done are L. rhamnosus GG, L. johnsonii LCI, L. casei Shirota and L. casei immunitass. A great deal more needs to be done to define microbiologically the content of probiotic preparations and with the advent of modern molecular-based techniques this can be done with accuracy. Probiotic trials should use the best methodologies available. One difficulty lies in the recovery of ingested strains from faeces by traditional methods involving culture. It is often

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difficult to separate the probiotic strains from the indigenous flora. Moreover, it would appear that a significant proportion of the human gut microbiota is 'non-culturable'. Consequently, there has been a recent move away from conventional cultural procedures coupled with phenotypic (morphological, biochemical) assessments of culture identity, towards more sophisticated molecular procedures. These include nucleic acid fingerprinting studies for reliable identification as well as the development of genetic probing systems such that predominant components of the gut flora can be quantified without resort to culture (Steer et al., 2000). The genotypic identity of gut bacteria can be investigated by polymerase chain reaction (PCR) 16S rRNA gene restriction fragment length polymorphism (RFLP) and partial gene sequence analysis. 16S rRNA consists of a mosaic of universal, semi-conserved and nonconserved regions and enables the molecule to span great, as well as to measure close, genetic relationships. The 'sequences' of the hypervariable moieties of the molecule are characteristic of different organisms and provide a rapid and reproducible means of determining genotype. Full 16S rRNA genes can be amplified from single colonies utilising primers to conserved regions proximal to the 5' and 3' termini of the gene. rDNA products will be subjected to RFLP analysis (restriction with endonuclease and electrophoretic analysis), and each bacterial type characterised by a simple, but highly specific, series of rDNA restriction patterns. A comprehensive rDNA signature database of high discriminatory potential is established against which isolates can be compared and identified. Isolates which give rise to unknown or novel rDNA patterns are subjected to detailed phylogenetic characterisation. rDNA products can be sequenced directly using automated Taq-cycle sequencing. New sequences are compared with sequences available in GenBank/EMBL Data libraries, and phylogenetic analyses (e.g. distance and parsimony methods) used to determine their precise identity and/ or whether they constitute hitherto unrecognised species/taxa. Molecular characterisation may also be performed using nested-probes. It is well established that on various taxonomic levels, phylogenetic groups are characterised by 'regional rRNA sequence idiosyncrasies', socalled signatures. Bacteria can be assigned to a variety of taxonomic groups by analysing such signatures. Oligonucleotide probes, ranging from high (e.g. domains Archaea) to low (e.g. genus, species) taxonomic levels are used to screen for such signatures. Applying such probes to parallel sub-samples in an ordered top-to-bottom approach (initially using domainspecific probes followed by probes of increasingly narrow specificity), increases refined information on community diversity. These kinds of technologies are now being applied to probiotics in order to check the efficacy of labelling as well as detecting the strains in mixed culture environments like faeces. They can also be coupled with genetic probing strategies to quantify the gut flora. One example is fluorescent in situ hybridisation (FISH). This involves the design of genotypic probes which specifically hybridise to a unique sequence(s) of the 16S rRNA of the target microorganism (Langendijk et al., 1995). An oligonucleotide sequence specific to a probiotic strain would facilitate the use of conventional PCR amplification techniques and exploitation of the FISH technique. For this, a specific oligonucleotide probe could be labelled with a fluorescent protein so as to mark the probiotic strain, thus facilitating differentiation from commensal strains. The FISH technique can be used to quantify the numerically predominant gut genera (Harmsen et al., 1999). Genotypic probes targeting the bifldobacteria (Bifl64: Langendijk et al., 1995), bacteroides (Bac303: Manz et al., 1996), clostridia

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(Hisl50: Franks et al., 1998) and lactobacilli (Labl58: 5'GGTATTAGCA(T/ C)CTGTTTCCA) are routinely used. The nucleic acid stain DAPI (4'6-diamidino-2phenylindole) is also useful for quantifying total bacterial counts.

Prebiotics Human breast milk is the classical (and original) prebiotic factor. This may be affecting the composition of the bifidobacterial flora in the gut of breast-fed infants. The specific entity responsible is probably a glycoprotein, either singularly or in combination. However, this has not been definitively determined and their purification and production in levels required to fortify foods is not economically feasible. Therefore, the search for prebiotic factors has moved towards other fermentable components in the diet, preferably involving substances that are readily available. Research on bifidobacteria-promoting substances began by screening a range of carbon sources for their ability to increase these organisms in pure culture. Much of the early work was carried out in Japan (e.g. Yazawa et al., 1978). A range of oligosaccharides and polysaccharides were screened for their ability to promote Bifidobacterium infantis and Bifidobacterium breve in pure culture and compared to the effect on Escherichia coli, Streptococcus faecalis and Lactobatillus acidophilus as representatives of other intestinal bacteria. Of all the substrates tested, B. infantis utilised a range of mono- and disaccharides, maltotriose (Glu otl-4 Glu ocl-4 Glu), raffinose (Gal al-6 Glu al-2f5 Fru), stachyose (Gal al-6 Gal al-6 Glu al-2(3 Fru), inulin (Glu al-2{(3 Fru l-2]n where n > 10) and oligosaccharides from cellulose, amylose, dextran and inulin. Of these, raffinose, stachyose, inulin of low molecular weight (< 4500) and tri- to penta-saccharides from dextran were useful substrates for bifidobacteria. Yazawa and Tamura (1982) suggested that the selectivity for bifidobacteria may be improved by a high molecular weight and a fructose molecule at the reducing end of the sugar. It has been concluded that bifidobacteria need sugar sources to supply energy and cellular components, and this results in the production of lactic and acetic acids which can inhibit the growth of potential pathogens (Tamura, 1983). Generally, di-, tri- and tetrasaccharides consisting of glucose, galactose and fructose monomers were well utilised by bifidobacteria (Tamura, 1983). Yazawa and Tamura (1982) concluded that raffinose was a good bifidobacterial-promoting factor. Minami et al. (1983) used a similar method to evaluate the fermentation of enzymatically synthesised isogalactobiose (Gal (3-(3 Gal), galsucrose (Gal a-f3 Fru) and lactosucrose (Gal P14oc Glu a 1-2 f3 Fru) in comparison to raffinose, lactose and glucose. Lactose and glucose were utilised by Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis, L. acidophilus, E. coli and S. faecalis but the other four sugars were used only by the bifidobacteria. Whilst such techniques are useful baseline studies, it is the selectivity of fermentation that is a major prerequisite for prebiotics. In this case, mixed culture experiments are required. Oligosaccharides as prebiotics A range of oligosaccharides have been tested using various in vitro methods, animal models and human clinical trials.

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Lactulose Lactulose is a synthetic disaccharide in the form Gal pl-4 Fru. Lactulose was originally used as a laxative as it is not hydrolysed or absorbed in the small intestine (Saunders & Wiggins, 1981). Lactulose has also received attention as a bifidogenic factor and has been administered as such (Tamura, 1983; Modler et al., 1990; Modler, 1994). Lactulose increased lactobacilli and bifidobacteria and significantly decreased bacteroides in a mixed continuous faecal culture (Fadden & Owen, 1992), although total bacterial numbers decreased. Bifidobacteria significantly increased while Cl. perfringens, Bacteroides, streptococci and Enterobacteriaceae significantly decreased on feeding lactulose to eight humans at 3 g/day for 14 days (Terada et al., 1992). In addition, decreases in the potentially harmful metabolites ammonia, indole, phenol, p-cresol and skatole, and enzymes (3glucuronidase, nitroreductase and azoreductase supported the beneficial claims made for lactulose. Although lactulose possesses prebiotic activity, it is not yet widely distributed as such. At high doses it is frequently used as a laxative. Inulin and Fructooligosaccharides Inulin is a polysaccharide of the form Glu al-2[|3 Fru 1-2]n where n > 10 (Crittenden & Playne, 1996). The structural relatives of inulin, fructooligosaccharides (FOS, a lower molecular weight version) have been the best documented oligosaccharides for their effect on intestinal bifidobacteria and are considered important prebiotic substrates. They are produced in large quantities in several countries and are added to various products such as biscuits, drinks, yoghurts, breakfast cereals and sweeteners (Mizota, 1996). The term 'fructooligosaccharides' may be used to represent two different preparations. Firstly, inulin extracted from chicory roots can be hydrolysed under controlled conditions by the enzyme inulinase (Crittenden, 1999) to produce short-chain inulin molecules known as oligofructose (OF) represented as Glu OC1-2[P Fru 1-2]n where n = 2—9. Inulin also occurs naturally in Western foods such as onion, asparagus, leek, garlic, wheat and artichoke, although to a lesser extent than in chicory (Gibson et al., 1994). Another FOS product known as 'Neosugar' or 'Meioligo' is a mixture of three oligosaccharides of different lengths, i.e. 1-kestose (Glu-Fru2), 1-nystose (Glu-Fru3) and lF-(3-fructofuranosylnystose (Glu-Fni4) (Hidaka et al., 1986). The mixture is enzymatically synthesised from sucrose by the transfructosylation action of p-fructofuranosidase from the fungus Aspergillus niger (Hidaka et al., 1986). Batch culture studies where faecal slurries were incubated with inulin, OF, starch, polydextrose, fructose and pectin for 12 hours (Wang & Gibson, 1993) showed the greatest increase in bifidobacteria with OF and inulin, indicating the prebiotic nature of these substrates. Continuous culture systems inoculated with faecal slurries were later used to investigate FOS fermentation (Gibson & Wang, 1994a, b). In accordance with earlier studies, bifidobacteria, and to a lesser extent lactobacilli preferred OF and inulin to glucose, whereas Bacteroides could not grow on OF. By varying parameters in the chemostat, the optimum conditions for growth of bifidobacteria but inhibition of Bacteroides, clostridia and coliforms were concluded to be: low pH (pH 5.5), high culture dilution rate (0.3/hour) and 1% (w/v) concentration of carbohydrate, i.e. similar to the physicochemical environment of the proximal colon. Three-stage chemostats, confirmed the enhanced proliferation of bifidobacteria by OF in conditions resembling the proximal colon (Gibson & Wang, 1994a; McBain & Macfarlane, 1997).

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A later single-stage chemostat study, with FOS (Sghir et al., 1998) demonstrated discrepancies between classical microbiological techniques and molecular approaches. Agar plate counts showed an increase in the combined populations of bifidobacteria and lactobacilli to reach 98.7% of the total bacterial flora by steady state. However, 16S rRNA genusspecific probes indicated an initial increase in the bifidobacterial population which decreased after six days, whilst lactobacilli thrived in the low pH fermenter (pH 5.2—5.4) maintaining a high population at steady state. Changes observed in the short-chain fatty acid (SCFA) profile corresponded well with the population data obtained through probe methods. Rats that were previously fed tyrosine and tryptophan (capable of producing putrefactive products) were administered a 10% (w/v) Neosugar diet, and this resulted in increased SCFA, decreased faecal pH and significantly decreased concentrations of the tyrosine derivatives phenol andp-cresol (Hidaka et al., 1986). Several studies have been conducted using human subjects although the dose, substrate, duration and volunteers vary. A general observation was the greater bifidogenic effect of substrates in subjects with a low initial bifidobacterial count (107/g faeces) than in those with high initial numbers (109.5/g faeces) (Hidaka et al., 1986). Also, a negative correlation between bifidobacteria and Cl. perfringens was observed suggesting that the former may inhibit growth of the latter in the intestine, supporting earlier studies (Wang & Gibson, 1993; Gibson & Wang, 1994c). There were large variations between the subjects in their microflora compositions and response to the substrates (Hidaka et al., 1986; Williams et al., 1994; Buddington et al., 1996), particularly between Western and Eastern subjects (Buddington et al., 1996). Another general observation was the decrease in bifidobacteria once administration of FOS ceased (Bouhnik et al., 1994; Buddington et al., 1996). Human trials with FOS and inulin, include those with a controlled diet (Gibson et a., 1995; Buddington et al., 1996; Kleesen et al., 1997), and demonstrate prebiotic activity of the substrates. Galactooligosaccharides Galactooligosaccharides are galactose-containing oligosaccharides of the form Glu al-4[p Gal 1-6}n where n = 2—5, and are produced from lactose syrup using the transgalactosylase activity of the enzyme [3-galactosidase (Crittenden, 1999). Three products are available with slightly differing compositions. Firstly, transgalactosylated oligosaccharides (TOS), are produced using (3-galactosidase from Aspergillus oryzae (Tanaka et al., 1983), and consist of tri-, tetra-, penta- and hexa-galactooligosaccharides. Oligomate 55, is prepared using (3galactosidase from A. oryzae and Streptococcus thermophilus (Ito et al., 1990) and contains 36% tri-, tetra-, penta- and hexa-galactooligosaccharides, 16% disaccharides galactosyl glucose and galactosyl galactose, 38% monosaccharides and 10% lactose. Finally, a transgalactosylated disaccharide (TD) preparation is produced using [3-galactosidase from S. thermophilus (Ito et al., 1993). Pure culture studies with TOS showed growth (as measured by acid production from TOS within 24 hours), of all the bifidobacteria strains tested, two Bacteroides fragilis strains, four lactobacilli strains and four enterobacteria (Tanaka et al., 1983). The remaining 48 strains showed acid production outside 24 hours or not at all, and TOS was therefore concluded to be a suitable bifidobacterial-promoting substrate. Adding 20 g/day TOS to a continuous culture (Durand et al., 1992) and 10 g/day TOS to a semi-continuous culture containing human faecal bacteria (Bouhnik et al., 1997) increased

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gas and SCFA. Although bacteria were not enumerated, increased lactate and acetate was suggested to be the result of a proliferation of lactic acid bacteria (lactobacilli and bifidobacteria) in response to TOS addition. Oligomate, TOS and TD have been studied in rats, HFA rats and humans to demonstrate their prebiotic effects. All three gave a prebiotic effect at doses of 10 g/day for Oligomate (Ito et at., 1990) and TOS (Tanaka et al., 1983), and 15 g/day for TD (Ito et al., 1993). The prebiotic effect was clear, although Teuri et al. (1998) showed no significant change in bifidobacteria but a significant increase in total bacteria on Mann—Rogosa—Sharp (MRS), a medium purportedly selective for lactic acid bacteria. In humans, 10 g/day TOS significantly reduced breath hydrogen (Bouhnik et al., 1997) whereas this increased in HFA rats fed 5% or 10% (w/v) TOS (Andrieux & Szylit, 1992). Contrary to TOS, Oligomate (10 g/day) increased gas production and bloating in humans according to symptoms recorded by the subjects. It is possible that a higher proportion of monosaccharides and lactose, and reduced content of higher oligosaccharides, may have led to elevated gas production. The results of the TOS studies demonstrate differences in response when using different subjects, substrates, doses and methodologies. Soybean Oligosaccharides The main oligosaccharides contained in soybeans are raffinose and stachyose which have been found to be good growth promoters of B. infantis but not E. coli, S. faecalis or L. acidophilus (Tamura, 1983). A soybean oligosaccharide extract (SOE) has been prepared from defatted soybean whey and consists of 23% (w/v) stachyose and 7% (w/v) raffinose which can be purified on an activated charcoal chromatography column, resulting in a refined soybean oligosaccharide product (SOR) containing 71% stachyose, 20% raffinose and 2% other sugars. In pure culture studies, SOR were fermented to a far greater degree by bifidobacteria than any other organisms tested (Hayakawa et al., 1990). The addition of a low concentration (0.1% (w/v)) of SOR to a two-stage continuous culture of faecal bacteria (Saito et al., 1992) resulted in a three-fold increase in the proportion of bifidobacteria in the total bacterial count. As only these bacterial groups were enumerated, any other changes that occurred in the microflora were overlooked. A significant decrease in azoreductase activity was recorded as well as large decreases in (3-glucosidase and (3glucuronidase. However, these results do not correspond fully with those from in vivo trials (Wada et al., 1992). Human trials have been carried out to assess the prebiotic activity of soybean oligosaccharides (Gibson et al., 2000). Again variation was observed between volunteers but overall, raffinose and SOE both showed prebiotic activity although less of the latter was needed to induce a response. Lactosucrose Lactosucrose is produced from a mixture of lactose and sucrose using the enzyme Pfructofuranosidase (Playne & Crittenden, 1996) and has been found to be bifidogenic in pure culture studies (Tamura, 1983; Fujita et al., 1991). A pure culture study compared lactosucrose with lactulose, FOS, SOR, raffinose and glucose for its utilisation by various intestinal bacteria (Hara et al., 1994). Six bifidobacteria and three lactobacilli strains grew to the same extent on lactosucrose and glucose, whereas all

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the other organisms tested preferred glucose. Contrary to the data of Fujita et al. (1991), lactosucrose did not appear to favour bifidobacterial growth. However, as the end pH was used to represent growth, this does not take into account the rate of utilisation or the fact that the amount or type of acid produced varies with different microorganisms. Studies in cats and humans have demonstrated the prebiotic potential of lactosucrose and associated desirable effects such as increased SCFA, lactate and acetate, decreases in putrefactive metabolites, reduction in detrimental enzymes known to produce toxins/carcinogens and lowering of faecal odour in cats. Although lactosucrose appears to have some prebiotic effects even at 3 g/day, there is a need for larger studies with adults, with particular regard to the minimum effective dose. Isomaltooligosaccharides Isomaltooligosaccharides (IMO) are composed of glucose monomers linked by a 1-6 glucosidic linkages. A commercial mixture known as Isomalto-900 has been produced by incubating a-amylase, pullulanase and a-glucosidase with cornstarch (Kohmoto et al., 1988). The major oligosaccharides in this mixture are isomaltose (Glu al-6 Glu), isomaltotriose (Glu al-6 Glu al-6 Glu) and panose (Glu al-6 Glu a 1-4 Glu). Pure culture studies showed panose, isomaltose, isomaltotriose and Isomalto-900 to be utilised as well as raffinose by all the bifidobacteria tested, with the exception of B. biftdum which gave no growth on any of the substrates (Kohmoto et al., 1988). Bacteroides species utilised all the sugars but fewer clostridia grew on the IMO than the raffinose. Overall, IMO appeared at least as selective, if not more so, for bifidobacteria than raffinose. Human studies have been conducted to determine the effect of IMO on the colonic microflora, to determine the minimum dose and to compare the disaccharide and trisaccharide fraction of IMO. These substrates had prebiotic potential and the minimum effective dose was 8-10 g IMO (13-15 g/day Isomalto-900) (Kohmoto et al., 1991). The IMO3 fraction had a greater prebiotic effect than IMO2 (Kaneko et al., 1994), although this was probably due to a greater hydrolysis of IMO2 than IMO3 by isomaltase in the human jejunum such that more substrate was available for the colonic microbiota. Although IMO do show prebiotic potential, there is a need for further studies to compare the effects of IMO2 and IMO3 on bacteria other than bifidobacteria. Our own studies with a three-stage continuous culture model of the gut have shown that IMO fermentation maintained a lactic acid flora whilst also allowing the generation of butyrate (Olano-Martin et al., 2000). As this is thought to be a desirable metabolite of colonic function, it may be that IMOs are effective prebiotics. Glucooligosaccharides A new oligosaccharide preparation has been enzymatically synthesised, using a glucosyltransferase from Leuconostoc mesenteroides, to transfer glucose molecules from the sucrose donor to an acceptor, namely maltose (Valette et al., 1993). The fructose from the sucrose molecule was released leaving a mixture of glucooligosaccharides (GOS) of various degrees of polymerisation (DP). The mixture composed of 18% mono-, di- and trisaccharides, 18% tetrasaccharides, 33% pentasaccharides and 31% hexa- and heptasaccharides comprising glucose units linked by al-6 and a 1-2 glycosidic bonds. GOS was poorly hydrolysed and digested in the intestinal tract of gnotobiotic rats (Valette et al., 1993).

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A defined mixed culture of Bact. thetaiotamicron, B. breve and Cl. butyricum incubated with 0.5% (w/v) GOS (Djouzi et al., 1995) resulted in no change in the Bact. thetaiotamicron population but increased the B. breve count, and reduced Cl. butyricum numbers. However, such data should be interpreted with caution, as the use of defined mixed culture does not necessarily indicate what would happen under in vivo conditions. Three organisms were inoculated separately (monoxenic) or jointly (trixenic) into gnotobiotic rats, half of which were fed a control diet and the other half a 2% (w/v) GOS diet for four weeks (Djouzi et al., 1995). In the trixenic rats, decreased butyrate and hydrogen production suggested that clostridia did not degrade the GOS to a great extent, as observed in a co-culture study (Djouzi et al., 1995). GOS was degraded equally by B. breve and Bact. thetaiotamicron in monoxenic rats, but not to the same extent by Cl. butyricum. In the trixenic rats, GOS had no prebiotic effect in terms of bacterial populations. Controlled human studies are needed to elicit the response of faecal bacteria, metabolites, SCFA, enzyme activity and gas production when GOS is administered. Xylooligosaccharides

Xylooligosaccharides (XOS) are chains of xylose molecules linked by Bl-4 bonds and mainly consist of xylobiose, xylotriose and xylo-tetraose. They are produced enzymatically by hydrolysis of xylan from birch wood (Campbell et al., 1997), oats (Jaskari et al., 1998) or corncobs (Playne & Crittenden, 1996). Few studies have been conducted on XOS fermentation by gut bacteria, although Okazaki et al. (1990) carried out a small trial in which five male subjects were fed 1 g and five were fed 2 g/day XOS for three weeks. The volunteers showed some variation in their response to the XOS but overall, both doses significantly increased faecal bifidobacteria and decreased bacteroides, the changes being more pronounced for the higher dose. When XOS administration ceased, the proportion of bifidobacteria decreased to its original level. A dose of 1 g/day XOS was sufficient to effect a bifidogenic response, although larger controlled human studies are needed to study a range of doses, to compare XOS to other prebiotics such as FOS, and observe the effect of XOS administration on normal gut function. Current status Three oligosaccharides are available in usable quantities in Europe and seem to have proven efficacy. These are fructooligosaccharides, /raTW-galactooligosaccharides and lactulose. Our studies have used molecular-based methodologies in human trials to confirm the prebiotic effect of FOS and lactulose (Tuohy et al., 2 00 la, 2002). The former has also been seen to be a highly effective prebiotic when incorporated into a biscuit product at 8 g/day (Tuohy et al., 200Ib). Other prebiotics, as mentioned above, are widely used in Japan. Some may have more desirable attributes than the currently recognised European forms and this is currently under evaluation. Moreover, the use of 'glycobiology' offers the deliberate manufacture of multi-functional prebiotics. This could include forms that have anti-adhesive capacities against common food-borne pathogens, types that persist into the distal bowel (the main site of colonic disorder), carbohydrates which attenuate the virulent properties of certain microorganisms and prebiotics that target individual species, not genera, of gut bacteria (Gibson et al., 2000). These so-called 'second generation' prebiotics are discussed below.

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Persistence of the prebiotic effect to distal regions of the colon As mentioned earlier, distal areas of the large intestine tend to be proteolytic environments and this may predispose towards an increased prevalence of chronic gut disorders, like bowel cancer (Rowland, 1992). Prebiotics have been postulated to be protective against the development of colon cancer, perhaps because of the metabolites formed (Rowland & Tanaka, 1993; Bouhnik et al., 1996; Buddingtonetal., 1996; Reddy etal., 1997; Hyllatf */., 1998). However, they should persist towards the distal colon to exert a maximal effect. This may be possible through the use of high molecular weight oligomers as well as chemical modification. Most prebiotic oligosaccharides are of relatively small DP (3-8), apart from inulin which can have a DP as high as 30. It is logical to suppose that the longer the oligosaccharide, the slower its fermentation and therefore this would increase the possibility that the molecule would not be fully degraded by bacteria in the proximal colon but would persist towards distal areas. Manufacturing techniques for controlled chain length distribution oligosaccharides are also being developed which can produce long chain versions. However, care needs to be taken to control the size fractionation as polymers are generally not selectively fermented by the gut flora, i.e. do not act as prebiotics. Chemical modification of residues in an oligosaccharide chain could result in a partial resistance to enzymic attack. Industrially, this has occurred with chemically modified starches and celluloses. The approach could be applied to prebiotics and may result in more recalcitrant forms.

Anti-adhesive activities against pathogens and toxins Prebiotic properties may be combined with anti-adhesive activities towards common gut pathogens. Many intestinal pathogens utilise monosaccharides or short oligosaccharide sequences as receptor sites in the gut and these may be chemically synthesised. Binding of pathogens to such receptors is the first step in the colonisation process (Finlay & Falkow, 1989; Karlsson, 1989). There is potential for developing prebiotics, which incorporate such receptor sequences. If present in the lumen, they may act as 'decoy oligosaccharides' in that they subvert the pathogen/toxin away from binding to the natural receptor sites on the gut wall.

Targeted prebiotics The current concept of a prebiotic is an oligosaccharide that is selectively fermented by bifidobacteria and lactobacilli (Gibson & Roberfroid, 1995). Due to the difficulties of characterising the colonic microflora at the species level, virtually all of the data on prebiotic properties of oligosaccharides is on microflora changes at the genus level. It would, however, be desirable to develop prebiotics, which are targeted at particular species of Bifidobacterium and Lactobacillus. Such targeted prebiotics might be considered as useful synbiotics (Gibson & Roberfroid, 1995). This refers to a combination of a probiotic and prebiotic. The most efficient form would have the oligosaccharide providing a highly selective substrate for the live microbe. However, as mentioned current prebiotics stimulate at the genus level and

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probiotics are bacterial species. As such, the degree of selectivity in current synbiotics may not be sufficiently sensitive. Species-targeted prebiotics might be developed through screening an enhanced range of oligosaccharide structures for their prebiotic properties. To this end, economical manufacturing technologies for complex oligosaccharides are required. These will allow the generation of wide structural diversity in candidate oligosaccharides for selectivity testing. A further method for generating such prebiotics depends upon the use of enzymes synthesised by the probiotics themselves. These enzymes will produce mixtures of oligosaccharide products, which are more readily hydrolysed by the cell-associated enzymes synthesised by the probiotic. Commercially, trans-galactooligosaccharides are manufactured using an industrial galactosidase (lactase) acting upon lactose which is used as a glycosyl donor and acceptor. We have repeated this process using similar enzymes from our culture collection of probiotics (Rabiu et al., 2001). Novel galactooligosaccharide mixtures were synthesised. Growth rate data showed that many of the probiotics had a higher growth rate on oligosaccharides synthesised by their own enzymes relative to other strains on the same substrate. These data need to be confirmed in mixed culture studies but are a very promising indicator that species-selective prebiotics have been manufactured. Commercial probiotics could theoretically be used in a similar manner to generate very useful synbiotics, which would metabolise a highly selective prebiotic in the gut and allow elevated chances of survival in this complex ecosystem. Attenuative properties The use of prebiotics to attenuate virulence of certain food-borne pathogens has also been a recent development in the area of prebiotics. For example the pathogenicity of Listeria monocytogenes is repressed in the presence of the plant-derived carbohydrate cellobiose (Park & Kroll, 1993). This microorganism is avirulent in its natural habitat of soil, where it is exposed to rotting vegetation and therefore the cellulose oligomer cellobiose. In the human body the absence of free cellobiose may allow virulence to be expressed, opening up the possibility of appropriate food supplementation.

Defined health outcomes of probiotics and prebiotics Several important health aspects have been claimed to result from gut flora modulation by probiotics and prebiotics. These are summarised below.

Improved tolerance to lactose Many populations worldwide are unable to digest lactose effectively. However, lactose in yoghurt is more readily digested than lactose in unfermented milk (Kolars et al., 1984). Experimental studies in rats indicated that the increased [3-galactosidase activity after yoghurt administration was microbial and not mucosal (Garvie et al., 1984). Lactase is destroyed at pH 3.0 and if the enzyme is to survive through the acid conditions of the stomach the cells must be intact (Martini et al., 1987). Heating and sonication to disrupt the

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cells results in increased maldigestion. Using strains of B. longum, it was found that the effect on hydrogen breath excretion depended on the strain used and its growth conditions (Jiang et al., 1996). Protection from gastroenteritis Acute gastroenteritis is something that probably affects everyone at one time or another. Usually it involves the ingestion of food or water contaminated with pathogenic microorganisms and/or their toxins (Zottola & Smith, 1990). The economic costs and medical aspects are therefore huge in spite of increased attention to food safety incidence. Typical causative agents include shigellae, salmonellae, Yersinia enterocolitica, Campylobacter jejuni, Escherichia coli, Vibrio cholera and Clostridium perfringens (Isolauri et al., 1999a). The gut microflora and the mucosa itself act as a barrier against invasion by potential pathogens (Isolauri et al., 1999b). Bifidobacteria and lactobacilli may inhibit pathogens like E. coli, Campylobacter and Salmonella spp. (Gibson & Wang, 1994a). The lactic microflora of the human gastrointestinal tract is thought to play a significant role in improved colonisation resistance (Gibson et al., 1997). There are a number of possible mechanisms in operation: Q

Q Q Q Q

metabolic end-products such as short chain fatty acids produced by these microorganisms may lower the gut pH, in a microniche, to levels below those at which pathogens can effectively compete competitive effects by occupation of mucosal colonisation sites direct antagonism by natural antimicrobial products competition for nutrients stimulation of an immune response.

This also has relevance for chronic diseases such as ulcerative colitis where the detailed pathogenesis is not known but would appear to have a bacterial aetiology. Coronary heart disease Coronary heart disease (CHD) is one of the major causes of death and disability in industrialised countries. Results from several epidemiological and clinical studies indicate a positive correlation between elevated total serum cholesterol levels, mainly reflecting the low-density lipoprotein (LDL) cholesterol fraction, and increased risk of CHD. There is evidence that lactic acid bacteria may be able to reduce total and LDL cholesterol levels (Delzenne & Williams, 1999). It is possible that some lactic acid bacteria may be able to directly assimilate cholesterol. This has been hypothesised from some in vitro experiments, but is a source of contention in that the data are conflicting and precipitation of the cholesterol with bile salts at a low pH may occur. It has also been suggested (Delzenne & Kok, 1999; Delzenne & Williams, 1999) that propionate produced by bacterial fermentation inhibits the formation of serum LDL cholesterol. However, the target genera for prebiotics (bifidobacteria, lactobacilli) are not propionate producers.

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Colon cancer The second most prevalent cancer in humans is colonic (Gibson & Macfarlane, 1994). It is thought that tumours arise 100 times more often in the large intestine compared to the small intestine (Morotomi et al., 1990). Many species of bacteria commonly found in the colon produce carcinogens and tumour promoters from food components that reach the colon. Examples are heterocyclic amines, polycyclic hydrocarbons, N-nitroso compounds, phenols, diacylglycerol and fecapentaenes. Some gut species also synthesise enzymes with genotoxic or toxic products. It is thought that probiotics and prebiotics may protect against the development of colon cancer through inhibiting those components of the flora responsible for carcinogen development, the mechanisms of effect being similar to those exerted against the pathogens responsible for acute gastroenteritis. Moreover, the fermentation end-product butyrate is known to stimulate apoptosis in colonic cancer cell lines and it is also the preferred fuel for healthy colonocytes (Prasad, 1980; Kim et al., 1982). Some prebiotics are known to have this effect (Olano-Martin et al., 2000), although lactobacilli and bifidobacteria do not produce butyrate. This indicates that our knowledge of prebiotic metabolism in the gut is far from complete and that more studies which unravel the full flora diversity are required. These would involve culture independent molecular procedures (Suau et al., 1999; Steer et al., 2000).

Vitamin synthesis Probiotic bacteria can synthesise various vitamins, largely of the B group. However, the relevance for this in the lower gut is debateable.

Irritable bowel syndrome Irritable bowel syndrome (IBS) probably occupies more GP time than any other disorder. IBS is characterised by irregular bowel movements and general malaise. Whilst certain hypotheses have linked stress with IBS, there is stronger evidence that gut dysfunction is involved. In particular, it is thought that the microbiota of the large intestine plays a key role in IBS onset and maintenance. Attention has been drawn to the relationship between carriage of yeasts such as Candida albicans and symptoms associated with the disorder. Often the antimicrobials (e.g. nystatin) used to treat yeast infections, e.g. Candida-'mduced recurrent thrush, may precipitate the onset of IBS. The role for probiotics and prebiotics would be to maintain indigenous levels of beneficial species. Moreover, some probiotics may have antagonistic activities towards problematic yeasts such as Candida spp.

Improved digestion and gut function The resident gut microbiota ferments substances that cannot be digested by the host in the small gut, these include resistant starch, non-digestible carbohydrates, oligosaccharides, proteins and mucins (Table 3.2). An active gut flora therefore helps to adequately digest the

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60-80 g of food that enters the adult colon each day. Gut bacteria, specifically probiotics, have a major role in carbohydrate degradation.

Immunomodulation Lactic acid bacteria administered by mouth can affect the immune system (Perdigon et al., 2001). The use of probiotics for immune stimulation is attractive because it provides a nonpathogenic carrier which will remain in the gut producing a chronic antigenic challenge. It has, therefore, potential as a vector for antigenic presentation related to pathogenic microorganisms. Probiotics may act as adjuvants able to stimulate non-specific host defences or capable of inducing a response to specific pathogens (Isolauri et al., 1991)- The response may be manifested as increased phagocytic activity or elevated antibody levels (e.g. secretory IgA). Effects on tumour development have also been reported (Kato, 2000). The effects of probiotics on the immune response will be discussed in more detail in other chapters in this book.

Mineral bioavailability There has been interest in the possibility of increasing mineral (particularly calcium and magnesium) absorption through the consumption of prebiotics and fibres. Although the small intestine is the principle site of mineral absorption in humans, it is thought that significant amounts are absorbed throughout the length of the gut, consequently, maximising of colonic effects is desirable. This may occur through carbohydrate metabolism by the gut flora. A reduced pH in the bowel following the generation of organic acids is thought to improve the sequestration of calcium and magnesium.

Conclusions It is important that the above-mentioned health aspects are supported by results from good clinical trials and underpinned with more knowledge of the mechanisms of effect. Because of the importance and ubiquity (gut diseases affect everyone at some time of their lives) of the many disorders mentioned, there is no doubt that probiotic and prebiotics will be around for long time and that their market will probably increase even more quickly over the next few years. It is imperative that such developments are supported by robust science that uses the best technologies available.

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A. Perez Chaia1,2 & G. Oliver1 1

Centro de Referenda para Lactobacilos (CERELA), Chacabuco 145, 4000 Tucuman, Argentina, and 2 Universidad Nacional de Tucuman, Argentina

The large intestine of the human adult harbours 10n-1012 bacteria/g comprising several hundred species the majority of which are strict anaerobes. Facultatively anaerobic bacteria such as enterobacteria and enterococci exist in highest numbers in the gastrointestinal tract of newborns. These bacteria may subsequently create a highly reduced environment that allows the growth of the strictly anaerobic species (Mackie et al., 1999). Numerically predominant anaerobes in adults are Gram-negative rods belonging to the genus Bacteroides and the Gram-positive rods Eubacterium and Bifidobacterium. Other groups, such as peptostreptococci, ruminococci, clostridia, lactobacilli and propionibacteria are present in significant numbers. A lower number of methanogens and dissimilatory sulfatereducing bacteria are also found in the human intestine. Different substrates such as dietary residues, mucus and lysis products from both epithelial and bacterial cells are available for use by the intestinal bacteria. However, the lower gut bacteria depend on the supply of dietary carbohydrates that have escaped digestion in the upper gastrointestinal tract to obtain energy for growth and maintenance of cellular function. The supply of carbohydrates is an important factor limiting bacterial growth in the colon, and bacteria able to transform the available substrates occur in the greatest numbers. Therefore, the complex mixture of carbohydrates that enter the colon resulting from dietary intake, nutrient digestion and absorption, have a great influence on the development of the different bacterial species and their metabolic activity.

Dietary carbohydrates Dietary carbohydrates may be divided into monosaccharides and disaccharides (sugars), oligosaccharides, with a degree of polymerization (DP) from 3 to 10, and polysaccharides with a DP higher than 10.

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Hydrolysis of dietary carbohydrates is necessary to allow nutrient absorption by the small intestinal enterocytes, the first step for entry into the bloodstream or lymphatic circulation. The digestion of carbohydrates takes place in the intestinal lumen and in the brush border membrane of enterocytes, and is mediated by a large number of hydrolases secreted in the gastrointestinal tract or localized in the brush border (Gudmand-Hoyer & Skovbjerg, 1996). Disaccharides form the major proportion of ingested carbohydrates in the small intestine and the digestion and transport systems for these sugars, except for lactose, are very efficient. The digestion of disaccharides and some oligosaccharides is undertaken by a number of small intestinal brush border enzymes: sucrase-isomaltase, lactase phlorizinhydrolase, maltaseglycoamylase and trehalase. Different disaccharide maldigestion syndromes have been described relating to the absence of some of these enzymes or the decline of their activity (Gupta et al., 1999; Kolho & Savilahti, 2000; Murray et al., 2000; Vesa et al, 2000). Oligosaccharides are a diverse group of soluble carbohydrates, many of which are not hydrolysed by gastrointestinal enzymes escaping digestion in the upper gastrointestinal tract. That is the case for fructooligosaccharides and galactooligosaccharides. The digestion of starch, the major dietary polysaccharide, begins with salivary and pancreatic amylase which breaks down oligosaccharides, dextrins, and maltose, which are in turn hydrolysed by the brush-border enzymes. Starch digestion depends on its physical form, the nature of the starch granule and the effects of food processing. As a consequence, starch may be rapidly digested, slowly digested or resistant (Cummings & Englyst, 1995). Non-starch polysaccharides (Prosky, 2000), like remnants of edible plant cells polysaccharides and lignin, the dietary fibre, as well as other carbohydrates like inulin, are all resistant to digestion. Therefore, dietary carbohydrates that escape digestion, and consequently absorption in the small intestine, include non-digestible oligosaccharides, resistant starch (RS), non-starch polysaccharides (NSPs) and sometimes maldigested disaccharides (Southgate, 1998). While the contents of the intestine are propelled by peristalsis, molecules to be taken from the centre of the intestinal lumen and absorbed at the epithelium surface must cross a diffusion barrier, the unstirred layer, in which molecules diffuse at a rate different from that predicted by the diffusion coefficient of water. The resistance of this barrier is modified by the agitation of the intestinal content becoming lower with the increase in the agitation of the bulk phase. The mucus contributes to the diffusion barrier increasing the resistance (Sanderson, 1999). The physicochemical properties of non-digested carbohydrates affect the physiology of the intestine. Water-holding capacity of some carbohydrates affects stool weight and intestinal transit time. Viscous polysaccharides can cause delayed gastric emptying and slower transit through the small bowel, resulting in the reduced rate of nutrient absorption in the small intestine and increased amount of carbohydrates entering the colon (Southgate, 1995). Colonic bacteria possess glycosidases able to hydrolyse the linkages of oligo- and polysaccharides non-digested or incompletely digested by the intestinal enzymes and are thus able to metabolize the resulting monosaccharides.

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Bacterial fermentation The process whereby strict and facultative anaerobes break down dietary and other substrates to obtain energy for growth and the maintenance of cellular function is known as fermentation. Fermentation is an anaerobic redox process in which adenosine triphosphate (ATP) is generated via substrate-level phosphorylation coupled to the partial oxidation of the substrate, rendering key metabolic intermediates. Electrons liberated in the first steps of the process are transported to an organic acceptor molecule, which is in turn reduced. By definition, fermentation may be a simple process, but the pathways of fermentation of most bacterial species are complex, usually involving several organic intermediates as electron donors and electron acceptors. Bacteria possess different pathways to convert hexoses or pentoses to either pyruvate alone or pyruvate and acetyl phosphate as intermediate metabolites. The Embden-Meyerhof pathway, the major colonic pathway for catabolism of hexoses, occurs in enterobacteria, clostridia, homofermentative lactic acid bacteria, propionibacteria and produces only pyruvate as a partial oxidation product. The phosphoketolase pathway, that occurs in heterofermentative lactic acid bacteria or the Bifidobacterium bifidum pathway, generates an additional molecule of acetyl phosphate (Fig. 4.1). Each bacterial genus transforms the intermediate metabolites by the reductive steps of the fermentation process in products of different type and molar yield (Table 4.1). In a mixed population, such as the intestinal microflora, carbohydrate breakdown involves more than one species. The fermentation products of some species are substrates for fermentation or incorporated as intermediate metabolites to the metabolic pathways of other species, resulting in substrates being sequentially fermented. Lactate, ethanol, pyruvate and succinate are fermentation products of some species, but in the bulk phase its concentration is diminished by subsequent bacterial utilization and short-chain fatty acid (SCFA) production. Therefore, the major end-products of sugar catabolism are SCFA, mainly acetate, propionate and butyrate that account for 85-95% of total SCFA in all the colon regions. Other fermentation end products, such as caproate and valerate, occur in lower amounts. Hydrogen and carbon dioxide are produced by the action of bacterial decarboxylases, oxidoreductases and hydrogenases and represent the main gases in the colon. In saccharolytic clostridia, hydrogen is generated from protons reduced during pyruvate oxidation by pyruvate: ferredoxin oxidoreductase, while in enterobacteria, hydrogen is produced by cleavage of formate obtained from pyruvate by pyruvate formate lyase (Fig. 4.1). Methane is a metabolic product in people who harbour large concentrations of methaneforming Archaea strains like Methanobrevibacterium smitbii that use H2 to reduce CO2 to CH4 (Miller & Wolin, 1982). The major acid anion product of intestinal fermentation is acetate because the bacterial genera that predominant in the colon are acetate producers. It can be formed by decarboxylation of pyruvate and further acetyl-CoA oxidation as in Bacteroides, Clostridmm, Enterobacterium, lactic acid bacteria and Propionibacterium and by acetylphosphate oxidation as in Bifidobacterium. However, nuclear magnetic resonance (NMR) analysis of the labelling of fermentation products obtained from isotopically labelled substrates provides evidence that the Wood—Ljungdahl pathway significantly contributes to the acetate pool in the human

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Fig. 4.1 Colonic pathways for catabolism of hexoses and the principal bacteria involved in the production of SCFA.

Intestinal Microflora and Metabolic Activity

Table 4.1

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Main bacteria found in the human large intestine Major products A P B L*

others

Bacteria

Nutritional characteristics

Bacteroides

Saccharolytic and proteolytic species

Eubacteria

Saccharolytic and proteolytic species Amino acid for energy supply

+ —+ +

Bifidobacteria

Oligosaccharides, disaccharides, hexoses and pentoses

+

+

Peptostreptococci

Saccharolytic and proteolytic species Amino acid for energy supply

+

+

Peptococci

Mainly amino acid for C, N and energy supply

+ - + +

Clostridia

Saccharolytic and proteolytic species Amino acid for energy supply

+ + + +

+ Ethanol, H2, C02

Lactobacilli

Oligosaccharides, disaccharides, hexoses and pentoses

+

+ Formate, ethanol

Propionibacteria

Oligosaccharides, disaccharides, hexoses and pentoses

+ +

Streptococci

Oligosaccharides, disaccharides, hexoses and pentoses

+

+

+ Ethanol

Enterobacteria

Oligosaccharides, disaccharides, hexoses and pentoses

+

+

Formate, ethanol, H2, C02

Metanobrevibacter

Utilize H2 and some species formate

Fusobacteria

Mainly amino acid for C, N and energy supply

+ + - +

+

Succinate

+ Formate, ethanol

± Succinate

CH4 + - + +

* A, P, B, L = acetic, propionic, butyric and lactic acids.

colon (Wolin & Miller, 1993; Miller & Wolin, 1996). Bacteria that use this pathway couple the oxidation of carbohydrates to acetate and CO2 to the reduction of CO2 to acetate. They use H2 produced by other bacteria to reduce CO2 to acetate competing with methanogenic bacteria and dissimilatory sulfate-reducing bacteria for molecular H2. Amino acids have a redox state similar to that of sugars and are fermented by a number of intestinal bacteria. Alanine, as electron donor and glycine, as electron acceptor are transformed by the Stickland reaction into NH3, CO2 and acetic acid. Fermentation of single amino acids may be possible. Some Clostridia use alanine as electron donor and acceptor and Eubacteria are able to ferment glycine. Hydrogen sulfide, branched-chain fatty acids and aromatic acids may be also formed from fermentation of amino acids.

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SCFA production Age may exert major influences on gut bacterial populations, their development and SCFA production. The intestinal microflora composition influences not only the production but also the profile of SCFAs. Measurements of overall fermentations and pathways by isotopic procedures may detect slight differences in the metabolic activity of colonic bacteria. Bifidobacteria use a unique fermentation pathway to produce primarily acetate and lactate. The fermentation yields 3 mol of acetate from 2 mol of glucose. Two of the acetates are formed from Cl and C2 of glucose and the third is formed entirely from C3 of glucose. NMR analysis can detect acetate formed from 3- C-glucose that contain C in both the methyl and carboxyl groups and produce a unique NMR signal (Wolin et al., 1998). Therefore, measurement of the amount of CH3 COOH produced from fermentation of 3- C-glucose by faecal suspensions has shown that the Bifidobacterium pathway can account for approximately 70% of acetate formed by fermentation of glucose in breast-fed infants. In humans up to three months old acetic and propionic acids are the principal SCFAs produced. Subsequently, the production of iso- and w-butyric, valeric, and caproic acids is dominant. After weaning, at more than two years of age, a population resembling the adult flora becomes established and the absolute amounts of all SCFAs, with the exception of nvaleric acid, reach adult values (Midtvedt & Midtvedt, 1992). Relative amounts of SCFAs are influenced by the feeding regime which selects specific bacterial groups able to transform the available substrates. Oligosaccharides, including N-acetylglucosamine and fucose oligomers or certain glycoproteins, which form a significant proportion of human breast milk, may be specific growth factors for bifidobacteria, which dominate the microflora of breast-fed infants. That ensures that acetate production is dominant in the intestinal content of infants before weaning. Adult diets containing trans-galactooligosaccharides (TOS), a mixture of oligosaccharides consisting of glucose and galactose, led to a significant decrease in breath hydrogen excretion and a significant increase in faecal concentrations of bifidobacteria (Djouzi & Andrieux, 1997; Roberfroid, 2001). Fructooligosaccharides (FOS), like oligofructose, and inulin as a replacement for sucrose in the diet cause a marked increase in bifidobacteria, whereas bacteroides, fusobacteria and clostridia decrease (Gibson et al., 1995; Gibson & Roberfroid, 1995; Gibson, 1999). Other bacteria tested such as lactobacilli, coliforms and Gram-positive cocci remain more or less unchanged. TOS, FOS and xylooligosaccharides (XOS) have been shown to be indigestible by human enzymes in the small intestine, but are extensively fermented in the large bowel to SCFA (Campbell et al. 1997). Fermentability depends on oligosaccharide structure, as shown by Van Laere et al. (1997) who studied the breakdown of short-chain carbohydrates with different sugar composition and molecular sizes. The fructans and XOSs were well fermented. Linear oligosaccharides were catabolized to a greater degree than were those with branched structures (Van Laere et al., 1997). The FOS-containing diets result in higher caecal butyrate concentrations when compared with XOS diet, but acetate is the acid produced in highest concentration when compared with control diets (Campbell et al., 1997). Recent studies showed that honey, FOS, GOS and inulin were especially effective in sustaining the growth of human intestinal bifidobacteria and the effects of honey on lactic

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and acetic acid production by intestinal Bifidobacterium spp. were similar to those of FOS, GOS, and inulin (Kajiwara et al., 2002). Fibre-containing diets produce higher concentration of SCFA than diets without fibre, but within a range of normal intake there are no variations in the amount of SCFA produced. Studies on the chemical composition and structure of NSPs allow us to predict its fermentation pattern because the nature and the amounts of each individual SCFA produced are closely related to the in vitro fermentation of the main sugars available (Salvador et al., 1993). Uronic acids are apparently involved in the production of acetic acid whereas glucose, and to a lesser extent xylose and arabinose, in the production of propionic acid. Xylose tends to have a greater impact than other sugars in the production of butyric acid. Colonic microorganisms produce a wide range of polysaccharide-degrading enzymes whose activity is the rate-limiting step in SCFA production. Polysaccharide solubility affects the rate of enzymatic hydrolysis. In that sense, it has been shown that soluble polysaccharides like pectin are broken down more rapidly than xylan, which is relatively insoluble (Cummings & Englyst, 1987). Acetate is the main fermentation product of pectin, while propionate is produced in great amount from arabinogalactan and guar gum and butyrate from starch. The amount of starch that reaches the colon is variable and depends on transit time, food manufacture, physical accessibility, amylase activity and interaction with other dietary components like protein and fat. It has been reported that RS fermentation in human large intestine stimulates specific groups of amylolytic bacteria such as Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio (Macfarlane & Englyst, 1986). The feeding of a diet containing potato starch resulted in a stimulation of bifidobacteria, lactobacilli, streptococci and enterobacteria in rats (Kleesen et al., 1997). The source and structure of starch determine the extension of its microbial fermentation (Norgaard & Mortensen, 1995); therefore, specific types of RS may be used to manipulate the intestinal bacterial metabolism (Bird et al., 2000). Raw or retrograded starch increases the caecal SCFA concentration in rats, but molar ratios of acids from potato diets compared to control of maize diet indicate that butyrate is mainly produced from raw potato starch and propionate from retrograded starch (Kleesen et al., 1997). Raw potato starch produces the highest increase on SCFA concentration mainly butyrate when compared with high-amylose maize starch (Ferguson et al., 2000). Recently, Cummings and co-workers reviewed digestion and fermentation of different carbohydrates some of which are established prebiotics (Cummings et al., 2001). Changes in fermentation patterns of intestinal bacteria associated with drug treatment have been also observed. Ascarbose, an oc-glucosidase inhibitor used to treat noninsulin-dependent diabetes mellitus, inhibits starch digestion in the small intestine and increases the amount of non-digested starch in the colon. This selects for growth of starch-using bacteria and increases the concentration of butyrate in faeces while the acetate and propionate contribution to the overall concentration of SCFA decrease (Scheppach et al., 1988). Antibiotic treatments may alter the SCFA production and excretion by acting upon the intestinal flora. Antibiotics present in high concentration in faeces and with activity against anaerobic bacteria may produce profound changes in bacterial metabolism. Neomycin, clindamycin, bacitracin and vancomycin have pronounced effects on faecal SCFA

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concentration. Ampicillin and erythromycin have moderate effects while nalidixic acid does not influence SCFAs excretion (Hoverstad et al., 1986). Measurements of faecal SCFA concentration cannot be directly related to its intestinal production as 95% of the acids generated in the colon are absorbed during transit of the digesta from the caecum and right colon where the more active sugar fermentation takes place, to the left colon where the main activity is proteolysis and amino acid fermentation. In vitro studies using chemostat models of the gut inoculated with intestinal microorganisms have been used to assess the amount of SCFA produced from different substrates in adults. Yields vary from 40 to 60 g of SCFA per 100 g of carbohydrate fermented, given molar ratios of acetate from 60 to 80, propionate from 14 to 22 and butyrate from 8 to 23. With a carbohydrate load of 15—60g/day and an average yield of 50%, it is possible to predict a daily production of between 100 and 450mmol (Cummings & Macfalane, 1991). Similar production values are obtained from measurements of SCFA in portal and arterial blood in surgical patients. Taking into account that SCFA, CO2, and H2 are the principal products of bacterial fermentation, the following stoichiometry has been proposed for a molar ratio of 60 : 20 : 18 for acetate, propionate and butyrate (Cummings & Macfarlane, 1997): 59 C6H12O6 + 38 H2O -> 60 CH3COOH + 22 CH3CH2COOH + 18 CH3(CH2)2COOH + 96 CO2 + 268 H+ The equation gives a yield of 63 g of SCFA from 100 g carbohydrate, close to that observed when starch is the substrate for fermentation. However, utilization of different substrates gives molar ratios different from the above and other fermentation balances have been proposed (Gibson & Roberfroid, 1995).

SCFA and electrolytes absorption Undissociated (HA) and dissociated (A - ) forms of SCFA are in equilibrium in the intestinal lumen. The HA form is lipid soluble and therefore readily diffuses through cell membranes, whereas A- requires specific transport proteins to cross into or out of a cell. A necessary equilibrium is established between the luminal, intracellular and basolateral compartment of the epithelium. Therefore, when one species of SCFA is transported into or out of a cell, it will affect the concentration of the other. The absorption of SCFA from the intestinal lumen may occur through non-ionic diffusion of the protoned form of the acids or through SCFA-/ HCO~3 exchange process, depending on animal species and the segment of intestine studied (Fig. 4.2). The non-ionic diffusion is the most important mechanism of SCFA absorption in the colon (Charney et al., 1998) whereas the SCFA~/HCO 3 exchange process may be present in the human ileum and proximal colon (Harig et al., 1991). The rate of SCFA absorption depends on the luminal pH and the concentration gradient from lumen to blood and is stimulated by decreases in bulk fluid or pH (Sellin et al., 1993; Sellin & DeSoigne, 1998). Evidence exists for an acid microclimate suitable for SCFA protonation on the surface of the intestinal epithelium, that allows an absorption of weak electrolytes much greater than

Intestinal Microflora and Metabolic Activity

Fig. 4.2

85

SCFA and ions transport across an intestinal epithelial cell.

expected from the bulk phase pH (Sanderson, 1999). This microclimate would be generated by the epithelial secretion of H ions and would be protected by the mucous coating from the variable pH of luminal contents. Mucus impedes the free diffusion of hydrogen ions into the bulk phase. Hydrogen ions are secreted by the epithelial cells in exchange for sodium ions by Na+ /H+ antiporters in the intestinal apical membrane (Sellin et al., 1993). Sodium diffuses into epithelial cells down its concentration gradient, causing hydrogen ions to be pumped into the apical space where they are trapped by the negatively charged mucopolysaccharide side chains (Fig. 4.2). For weak electrolytes, such as SCFA, sodium removal would indirectly alter absorption by increasing the pH, which normally maintains the weak acid in its protonated form. Stimulation of Na+ /H+ exchange across the apical membrane by epinephrine has been shown to increase SCFA absorption in rabbit proximal colon. Inhibition of Na + /H + exchangers by amiloride or theophylline in turn decreases SCFA absorption (Sellin et al., 1993). However, neither removal of Na+ to inhibit Na+ /H+ exchange, nor luminal or serosal ouabain, that inhibits active transport processes like the K+ /H+ -ATPase from apical

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colonic membrane, affected SCFA transport in rat colon tissues as assayed with Ussing chambers (Charney et at., 1998). Therefore, the mechanisms whereby requirements for the transport are covered seem to be species specific. Some experimental models have shown that absorption of SCFA increases with increasing chain length and the concentration gradient from lumen to blood (Oltmer & von Engelhardt, 1994). These observations are consistent with non-ionic diffusion of SCFA across the apical cell membrane but are also compatible with SCFA absorption through anion exchange processes. However, transepithelial flux of SCFA, in vitro measured in rat colon segments, increases in a linear way as concentrations of acids increase and is non-saturable at concentrations up to 100 mM (Sellin & DeSoignie, 1990; Charney et al., 1998). These findings would not be expected for a carrier-mediated epithelial transport. Therefore, the main mechanism of SCFA absorption in rat colon seems to be non-ionic diffusion, a passive process driven by pH or proton gradient. A partial recycling of SCFA across the luminal membrane occurs via Cl-/SCFA- exchange. To establish the presence of a Cl-/butyrate exchange, Rajendran and Binder (1994) studied the Cl and C-butyrate uptake across apical membrane vesicles of rat distal colon. An outward butyrate-gradient stimulated transient accumulation of 36C1 uptake that was not inhibited with valinomycin (a K ionophore) or FCCP (a proton ionophore), but was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor. The transport was saturable by both increasing extravesicular Cl concentration and increasing intravesicular butyrate concentration. The results confirmed the presence of an anion exchanger for recycling SCFA. The Cl-/SCFA- exchange acts in concert with Na+ /H+ exchange to stimulate electroneutral Na+ /Cl- absorption and a bi-directional flux of SCFA is established. A number of studies with animals have shown that NH3 diminished Na+ absorption in the rat proximal colon interacting with the apical Na+ /H+ exchanger (Cerma et al., 2000). However, active fermentation of non-digestible carbohydrates limit protein breakdown for energy supply and increase the consumption of nitrogen by biomass production. That reduces ammonia concentration in the colon (Sakata et al., 1999) and may indirectly favour NaCl absorption. Differences of pH between lumen and the epithelial cells determine that SCFA that enter the cells in their protonated form suffer a rapid dissociation and release protons. That decreases the inner pH near the apical membrane and stimulates Na+ /H+ exchange (Gonda et al., 1999). It in turn increases intracellular Na+ concentration and cell volume. Absorption of Na and Cl- drive water absorption from intestinal lumen. Therefore, the effect of SCFA on electrolyte movements is of great importance in the treatment of diarrhoea induced by cholera toxin or osmotic effects of non-digested carbohydrates that alter fluid and electrolyte balances (Rabbani et al., 1999). Over a physiological range of concentration, Ca is absorbed in the human rectum and distal colon by a non-saturable diffusion process, stimulated by acetate and propionate absorption (Trinidad et al., 1999). Inulin and RS ingestion led to considerable caecal fermentation and increased the intestinal absorption and balance of Ca and Mg, without significantly altering the plasma level of these two minerals (Scholz-Ahrens et al., 2001; Younes et al., 2001). Effects on

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mineral absorption seem to be related to pH reduction in the colon and increase of mineral solubility. The production of organic acids different from SCFA such as lactic and succinic acids are in the main responsible for pH reduction in the intestinal lumen by the fermentative activity of microflora. As this also favoured SCFA absorption, a relation between minerals and SCFA absorption would be found. Intracellular concentration of SCFA is regulated by the limited apical permeability, cellular metabolism and transport through basolateral membranes. Exit from the epithelial cells would be mediated by a basolateral SCFA-/HCO3exchanger similar to the apical anion exchanger but operating in a different direction. Another mechanism would be the metabolism of SCFA to HCO3- and the apical Cl- /HCO3exchange. A basolateral Na+/HCO3- or Na+ /SCFA- cotransporter may be also responsible for the reduction of SCFA intracellular concentration (Praetorius et al., 2001).

Colonic metabolism Part of the absorbed SCFA does not reach plasma because it is metabolized in the gastrointestinal epithelium to CO2, acetoacetate, 3-hydroxybutyrate and ketone bodies (Fitch & Fleming, 1999). The principal metabolic pathway for all the SCFA is catabolism to CO2 which provides energy to the epithelial cells. The CO2 may be used to generate HCO3~ by the carbonic anhydrase, which can return to the intestinal lumen by apical C1~/HCO3~ or basolateral Na /HCO3~ exchangers. Colonic epithelial cells oxidize butyrate more readily than other potential substrates such as acetate, propionate, glucose or glutamine. In substrate competition experiments, they utilized SCFA in a preferential order of butyrate > propionate > acetate. Moreover, butyrate suppressed the oxidation of the other SCFA (Clausen & Mortensen, 1994). Colonic segments of rats perfused with C-labelled SCFA, showed that the main metabolites CO2, 3hydroxybutyrate and lactate were produced from butyrate even when a mixture of acetate and butyrate were used in the perfusion fluid (Fitch & Fleming, 1999). Oxidation of propionate or acetate, however, may provide the energy needed for cellular functions when the luminal supply of butyrate is limited. Thus, SCFA are an important energy source for the gut mucosa itself. The fermentation products generated by the colonic bacteria provide almost 70% of the energy consumed by epithelial cells.

Physiological consequences of SCFA absorption SCFA are frequently implicated as trophic factor for the colonic mucosa. Infused into the colon, these compounds stimulate mucosal growth when basal levels of SCFA are low (Kripke et al., 1989). In that sense, fibre diets have been assayed to promote colonic mucosal growth by increasing SCFA production. Studies with different fermentable substrates have failed to find a correlation between mucosal growth and SCFA production and both effects seem to be simultaneous physiological adaptations to alterations in colonic luminal content (Fleming et al., 1992; Whiteley et al., 1996).

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Enhanced cell proliferation or increased colon size that result in higher total number of proliferating cells, has been associated with increased risk of developing colonic cancer. However, proliferation, differentiation and apoptosis are tightly regulated in rapidly renewing tissues like the colonic mucosa. Infused into the colon, the three major SCFA, acetate, propionate and butyrate, stimulate proliferation of normal crypt cells (Sheppach et al., 1995; Ichikawa & Sakata, 1997), but also favour differentiation and induce growth arrest and apoptosis in various tumour cell lines (Barnard & Warwick, 1993; Heerdt et al., 1994, 1997). The unbranched butyrate showed to be more effective on differentiation and apoptosis of colonic carcinoma cell lines than branched isobutyric or heptafluorobutyric acids suggesting that the effects are linked to SCFA structure and utilization (Heerdt et al., 1994). However, some carcinomas may evolve mechanisms to protect the cells from the induction of apoptosis at physiological concentrations of butyrate. This fact must be taken into account when evaluating the effects on colon cancer (Hague et al., 1995). It has been reported that butyrate modifies gene expression via several mechanisms, including inhibition of histone deacetylase (Boffa et al., 1978; Hinnebusch et al., 2002) and DNA hypermethylation (Parker et al., 1985). Previous studies have identified butyrate response elements from the 5' promoter region of various genes related to differentiation and growth regulation. Recently, it has been demonstrated that butyrate inhibits intestinal trefoil peptide gene expression, related to migration of both non-transformed intestinal epithelial cells and colon cancer cell lines (Tran et al., 1998). The ability of butyrate to inhibit ITF gene expression is related to the inhibition of molecules that promote cell invasiveness, which indicates that butyrate has an important role in colon cancer control. Propionate caused growth arrest and differentiation in human colon carcinoma cells, but produced a lesser degree of histone hyperacetylation compared with butyrate (Hinnebusch et al., 2002). Recent works have demonstrated that pronionic acid induced colorectal carcinoma apoptosis via effects on the mitochondrial adenine nucleotide translocator (Jan et al., 2002). The most studied effects of propionate are those related to lipids and glucose metabolism. It has been proposed that certain dietary supplements, fibre and probiotic microorganisms, lower plasma cholesterol concentration through inhibition of hepatic cholesterogenesis by the propionate produced by the intestinal flora (Chen et al., 1984; Perez Chaia et al., 1995). Propionate-supplemented diets lower blood cholesterol in rats and pigs either by inhibition of hepatic synthesis or redistribution of cholesterol from plasma to liver (Thacker et al., 1981; Illman et al., 1988). Studies in isolated rat hepatocytes indicated that propionate inhibits cholesterol synthesis at 0.25 mM concentrations (Wright et al., 1990) and in perfused livers inhibition occurred at 18 [imol/mL (Illman et al., 1988). Infused into rat caecum or stomach, propionic acid, but not propionate, moderated the cholesterol increase caused by a hypercholesterolaemic diet at 100 mM (Ebihara et al., 1993). Some studies in humans have failed to find a correlation between dietary propionate and cholesterol reduction (Venter et al., 1990; Zhang et al., 1992). It has been proposed that dietary propionate and propionate produced by the intestinal flora do not produce the same effects (Chen et al., 1984). Similarly dietary or infused propionate produce different responses (Ebihara et al., 1991, 1993). Propionate had no effects in short-term studies and different responses were also obtained in male and female individuals indicating a hormonal influence.

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The consumption of propionate in capsule form for several weeks decreased fasting serum glucose and reduced maximum insulin increments during the glucose tolerance test (Venter et al., 1990). Consumption of white bread mixed with sodium propionate also depressed blood glucose response (Todesco et a/., 1991). A restriction in dietary calories has been shown to cause a reduction in hepatic synthesis of cholesterol (Grundy, 1978). This may be a consequence of reduced fed intake or lower digestibility of dietary carbohydrates when propionate is included in the diet. Sodium propionate mixed with white bread resulted in inhibition of salivary amylase (Todesco et al., 1991). Therefore, inhibition of human amylolytic activity and lower absorption of dietary carbohydrates may be the mechanism whereby propionate affects both cholesterol synthesis and blood glucose reduction. Acetate did not cause histone hyperacetylation and also had no appreciable effects on cell growth or differentiation in human colon carcinoma cells (Hinnebusch et al., 2002). Acetate absorbed from the small bowel is always found in human blood in a basal level but rises to 100—300 (imol/L after consumption of fermentable carbohydrates (Scheppach et al., 1991), it is rapidly cleared from the blood and metabolized by skeletal and cardiac muscle and brain, as secondary fuel after carbohydrates absorbed from the small bowel. In the large bowel dietary carbohydrates that escape digestion, and consequently absorption in the small intestine, have the opportunity to be digested by microbial enzymes and converted to absorbable SCFAs avoiding the energy contained in their molecules being lost in faeces. In healthy humans, SCFAs contribute 5—10 % of the energy requirements when gastrointestinal length and function are normal. However, bacterial colonic digestion has a great value in short-bowel patients. Acetate and butyrate may substitute for fat by their conversion to acetyl-CoA, and propionate can substitute for carbohydrates because it is converted to gluconeogenic succynil-CoA (Nordgaard et al., 1996).

Probiotics and the intestinal metabolism of carbohydrates Among the claimed benefits of probiotic consumption, improvement of lactose maldigestion is one of the most studied (de Vrese et al., 2001). It is the most common disorder of intestinal carbohydrate digestion in humans produced by the reduction or loss of lactase activity in the intestinal brush border. Lactose maldigestion is defined by an increase in blood glucose concentration of < 1.12 mmol/L or in breath hydrogen of > 20ppm after ingestion of 1 g/ kg body weight or 50 g lactose (Bayless, 1981). Ingestion of lactose by a person with lactose maldigestion may lead to abdominal bloating, flatulence and diarrhoea; these are characteristic symptoms of lactose intolerance. Bloating and flatulence are the result of undesirable fermentations of the non-digested disaccharide by colonic microorganisms that produce large amounts of gas (H2, CO2 and sometimes CH^. Fluid loss by diarrhoea is a consequence of the osmotic effect of nonabsorbed carbohydrates. Different therapeutic treatments have been proposed to reduce symptoms of lactose intolerance including consumption of prehydrolysed milk, addition of purified lactase to milk, addition of microorganisms with high p-galactosidase activity to non-fermented milk or consumption of yoghurt or other fermented milks (Paige et al., 1975; Rosado et al., 1984; Lin et al., 1991; Gaon et al., 1995; Jiang et al., 1996; Mustapha et al., 1997). All of them

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focus on improving digestion in the upper gastrointestinal tract and reducing the load of lactose that enters the colon. Probiotic bacteria in fermented and unfermented milk products improve lactose digestion and eliminate symptoms of intolerance in lactose maldigesters, but they normally promote lactose digestion in the small intestine less efficiently than do yoghurt cultures. Although most adults are lactose intolerant, large quantities of yoghurt are consumed by some of these lactose-maldigesting populations. The fermentation process reduces the lactose content of milk which becomes lower as the storage time is prolonged. Moreover, the lactose in yoghurt is better digested than the lactose in milk. It is believed that the enhanced digestion of lactose in yoghurt is a result of intraintestinal digestion of lactose by Pgalactosidase from yoghurt-producing organisms. However the free enzyme does not resist the gastric acidity (Kotz et al., 1994). Lactose digestion in lactose malabsorbers can be significantly improved only if a milk product contains active microbial P-galactosidase. Intact cell walls are required to act as a mechanical protection of the enzyme during gastric transit. Recently, it has been shown that Streptococcus thermophillus was able to produce an active P-galactosidase in the digestive tract, although it did not multiply during its transit (Drouault et al., 2002). Employment of strains provided with enzymes that resist the conditions of the gastrointestinal tract may improve lactose utilization but only while the milk product is being ingested causing a reduction in the amount of lactose that reaches the colon; long-term effects would not be achieved. Lactulose, a disaccharide of glucose and fructose, also escapes digestion in the small bowel and enters the colon where it is fermented by the resident flora. Lactulose has been used to study the bacterial adaptation phenomenon in the colon. Breath hydrogen excretion lowers after some days of lactulose ingestion while remnants of the disaccharide in the colon are diminished. The same results have been reported for a daily feeding of lactose (Vesa et al., 1996a). Colonic adaptation involves changes in the metabolic activity of the ecosystem by induction of P-galactosidase activity from the colonic flora. The pH reduction due to fermentative activity favours the growth of acid-resistant bacteria such as lactobacilli which are able to utilize lactose without hydrogen production (Hertzler & Savaiano, 1996). It has been hypothesized that ingestion of a strain of Lactobacillus acidophilus with properties of high P-galactosidase activity and avid intestinal adherence would lead to prolonged intestinal survival of lactobacilli and possibly the conversion from a lactose-intolerant to a lactose-tolerant state (Saltzman et al., 1999). Even when the Lactobacillus survived passage through the entire gastrointestinal tract, it was not detected in the upper intestine 12 h after ingestion, inspite of its ability to adhere to epithelial cells which had been demonstrated in in vitro tests. This was reflected by the lack of improvement in overall breath hydrogen excretion or changes in symptom scores after lactose ingestion. However, another study using fermented milk containing a strain of Lactobacillus acidophilus and one of Lactobacillus cam with adherent properties gave better results (Gaon et al., 1995). Jiang and Savaiano (1997a) studied the effect of supplementation with 10 cells of Lactobacillus acidophilus LA-1 to modify colonic fermentation of lactose and SCF A production in an anaerobic continuous culture. They showed that rapid adaptation of colonic bacteria to lactose occurred within 1-2 days, with an efficient utilization of lactose. L. acidophilus supplementation may enhance lactose fermentation during early periods when the adaptation is not established.

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Continuous consumption of lactose-containing foods may promote the colonic adaptation phenomenon. Effects of chronic consumption of fresh or heated yoghurt on plasma glucose, insulin and fatty acid and cholesterol concentrations were studied by Rizkalla et al. (2000) in healthy men with or without lactose malabsorption. In subjects with lactose malabsorption, the production of breath hydrogen was lower after fresh yoghurt consumption than after heated yoghurt consumption. The study showed that chronic consumption of yoghurt containing live bacteria increased plasma butyrate and propionate concentrations without significant changes in plasma acetate. The authors concluded that lactose maldigestion is manifested by increased hydrogen excretion, whereas alleviation of lactose maldigestion might be associated with increased plasma SCFA. Therefore, manipulation of colonic fermentation to increase production of SCFA may be used to prevent lactose intolerance. Therefore, Lactobatillus addophilus and Bifidobacterium bifidum have been assayed for the ability to modify fermentative processes in faeces and in ileostomy effluents (Hove et al., 1994). No changes in the concentration of SCFAs and DLlactate were observed in ileostomic outputs after ingestion of 10 bifidobacteria. Increased organic acids production was obtained under in vitro conditions after incubation of ileostomic outputs with lactose, but similar results were obtained before and after B. bifidum intake. Faecal samples inoculated with mixtures of L. addophilus and B. bifidum showed increases on SCFA and lactate when lactose was used as a substrate. However, the consumption of capsules containing both microorganisms did not ameliorate lactose malabsorption measured by the breath hydrogen test in lactose malabsorbers. The authors concluded that ingested lactic acid bacteria failed to improve lactose digestion. Supplementation with Bifidobacterium longum has been proposed to modify colonic fermentation with production of SCFA and lactate (Jiang & Savaiano, 1997b). The presence of bifidobacteria in a culture medium simulating ileostomy effluent reduced lactose concentration to a different degree depending on pH. Greater amounts of total SCFA and lactate were produced at pH 6.2 than pH 5.7, but the p-galactosidase activity and lactate concentration were higher at pH 6.7. Maximal bacterial carbohydrate metabolism occurs at around neutral pH and inhibitions may be observed at an acidic pH. The activity of some intestinal bacteria different from the probiotic strain assayed would be altered, as butyrate was almost absent in the media at pH 6.7 while propionate concentration remained unchanged. Acetate and lactate were the fermentation products in higher concentration in these cultures. Lactic acid is the main fermentation product of homofermentative lactic acid bacteria under non-limiting substrate conditions and one of the products of the bifidum pathway. Lactate is also a substrate for many other bacteria such as Bacteroides and Propionibacterium that ferment it to SCFA and other products. Lactate is slowly absorbed from the lumen by the colonic mucosa compared to SCFA and tends to accumulate in patients with short-bowel syndrome and inflammatory bowel disease. Excretion of high amounts of lactate in faeces increases the osmotic charge and contributes to diarrhoea. Therefore, treatments of lactose intolerance should be focused on the production of acids which are better absorbed from the intestinal lumen. Recently, it has been proposed that dairy propionibacteria may be used as probiotics for improving intestinal metabolism of lactose. Selected strains with high (3-galactosidase activity (Zarate et al., 2000), resistant to gastrointestinal digestion (Zarate et al., 2001b)

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and able to adhere to epithelial cells (Zarate et al., 2001a, 2002) were studied for the production of SCFA from lactose in mice fed with milk (Zarate, 2001). Different strains provided (3-galactosidase to the caecal content and all of them increased in vitro SCFA production. Different molar ratios were obtained from lactose fermentation in caecal homogenates depending on the studied species. Whilst Propionibacterium addipropionici increased the molar ratio of propionate and slightly reduced the acetic and butyric acids ratio, Propionibacterium freudenreichii strains increased propionate at the expense of lactate. In the former, lactose was utilized by propionibacteria to produce propionic and acetic acids, but with Propionibacterium freudenreichii sequential carbohydrate consumption was achieved with SCFA produced from the lactate generated by other species during lactose fermentation. Consumption of dairy propionibacteria for almost seven days produced changes in the intestinal flora composition (Perez Chaia et al., 1995, 1999; Zarate et al., 1999) with increases on total anaerobes, higher ratio of anaerobes/aerobes and reduction in the counts of enterobacteria. SCFA were well absorbed from the intestinal lumen as there was no accumulation in the caecal contents at the end of the feeding period. Therefore, dairy propionibacteria could be used to modify colonic fermentation in lactose malabsorbers and to prevent intolerance symptoms. Modifications of specific bacterial groups and SCFA profiles, and increase of lactic acid production, were some of the results obtained, in the last years, both in vivo and in vitro with probiotic strains (Siigur et al., 1996; Djouzi et al., 1997; Alander et al., 1999). The health implications of SCFA produced in the large intestine are significant in terms of modifying risk factors for disease and for disease prevention. It is therefore important that we study the effect of different probiotic strains, individually or combined, on the metabolic activity of the intestinal flora.

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of the International Symposium, Wageningen Graduate School VLAG. Wageningen, Netherlands: Wageningen Graduate School VLAG, 37—46. Venter, C.S., Vorster, H.H. & Cummings, J.H. (1990) Effect of dietary propionate on carbohydrate and lipid metabolism in healthy volunteers. American Journal of Gastroenterology, 85, 549-53Vesa, T.H., Korpela, R.A. & Sahi, T. (1996a) Tolerance to small amounts of lactose in lactose maldigesters. American Journal of Clinical Nutrition, 64, 197-201. Vesa, T.H., Marteau, P., Zidi S., Briet, F., Pochart, P. & Rambaud, J.C. (1996b) Digestion and tolerance of lactose from yoghurt and different semi-solid fermented dairy products containing Lactobacillus acidophilus and bifidobacteria in lactose maldigesters—is bacterial lactase important? European Journal of Clinical Nutition, 50, 730—3. Vesa, T.H., Marteau, P. & Korpela, R. (2000) Lactose intolerance. Journal of the American College of Nutrition, 19, 165S-75S. Whiteley, L.O., Higgins, J.M., Purdon, M.P., Ridder, G.M. & Bertram, T.A. (1996) Evaluation in rats of the dose-response relationship among mucosal growth, colonic fermentation, and dietary fiber. Digestive Disease Science, 41, 1458—67. Wolin, MJ. & Miller, T.L. (1993) Bacterial strains from human feces that reduce CO2 to acetic acid. Applied and Environmental Microbiology, 59, 3551-6. Wolin, MJ., Zhang, Y., Bank, S., Yerry, S. & Miller, T.L. (1998) NMR detection of CH3 COOH from 3- C-Glucose: A signature for Eifidobacterium fermentation in the intestinal tract. Journal of Nutrition, 128, 91—6. Wright, R.S., Anderson, J.W. & Bridges, S.R. (1990) Propionate inhibits hepatocyte lipid synthesis. Proceedings of the Society of Experimental Biology and Medicine, 195, 26—9. Younes, H., Coudray, C, Bellanger, J., Demigne, C, Rayssiguier, Y. & Remesy, C. (2001) Effects of two fermentable carbohydrates (inulin and resistant starch) and their combination on calcium and magnesium balance in rats. British Journal of Nutrition, 86, 479-85. Zarate, G. (2001) Propionibacterias como probioticos en el metabolismo intestinal de lactosa. Doctoral thesis, Universidad Nacional de Tucuman, Argentina (in Spanish). Zarate, G., Perez Chaia, A., Gonzalez, S. & Oliver, G. (1999) Effect of feeding with propionibacteria on intestinal microflora composition. Biocell, 23 (Abstr). Zarate, G., Perez Chaia, A., Gonzalez, S. & Oliver, G. (2000) Effect of bile on the (3galactosidase activity of dairy propionibacteria. Le Lait, 80, 267-76. Zarate, G., Morata de Ambrosini, V., Perez Chaia, A. & Gonzalez, S. (2001a) Adhesion of dairy propionibacteria to intestinal epithelial tissue in vitro and in vivo. Journal of Food Protection, 65, 534-9. Zarate, G., Perez Chaia, A. & Oliver, G. (2001b) Viability and (3-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids. Journal of Food Protection, 63, 1214—21. Zarate, G., Morata de Ambrosini, V., Perez Chaia, A. & Gonzalez, S. (2002) Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells. Canadian Journal of Microbiology, 48, 449—57. Zhang, J.X., Hallmans, G., Anderson, H. et al. (1992) Effect of oat bran on plasma cholesterol and bile acid excretion in nine subjects with ileostomies. AmericanJournal of Clinical Nutrition, 56, 99-105.

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C.M. Riera, M. Maccioni & C.E. Sotomayor Departamento de Bioquimica Clinica, Facultad de Ciencias Quimica, Ciudad Universitaria, Universidad Nacional de Cordoba, 5000, Cordoba, Argentina

Overview of the immune system Introduction The immune system is a collection of cells and organs that can recognize any foreign substance (antigen),* usually an infectious microorganism. We are all born with certain natural or innate protective mechanisms against infectious disease, including the physical and physiological barriers such as skin, mucus, tears, saliva, respiratory tract, cilia, stomach acid, and antibacterial agents such as lysozyme that prevent the entry of infectious agents. Innate immunity also includes inflammation, pain, fever, coughing and healing. Innate immunity begins when antigens enter the body and operate against many but not all pathogens. It is triggered by commonly shared molecules on pathogens and is not specific to any particular microorganism. The speed and magnitude of the innate response is not affected by prior exposure to the same microorganism. In contrast, adaptive immunity develops over several days or weeks after exposure to antigens and provides highly sophisticated mechanisms for recognition of antigens, because it is able to discriminate among different molecular entities presented to it and to respond to those uniquely required, rather than making a random undifferentiated response. This phenomenon is known as specificity of the immune response. Adaptive or acquired immunity came into play relatively late, in evolutionary terms, and is present only in vertebrates. The main players involved in adaptive immunity are lymphocytes B and T and other types of cells that can also participate in the innate immunity, the antigen-presenting cells (APC). Lymphocytes are antigen-specific leukocytes responsible for adaptive immunity * Antigen: any molecule that reacts with antibodies (its names come from their capacity to generate antibodies). Not all antigens are capable of eliciting an immune response; those that are capable of doing so are called immunogens. An epitope is a site on an antigen recognized by an antibody; epitopes are also called antigenic determinants.

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which express on their surface a wide variety of antigen receptors induced by gene rearrangement (a hallmark of the adaptive response). Each individual lymphocyte (B or T) presents on its surface a unique type of receptor with a determined specificity for antigen. Due to the vast array of potential antigens, the immune system has evolved to produce a great repertoire of distinct receptor proteins on B or T cells. So, whereas collectively lymphocytes exhibit great diversity, in terms of the molecules which they can recognize, each individual clone possesses only one specific receptor. Lymphocytes recognising many diverse antigens are produced continually even in the absence of antigen exposure. When a lymphocyte encounters its specific antigen and receives the proper co-stimulatory signals, it proliferates and differentiates into a clone of effector cells with the same antigen specificity. This phenomenon is called clonal expansion. The adaptive immunity has the potential to recognize virtually any strange molecule, even those that could have previously been unseen on earth. However, the immune system normally does not react against self constituents (self-tolerance) and this is another extraordinary feature of the acquired immune response. Moreover, once the immune system has responded against an antigen, it has the ability to 'recall' this first contact and, in case of a second encounter with the same antigen, it will respond faster and more effectively. This is called immunological memory. Table 5.1 summarizes the most important features and elements of innate and adaptive immunity. Table 5.1

Main features of innate and adaptive immunity Innate immunity

Early induced immunity

Adaptive immunity

Features

No specificity No memory

Limited specificity No memory

Specificity Memory Autotolerance

Elements

Normal flora Local chemical factors Phagocytes, NK Complement Anti-microbial peptides Cytokines

B-l B cells MZ B cells 8y T cells CDld-restricted NKT

CD4+ T cells CD8+ T cells B cells Antibodies

Time (hours)

O A \J *t

^

96

Innate immune response Innate immunity that protects us against pathogens in the tissues and circulation is mediated by a diverse variety of soluble substances, such as cytokines, chemokines, different types of anti-microbial peptides, the complement system and a variety of effector cells, mainly phagocytes and natural killer (NK) cells (Table 5.1). Cytokines are proteins that are secreted by cells and exert actions on either the cytokineproducing cell (autocrine actions) or on other target cells (paracrine actions). They exert their function by interacting with and transducing signals through specific cell surface receptors.

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Chemokines (chemoattractant cytokines) comprise the most diverse and largest subset of cytokines identified to date. They are characterized by their capacity to induce the directional migration and activation of leukocytes, and thus play a major role in inflammation (Luster, 2002). The cytokines are important elements that participate in the innate immunity. For example, the innate immune response to viral infection is primarily through the induction of type I interferons (IFN-a and IFN-B) and the activation of NK cells. Double-stranded RNA (dsRNA) produced during the viral life cycle can induce the expression of IFN-a and IFN-(3 by the infected cell. Macrophages, monocytes and fibroblasts also are capable of synthesising these cytokines, but the mechanisms that induce the production of type I interferons in these cells are not completely understood. Both interferons can induce an antiviral response or resistance to viral replication by binding to the IFN-a/B receptor. Once bound, IFN-a and IFN-B activate the JAK-STAT pathway, which in turn induces the synthesis of several genes. One of these genes encodes an enzyme, which activates a ribonuclease that degrades viral RNA. Other genes activated by IFN-a/(3 binding induces a dsRNA-dependent protein kinase, which inactivates protein synthesis, thus blocking viral replication in infected cells. The binding of IFN-OC/(3 to NK cells induces lytic activity, making them very effective in killing virally infected cells. The activity of NK cells is also greatly enhanced by interleukin 12 (IL-12), a cytokine that is produced very early in a response to viral infection. Infectious agents trigger IL-12 production by dendritic cells and macrophages and this cytokine is an initiator of the activation of innate immunity (Kadowaki et al., 2000). Another important cytokine involved in the innate immune response is tumour necrosis factor a (TNF-a). It is secreted mainly by macrophages and mast cells. Its major biological functions are directed to tumour and inflammatory cells inducing cytotoxicity and cytokine secretion respectively. TNF-a activates vascular endothelium to express E-selectin and, together with IL-8, activates neutrophils to be more cytotoxic. Interleukin 1 (IL-1), IL-6 and TNF-a cause fever that signal the liver to produce acute phase proteins. On the other hand, there is a wide spectrum of antimicrobial peptides and proteins which are components of the innate defence system. Proteins with antibacterial activities include phospholipase A2, lysozyme and granzyme B. Antimicrobial peptides include defensins, histatins and cathelicidins, some of which are in development as new antimicrobial agents. Some antimicrobial peptides and proteins are constitutively expressed, whereas others are induced during inflammation or by specific cytokines. Antimicrobial peptides, such as defensins, which are enriched in arginine and lysine residues leading to a net positive charge, bind to anionic components in the target membrane and kill the microorganisms by pore formation and increased permeability of the cell membrane. Schittek et al. (2001), isolated a gene encoding a new antimicrobial peptide, dermcidin. The protein is specifically and constitutively expressed in sweat glands and secreted into sweat. Dermcidin is proteolytically processed, and the purified peptide showed broad activity against pathogenic bacteria and fungi, which was maintained over a broad pH range and in high salt concentrations. These results show that human sweat contains at least one antimicrobial protein that might have a role in the regulation of skin flora and in innate immune responses. There is another group of molecules that participate actively in the innate immune response called the complement system. Biological activities of this system impact on both innate and acquired immunity and reach far beyond the original observations of antibodymediated lysis of bacteria and red blood cells. After initial activation, the various comple-

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ment components interact, in a highly regulated cascade, to carry out a number of basic functions including: (1) lysis of cells, bacteria and viruses; (2) opsonization, which promotes phagocytosis of particulate antigens; (3) binding to specific complement receptors on cells of the immune system; (4) triggering activation of immune responses such as inflammation and secretion of immunoregulatory molecules that amplify or alter specific immune responses; and (5) immune clearance, which removes immune complexes from the circulation and deposits them in the spleen and liver. The proteins and glycoproteins that compose the complement system are synthesized mainly by liver hepatocytes, although significant amounts of them are also produced by blood monocytes, tissue macrophages, and epithelial cells of the gastrointestinal and genitourinary tracts. These components constitute 5% of the serum globulin fraction and most circulate in the serum in functionally inactive forms. Complement components are designated by numerals (C1-C9), or by letter symbols. The complement activation, can occur by the classical pathway, the alternative pathway, or the lectin pathway. The final steps that lead to a membrane attack are the same in all pathways. Phagocytic cells, which engulf and destroy pathogens include two types of leukocytes (white blood cells): monocytes, also called macrophages when they leave the circulation and enter the tissues, and polymorphonuclear leukocytes, including neutrophils, eosinophils, and basophils. Macrophages and polymorphonuclear cells release proteolytic enzymes, strong oxidants, and other bactericidal molecules. They also stimulate inflammation with vasoactive compounds that allow fluid movement from the circulation into the tissues and chemotactic molecules that attract leukocytes. As mentioned above, blood complement proteins promote pathogen phagocytosis and lysis and also innate immunity. NK cells recognize and destroy virus-infected cells without engulfing them. They participate in innate immune responses, killing intracellular pathogens and malignant tumours through the prompt secretion of cytokines and the ability to lyse virally infected cells or tumour cells. NK cell-secreted cytokines modulate haematopoiesis, control monocyte and granulocyte cell growth and function and influence the type of subsequent adaptive responses (Moretta et al., 2002). NK cells were originally identified on a functional basis because of their ability to lyse certain tumours in the absence of previous stimulation. They represent a lymphoid population that, in contrast to T or B lymphocytes, does not express clonally distributed receptors for antigen. Under normal conditions, NK cells are mostly confined to peripheral blood, spleen and bone marrow, but can migrate to inflamed tissues in response to different chemokines. Once thought to be promiscuous killers, it is now known that NK cells possess an elaborate array of receptors that regulate NK cytotoxic and secretory functions upon interaction with target cell specialized surface proteins: the major histocompatibility complex (MHC class I proteins. These NK receptors, known as killer cell immunoglobulin-like receptors (KIRs) in humans, and Ly49 receptors in the mouse, have become the focus of intense study in an effort to discern the underlying biology of these large receptor families. These receptors deliver signals that suppress, rather than activate, NK cell function (Fig. 5.1). Lack of engagement of such MHC-specific receptors leads, in most instances, to target cell killing, NK cells kill those target cells that have lost or express insufficient amounts of MHC class I, a frequent event in tumour or virus-infected cells. However, recognition of MHC class I by NK cells is not simply due to the existence of a universal receptor that allows detection of any MHC class I molecules. In humans, NK cells have much more complex inhibitory

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Fig. 5.1 Natural killer (NK) cell activation is the final result of the engagement of a number of receptors that have opposite functions. In the absence of inhibitory signals (Ly49 receptor), activating NK cell receptor (NKR-P1) ligation with molecules on the target cell results in NK cell triggering and target cell lysis. This event occurs in MHC class I HLAdefective cells, such as tumours or virus-infected cells. In the case of normal cells that express MHC class I, the interaction between inhibitory receptors and MHC class I delivers signals that overcome NK cell triggering, thus preventing target cell lysis. receptors. They recognize different allelic groups of MHC class I molecules and remarkably, each type of KIR is expressed only by a subset of NK cells. Although all mature NK cells express at least one receptor specific for self-MHC, the co-expression of two or more selfreactive receptors is a relatively rare event. This allows the whole NK cell compartment to sense the loss of even a single MHC class I allele on self-cells. The various inhibitory receptors are characterized by their ability to recruit and activate SHP-1 and SHP-2 phosphatases through ITIMs (immunoreceptor tyrosine-based inhibitory motifs) present in their cytoplasmic tails. Thus, a general inhibitory receptor mechanism is used to turn off the activating signalling cascade initiated via a number of different triggering NK cell receptors. Early induced immune response If the microorganism evades or overwhelms the innate defences, the infection may still be contained by a second wave of responses involving the activation of a variety of humoral and cell-response mechanisms that are strikingly similar to those involved in adaptive immunity. This is the early induced responses. Unlike the adaptive responses, these early induced responses to pathogens involve recognition mechanisms that are based on relatively invariant receptors, and they do not lead to the lasting protective immunity against the inducing pathogen that is the hallmark of adaptive immunity.

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The early induced but non-adaptive responses are important because they can repel a pathogen or, more often, hold it in check until an adaptive immune response can be mounted. The early response occurs rapidly because they do not require clonal expansion, whereas adaptive responses have a latent period of clonal expansion before the proliferating lymphocytes mature into effector cells capable of eliminating an infection. Innate and adaptive immune responses rely on radically different strategies for the recognition of pathogens. Early induced response involves what has been called innate B and T lymphocytes, which resemble NK cells more than they do conventional adaptive lymphocytes (Table 5.1). These innate B and T lymphocytes include B-l B cells, marginal zone (MZ) B cells, y5 T cells and CD Id-restricted natural killer T (NKT) cells. Such lymphocytes often show characteristics that distinguish them from conventional lymphocytes. Their defining hallmark is the limited diversity of their antigen receptors. This indicates that they recognize conserved structures, rather than the diverse set of antigens recognized by lymphocytes of the adaptive immune system. Surprisingly, many of the target structures seem to be of self-origin, which are induced or exposed in conditions of stress or tissue damage (Bendelac et al., 2001; Wardemann et al., 2002). The innate B and T cells represent a substantial proportion of the total pool of lymphocytes, ranging from 5 % or more. Apart from their specificity, these lymphocytes differ in many other ways from conventional adaptive lymphocytes. Innate lymphocytes express distinct phenotypic and morphological features, and reside in tissues rather than in lymph nodes. Characteristically, they have a natural activated/memory or effector phenotype, and are poised to unleash their effector function, such as antibody production, cytokine and chemokine release, and cytolytic activity, explosively and at short notice. The natural memory phenotype was found to be conserved in germfree conditions. Adaptive immune response Adaptive immune response arises when these receptors on individual lymphocytes interact with antigen and initiate a cascade of biochemical events that will end up in the activation of numerous important cellular functions, such as secretion of molecules (cytokines, antibodies) and proliferation, expansion and differentiation of these lymphocytes into effector cells, generating also what is called immunological memory. B lymphocytes respond by becoming plasma cells that secrete antigen-binding proteins called antibodies. Antibodies bind extracellular pathogens and bacterial toxins to inactivate them; they also opsonize the pathogens to promote phagocytosis. Some T lymphocytes respond to intracellular pathogens like viruses by becoming cytotoxic T lymphocytes, which destroy infected cells. Other T lymphocytes become active helper T cells, which stimulate antibody synthesis and macrophage activation by secreting cytokines. Both B and T lymphocytes also respond to antigen by differentiating into memory cells, which are long-lived and respond more quickly than naive lymphocytes when re-exposed to antigen (Table 5.1). B lymphocytes and the immunoglobulim The ultimate function of B lymphocytes is to differentiate into plasma cells and secrete antigen-specific soluble glycoproteins, known as antibodies. Collectively, they form a family of plasma proteins known as immunoglobulins (Igs). Although they were first discovered in

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their secreted form as proteins that appear in plasma after immunization or infection, it is now known that these Igs, in association with at least two other membrane polypeptide chains (IgOC and Ig(3), are expressed on the membrane of B cells and function as B-cell antigen receptor (BCR). The IgOC and Ig(3 polypeptides play a critical role in transducing signals to the interior of the cell, whereas the Ig molecule is in charge of recognizing the antigen. Usually, the terms cell-surface Ig and BCR are used interchangeably, although it should be kept in mind that the BCR is a complex of proteins that include the Ig molecule (Goldsby et al., 2000). The Ig molecule is made up by four polypeptide chains, comprising two identical heavy chains of molecular weight 50 000 or more (H chains) and two identical light chain (L chains) of about 25 000 molecular weight and can be thought of as forming a Y-shaped structure (Fig. 5.2). Upon engagement of the BCR by a specific antigen, B cells become activated, differentiate into plasma cells, and produce large amount of Igs to be secreted into the blood. These secreted Igs have exactly the same specificity as the cell-surface

IgAntibodies are the main players of what is known as humoral immune response. Nowadays, it is clear that all antibodies are constructed in the same way, from four polypeptide chains, as described above. There are only two types of light chains, which are termed lambda (k) and kappa (K), and each Ig molecule has either one or the other. So far, no functional differences have been found between antibodies having A, or K chains. By contrast, there are five heavy chain classes termed isotypes, and for that reason, five classes of Igs can be distinguished biochemically as well as functionally. Each class of Ig is designated by a capital Roman letter (IgM, IgG, IgD, IgA and IgE) and each of them differs in the heavy chain used, designated by the corresponding lowercase Greek letter (|J,, X, 5, a and £ heavy chains, respectively). Each antibody molecule, no matter which isotype, will have either a K of A, light chain, but never both. Therefore, all the Ig classes use two L chains of the same type and two H chains of the same class. There is enough sequence homology between the classes (approx. 30%) to think of a common evolutionary origin for the H chains. There are also subtle differences in the H chain of a determined class (approx. 95% of homology), and because of them, in humans there are four known subclasses of IgG called IgGl, IgG2, IgG3 and IgG4 and two subclasses of IgA, IgAl and IgA2. The antibody itself has two separable functions. One is to bind specifically an area of pathogen that is eliciting the immune response and the other is to engage different effectors mechanisms (e.g. complement fixation, crossing of the placenta, activation of mast cells). Therefore, while one part of the molecule must be adapted to allow tremendous variability to accommodate the large number of epitopes that can be recognized, another part must be adapted to allow the Ig to exert biological activities common to many antibody molecules. Comparison of amino acid sequences obtained for individual antibodies reveals this fact: the amino-terminal first 110 amino acid sequences of both the heavy and light chains vary greatly between different antibody molecules. This region in the molecule is known as variable region (V) and is the zone of the molecule that will interact with the antigen. Within each unit, an intrachain disulfide bond forms a loop of about 60 amino acids, termed a domain. The carboxy-terminal sequences are constant between Ig chains, either light or heavy of the same isotype, and it is called the constant region (C) (see Fig 5.2). Light chains contain one variable domain and one constant domain, heavy chains contain one variable domain and either three or four constant domains, depending on the antibody class. The

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Fig. 5.2 Structure of an immunoglobulin (Ig) molecule and biological function of various isotypes. The Ig molecules are composed of two different polypeptide chains joined by disulfide bridges; the heavy chain (which can be u, y, a, 5 and &) and the light chain (which can be K or A,). The amino-terminal domains of both chains are variable in sequence (VL and VH) and bind the antigen. The remaining domains of both chains are constant (CL and CH) and are involved in the biological activity. The CH region is further divided into three distinct domains CHI, CH2 and CHS. Depending on which subclass of heavy chain is being used, the biological properties of the Ig molecule will vary as can be seen in the right panel of the figure.

light chains are bonded to the heavy chains by disulfide bridges and the variable regions of the heavy and light chains pair to generate two identical antigen-binding sites (Fab), which lies at the tips of the arms of the Y. So, an antibody molecule is said to be bivalent. The leg of the Y is composed by the two carboxy-terminal of the two heavy chains (Fc). It is part of the constant region and the effector function of each different Ig subclass resides in this area of the molecule.

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The variable region of the Ig: the generation of diversity

If a given antibody-producing cell within an animal makes antibody molecules of only one specificity and that animal can respond to 10 potential antigens, how is the genetic information encoded and distributed? The amount of DNA required to be conserved and transmitted would be too great to make biological sense. Nonetheless, the mammalian immune system has evolved unique genetic mechanisms that enable it to generate an almost unlimited number of different light and heavy chains by joining separated gene segments together before they are transcribed. The genes for the C and the V domains of an Ig chain are separated by a great distance in germline DNA and in all the cells of the body except for lymphocytes of the B-lineage. Putting together the C and V region genes occurs by a process known as somatic recombination and it is a necessary event for the expression of the Ig gene, since expressed genes are always rearranged in mature B lymphocytes, whereas the same genes in other tissues are always in germline configuration. Two genes have been identified that allow this process: RAG-1 and RAG-2, for recombination activator genes. Their products are enzymes that catalyse these breakage and rejoining events. The mechanism by which V and C region genes associate has been worked out over the last few years and the key finding by Tonegawa (1983) (who won the Nobel Prize for it) was that antibody genes can move and rearrange themselves within the genome of a differentiating B cell. It was found that a single C gene segment encodes the C region of an Ig chain, but two or more gene segments are combined to encode each V region. Each light V region is encoded by a DNA sequence assembled from two gene segments: a long V gene segment and a short J (joining) gene segment. Each heavy chain V region is encoded by the DNA sequence that is obtained after bringing together three gene segments: a V segment, a D (diversity) segment and a J segment (Fig. 5.3). More than 100 VH segments exist; there are more than 10 DH elements and a small number of JH elements. Although it is likely that the choice of VH, DH and JH that are assembled is not entirely random, the combinatorial process allows the creation of a very large number of distinct H-chain-V region genes and Lchain-V region genes. Additional diversity is created by the junctional imprecision of the joining events. As it will be explained later in this chapter, all these processes take place in the bone marrow in complete absence of antigen, during the differentiation of the B cell progenitors into mature B cells. The constant region and the biological properties of the Ig subclasses

The protective effect of an antibody is not achieved simply by binding to the antigen, using its variable regions (tips of the Y) (Goldsby et al., 2000). After the interaction with antigens, they promote their removal from the blood. Antibodies can neutralize toxins and viral particles, inhibiting their binding to the host cellular receptors; they can fix serum complement proteins activating a cascade of proteolytic events that can kill the invading pathogen; they can facilitate the phagocytosis of the antigenic particles by macrophages or other phagocytic cells. All these effector functions are performed by the constant region of the heavy chains (the tail of the Y). Not all antibody isotypes have the same performance in exerting these biological functions, even when they could be recognizing the same epitope on the pathogen. Figure 5.2 shows the main features of the various Ig isotypes.

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Fig. 5.3 Gene rearreangement, transcription and synthesis of heavy and light immunoglobulin chains. For light chain a complete variable-region gene is created by somatic recombination of a V and J gene segments. Immunoglobulin chains are extracelluar proteins and the variable segment is preceded by an exon encoding a leader peptide (L), which directs the protein into the cell secretory pathways and is then cleaved. The constant region is also encoded in a separate exon and is joined to the variable domain by RNA splicing. For heavy chain a complete variable-region gene is created by somatic recombination, first fusing the D and J segments, then joining the V segment to the combined DJ segments. The leader peptide (L) and the constant-region sequences are encoded in separate exons. The C segments are joined to the variable domain by RNA splicing. IgM is found in serum as a pentamer of monomeric units composed of u, and L chains and accounts for approximately 10% of normal human serum Ig. It is the first isotype to be produced in an immune response and it is a potent activator of complement. IgD acts virtually exclusively as a membrane receptor for antigen. It is present in association with IgM on the surface membrane of B lymphocytes. A cell that express IgM and IgD is a mature but virgin or naive B cell. The IgM and IgD on the same cell have the same antigen-binding specificity. IgG is the major class of Ig, constituting approximately 70% of the total Ig and has the

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longest half-life. As mentioned above, four subclasses of IgG exist each of them having somewhat different biological functions. The IgG subclasses are able to cross the placenta; therefore they are the subclasses of maternal antibodies that will protect the newborn. After the first exposure to the antigen (primary immune response), IgG is made later than IgM, but in a memory response, it is produced more rapidly. IgG efficiently opsonizes pathogens, which means that IgG—antigen complexes can bind to receptors for the Fc portions of the Ig molecule (FcR) which are present on the phagocyte membranes, thus making the process of phagocytosis more efficient. IgA is mostly found, not in serum, but in secretions such as tears, saliva, colostrum, sweat and mucus of the bronchial, genitourinary, and digestive tracts. In serum, IgA exists as a monomer, but can also be found in polymeric forms. The IgA of the external secretions is found as a dimer and, consists of two monomers of IgA joined by a J (joining)-chain polypeptide and a polypeptide called secretory protein. It does not contain a receptor for complement and thus, it is not a complement activating or complement fixing isotype. However, it serves an important function for the mucous membrane, because its binding to bacterial and viral surface antigens prevents attachment of the pathogens to the mucosal barrier, which is the main site of entry for most microorganisms (NaglerAndersen, 2001). IgE is the least abundant isotype in serum. Its constant region binds to receptors expressed on the membranes of blood basophils and mast cells; as a result, crosslinkage of receptorbound IgE by antigens (usually called allergens) induces degranulation of basophils and mast cells. For that reason, IgE is involved in allergic reactions, asthma, hives and anaphylactic shock. Localized mast cell degranulation induced by IgE facilitates the recruitment of various cells important for antiparasitic defence. T lymphocytes and the T cell receptor complex T cells recognize antigens through a special structure that shares many features with the Ig molecule: the T cell receptor (TCR) (Goldsby et al., 2000). Unlike the antibodies made by the B cells, the TCR exists only in a membrane-bound form and they are never secreted. They are composed by two disulfide linked polypeptide chains, normally the a and (3 chains, although they can also be heterodimers formed by y and 5 chains. All of them, a, (3, y, and 5 belong to the Ig supergene family. The majority of T cells express receptors made up of a(3 heterodimers. As in the antibody structure, they also have a variable domain, found in the amino-terminal region of the molecule which interact with the antigen and a constant domain localized in the carboxylterminal region. Furthermore, similar recombinational mechanisms are used to assemble Vregion genes of the TCR, showing that the strategies for creation of a very large number of distinct genes by combinatorial assembly are the same. The TCR is associated with a set of transmembrane proteins, collectively designated the CD 3 complex. The CD 3 complex plays a critical role in transducing signals to the interior of the cell. It consists of the y, 5 and 8 chains and is associated with an homodimer of two £, chains or an heterodimer form by £, and T) chains (Fig. 5.4). Among the a(3 T cells are two important sublineages: those that express the co-receptor molecule CD4 (CD4 + T cells) and those that express the co-receptor CDS (CDS + T cells). These cells differ mainly in how they recognize antigen but also tend to mediate different

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Fig. 5.4 Structure of the T cell receptor-CD3 complex and their expression on T cell populations. The receptor for antigen on the surface of T cells is composed of two polypeptide chains: the heterodimer a: (3 or y: 5. The CD3 complex are formed by five chain (e, 5, y and ^ or ^-r|) and are involved in the intracellular signalling. According to the presence of different TCR-CD3 complexes and the expression of CD4 or CDS co-receptor molecules, T cells could be divided into three different subsets of effector cells.

types of regulatory and effector functions. CD4+ T cells are also known as helper T cells and CD8+ cells are usually designated cytotoxic T cells. T cells bearing y5 TCR are a distinct lineage of cells. In peripheral lymphoid tissues, only a very small percentage (generally 1—5%) of CD3+ cells express this receptor. However, in epithelial tissues, especially in the epidermis and small intestine of the mouse, the majority of the T cells express a y8 TCR. The receptor of these cells show extremely restricted variability. As already mentioned, this T cell population is involved in the early immune response and has also been called innate T lymphocytes. APCs and the MHC molecules Not all pathogens grow in extracellular spaces where they are accessible to antibodies. Viruses and many bacterial pathogens reside inside the cells, where they multiply and are safe from antibody attack. For this type of invader, a different system of recognition exists,

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which is called cell-mediated immune response and is carried out by T cells, so named because they mature in the thymus. Like the humoral immune response, T cell responses are exclusively antigen specific. However there are major differences between both types of responses: whereas antibodies recognize antigens in their intact (native) form, generally on soluble molecules; T cells are specialized to recognize very small peptide fragments of antigens that have been internalized, partially degraded and then displayed on the surface of cells named APC. In that way, T cells detect microorganisms that live inside the cells as well as extracellular pathogens that have been ingested. The main APC for T cells are dendritic cells, macrophages and B cells (Germain, 1999)- The presence of these antigenic peptide fragments on the surface of the APC will depend on a special kind of protein that binds these fragments, carries them to the cell surface and presents them to the T cells. These special kinds of protein are called MHC molecules, because thay are encoded by a complex of genes called MHC. MHC molecules are expressed on the cells of all higher vertebrates. In humans they are called HLA (human leucocyte-associated antigens). Originally, as it names implies, the MHC occupied the attention of immunologists because of its influence on transplantation rejection. However, their main role is not to prevent the work of transplant surgeons, but to bind peptide antigens and present them as a complex MHC molecule/peptide to T cells. There are two structurally and functionally distinct classes of MHC molecules: MHC class I molecules, which present foreign peptides to cytotoxic CD8+ T cells and MHC class II molecules which present antigenic peptides to helper CD4+ T cells (Fig. 5.5). These distinct types of MHC molecules deliver peptides that are degraded in different intracellular sites of the cell. Cytotoxic T cells attack mainly cells that make foreign antigens, in other words, cells that are infected by pathogens (mainly viruses) that employ the cell machinery to produce their own proteins (Fig. 5.6). These antigens are commonly known to be processed by the endogenous pathway, since they are synthesized within the cell and are degraded in the cytosolic compartments or in the endoplasmic reticulum (not in acidified vesicles) and interact with recycling MHC class I molecules in this milieu, before appearing on the cellular surface. The fact that class I molecules are expressed on virtually all nucleated cells (although their level of expression can vary) provides an essential mechanism of defence against viruses that potentially can infect any cell of the body. The only way to eliminate this type of pathogen is to destroy the infected cell. Therefore, the main role of CDS + cytotoxic T cells is to kill cells that present foreign peptides associated with MHC class I molecules (Goldsby et al., 2000; Hwang et al., 2001). In contrast, MHC class II molecules present antigens which are primarily processed by the exogenous pathway, in other words, antigens that are ingested by the cell by phagocytosis, and degraded into peptide fragments of 22-25 aa. This process occurs in membrane-limited endosomal or lysosomal vesicles (Fig. 5.6). In these compartments they interact with class II molecules, whose biosynthetic and recycling pathways take them through the same vesicles. Class II molecules are normally confined to the surface of a few specialized cells (also known as professional APC) such as B cells, macrophages and dendritic cells, but not on other tissue cells. Dendritic cells, for example, are amongst the most potent activator of T cells, since they express constitutively high levels of MHC class I and II molecules. The complexes formed by class II molecules/peptide fragments are recognized by specific helper CD4+ T cells, whose main function will be to activate other types of cells, mainly lymphocytes B and

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Fig. 5.5 The structures of MHC class I and class II molecules and their co-dominant expression, (a) MHC class I molecule is an heterodimer of a chain, non-covalent associated with (32 microglobulin. The a chain folds in three domains al, a2 and a3. The a3 domain binds P2 microglobulin. The folding of the al and a2 domains creates a long cleft that is the site at which peptide antigen bind to the MHC molecules. The MHC class II molecule is composed of a and (3 chains; each of them has two domains (al, a2 and (31, (32), the al and (31 domains forming the peptide-binding cleft. MHC molecules are codominantly expressed, with three MHC class I (A, B, and C-HLA molecules) and four potential sets of MHC class II (DP, DQ and DR-HLA molecules). Every heterozygous nucleated human cell will express six different MCH class I and, if it is an antigenpresenting cell (APC) it will also express eight different MHC class II molecules. The display of them over the cell is shown in fibroblast (b) and in APC (c).

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Fig. 5.6 Processing and presentation of antigen on MHC class I or class II molecules. Antigen that binds MHC class II molecules is internalized by endocytosis or phagocytosis by ARC (exogenous pathway), passes into the interior of the cell and is processed inside membrane-limited endosomal vesicles. Transported peptide can be recognized by appropriate CD4 T cells. The antigen that binds to MHC class I molecules is generally synthesized within the cell (endogenous pathway). Its processing occurs in the cytosolic compartment or endoplasmic reticulum. Transported peptide can be recognized by CD8 T cells.

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macrophages (depending on which cell type the antigen is being recognized) to produce antibodies and to destroy the macrophage ingested microbes, respectively. The expression of the co-receptor glycoproteins CD4 and CD8 on the T cell does not only allow immunologists to distinguish these two functionally different subpopulations of T cells, but it also helps to stabilize the interaction between a T cell and an APC, increasing the overall strength of the cell-cell adhesion.

Box 5.1

MHC class I and class II molecules have similar overall structures: they are both transmembrane heterodimers whose extracellular face has the form of a pocket where the peptide will be inserted. What would happen if specific antigenic peptides are unable to bind to the MHC molecules? It is well known that there is strong selective pressure in favour of any pathogen to somehow escape presentation by MHC molecules, allowing it to survive undetected by the host immune system. However, there are two separate mechanisms that ensure that usually any type of peptide can be bound to an MHC molecule and presented to a T cell: (1) MHC is highly polymorphic, in fact these genes are the most polymorphic genes known so far. This means that, within a species there is an extraordinary large number of alleles (alternative forms of the same gene) at each locus (more than a 100 for some genes). For that reason, it is very rare for two individuals to have an identical set of MHC molecules. Moreover, most individuals are heterozygous at these loci. This is an evolutionary strategy, since there will always be at least one individual capable of fighting an infection, thus, assuring the survival of a given species. (2) MHC is polygenic: several genes code for different MHC class I and class II products with different ranges of peptide-binding specificities. MHC class I molecules consist of two polypeptide chains: an a chain encoded in the MHC and a smaller, non-covalently associated chain, (32 microglobulin, which is not encoded in the MHC. In humans, there are three classes of class I a genes, called A, B and C. Therefore, there will be three different types of MHC class I molecules on the cell surface. Similarly, MHC class II molecules consist of a non-covalent complex of two chains, a and (3, but in this case, both of them are encoded in the MHC. There are also three pairs of MHC class II a and P genes in humans, called HLA DR (DRa and DRp), DP (DPoc and DPP) and DQ (DQa and DQ[3). Moreover, the DR cluster contains an extra DR[3 chain gene, whose product can pair with the DRoc chain. (3) Furthermore, MHC class I and II molecules are codominantly expressed, that is each cell expresses MHC proteins which are transcribed from both maternal and paternal chromosomes. Thus, with three MHC class I genes and four potential sets of MHC class II genes on each chromosome, a typical heterozygous human cell will express six different MHC class I molecules and eight different MHC class II molecules (Margulies, 1999) (Fig. 5.5b and c).

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Education of lymphocytes in the primary lymphoid organs Education of T lymphocytes in the thymus Lymphoid progenitors that have developed from haematopoietic stem cells in the bone marrow migrate to the thymus to complete their maturation into functional T cells. The thymus is a central (primary) lymphoid organ that provides the unique and specialized microenvironment (humoral and cellular) for T cell maturation. Two clear architecturally organized areas could be detected in this organ, an outer cortical region, the thymic cortex and an inner medulla. During the migration through the thymic epithelial network known as thymic stroma, the immature T cells or thymocytes, proliferate and differentiate along several different developmental pathways. Most steps of differentiation occur in the cortex, whereas the medulla contains only the mature T cells that later could be exported to the periphery. These mature and functional T cells that exit from the thymus and begin to recirculate in the periphery are called naive T cells. Naive T cell have never been exposed to foreign antigens. All these mature emerging T cell populations express the TCR-CD3 complex (c/P or y§) and can express the co-receptor molecules, that could either be CD4 or CDS. Therefore, in periphery, there will be found at least, three different populations of mature T cells: CD4-CD8-TCRy8, and CD4+TCRap or CD8+TCRap\ Several important events occur during T cell development. They are summarized below. (1)

(2)

(3)

(4)

Generation of a primary T cell repertoire by gene rearrangements that produce the TCR (explained previously). These processes involve the activity of several enzymes, such as the terminal deoxynucleotide transferase (TdT), and the recombinases RAG1 and RAG2. All these processes take place in an antigen-independent way. Positive selection that permits the survival of only those thymocytes whose TCRs recognize self-MHC molecules with low avidity. This process is responsible for the creation of a self-MHC-restricted repertoire of T cells (MHC restriction). Negative selection that eliminates thymocytes bearing high-affinity TCRs for self-MHC molecules alone or self-antigens presented by self-MHC. These processes generate a primary T cell repertoire that is self-tolerant (self-tolerance). Expression of cell surface proteins, known as cell markers or CDs (cluster of differentiation antigens). Different cell populations with divergent functions can be identified by the particular expression of these CDs. Also, the expression of few of them are lineage restricted and others appear at distinct developmental stages of the cell.

During T cell development, the thymocytes have a series of discrete stages that can be identified by distinctive patterns of expression of various CDs, activity of genes involved in the TCR rearrangement (RAG-1 and RAG-2 genes) and also, the expression of a functional TCR on the membrane (Durkin & Waksman, 2001). The T cell precursors arrive at the outer cortex of the thymus and slowly begin to proliferate (Fig. 5.7). These progenitors do not express the cell surface molecules characteristic of T cells, such as CD4 and CDS; they do not even express the TCR complex (TCR-CD3). Since these cells are negative for the presence of these co-receptors on their surface, they are referred to as double-negative (DN) cells. Even though these molecules are not expressed in this early phase of the development, other surface molecules such as c-Kit, CD44 and CD25

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Fig. 5.7 T-cell development in the thymus. Framework cells of the thymus are shown in the schema. After T cell precursor arrival to the thymus, several developmental stages could be defined by the presence or absence of CD4 and CD8 co-receptors: double-negative (DN); double-positive (DP) and single-positive (SP) thymocytes. The rearrangement of y and 5-chain gene of TCR and the expression of surface markers are shown on the left. The rearrangement of a and (3 chain genes of TCR and the expression of surface markers are shown on the right. After positive and negative selection the mature naive T cells are exported to the periphery.

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are useful markers of the progression of the DN population. Thymocytes begin to rearrange their TCR genes only after c-Kit expression has stopped and CD44 expression is downmodulated. Some thymocytes make productive rearrangements of both y and 5 chain genes and develop into DN CD 3+ yS T cells that could be exported to the periphery (Goldsby et al., 2000; Krangel et al., 2000). However, most DN cells progress to a different pathway: they stop proliferating and begin to rearrange the TCR-(3 chain gene. If this rearrangement is productive, they will transcribe the rearranged (3 chain gene and express it on the cell membrane. At this stage, the cell expresses a pre-TCR complex on its surface which also involves the CD3 group of polypeptides, essential for signal transduction. Once this pre-TCR complex is presented on the membrane of maturing thymocytes, it can deliver signals to the interior of the cell which will enhance the rearrangement of TCR OC chain genes and promote the expression of both CD4 and CD8 molecules. The thymocytes at this stage are called double-positive (DP) cells. DP thymocytes are small cells and correspond to 80% of the lymphoid thymic population. The possession of TCR enables DP cells to undergo the rigors of positive and negative selection guided by interactions with self-MCH/peptide complexes expressed on thymic or bone marrow derived APC. A high percentage of these cells die by apoptosis within the thymus either because they fail to make a productive TCR-gene rearrangement or because they fail to survive the selection processes. DP thymocytes that express the a(3 TCR-CD3 complex and survive the selection develop into either single-positive CD4 + thymocytes and single-positive CDS + thymocytes (SP). The cell surface phenotype of the mature T cell is determined during the positive selection and involves cortical epithelial cells expressing class I or class II MHC molecules. The class II molecules are required for CD4 T cell development, while class I are required for CDS T cell development. These events decide the expression of the co-receptor molecule on mature T cell and the MHC-restricted peptide presentation. Education of B lymphocytes in the bone marrow Resembling what happens during the T cell differentiation in the thymus, B lymphocytes derive also from haematopoietic stem cells by a complex set of events which are not completely understood. They occur in the fetal liver and, in adult life, principally in the bone marrow, the other important primary lymphoid organ. Here again, interaction with the specialized stromal cells and the presence of cytokines such as IL-7 seem to be fundamental to the normal regulation of this process (Frazer & Capra, 1999). The first stage of development is characterized by a cell designated as pro-B. The main feature of these cells is that its DNA has begun the process of Ig gene rearrangements, largely limited to H chain genes. It also exhibits on its membrane certain typical surface molecules, markers of B cell lineage, such as the CD45 R and CD 19 markers (Fig. 5.8). The successful completion of VHDJn rearrangement and expression of (J.H chain, signals the end of the H-chain rearrangement and the onset of L-chain gene rearrangement. It marks the boundary of pro-B cell and pre-B cell states. As T cells, pre B cells express on their surface a pre-BCR made up by the JJ. chain and a surrogate L chain. It is generally believed that this complex jl chain/surrogate L chain interacts with ligands within the bone marrow and that the binding with this ligand determines whether the cell continues its differentiation process, starting the rearrangement of the Ig K and X loci. Once an L chain gene has

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Fig. 5.8 B cell development in the bone marrow. The gene rearrangement events are shown on the left at the B cell developmental stage in which they occur. On the right, changes in the expression of several surface markers are shown. CD45R and CD19 are important in signalling during B cell activation. The function of CD20 is not well known.

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been successfully rearranged, it is transcribed and the resultant IgM will be expressed on the surface. The cells, now classified as immature B cells, express CD20 marker. The newly differentiated B cell initially expresses surface Ig solely of the IgM isotype. A B cell is considered to be a fully mature B cell, when it expresses on its membrane a second class of Ig, the IgD, composed of a 8 chain with the same variable region (VDJ) as the u, chain, but a different constant region. The C|i and the C8 genes lie close together in the DNA. B cells expressing IgM and IgD produce two different primary RNA transcripts: in one, the transcript is terminated after the C|I gene; in the other both the C|I and C5 are transcribed and the transcript is terminated after the C5 gene. Alternative splicing of these transcripts will generate mRNA encoding for either Ji or 5 heavy chain. Again, there are many similarities between the processes of differentiation of T and B cells that occur in the thymus and bone marrow respectively. Self-reactive B cells are also eliminated from the repertoire before emerging from bone marrow, although there are different ways of controlling B cell autoreactivity: (1)

(2)

(3)

B cell specific for ubiquitous multivalent ligands such as MHC molecules are deleted early in the development, just after antigen receptors are first expressed (Tsubata, 2001). B cell specific for soluble self-antigens are rendered unresponsive or anergic. Anergy is generally defined as a state of non-responsivness to antigen. These anergic B cells lost the expression of surface IgM at the cell surface (although it is retained within the cell), but they express normal levels of IgD. However, they are unable to become activated by antigen in the periphery, they do not differentiate into plasma cells and usually they die in the peripheric lymphoid organs. Secondary L chain gene rearrangements may, in fact, be induced by autoantigen present in the bone marrow. Interaction of immature B cells expressing a surface IgM with an autoantigen, may induce further rearrangement in the L chain gene, as measured by increased levels of A, L over K L-chain gene rearrangement. The subsequent expression of this newly generated L chain gives an autoreactive B cell a second chance to survive by losing its specificity for the autoantigen, since the V or V and C regions of the L chain changes. This process is known as receptor editing and has been studied mainly in transgenic models (Fig. 5.8).

The immune system functioning Peripheral lymphoid tissue, a place where lymphocytes meet the antigen After the lymphoid progenitor cells differentiate in the primary lymphoid organs, they migrate to peripheral lymphoid tissue. Lymph nodes and spleen are the most highly organized of peripheral lymphoid organs. Less organized, the collectively called mucosalassociated lymphoid tissue (MALT), is found in various body sites. MALT includes Peyer's patches, the tonsil and the appendix as well as numerous follicles within the lamina propria of the intestine and in the mucous membranes lining the upper airways, bronchii and genital tract (lijima et al., 2001). Although remarkably different in appearance, the lymph nodes, spleen and MALT, all

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share the same basic architecture, with discrete areas where B lymphocytes are localized (the follicles or the B cell corona) next to areas in which T cells predominate. In each tissue, there are specialized sites (germinal centres) where B cells proliferate and differentiate into antibody-secreting plasma cells, and these are usually found at the junction between T and B cell areas. In these organs, several cells have the ability to operate as APC. Each anatomical site operates on the same principle and function to capture and concentrate the antigen. The lymphatic system drains the tissue space and interconnects many organized lymphoid tissues. Lymph nodes are specialized to trap antigen from regional tissue spaces, whereas the spleen traps blood-borne antigens. Lymphoid tissues of MALT have the ability to concentrate and respond to local antigens. The cutaneous-associated lymphoid tissue constitutes the most important tertiary lymphoid tissue. The foreign antigen can arrive at peripheral lymphoid tissues from blood or lymph, or be carried by migratory APC, such as dendritic cells. Free antigens are taken by APC, processed, and then displayed as peptide fragments associated to MHC molecules. At this point, the APC is ready to encounter the T cell bearing the appropiate TCR, specific for the MHC/ peptide complexes on its surface. Naive T cells, recently emerging from the thymus, will migrate through the lymph node, scanning these APC loaded by foreign antigens through very loose interactions. Occasionally, few T cells will engage their TCR with the MHC/ peptide complexes displayed on an APC in a highly specific interaction. These T cells will become activated, starting an adaptive immune response. It is in the secondary lymphoid organs where the three major steps of adaptive immune response take place: (1) recognition; (2) clonal proliferation; and (3) differentiation into effector cells. The majority of the T cells, however will just bind loosely to the APC and will continue migrating through the lymph node, eventually leaving it via the efferent lymphatic vessels to re-enter the blood and continue to recirculate. Lymphocyte traffic Lymphocytes undergo constant recirculation between the blood, lymph, lymphoid organs and tertiary extralymphoid tissue. This extensive recirculation significantly increases the chances that the small number of lymphocytes carrying a specific receptor will encounter the appropriate antigen when it is present in the body. The mature naive T cells are activated to proliferate and differentiate into armed effector T cells the first time that they encounter their specific antigen on the surface of a professional cell. The migration of the cells through the lymphoid organ and the initial interaction with APC involves antigen non-specific interactions of lymphocytes with other cells (Moser & Loetscher, 2001). Similar interactions eventually guide the effector T cells into peripheral tissues and play an important part in their interaction with the target. These binding reactions are mediated by several molecules collectively named cell adhesion molecules (CAMs), that recognize a complementary array of molecules on the surface of two interacting cells. Most of these CAMs fall into one of four protein families: the selectins, the mucine-like family, integrins, or the Ig superfamily. Each of the secondary lymphoid organs, with the exception of the spleen, have a vascular endothelium in the postcapillary venules composed of specialized cells with a cuboidal ('high') shaped, called high-endothelial venules or HE Vs. During the migration from the primary lymphoid organs, naive cells do not exhibit preference for a particular type of

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secondary lymphoid tissue and circulate indiscriminately among these organs throughout the body by recognizing CAMs on HEVs. After contact with the antigen in the secondary lymphoid organ, the effector and memory lymphocytes have the ability to migrate differentially into different tissues. This process is also called trafficking and the CAMs involved are named homing receptors. Particularly interesting is the fact that memory and effector populations exhibit tissue-selective homing behaviour. Such tissue specificity is imparted by different combinations of adhesion molecules. For example the mucosal homing subset of molecules are different from skin homing molecules. The process of lymphocyte migration can be divided into four sequential steps: (1) rolling; (2) activation; (3) arrest and adhesion; and (4) transendothelial adhesion. In the first steps, the cells attached loosely to the endothelium by low-affinity selecting mucine-like CAMs interaction, but the shear force of the circulating blood soon detaches the migratory cells from the endothelial cells. The first groups of molecules again tether the lymphocytes and this process is repeated so that the cells tumble end-over-end along the endothelium; a type of binding called rolling. The second step, activation, involves the binding between chemokines and their receptors on the membrane of migrating cells, that is followed by activating signals mediated by G protein associated with the receptor (Luster, 2002). These signals induce a conformational change in the integrin molecule in the migratory cell, increasing their affinity for the Ig superfamily CAM molecules on the endothelium. Subsequent interaction between integrins and Ig superfamily CAMs stabilize the adhesion and the cells adhere firmly to endothelial cells and finally the cells migrate through the vessel wall. The overall process of leukocyte migration into inflamed tissue is similar to that previously described. During the inflammation, cytokines and other inflammatory mediators act upon the local blood vessels, inducing increased expression of endothelial CAMs. Recirculating lymphocytes, monocytes and granulocytes bearing receptors that bind to CAMs in vascular endothelium, are now able to extravasate into the tissue and do their work. Activation of T cells Naive T cells can live for many years without dividing. Upon encounter with the specific antigen, naive T cells must re-enter the cell cycle and divide rapidly to produce large numbers of progeny, in a process already mentioned as clonal expansion. Also, they will differentiate into armed effector cells. These two essential events for the initiation of an adaptive immune response are driven by an important cytokine called IL-2. T cell activation is dependent on the interaction of the TCR—CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule (Judd & Koretzky, 2000). The co-receptor molecules CDS or CD4 also participate in the binding to the MHC class I or II molecules and act synergistically with the TCR in signalling, resulting in about a 100-fold increase in the sensitivity of T cells to antigen. This antigen-specific signal (often known as first signal) will induce the synthesis of IL-2 along with the induction of the expression of high-affinity IL-2 receptors (IL-2R) on the membrane of the T cell. However, ligation of the TCR and the coreceptors on their own are not sufficient to initiate the stimulation of the T cell and promote the clonal expansion and differentiation into an effector cell. It is necessary to engage another pair of molecules which will provide a second or co-stimulatory signal. The best characterized

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co-stimulatory molecules on the APC are the structurally related glycoproteins B7.1 (DC80) and B7.2 (CD86). The counterpart molecule on the T cell surface that will interact with these glycoproteins is CD28 (Holford et al., 2000; Sharpe & Freeman, 2001). Dendritic cells in lymphoid organs constitutively express MHC class I, class II and B7 — for that reason they are also called professional APC. Macrophages and B cells have low constitutive levels of B7 expression, but after they contact with microorganisms its expression is up-regulated. The main effect of signalling through CD28 is thought to be the stabilization of IL-2 mRNA, which in turn permits a significant increase in the amount of IL-2 produced. When a T cell recognizes antigen in the absence of the co-stimulatory signal, very little IL-2 is produced and the T cell does not induce its own proliferation and the cells enter in a state of anergy. After their activation, T cells begin to express another surface molecule, CTLA-4 instead of CD28. CTLA-4 is a high-affinity receptor for B7. The interaction between CTLA-4 and its ligand B7 on APC, triggers inhibitory signals in the activated T cells, which contribute to limit the expansion of this population. Thus, antigen, co-stimulatory, and IL-2 signals cause T cells to divide and differentiate into a clone of armed effector cells that do not need further co-stimulation to provide cytotoxicity. Activated T cells now express a particular pattern of CAMs that direct them to a determined site of the body where they execute their function. When the work is finished, several mechanisms are triggered in order to maintain cellular homeostasis and to protect activated mature T cells from continued secretion of potentially harmful levels of cytokines. One of the most important mechanisms is the elimination from circulation of activated T cells by induction of apoptosis. This occurs via Fas and Fas ligand (Fas-L) interaction. Another fraction of activated T cells will form the memory T cell pool. Memory T cells are longer lived and have reduced requirements for co-stimulation than naive T cells. Cell-mediated Immune response: generation of armed effector T cells After antigen and co-stimulatory signals activate naive T cells, they begin to synthesize and secrete IL-2, increasing the expression of high affinity IL-2R. These signals cause T cells to divide and differentiate into clones of armed effector cells that do not need further costimulation to provide their function. The type of activated armed effector cells that will be generated depends on the mechanism used by the immune system to control and eliminate the foreign antigen. Two major categories of effector cells can be defined. One group contains effector cells that have direct cytotoxicity activity and their target are allogeneic cells, malignant cells and virus infected cells. Most of the specific cytotoxic T lymphocytes (CTL) are CD8+ and express ap TCR. The other subpopulation of effector T cells is constituted by CD4+ lymphocytes, which also express mainly oc|3 TCR and are usually designated as helper T cells. They can be further subdivided into inflammatory T helper 1 (Thl) cells or T helper (Th2) cells. Generation of Thl and Th2 cells Once a naive CD4 T cell contacts the peptide/MHC class II complex appropriately, they are thought to go through an intermediate stage, known as Th0 (Fig 5.9). ThO cells express some differentiated effector functions shared by both the inflammatory and helper T cells. The factors that determine whether proliferating CD4 T cells will differentiate into the two

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Fig. 5.9 Th1 and Th2 paradigm. CD4 T cell could differentiate into ThO, Th1 and Th2. Cytokine environment drives the differentiation. Interferon-y (INF-y) and IL-4 produced by Thl and Th2, respectively, can also act as autocrine growth factors as well as inhibitory factors for the opposite subset. On scale plates, cytokines released by each subset are shown. Thl enhances lgG2 synthesis by B cell and promotes the destruction of intracellular pathogen and organ-specific autoimmunity. Th2 induces IgE and IgGl production and promotes the anti-helminthic and allergic reaction.

alternative subpopulations mentioned above are not fully understood. It seems that both the cytokine microenviroment and the density of peptide : MHC ligands have an effect. However, the consequences of this decision are profound: selective activation of inflammatory CD4 Thl cells leads to cell-mediated immunity, while selective production of helper CD4 Th2 cells provides primarily humoral immunity (Dong & Flavell, 2001). Thl and Th2 cells

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release different but overlapping set of cytokines, which define their distinct actions in immunity; ThO cells, from which both of these functional classes are derived, also secrete cytokines and may have some effector functions. Th2 cells secrete IL-2, IL-4, IL-5, IL-6 and IL-10, all of which are cytokines that are potent in promoting B cell activation and differentiation. Thl or inflammatory T cells secrete IL-2, INF-y, which is the main macrophage-activating cytokine, and TNF-(3 or lymphotoxin (LT) which has a direct cytotoxic effect on some cells. The critical cytokine patterns produced by Thl and Th2 subsets, not only promote different effector arms of adaptive immunity, but also have two characteristic effects on the subset development. First, it induces the growth of the subset that produces them, and second, it inhibits the development and activity of the opposite subset, a phenomenon known as cross-regulation. Another T cell population was described, the Th3, that has the ability to produce a high concentration of TGF-(3. Cell-mediated immunity involves the delayed-type hypersensitivity (DTH) response, which plays an important role in host defence against intracellular pathogens. DTHassociated T cells, secrete a number of Thl-like cytokines that cause macrophages, to accumulate and to become activated. The activated macrophages, which are more effective killers of intracellular pathogens, are the primary effector cells in the DTH response. The presence of a DTH reaction can be measured by injection of an antigen intradermally (PPD or candidina) and the development of a skin lesion can be observed 24 or 48 hours later. The activation of the Th2 subset of lymphocytes involves the modulation of the humoral immune response; the pattern of cytokines they release promotes B cell differentiation and induces isotype switching. Th2 subset also stimulates eosinophil activation and differentiation; promotes the production of a relatively large amount of IgM, IgE and noncomplement-activating IgG isotypes. The Th2 subset also supports the allergic reactions and the protection against several helminth infections. Cytotoxic T cells Most of the CTL cells express the CDS surface marker and recognize antigenic peptides associated with the MHC class I proteins on the surface of APC. Some effector CTLs can have CD4 phenotype and interact with antigenic peptides associated to MHC class II molecules. However, in order for a CTL cell to acquire cytotoxic activity, it must receive an additional signal. Besides the specific and co-stimulatory signals that involve TCR and CD28 molecules, CDS + cells need the induction of high-affinity IL-2R, which is followed by IL-2 binding onto this receptor (Goldsby et al., 2000). These events promote the development and final maturation of CTL cells and the appearance of enzyme-containing granules in their cytoplasm, allowing the CTL cells to initiate their cellular cytotoxic activity (Fig. 5.10a). Mature CTL exert their cytotoxic activity by binding onto target cells that bear peptide/ MHC class I complexes. Once this first recognition step has been done, CTL cells reorient their cytoskeleton, focusing it to the site where the interaction is taking place and releasing a set of effector molecules towards the target cells that will induce its death by apoptosis. Programming the target cells to die requires approximately 5 minutes of contact between CTL and the target, although they appear viable for much longer. After that, CTL cells dissociate from the target and prepare for killing other target cells presenting the same epitope. During the maturation into effector CTL, CD8+ T cells synthesize molecules called

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perforin and granzymes and store them in cytoplasmatic granules. These are the effector molecules that will be released into the space between the CTL and the target. This directional release prevents killing of nearby uninfected cells. Perforin, similar in sequence to complement C9, polymerize to form a pore-like channel that will permit intracellular contents leakage, and allows the granzymes to enter into the target cells and promote its death. Granzymes activate a sequence of biochemical signals that induce the apoptosis of the cell. This pathway of killing is also called the secretory pathway (Fig. 5.10b). CTL cells possess another alternative mechanism that involves the binding of CTL membrane Fas-L to target cell Fas (a member of the TNF receptor family). This interaction induces target cell apoptosis in the absence of performs and granzymes and it is called nonsecretory pathway (Fig. 5.10c). Because several subsets of activated CD4+ T cells express Fas-L, they may also be cytotoxic as described earlier. Besides classical cytotoxic activity, CD8+ CTL cells can also modulate immunity by the release of cytokines, such as INF-y, TNF-P and TNF-OC. INF-y inhibits viral replication, activates IL-1 and promotes antigen processing.

Humoral immune response Activation ofB cells in the secondary lymphoid organs Once B cells complete their maturation in the bone marrow, they migrate through the blood into the lymphoid follicles of lymph nodes and spleen and other peripheral lymphoid tissues. Like T cells, it is also in the secondary lymphoid organs where B cells will encounter their antigen, get activated and differentiate into plasma cells. The activation of a B cell involves a first essential step: the crosslinking (aggregation) of their surface Ig (BCR) by the antigen to induce a cascade of biochemical events inside the cell (Tsubata & Wienands, 2001). In the case of a certain type of antigen, this first event is sufficient to promote the differentiation into plasma cells and production of antibodies; others need a further cooperative signal. Indeed, there are antigens that only elicit antibody responses in animals or people that have T cells; they need the cooperation of T cells to provide these further signals and are called thymus-dependant antigens (TD antigens). Other antigens can elicit a humoral response in the absence of T cells and are called thymusindependent antigens (TI antigens) (Fagarasan & Honjo, 2000). There are two types of TI antigens; TI-1 antigens, like components of bacterial walls like LPS, peptidoglycan, lipoprotein, porin, etc., which have intrinsic B cell activating activity, being potent polyclonal stimulators. As its name implies, polyclonal B cell activators are directly mitogenic, irrespective of antigen specificity, inducing B cells to differentiate into antibody-secreting cells. On the other hand, TI-2 antigens are typically repeated polymers like bacterial polysaccharides which appear to activate B cells by having multiple identical epitopes that crosslink the BCR. Most proteins are TD antigens. The activation of B cells by these antigens would typically happen in the T cell areas of the lymphoid tissues and requires Th2 cells. Antigen is brought there by macrophages and/or dendritic cells and it is the antigen contact which causes the B cell to stop migrating and remain in the T cell zone. Naive B cells can bind antigens that bear epitopes complementary to their cell surface Ig and this antigen contact will trigger the

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Fig. 5.10 Recognition and killing by CD8+ cytotoxic T cells, (a) Molecular recognition and signalling that promote the maturation of cytotoxic T cells to mature effector T cells. Effector cells kill the target by apoptosis through secretory and non-secretory pathways, (b) In the secretory pathway, perform and granzymes act in a Ca++ dependent manner, (c) In the non-secretory pathway the Fas and Fas-L interaction triggers the death signal.

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transmembrane signalling function of the BCR, which in turn induces early events in B cell activation including increasing expression of class II MHC molecules. Even if the antigen cannot cross-link the receptor, because it does not have more than one copy of the recognized epitope, it will be endocytosed and enter late endosomes and lysosomes, where it will be degraded into peptides. Some of these antigenic peptides will be loaded into class II MHC molecules and brought to the cell surface, where they can be recognized by T cells that bear receptor specific for the B cell's peptide/MHC class II protein complex. This interaction allows the activation of the T cell and it is usually designated as linked recognition. The fact that both T and B cells become activated by the same antigen does not mean that they are recognizing the same epitope on it. As it has been stated in previous sections, T cells recognize internal peptides, products of protein degradation, whereas B cells usually interact with surface epitopes. In the case of more complex antigens such as viruses, they may not even recognize the same protein. The recognition of peptide/MHC class II complexes on B cells by CD4 + helper T cells induces them to form a tight and long-lasting interaction with the B cell and to provide B cells with important signals necessary for their growth and differentiation. These important signals are given by: (1) cytokines which are secreted directly into the narrow space between the interacting cells, where the most important cytokine involved in this initial phase of B cell activation is interleukin 4 (IL-4); and (2) CD40 ligand (CD40-L) (Bishop & Hostager, 2001). Upon activation, CD4+ Th cells express a T cell surface molecule known as the CD40-L because it binds to the counterpart molecule CD40 expressed on the surface of the interacting B cell. CD40-L is a glycoprotein expressed primarily by activated T cells, but certain other cells can express it as well. IL-4 and CD40-L synergize and drive the clonal expansion that precedes antibody production in vivo. This event is responsible for the first IgM produced in a primary antibody response.

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Instead of terminally differentiating in situ, some of the B cells stimulated in this way migrate into the follicular region and initiate the germinal centre reaction (Fig. 5.1 la). These primary follicles contain resting B cells clustered around a dense network of processes extended from a specialized cell type known as follicular dendritic cell (FDC), which is thought to play a central contribution in the next steps that underlie the antibody response. When an activated B cell enters the primary follicle, they start to divide forming a germinal centre. These cells divide once every six hours and expand to about 10 in a few days. They have morphological characteristics typical of blast cells and for that reason they are called centroblasts. The region of centroblast proliferation is also called the dark zone. The remaining resting B cells, not specific for the antigen are pushed outside and form the mantle zone. In this specialized microenviroment of the germinal centre, helper T cells will also control the isotype switching and play a role in initiating somatic hypermutation of antibody variable-region genes which occurs during the course of a humoral immune response. As described before, one B cell makes antibody of just one specificity that is fixed by the nature of VJ and VDJ rearragements. However, during the life-time of this cell, it can switch to make a different class of antibody, such as IgG or IgE, while retaining the same antigenic specificity. This phenomenon is known as isotype switch and it should be differentiated from the alternative splicing of the mRNA that originates either surface IgM or IgD. Isotype switching involves further DNA rearrangement, juxtaposing the rearranged VDJ genes with a different heavy chain C region gene. In contrast to the previously described VJ and VDJ rearrangements, class switching is dependent on antigenic stimulation of the cell and the presence of cytokines released by T cells. Figure 5.11b summarizes the present knowledge about Th cytokines and their influence in isotype switching. The process known as somatic hypermutation involves the introduction of point mutations into the variable regions of the heavy and light chains at very high rate (one base pair per 103 per cell division). Other genes expressed in the B cells do not undergo mutagenesis at this elevated rate and in particular, the Ig constant region genes are not affected. The mechanisms by which this process occurs is poorly understood. The resulting mutant receptors are expressed on the progeny of these rapidly dividing centroblasts, which are small cells called centrocytes, all derived from the same progenitors that founded the germinal centre. Most of these mutations are likely to be deleterious, with only rare mutations increasing the affinity of Ig for the antigen. There exists a powerful mechanism to select those cells with favourable mutations against those with harmful or useless mutations. This selection process seems to occur in the centrocyte stage. Centrocytes will move into an adjacent area, called the light zone, densely packed with the processes of the follicular dendritic cells. A striking feature of the FDC is their ability of holding antigen in their native form for periods that can be longer than a year. In a Darwinian process, only those centrocytes whose mutated cell-surface Igs have improved their affinity for the antigens presented by the FDC will survive. The rest will die by apoptosis; a typical feature of the germinal centre is the presence of macrophages engulfing apoptotic cells. Thus, contact between FDC and the centrocytes is an important element in the survival of centrocytes. This gradual increase in the affinity of antibodies for the inducing antigen that is seen in the course of an immune response is known as affinity maturation. Finally, germinal centre B cells either terminally differentiate into plasma cells or become quiescent memory cells (Calame, 2001). The former usually migrate to the gut or the bone

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Fig. 5.11 The B cell activation in the germinal centre and immunoglobulin isotype switching, (a) Germinal centres are formed when activated B cells enter lymphoid follicles where they start to proliferate rapidly (centroblasts) and form the dark zone. As these centroblasts mature, they stop dividing and become small centrocytes, moving out into the light zone where the centrocytes make contact with follicular dendritic cells. Resting B cells are pushed outside in the mantle zone, (b) The cytokines and their influence in isotype switching.

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marrow, where they are quite long lived (three to six weeks). It seems that the adoption of a memory cell fate requires continuous delivery of CD40-L signal to B cells. Once generated, memory B cells can persist in a non-proliferative state for an extended period of time. Their survival requires the continued presence of antigen, probably kept on the surface of FDC. Kinetics of the immune response The first exposure of an individual to a particular antigen is referred to as the priming event. Sometimes this priming event is deliberately provoked and in this case it is called immunization. It is followed by a latent phase of about one or two weeks in which antibodies are undetectable in the serum. During this time, both the innate and early induced immunity is operating, whereas the initial contact of T and B cells with the antigen is taking place in the secondary lymphoid organs, and the processes of clonal expansion and generation of effector cells start. The first antibody isotype detected in the blood is generally IgM, which in some instances may be the only class of Ig that is made. Sometimes it can be followed by a low peak of IgG or some other isotype. Generally, this first wave of antibodies generated after a first contact with the antigen has considerably low affinity and titre, and it is called the primary immune response. The concentration of circulating antibodies starts to decline due to their degradation and within a few weeks the production of them may cease completely. However, the immunized individual is left with immunological memory and if he comes into contact again with the same antigen, the response has a considerably shorter latent phase and achieves a markedly higher plateau level, producing antibodies of higher affinity. These features are typical of a secondary immune response, which shows a remarkable change in the quality and type of antibodies produced, with IgG (or other isotypes) appearing at higher concentrations and greater persistence (months or even years later). This phenomenon is usually known as maturation of the immune response and it is the result of the mechanism of isotype switching and somatic hypermutation that have been explained in the previous section. Furthermore, immune response to TI antigens do not present these characteristics, it does not show a change in the isotype produced or in the affinity of the antibodies; they do not elicit immunological memory and promote mainly IgM circulating antibodies.

Endogenous regulation of the immune response The basis of a functional immune system is genetic: the ability to make mature lymphocytes and accessory cells bearing the required antigen-specific receptors, co-receptors, MHC, and co-stimulatory molecules. Functional genes for complement, inflammatory mediators and cytokines are also required. However, other factors which are not genetic affect immune responsiveness: nutrition, the presence of chronic disease and lifestyle, for example, are among the most important of them. Ageing also has a great consequence on the performance of the immune system. As we age, our immune systems works less efficiently; thymus atrophy results in fewer circulating and functional T cells, weaker primary responses, and more infections, cancer, hypersensitivity, and autoimmunity. Thus, in a system as complex as this which produces an immune response, it is to be expected that multiple levels of control exist. These levels of control are in charge of limiting an ongoing response, in order to get

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back to homeostasis once the large deviation of clonal expansion and secretion of cytokines has taken place. This includes internal mechanisms for self-regulation. Once the antigen stimulates adaptive immunity by activating lymphocytes (which in turn make antibody to activate complement and cytokines to increase antigen elimination and recruit additional leukocytes), secreted antibodies compete with B cells for antigen. Immune complexes bind Fc receptors on B cells; at low levels they increase the sensitivity of B cells to antigen and increase responsiveness, whereas at high levels they give negative signals to B cells to reduce humoral immunity. On the other hand, when the effector T cells finish their functions, several mechanism are triggered in order to maintain cellular homeostasis. During activation-induced cell death the circulating activated T cells are eliminated through apoptosis induction via Fas and Fas-L interaction. At another level, IL-1 induces sleep and fever and stimulates release of pituitary hormones (corticotrophin, thyroid-stimulating hormone, and growth hormone) (Artz et al., 2000). These hormones together with thymic, steroid, and peptide hormones also influence our ability to make immune responses. There are also multiple levels of control during a lymphocyte lifetime which are in charge of controlling the appearence of self reactive clones that could potentially be harmful for the organism. The capacity of the system to ignore host antigens is an active process involving the elimination or inactivation of cells that could recognize self antigens through a process designated immunological tolerance. Tolerance to 7 cell repertoire Central or clonal tolerance results from clonal deletion of self reactive T cells during development. This process involves the deletion of cells that express receptors that bind with high affinity to complexes of self-peptides with self-MHC molecules. This is a major mechanism through which the T cell compartment develops immunological unresponsiveness to self antigens. As explained elsewhere in this chapter, the particular MHC alleles available to present self antigen to developing T cells influence which peptides are presented well enough to induce clonal deletion. The thymic development of T cells results in the acquisition of both immunological tolerance to self and reactivity to foreign antigenic stimuli (Sprent & Kishimoto, 2001). However, studies in animal models of T cell mediated autoimmunity show that these processes in the thymus may not be sufficient for controlling self reactive T cells. For example, analysis of self reactive TCR transgenic mouse strains have established that as many as 25-40% of autoreactive T cells escape clonal deletion even in the presence of the deleting ligand. Most self reactive T cells are not completely deleted by negative selection in the thymus. Low-avidity T cells escape thymic deletion and are retained in the mature naive T cell population and will be subject of peripheral tolerance. Peripheral tolerance is an extrathymic tolerance and occurs in the periphery. It maintains tolerance to self antigen not seen or inactivated in the thymus and marrow (Walker & Abbas, 2002). It is believed to be a mechanism of security controlling these autoreactive T cells that could escape to the negative selection, and encounter unprocessed antigen or processed antigen in the absence of co-stimulatory signals. One of the most important mechanisms is the lack of co-stimulatory signals and presence of inhibitory receptors. T cell activation requires recognition of specific antigenic peptides by the TCR, as well as additional

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co-stimulatory signals provided by accessory surface molecules on T cells. CD28 is the best characterized and most effective co-stimulatory molecule expressed by naive and primed T cells and binds B7-1 (CD80) and B7-2 (CD86) on APCs. TCR binding in the absence of CD 2 8 ligation results in a state called anergy. Such anergic T cells are incapable of producing IL-2 or proliferating on subsequent stimulations. It is an important way of inducing tolerance in mature T cell populations. Thus, the recognition of autoantigens on resting tissue APCs in the absence of co-stimulatory molecules results in the induction and maintenance of T cell tolerance to self antigens. Conversely, the aberrant expression of co-stimulators on APCs could activate self reactive T cells, an hypothesis to explain autoimmunity. In addition to positive co-stimulatory molecules, inhibitory receptors have also been identified on T cells. These include CTLA-4 (CD 152), programmed death 1 (PD-1). CTLA4 is expressed on activated T cells, is approximately 30% homologous with CD28, albeit with a much higher affinity. This suggests that CTLA-4, as it becomes up-regulated on activated T cells, might preferentially interact with CD80 and CD86 and, therefore aid in the termination of immune responses. CTLA-4 inhibits T cell activation by reducing IL-2 production and IL-2 receptor expression, and by arresting T cells at the Gl phase of the cell cycle. PD-1 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor expressed upon T cell activation. PD-1 animals develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses. Members of the B7 family, PDLl and PD-L2, are ligands for PD-1 (Carter et al., 2002). However, because naive autoreactive T cells have limited access to non-lymphoid tissues, it is very unlikely that they would be rendered anergic by recognition of tissue-specific antigens in the absence of a co-stimulatory signal. In fact, it has been shown that the immune system in its resting state possesses T cells capable of recognizing tissue-specific antigens, but these cells are neither activated nor inactivated by it. This situation has been called immunological ignorance (Ohashi etal., 1991). It is believed that these autoreactive T cells exit the thymus because their avidity for the MHC—self peptide complexes is too low to induce clonal deletion. Most self-proteins on any tissue are expressed at levels too low to serve as targets for lymphocyte recognition and activation. However, under situations such as viral infection, it seems reasonable to assume that the local expression of TNF-OC could affect the presentation of these self-proteins and activate these otherwise ignorant T cells. These results are most compatible with the view that tolerance to peripheral antigens results from immunological ignorance rather than specific unresponsiveness. Tolerance to B cell repertoire In the process of maturation from marrow stem cells, B cell precursors undergo genetic rearrangement leading to insertion of an antigen-specific receptor (slgM) on the surface. At this early immune stage (immature B cell), experimental evidence suggests that if they interact with antigen, even at relatively low dosages, they become unresponsive rather than activated (Iglesias, 2001). The unresponsiveness of the B cells may be due either to deletion for apoptosis or to anergy. In the anergic state, the antigen-specific cell is still present but has been inactivated. The molecular mechanisms underlying differential sensitivity of mature and immature B cells to tolerance induction are poorly defined. An attractive hypothesis holds that antigen receptor-mediated signal transduction pathways are qualitatively or

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quantitatively different in these cells, and, as a consequence, trigger differentiation stageunique responses such as receptor editing, apoptosis, and activation. Following acquisition of IgD on the surface, the now-mature B cells are much more resistant to tolerance induction, requiring much higher doses of antigen to achieve an unresponsive state. The mature B cells could be eliminated or inactivated in the periphery due to the lack of T cell cooperation. Immunoregulation In the past few years it has become clear that many states of unresponsiveness observed following the introduction of antigen to a mature immune system are actually the result of negative regulation of one type of immune response by another (Chatenoud et al., 2001). Regulatory T (Tr) cells

Although negative selection in the thymus and induction of anergy in the periphery have been widely accepted as mechanisms for controlling autoreactivity, much less attention has been devoted to the role of suppressor or regulatory T cells in mediating dominant immunological self-tolerance (Annacker et al., 2001). In 1995, Powell et al. were the first to describe that potent Tr cells were present in the thymus of the adult rat. PVG rats develop autoimmune diabetes after adult thymectomy and split-dose irradiation. Sakaguchi et al. (1995) made the seminal observation that the transfer of CD4+ T cells which had been depleted of the minor subpopulation (10%) of cells that coexpressed the IL-2R a chain (CD25) to nu/nu recipients induced organ-specific autoimmune disease in the majority of recipients. The CD4+CD25+ population was also shown to be solely responsible for the prevention of autoimmunity observed after mice are thymectomized on the third day of life. Further studies demonstrated that the CD4+CD25+ T cells act through an APCindependent mechanism and their development is dependent on MHC class II-positive thymic cortical epithelium. Induction of suppressor activity requires that the CD4+ CD25 + cells be activated through their TCR, but once activated, they suppress T cell activation in an antigen-independent manner without a requirement for reactivation through their TCR. The CD4+ CD25+ cells could represent a unique lineage of CD4+ T cells that function as 'professional suppressor cells' (Shevack, 2001). The major consequence of CD25 + -mediated suppression is the induction of cell cycle arrest in the CD25 — responders and cell cycle arrest is normally followed by apoptotic cell death. It is highly likely that thymic-derived CD4+ CD25+ T cells represent only one of potentially multiple types of regulatory/suppressor T cells. These inhibitory effects only require CTLA-4 to be present on the CD25 + T cells, which indicates that CTLA-4 engagement on Tr cells might confer a suppressor function. As can be seen the cytokines play an important role in the regulation of the immune response. IL-10, IL-4, and TGF-(3 have been implicated as mediating suppression of some, but not all autoimmune diseases by Tr cells (Gorelik & Flavell, 2002). While IL-10 is produced by the Tr cells, it has not yet been shown that IL-4 or TGF-P are actually produced by CD25+ regulatory cells in vivo. Thus, it is difficult to conclude if these cytokines are responsible for the suppressive effects of the Tr cells or if they play a role in the differentiation of the Tr cells.

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Thl—Th2 balance The cytokines play an important role in the regulation of the immune response. It was observed that CD4+ subsets can inhibit one another's production and function: Thl cells stimulate cellular immunity and suppress humoral immunity, whereas Th2 cytokines have the opposite effect. Th2 cells make TGF-fJ, which inhibits the growth of Thl, and IL-10, which acts on macrophages to inhibit Thl activation, by blocking macrophage IL-12 synthesis (Dong & Flavell, 2001). Thl cells make IFN-y, which blocks the growth of Th2 cells. These effects allow either subset to dominate a response by suppressing outgrowth of cells of the other subset. Network of idiotypic interactions

A general type of regulatory interaction between lymphocytes can occur when the receptors on one lymphocyte recognize the receptors on a second lymphocyte. The immune system can respond specifically to an almost infinite array of epitopes. In responding to a single epitope, many different antibodies are made, each of which carried many different epitopes on the variable region of the antibody called, idiotopes, since they are epitopes of the antigenbinding region of a given receptor; the idiotype is the sum of idiotopes on that receptor. When antigen activates the proliferation of a set of responding cells, the receptors on this set of cells can activate two further sets of cells via receptor—receptor interactions. Some cells are the internal image set, whose receptors carry epitopes that resemble the antigen and can stimulate antigen-specific cells. The second set of cells called the anti-idiotypic regulator set, bear receptors that recognize the idiotype of antigen-specific receptors but do not mimic the original antigen. The elements of an idiotypic network exist and form an essential part of the normal functioning of the immune system.

References Annacker, O., Pimenta-Araujo, R., Burlen-Defranoux, O. & Bandeira, A. (2001) On the ontogeny and physiology of regulatory T cells. Immunology Review, 182, 5—17. Arzt, E., Kovalovsky, D., Igaz, L.M. et al. (2000) Functional cross-talk among cytokines, Tcell receptor, and glucocorticoid receptor transcriptional activity and action. Annals of the New York Academy of Sciences, 917, 612—1. Bishop, G.A. & Hostager, B.S. (2001) Signaling by CD40 and its mimics in B cell activation. Immunology Research, 24, 97-109. Bendelac, A., Bonneville, M. & Kearney, J.F. (2001) Autoreactivity by design: innate B and T lymphocytes. Nature Reviews, 1, 177-86. Calame, K.L. (2001) Plasma cells: finding new light at the end of B cell development. Nature Immunology, 2, 1103—8. Carter, L., Fouser, L., Jussif, J. et al. (2002) PD-1:PD-L inhibitory pathway affects both CD4( + ) and CD8( + ) T cells and is overcome by IL-2. European Journal of Immunology, 32, 634-43. Chatenoud, L., Salomon, B. & Bluestone, J.A. (2001) Suppressor T cells — they're back and critical for regulation of autoimmunity! Immunology Review, 182, 149—63. Dong, C. & Flavell, R.A. (2001) Thl and Th2 cells. Current Opinion in Hematology, 8, 47-51.

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Durkin, H.G. & Waksman, B.H. (2001) Thymus and tolerance. Is regulation the major function of the thymus? Immunology Review, 182, 33-57. Fagarasan, S. & Honjo, T. (2000) T-independent immune response: new aspects of B cell biology. Science, 290, 89-92. Fowell, D., Powrie, P., Saoudi, A. Seddon, B., Heath, V. & Mason, D. (1995) The role of subsets of CD4+ T cells in autoimmunity. Ciba Foundation Symposium, 195, 173—85. Frazer, J.K. & Capra, J.D. (1999) Immunoglobulins: structure and function. In: Fundamental Immunology, 4th edn (ed. W.E. Paul), pp. 37—74. Lippincott-Raven Publishers, Philadephia. Germain, R.N. (1999) Antigen processing and presentation. In: Fundamental Immunology, 4th edn (ed. W.E. Paul), pp. 287-340. Lippincott-Raven Publishers, Philadephia. Goldsby, R.A., Kindt, TJ. & Osborne, B.A. (eds) (2000) Immunology, 4th edn. W.H. Freeman and Company, USA. Gorelik, L. & Flavell, R.A. (2002) Transforming growth factor-beta in T-cell biology. Nature Review Immunology, 2, 46—53. Holford, A.D., Kanagawa, O. & Shaw, A.S. (2000) CD28 and T cell co-stimulation. Review of Immunogenetics, 2, 175—84. Hwang, L.Y., Lieu, P.T., Peterson P.A. & Yang, Y. (2001) Functional regulation of immunoproteasomes and transporter associated with antigen processing. Immunology Research, 24, 245-72. Iglesias, A. (2001) Maintenance and loss of self-tolerance in B cells. Springer Seminar of Immunopathology, 23, 351—66. lijima, H., Takahashi, I. & Kiyono, H. (2001) Mucosal immune network in the gut for the control of infectious diseases. Review Medical Virology, 11, 117—33. Judd, B.A. & Koretzky, G.A. (2000) Antigen specific T lymphocyte activation. Review of Immunogenetics, 2, 164—74. Kadowaki, N., Antonenko, S., Lau, J.Y. & Liu, YJ. (2000) Natural interferon a/(3 - producing cells link innate and adaptive immunity. Journal of Experimental Medicine, 192, 219-226. Krangel, M.S., McMurry, M.T., Hernandez-Munain, C, Zhong, X.P. & Carabana, J. (2000) Accessibility control of T cell receptor gene rearrangement in developing thymocytes. The TCR alpha/ delta locus. Immunology Research, 22, 127—35. Luster, A.D. (2002) The role of chemokines in linking innate and adaptive immunity. Current Opinion in Immunology, 14, 129—35. Margulies, D.H. (1999) The major histocompatibility complexes. In: Fundamental Immunology, 4th edn (ed. W.E. Paul) pp. 263-85. Lippincott-Raven Publishers, Philadephia. Moretta, A., Bottino, C., Mingani, M.C., Biassoni, R. & Moretta, L. (2002) What is a natural killer cell? Nature Immunology, 3, 6—8. Moser, B. & Loetscher, P. (2001) Lymphocyte traffic control by chemokines. Nature Immunology, 2, 123—8. Nagler-Anderson, C. (2001) Man the barrier! Strategic defences in the intestinal mucosa. Nature Review Immunology, 1, 59—67. Ohashi, P.S., Oehen, S., Buerki, K. et al. (1991) Ablation of'tolerance' and induction of diabetes by virus infection in viral antigen transgenic mice. Cell, 65, 305—17. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. & Toda, M. (1995) Immunologic self-

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tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. Journal of Immunology, 155, 1151—64. Schittek, B., Hipfel, R, Sauer, B. et al. (2001) Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nature Immunology, 2, 1133-7. Sharpe, A.H. & Freeman, G.J. (2001) The B7-CD28 superfamily. Nature Review Immunology, 2, 116-26. Shevach, E.M. (2001) Certified professionals: CD4 + CD25+ suppressor T cells. Journal of Experimental Medicine, 193, F4l—6. Sprent, J. & Kishimoto, H. (2001) The thymus and central tolerance. Transplantation, 72, S25-8. Springer, T.A. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell, 76, 301-4. Tonegawa, S. (1983) Somatic generation of antibody diversity. Nature, 302, 575—81. Tsubata, T. (2001) Molecular mechanisms for apoptosis induced by signaling through the B cell antigen receptor. International Review of Immunology, 20, 791—803. Tsubata, T. & Wienands, J. (2001) B cell signaling. Introduction. International Review Immunology, 20, 675—8. Walker, L.S. & Abbas, A.K. (2002) The enemy within: keeping self-reactive T cells at bay in the periphery. Nature Review Immunology, 2, 11—19. Wardemann, H., Boehm, T., Dear, N. & Carsetti, R. (2002) B-la B cells that link the innate and adaptive immune responses are lacking in the absence of the spleen. Journal of Experimental Medicine, 195, 771-80.

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A. Marcos, E. Nova and S. Lopez-Varela Instituto de Nutricion y Bromatologia, Consejo Superior de Investigaciones Cientificas, Spain

Relationship between nutrition and the immune system The defence of the organism The immune system is very complex and consists of a number of organs, tissues, cells types, and molecules that are scattered throughout the body. The cells of the immune system synthesize and recognize a variety of molecules, including antibodies, complement proteins, cytokines, growth factors, and receptors for these molecules. Many of these molecules have pleiotropic and synergistic effects. Overall protection of the organism is provided by an interaction between the various cells and molecules of the immune system. Immunity is a state of resistance to or protection from pathogenic microorganisms. There are two types of immunity, innate and adaptive. Innate, or non-specific, immunity is present at all times in normal individuals and is thus fully functional before infectious agents enter the body. Adaptive immunity involves the specific reaction of the animal body to challenge by immunogen. It is activated after the pathogen has evaded the innate response and entered the body. There are two types of adaptive immunity: cellular immunity, mediated by T lymphocytes, and humoral immunity, mediated by B lymphocytes. However, it is important to highlight that all these cells collaborate among each other together with the substances they secrete in order to achieve immune homeostasis (Fig. 6.1). Allergy, or hypersensitivity, is an immune-mediated reaction to a foreign antigen (allergen) that causes tissue inflammation and organ dysfunction. Oedema, redness, pain, and heat are the four cardinal symptoms of inflammation. Cytokines play a critical role in the pathogenesis of inflammatory and allergic diseases. An imbalance between pro- and antiinflammatory cytokines leads to inflammatory or allergic diseases. Among immunocompetent cells, polymorphonuclear cells (PMN) are involved in acute inflammation processes while mononuclear cells (macrophages, T lymphocytes, and plasma cells) are in charge of chronic inflammation mechanisms. Some aspects of inflammation will be discussed further in this chapter.

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Fig. 6.1 The collaboration among immunocompetent cells and the substances secreted to get immune homeostasis after an immune response has been triggered. CK, cytokines.

Imume system behaviour under malnutrition The development, maintenance and optimal functioning of the immune system depend on balanced and adequate nutrition. Both deficiency and excess of a number of nutrients adversely affect the number and activity of immune cells (Chandra, 1997). In this sense, many metabolic pathways require specific nutrients as cofactors. The antioxidative defence mechanisms employed by the body are also heavily dependent on the nutritional intake of the individual and involve a variety of vitamins (e.g. vitamins C and E), trace elements (e.g. zinc and selenium) and amino acids (e.g. cysteine). Multiple- rather than single-nutrient deficiencies are often the causes for a compromised immune system and an increased risk of infection, as observed in patients with protein-energy malnutrition (PEM) (Chandra, 2001). In malnourished subjects anatomical changes involve atrophy of primary as well as secondary immune organs such as the thymus and spleen, respectively (Chandra, 1991). Malnutrition impairs mainly cell-mediated immunity, which is altered at an early stage in the development of undernutrition. Nevertheless, some alterations in phagocytosis and the complement system have also been described in PEM (Chandra, 1997). B cell function has so far not been shown to be affected by PEM to the same extent as that of T lymphocytes. In addition, the production of several cytokines, such as interleukin-1 (IL-1), interleukin-2 (IL2), and interferon-y (IFN-y), is decreased. Moreover, malnutrition alters the ability of T

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lymphocytes to respond appropriately to cytokines. There is little work on the effect of malnutrition on the integrity of physical barriers, quality of mucus, or several other innate immune defences. For example, lysozyme concentrations are decreased, largely as the result of reduced production in monocytes and neutrophils and an increased excretion in urine (Chandra et al., 1977). Effects of micronutrients on the immune system Several trace elements and vitamins have an essential role in key metabolic pathways of immune cells functions. Isolated deficiencies of micronutrients are rare with the exception of zinc, iron, and vitamin A. Observations in laboratory animals and findings in the rare patient with a single nutrient deficiency have confirmed the crucial role of several vitamins and trace elements in immunocompetence (Benedich & Chandra, 1990). Alterations in immune responses occur early in the course of reduction in micronutrient intake, and the extent of immunological impairment depends on the type of nutrient involved, its interactions with other essential nutrients, the severity of the deficiency, the presence of concomitant infection, and the age of the subject. Thus, tests of immunocompetence are useful in titration of physiological needs and in assessment of safe lower and upper limits of micronutrient intakes. Many studies have pointed out that micronutrients such as zinc, selenium, iron, copper, (3carotene, vitamins A, C, and E and folic acid can influence several components of the nonspecific immunity (Erickson et al., 2000). Because of space limitation, only the nutrients that have most pronounced effects on the immune system, such as the minerals zinc, copper, selenium and iron, and vitamins A, C and E are reviewed here. Zinc The essentiality of zinc for humans was already first documented in the 1960s. In human subjects body growth and development are strictly dependent on zinc. The nervous, reproductive and immune systems are particularly influenced by zinc deficiency, as well as by increased levels of zinc (Rink & Gabriel, 2000). More than 300 metalloenzymes have been identified as being zinc (Zn) dependent. Many of these are critical for cellular metabolic pathways, including those that mediate phagocyte and lymphocyte functions. Zinc deficiency is associated with lymphoid atrophy, decreased delayed-type hypersensitivity (DTH) cutaneous responses, delayed homograft rejection, and a lower thymulin (thymic hormone) activity (Table 6.1). It is not surprising then that zinc deficiency results in profound immunodeficiency (Thurnham, 1997). The salient changes observed are in (a) phagocytes: reduced ingestion of microorganisms, impaired chemotactic migration, decreased activity of reduced oxidase, which is a cofactor for phospholipases A2 and C and instability of cell membranes possibly due to oxidation of arachidonic acid by iron complexes; (b) cell-mediated immunity: imbalance between T helper type 1 (Thl) and Th2 functions (Prasad, 2000), reduced lymphocyte proliferation response, decreased CD4 : CDS cell ratio and helper T cell function, impaired natural killer (NK) cell function, reduced thymulin activity (a thymic hormone); and (c) humoral immunity: decreased antibody production after challenge with T cell dependent antigens and alloantigens (Prasad, 1998). A slightly excessive intake of certain nutrients such as zinc may be associated with enhanced immune responses. However, there is evidence that almost all nutrients given in

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Table 6.1

Effects of mineral deficiencies on the immune response

Micronutrient

Effect of deficiency

Zinc

Thymus atrophy Cell-mediated immunity functions j Delayed cutaneous hypersensitivity | Phagocytic function j Wound healing | Leukopenia Morbidity, mortality during infection |

Selenium

Antioxidant capacity j Cell-mediated immunity j NK cell function j. Immunoglobulin production |

Copper

Humoral response, B cell function J. Thymus atrophy Cell-mediated immunity functions | T lymphocyte response J, Phagocytic function | Morbidity during infection | Cytokine and lymphokine function or production |

Iron

Thymus atrophy Lymphocyte proliferation j Delayed cutaneous hypersensitivity J. Phagocytic function | NK cell function j Cytokine and lymphokine function or production |

Up and down arrows indicate, respectively, an increase or decrease of the parameter mentioned.

quantities beyond a certain threshold can reduce the immune response. This outcome has been shown for zinc regarding both phagocyte and lymphocyte functions. The mechanisms of immunotoxic effects are not clear, but for Zn overdose, alterations in serum and cellbound low-density lipoproteins, reduced concentrations of other nutrients, changes in membrane structure and receptor expression have been described as possible causes (Chandra, 1984; Chandra & Au, 1980). The moderate deficiencies in zinc noted in sickle cell anaemia, renal disease, chronic gastrointestinal disorders, acrodermatitis enteropathica, virus-associated immunodeficiency, diarrhoeas, and in elderly persons can greatly alter host defence systems, leading to increases in opportunistic infections and mortality rates (Franker et al., 2000). Conversely, short periods of Zn supplementation substantially improve immune defence in individuals with these diseases.

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Selenium

The trace mineral selenium is an essential nutrient of fundamental importance to human biology. As selenocysteine, the 21st amino acid, selenium (Se) is a component of selenoproteins, some of which have important enzymatic functions (Sunde, 1997). In the active site selenium functions as a redox centre; the best-known example of this redox function is the reduction of hydrogen peroxide and damaging lipid and phospholipid hydroperoxides to harmless products (water and alcohols) by the family of selenium-dependent glutathione peroxidases (Diplock, 1994; Sunde, 1997; Allan et al., 1999). This function helps to maintain membrane integrity, protects prostacyclin production, and reduces the likelihood of propagation of further oxidative damage to biomolecules such as lipids, lipoproteins, and DNA with the associated increased risk of conditions such as atherosclerosis and cancer (Diplock, 1994; Neve, 1996). Selenium has additional important health effects particularly in relation to the immune response and viral disease (Rayman, 2000). This trace mineral is normally found in significant amounts in immune tissues such as liver, spleen, and lymph nodes. Both cellmediated immunity and B cell function can be impaired in selenium deficiency (Spallholz et al., 1990) (Table 6.1). By way of contrast, supplementation with selenium, has marked immunostimulating effects, including an enhancement of proliferation of activated cytotoxic T cells and an increased NK cell activity (Kiremidjian-Schumacher & Roy, 1998). These effects have been related to the ability of selenium to enhance interleukin-2 receptor (IL-2R) expression on the surface of activated lymphocytes and NK cells (Kiremidjian-Schumacher & Roy, 1998). Immunocompetent cells may have an important functional need for selenium. Activated T cells show up-regulated selenophosphate synthetase activity (Guimaraes et al., 1996), directed towards the synthesis of selenoproteins, which shows the importance of selenoproteins to activate T cell function and the control of the immune response. The mRNAs of several T cell associated genes (e.g., IL-2R a-subunit, CD4) have the theoretical capacity to encode functional selenoproteins, suggesting that the roles played by selenium on the immune system may be more diverse than previously suspected (Taylor & Nadimpalli, 1999). Copper

Copper is known to play an important role in the development and maintenance of the immune system but its exact mechanism of action is not yet known (Percival, 1998). Animal models and cells in culture have been used to assess copper's role on the immune response. The immune effects of copper deficiency are listed in Table 6.1. They include both B cell and T cell related deficiencies. Impaired antibody formation, inflammatory response, phagocytic killing power, and lymphocyte stimulation responses, as well as thymic atrophy, have been well documented (Kelley et al., 1995; Zhavoronkov & Kudrin, 1996; Failla, 1998; Erickson et al., 2000). Some of the previous research showed that IL-2 is reduced in copper deficiency (Blakley & Hamilton, 1987; Kelley et al., 1995) and is likely the mechanism for T cell proliferation reduction (Lukasewcycz & Prohaska, 1990; Kelley et al., 1995). Similarly, the number of neutrophils in human peripheral blood and their ability to generate superoxide anion and kill ingested microorganisms is reduced in both overt and marginal copper deficiency (Failla &

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Hopkins, 1998). In addition, infections decrease both serum copper and zinc (Castillo-Duran et al., 1988) aggravating the situation and further impairing the defence system. Neutrophillike HL-60 cells accumulate copper as they differentiate into a more mature cell population and this accumulation is not reflected by increases in Cu/Zn superoxide dismutase or cytochrome-c oxidase activities. The identity of copper-binding proteins in neutrophil-like HL-60 may be useful in learning new functions of copper (Percival, 1998). Iron

Iron deficiency is the most widespread nutrient deficiency in the world today. There is a large body of evidence accumulated from animal and human studies (Sherman, 1990; Chandra, 2001) to indicate that iron deficiency states are associated with alterations in cellular function, growth, motor development, behaviour and cognitive function. No controversy exists about the deleterious effects of iron deficiency on immune responses (Chandra, 1991; Brock & Mulero, 2000); almost all published studies indicate that individuals with iron deficiency show impairment of cell-mediated immunity (DTH responses, T lymphocyte proliferation response to mitogens and antigens, production of cytokines such as IL-2 and IFN-y). In addition, phagocyte microbicidal function, NK activity and mucosal immunity are impaired (Table 6.1). Apparently B cell and antibody formation are not affected. These alterations may well be linked to changes in the activity of iron-dependent enzymes such as myeloperoxidase and ribonucleotide reductase. In addition, physical changes in the mucosal epithelia may also be important. A more recent study indicates that iron presence can help monocytes to suppress Mycobacterium tuberculosis growth (Byrd, 1997). Iron-mediated growth suppression was correlated with selective suppression of tumour necrosis factor-a (TNF-a) release from infected monocytes and decreased monocyte sensitivity to exogenously added TNF. Any discussion of the effect of dietary iron on immunity is incomplete without discussion of the biological mechanisms for withholding iron from invading organisms. This micronutrient is needed for a wide variety of biochemical functions not only by the host but also by the infectious agent (Weinberg, 1974). Transferrin is found not only in blood but also in all body fluids and is the normal mechanism for withholding iron from the infectious agent, as is lactoferrin. Conalbumin and lactoferrin have stronger iron binding properties than do most bacterial siderophores and are normally highly unsaturated. When molecules of lactoferrin become * 40% saturated with iron, they are assimilated by macrophages that have been attracted to the site of infection and much of the iron is incorporated into ferritin. Ferritin functions as an iron withholding rather than an iron-transport agent. In an iron-deficient host with reduced immune function, lack of available iron for agent replication is protective. When individuals whose resistance to infection is compromised by iron deficiency are given parenteral iron or large doses of oral iron, a disastrous exacerbation of the infection and death may occur (McFarlane et al., 1970; Brock, 1993). This happens because the agent is supplied with iron for replication before the host immune system has had time to recover. However, in field studies, supplementation of poorly nourished adults with physiological amounts of up to l00mg Fe/day and proportionately less for children, consistently results in decreased morbidity from infectious diseases. It is important to recognize that there is a fairly large range of iron intakes over which immune system can function normally.

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In summary, when prevention or treatment of iron deficiency may be expected to lower incidence of common infections, oral iron administration in conventional doses is safe and effective (Walter et a/., 1997; Hershko et al., 1998). Antioxidant vitamins The oxidant/antioxidant balance is an important determinant of immune cell function, including maintaining integrity and functionality of membrane lipids, cellular proteins, nucleic acids, and for control of signal transduction and gene expression on immune cells (Meydani et al., 1998). Thus, optimal function of the host defence system depends upon an adequate supply of antioxidant vitamins and, on the other hand, impaired host defence activity can act as a very early and sensitive marker of marginal deficiency of antioxidant vitamins (Schmidt, 1997). In humans, dietary supplementation with ascorbic acid, tocopherols and vitamin A has been shown to enhance a number of aspects of immunity and resistance to disease (Grimble, 1997; Meydani et al., 2001). Vitamin A Atrophy of lymphoid organs, including spleen, thymus, and lymph nodes, has been reported in vitamin A-deficient animals. However, some of these effects may be caused by loss of appetite and decreased food intake. Changes in spleen cell number are observed in the early stages of vitamin A deficiency and might be a more sensitive indicator of vitamin A deficiency (Ross, 1992), which can also affect the function of different cells of the immune system (Blumberg & Hughes, 2001) (Table 6.2).

Table 6.2

Effects of vitamin deficiencies on the immune response

Vitamin A

Thymus atrophy Lymphocyte proliferation [ Immunoglobulin profuction [ Bacterial adherence to host epithelium f Cytokine and lymphokine function or production j

Vitamin E

Cell-mediated immunity [ Immunoglobulin production j. Antioxidant capacity J, Phagocytic function j Delayed cutaneous hypersensitivity J. Cytokine and lymphokine function or production [

Vitamin C

Locomotion macrophages j. Bactericidal capacity macrophages | Antioxidant capacity j Delayed cutaneous hypersensitivity J.

Up and down arrows indicate, respectively, an increase or decrease of the parameter mentioned.

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Different studies have reported defects in phagocytic activity (defect in chemotaxis, adhesion and the ability to generate reactive oxygen metabolites in neutrophils) (Twining et al., 1997) and impairment of T and B cell function (Ross, 1992; Semba, 1998). In addition, vitamin A deficiency reduces NK activity, and is associated with a lower production of interferon (Ross, 2000; Austenaa & Ross, 2001). Vitamin A is essential for the maintenance of epidermal and mucosal integrity, thus low plasma vitamin A concentrations have deleterious effects on membrane integrity and mucosal function (Thurhman, 1997). It is not surprising that impaired antibody response to viral and parasitic antigens has also been pointed out in vitamin A deficiency in animals (Ross, 1992). It is well-known that impaired intestinal immunoglobulin A (IgA) production is attributed to impairment of gut-associated immune response (Chandra, 2001). Conclusive information related to vitamin A deficiency and antibody production in humans, as well as evaluation of DTH skin test response, independent of other nutritional deficiencies, is not available. As vitamin A deficiency has been associated with an increased morbidity and mortality from infectious diseases (diarrhoea and respiratory infections), several investigators have attempted to improve the immune response and, thus, resistance to infection by vitamin A supplementation in vitamin A-deficient subjects (Moriguchi, 1999). The outcome of these studies has varied, depending on the vitamin A status of the host, the type of infectious agent, and the immune response evaluated. In general, both an improvement of the immune function and an increased resistance to infection have been observed when vitamin A supplementation has been given to vitamin A-deficient hosts (Meydani et al., 2001). Vitamin E Vitamin E is the major peroxidation chain-breaking antioxidant in membranes. Membrane phospholipids of immunocompetent cells have a high content of polyunsaturated fatty acids and are prime targets for free radical reactions. Release of reactive oxygen species by phagocytes on encountering pathogens and rapid lymphocyte proliferation following antigenic stimulation expose the immune cells to high levels of oxidative stress. Thus, it is not surprising that cells of the immune system have a higher vitamin E content than other cells of the body (Hartman & Kayden, 1979). Both deficiency and supplementation of vitamin E have been shown to alter the immune response and resistance against infection (Meydani et al., 2001). The influence of vitamin E on the immune function has been shown to affect different aspects of immune functions including T cell response, antibody production, NK cell activity, phagocytic activity, and the production of immunoregulatory molecules (Moriguchi & Muraga, 2000) (Table 6.2). In humans, primary deficiency of vitamin E rarely occurs, whereas secondary deficiency is observed as a consequence of certain diseases such as primary cirrhosis, intestinal malabsorption disorders (Munoz et al., 1989), and several viral hepatitis and human immunodeficiency virus (HIV)-l infection (Periquet et al., 1995). In addition, vitamin E deficiency is associated with increased infectious diseases and the incidence of tumours (Moriguchi & Muraga, 2000). In contrast, vitamin E is one of the few nutrients for which supplementation at higher than established dietary requirements has been shown to enhance immune response and resistance to disease (Meydani et al., 2001), and this seems to be especially important in the aged. The beneficial effects of

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supplementation on the host immune system include enhanced humoral and cell-mediated immunity and increased efficacy of phagocytosis in humans (Beharka et a/., 1997; Moriguchi & Muraga, 2000). Vitamin C Vitamin C appears to affect most aspects of the immune system. It is found in high concentrations in leukocytes, it is rapidly utilized during infection, and reduced plasma levels are often associated with reduced immune function (Hughes, 1999). However, the belief that high intakes of vitamin C will prevent the onset of common cold has not been scientifically substantiated. A decrease in the duration of cold episodes and the severity of symptoms seem more plausible, although the benefits that have been observed in different studies show a large variation. The explanation for this amelioration of symptoms could be derived from the decrease in the inflammatory effects due to the reaction of vitamin C with the phagocytederived oxidants released extracellularly during infection episodes, thus being neutralized (Anderson & Lukey, 1987; Hemila, 1992). In a placebo-controlled, double-blind intervention study, responses to DTH skin tests were significantly reduced in healthy men submitted to a vitamin C-deficient diet for 60 days (Jacob et al., 1991). This study showed that moderate vitamin deficiency reduces cellmediated immunity. On the other hand, supplementation has been shown to increase neutrophil motility and mitogen-stimulated lymphocyte proliferation in young males (Anderson et al., 1980), as well as decrease the incidence of post-race infections in marathon runners (Peters et al., 1993). Vitamin C is one of the food components with higher antioxidant properties as demonstrated, for instance, on the protection of lipids in plasma and low-density lipoproteins (LDL) against detectable peroxidative damage (Frei, 1991). Vitamin C also serves as an electron donor to vitamin E radicals generated in the cell membrane during oxidative stress (Ji, 1995). Some effects of vitamin C, such as the decrease in DNA damage by reactive oxygen species after supplementation, still remain to be established in humans. However, their function as antioxidant may be associated to atherosclerosis and cancer prevention. The effect of simultaneous supplementation with several vitamins over the immune system has been addressed many times. Supplementation of vitamins C (Ig/day) and E (400mg/day) in healthy adult volunteers has been found to enhance lipopolysaccharide (LPS)-induced IL-1(3 and TNF-oc production by peripheral blood mononuclear cells (PBMCs). This effect coincided with peak plasma a-tocopherol and ascorbate concentrations and the lowest plasma lipid peroxide concentrations on day 14 (Jeng et al., 1996). Chandra (1992) showed a significant decrease in the number of sick days and in the use of antibiotics, as well as an increased antibody response to the flu vaccine, in a group of healthy elderly subjects who supplemented their diets with a multivitamin supplement that contained 100% of the recommended daily allowance (RDA) of most vitamins and moderately higher amounts of antioxidant vitamin C (80 mg/day), vitamin E (44 mg/day), and (3-carotene (I6mg/day). The importance of maintaining adequate amounts of antioxidants throughout life, is based on a potential long-term effect of prevention of the accumulative damage caused by reactive oxygen species being made manifest in later years (Hughes, 1999).

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The immune system in eating disorders Eating disorders as a clear example of malnutrition The syndrome of anorexia nervosa (AN) is characterized by an intense fear of fatness and the individual's refusal to eat with the purpose of losing weight or maintaining it below normal standards. Fear of fatness is shared by bulimia nervosa (BN) patients, but the actual behaviour towards food is dominated by bingeing episodes and compensatory mechanisms such as exercise, vomiting, or abuse of laxatives and/or diuretics. As a result, a normal weight is commonly shown in BN patients as compared with the extremely slim or emaciated AN patient. This behaviour, deeply influenced by aesthetic patterns, is so completely ingrained in the patients that severe situations of malnutrition develop over time and high mortality rates are associated with eating disorders (Marcos, 2000). Studies on immunohaematological variables in patients suffering from AN and BN suggest an aberrant immunological status (Marcos, 2000). In these patients, a tendency to leukopenia together with relative lymphocytosis and a decreased DTH skin test response have been demonstrated (Cason et al., 1986; Varela et al., 1988; Marcos et al., 1993). However, when the diagnosis of BN is made early, neither alteration in the neutrophil or monocyte counts nor a relative lymphocytosis occur. Nevertheless, lymphocyte counts are lower than in controls, which is a sign that malnutrition is occurring in bulimia patients (Marcos et al., 1997b). It is generally accepted that malnutrition affects in the first term cellular immunity rather than humoral immunity, and similar findings have been also described in AN. A one-year follow-up of 16 female adolescents with restricting-type AN revealed that in addition to lower leukocyte counts than their matched controls, the lymphocyte subset counts were lower and reflected the fluctuations in the nutritional status (Marcos et al., 1997a). A marked reduction in the percentage and absolute number of memory CDS + T cells has been found in AN patients by other authors (Mustafa et al., 1997). However, in other studies the anomalies found in the T cell subpopulations are a reduction of CD4+ CD45RA+ together with an increase of CD8 + (Allende et al., 1998). It has been speculated that these findings might lead to a reduced frequency of lymphocytes capable of recall responses and this could be related to the perceived lack of symptomatic common viral infections that have been frequently reported in underweight anorexic patients.

Cytokine participation over the lack of infection in eating disorders Despite these signs of immune impairment, a great deal of controversy exists, because on several occasions it has been noted that underweight AN patients are surprisingly free from common viral infections (Armstrong-Esther et al., 1978; Golla et al., 1981; Wade et al., 1985). In this regard, consideration should be paid to the nutritional differences between AN and PEM. The high relative protein intake which is usually found in AN patients, in contrast with more typical situations of nutritional deprivation, could contribute towards maintaining the immune function in underweight AN patients better preserved than might be expected (Marcos, 1997). Cytokines are among the more important factors regulating immune function and the

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extent to which these are involved in the pathogenesis of the AN syndrome seems to be relevant. Since several cytokines including TNF, IL-1, IL-6, IL-8, and IFN-y have been shown to induce anorexia and cachexia, hypotheses have been developed conceiving cytokines as the fundamental regulators of body metabolism in AN and BN (Holden & Pakula, 1996). However, according to the different immune parameter values observed in several reports, it is evident that not all AN patients respond similarly. The proliferative response and cytokine production by PBMCs in response to a mitogen are variable among anorectic subjects (Polack et al., 1993). Since complex interactions occur between cytokines and the central nervous system, differences in the capacity of AN patients to evoke a compensatory mechanism through either the neuroendocrine system or the autonomic nervous system could explain the variability of the results found (Marcos, 2000). As part of the normal acute-phase response to infection, proinflammatory cytokines activate the hypothalamic-pituitary-adrenal axis (HPAA) and directly stimulate adrenocorticotrophic hormone (ACTH) secretion as well. In turn, glucocorticoids alter the production of these cytokines in a feedback regulation. However, in the particular case of AN, these regulatory mechanisms seem to be altered. Firstly, there is an apparent failure to mount the typical acute-phase response to infection, since some of its symptoms, such as fever, are lacking. On the other hand, some authors have reported a spontaneous and elevated production of proinflammatory cytokines such as IL-1, IL-6, and TNF-a, contributing to weight loss, cachexia and osteoporosis in AN (Holden & Pakula, 1999; Allende et al., 1998). In relation with those spontaneously elevated levels of proinflammatory cytokines, it has been suggested that a cell that has been stimulated to a certain extent to produce a cytokine will respond poorly to further stimulation. According to this fact, an impaired capacity to mount an acute-phase response to infection, under an extra stimulus, could be expected (Fig. 6.2). In agreement with this hypothesis, we have found a decreased PHA-stimulated TNF-OC and IL-6 production by PBMC from a group of AN patients admitted to hospital for refeeding, in comparison with matched controls (Nova et al., 2002). Elevated levels of serum cortisol and a positive correlation between cortisol and in vitro secretion of IL-1 (3 by PBMC of AN patients have been reported (Limone et al., 2000). In agreement with what has been previously mentioned, the authors of this study consider that the normal relationship between IL-1 (3 release and cortisol secretion is deranged in AN patients. On the other hand, the in vitro IL-1 (3 production might be very different from the in vivo production, where a broad array of mediators could be affecting the immune system. In this sense, since macrophage production of IL-1 (3 is regulated by a cortisol receptor in monocytes (Paez-Pereda et al., 1996), high serum cortisol levels could result in an impaired IL-1 [3 production in response to an infection (Nova et al., 2002). A decreased release of this pyrogen could explain the inability of AN patients to get fever. Thus, according to what has been reported above, the altered production of cytokines depicts another possible mechanism to explain the absence of symptoms of infection in patients with eating disorders. The interrelationship between leptin and cytokines Moreover, there is an interrelationship between cytokines and leptin levels. Leptin, is a protein encoded by the ob gene, that is known to regulate appetite and energy expenditure. It

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Fig. 6.2 Proinflammatory cytokines and their relationships with cortisol and leptin alterations in AN. Hypothetical implications during infection processes. AN patients typically show a decreased plasma leptin concentration and an increased cortisol concentration. Proinflammatory cytokines (IL-1, TNF-a, IL-6) seem to be maintained at high levels in AN patients, which might indicate that the negative feedback mechanism mediated by cortisol is not working. However, when an infection occurs, high plasma cortisol levels could regulate IL-lp production through a cortisol receptor in monocytes, i.e. preventing the normally increased IL-1(3 secretion in response to an infection. Also during infection episodes, leptin levels are normally increased in plasma, which in turn seem to activate proinflammatory cytokine production by macrophages. However, it could be hypothesized that an incapacity to increase leptin due to neuroendocrine and BMI alterations in AN patients would result in a suppression of the expected increase of these cytokines and consequently to the surprising lack of infection symptoms described in AN patients.

is usually positively correlated with body mass index (BMI) in obese, normal and slim individuals and also in AN and BN patients (Monteleone et al., 2000). In addition to its role regarding weight control and energy metabolism, leptin has been suggested to function as a prominent regulator of immune system activity, linking the function of T lymphocytes to nutritional status. In starvation, the dramatic reduction in leptin seems to be a key mediator of thymic atrophy and immune suppression, involving for instance, an inhibitory effect on CD4+ T lymphocyte activation (Matarese, 2000). In addition, exogenous leptin has been reported to stimulate phagocytic function and activate macrophages to produce

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proinflammatory cytokines, such as TNF-OC, IL-6, and IL-12 in ob/ob mice (Loffreda et al., 1998). Therefore, there is a novel function for leptin as an up-regulator factor of inflammatory immune responses. Moreover, leptin production is acutely increased during infection and inflammation (Fantuzzi & Faggioni, 2000), thus, an impairment in this acute increase in leptin production in AN patients might be related to the lack of infection symptoms in these patients. In BN patients, basal leptin concentration is decreased as well, despite weight values being similar to those in age-matched controls (Monteleone et al., 2000). Thus, factors other than body weight may play a role in the determination of leptin changes in eating disorders. Another aspect to consider is the relationship between leptin and cortisol. Prior to refeeding, the semistarvation state in AN patients is associated with a quantitative alteration in the circadian rhythm of leptin and cortisol levels and an alteration in the temporal coupling between cortisol and leptin. After weight gain, leptin increments precede cortisol increases by eight hours, while no temporal relationship has been found between both hormones in the semistarvation state (Herpertz et al., 2000).

Conclusions According to the immunological parameters, patients with AN and BN show immunodeficiency which is secondary to a situation of malnutrition. Due to the dietary restriction which these patients are voluntarily submitted to, nutrient intakes are lower than recommended ones. It is not surprising then that these patients show a depleted immune response, however it should be noted that more than a deteriorated immune function is required, it must be distorted, in order to adapt the homeostasis of the immune system to a new situation where neuroendocrine and the central nervous system may be involved (Marcos et al., 2001). The outcomes so far suggest that the interrelationships among cortisol, leptin and cytokines, all of them repeatedly found to be altered in eating disorders, may play an important role in the regulatory mechanism triggered in individuals with these syndromes. These compensatory mechanisms may enable the patients to adapt to these atypical situations of malnutrition and might also provide a clue to explain some of the immunological findings, such as the lack of infections in AN patients. Future research which will increase our knowledge about the bi-directional communication pathways between the neuroendocrine and the immune systems, will help us to perform further studies in patients with eating disorders trying to define the role of those mediators in the pathogenesis of these syndromes.

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Kelley, D.S., Daudu, P.A., Taylor, P.C., Mackey, B.E., Turnlund, J.R. (1995) Effects of lowcopper diets on human immune response. American Journal of Clinical Nutrition, 62, 412-16. Kiremidjian-Schumacher, L., Roy, M. (1998) Selenium and immune function. Zeitschrift Ernahrungswiss, 37 (Suppl. 1), 50—6. Limone, P., Biglino, A., Bottino, F. et al. (2000) Evidence for a positive correlation between serum cortisol levels and IL-lbeta production by peripheral mono nuclear cells in anorexia nervosa. Journal of Endocrinology Investigation, 23, 422—7. Loffreda, S., Yang, S.Q., Lin, H.Z. et al. (1998) Leptin regulates proinflammatory immune responses. FASEBJ, 12, 57-65. Lukasewcycz, O.A., Prohaska, J.R. (1990) The immune response in copper deficiency. Annals of the New York Academy of Science, 587, 147—59. Marcos, A. (1997) The immune system in eating disorders: an overview. Nutrition, 13, 853-62. Marcos, A. (2000) Eating disorders: a situation of malnutrition with peculiar changes in the immune system. European Journal of Clinical Nutrition, 54, 61IS—4S. Marcos, A., Varela, P., Santacruz, I., Munoz-Velez, A., Morande, G. (1993) Nutritional status and immunocompetence in eating disorders. A comparative study. European Journal of Clinical Nutrition ,47,787-93. Marcos, A., Varela, P., Toro, O. (1997a) Interactions between nutrition and immunity in anorexia nervosa. A one year follow-up. American Journal of Clinical Nutrition, 66, 485S-90S. Marcos, A., Varela, P., Toro, O., Nova, E., Lopez-Vidriero, I., Morande, G. (1997b) Evaluation of nutritional status by immunological assessment in bulima nervosa. Influence of BMI and vomiting episodes. American Journal of Clinical Nutrition, 66, 49 IS—7S. Marcos, A., Montero, A., Lopez-Varela, S., Morande, G. (2001) Eating disorders (obesity, anorexia and bulimia), immunity and infection. In: Nutrition, Immunity and Infection Disease in Infants and Children, 45th Nestle Nutrition Workshop (ed. K. Tontisirin & R. Susking). Bangkok, Thailand. Nestle Nutrition Services, 45, 243—79. Matarese, G. (2000) Leptin and the immune system: how nutritional status influences the immune response. European Cytokine Network, 11, 7—13. McFarlane, H., Reddy, S., Adcock, K.J., Adeshina, H., Cooke, A.R., Akene, J. (1970) Immunity, transferrin and survival in kwashiorkor. British Journal of Medicine, 4, 268—70. Meydani, S.N., Santos, M.S., Wu, D., Hayek, M.G. (1998) Antioxidant modulation of cytokines and their biologic function in the aged. Zeitschrift Ernahrungswiss, 37, 35—42. Meydani, S.N., Fawzi, W.W., Sun, N.H. (2001) The effect of vitamin deficiencies (E and A) and supplementation on infection and immune response. In: Nutrition, Immunity and Infection Disease in Infants and Children. 45th Nestle Nutrition Workshop, (ed. K. Tontisirin & R. Suskind) Nestle Nutrition Services, 45, 213-41. Monteleone, P., Di Lieto, A., Tortorella, A., Longobardi, N., Maj, M. (2000) Circulating leptin in patients with anorexia nervosa, bulimia nervosa or binge eating disorder: Relationship to body weight, eating patterns, psychopathology and endocrine changes. Psychiatry Research, 94, 121-9Moriguchi, S. (1999) Cellular immunity and vitamins. Nippon Rinsho, 57, 2313—18. Moriguchi, S., Muraga, M. (2000) Vitamin E and immunity. Vitamin Hormone 59, 305—36.

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M.E. Roux1, N.H. Slobodianik2, P. Gauffin Cano3 & G. Perdigon3'4 1

Laboratorio de Inmunologia Celular, Departamento de Ciencias Biologicas, Fac. de Farmacia y Bioquimica, UBA, Junin 956, 5° piso, (1113), Buenos Aires, Argentina, 2 Departamento de Nutricion, Fac. de Farmacia y Bioquimica, UBA, Buenos Aires, Argentina, 3 Centra de Referenda para Lactobacilos (CERELA), Chacabuco 145 (4000), Tucuman, Argentina, and 4 Catedra de Inmunologia, Fac de Bioquimica, Quimica y Farmacia, UNT, Tucuman, Argentina

Mucosal immunity Introduction The mucosal surfaces of the respiratory and digestive systems represent vast surface areas covered by delicate epithelial barriers (Neutra et al., 2001). Mucosal surfaces are protected by epithelial secretion products such as mucins, defensins and secretory antibodies - especially immunoglobulin A (IgA) (Lamm, 1997) and by epithelial specializations that include tight junctions and apical surface coats. On most mucosal surfaces, however, only a single layer of epithelial cells separates the outside world from interstitial tissues. Thus it is not surprising that mucosal tissues are sites of intense immunological activity. Despite their protective function, epithelial barriers provide the mucosal immune system with a continuous stream of information about the external environment. Effective immune surveillance of the mucosal surface requires transport of intact macromolecules and microorganisms across the epithelial barrier to cells of the mucosal immune system. This transport, which occurs along the gastrointestinal tract and the airways, is concentrated at sites that contain organized mucosal lymphoid follicles. At these specialized sites the collaboration of epithelial cells with antigenpresenting and lymphoid cells is highly developed (Brandtzaeg et al., 1999). Although consisting of only a single layer of cells, the intestinal epithelium must control the access of potential antigens and pathogens, and, at the same time, function in the digestive absorption of dietary nutrients. It is aided in this dual role by intracellular tight junctions that restrict the passage of even very small (2-kDa) molecules. The intestinal epithelium also boasts a number of specialized protective adaptations that are not found in other sites. This includes antimicrobial peptides (defensins), secretory immunoglobulin A,

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mucins and trefoil peptides. The apical surface of the enterocyte, which faces the intestinal lumen, is ideally suited for the terminal digestion of nutrients because of its dense coating with absorptive microvilli. At the tips of the microvilli, a layer of membrane-anchored glycoproteins forms the filamentous brush border glycocalyx (FBBG). Digested nutrients can gain access to the body through the FBBG, but it is relatively impermeable to macromolecules or bacteria (Neutra et al., 2001).

Organized mucosal lymphoid tissues Transport, processing and presentation of foreign antigens, as well of induction and clonal expansion of antigen-specific effector lymphocytes, occurs at specific 'sentinel' sites in the mucosa that are marked by the presence of organized lymphoid tissues and specialization of the overlying epithelium (Brandtzaeg et al., 1999; McGhee et al., 1999). The distribution of these sites in the body generally reflects the local abundance of foreign material and microorganisms. In humans, for example, the oral and nasal pharynx is monitored by a ring of mucosal lymphoid tissues, the palatine and lingual tonsils and the adenoids, and lymphoid follicles are present in the bronchii of some species (Bienenstock et al., 1999). Single lymphoid follicles are distributed throughout the intestine and increase in frequency in the distal ileum and colon where the microbial flora is abundant and diverse. In the distal ileum, lymphoid follicles are grouped in large patches (Peyer's patches) that are visible to the naked eye. However, in humans the greatest frequency of follicles and follicle-associated epithelium (FAE) occurs in the rectum and in dead-ended extensions of the intestinal lumen, such as the caecum and the appendix, where the microflora is particularly abundant. The hallmark of organized mucosal-associated lymphoid tissues (MALTs) is the presence of lymphoid follicles. The composition and distribution of these tissues have been reviewed (Roux et al., 2000).

Antigen uptake Role of epithelial, toll-like receptors and dendritic cells Soluble proteins and microbes do cross the epithelial barrier. Enterocytes are the primary cell type in the epithelial monolayer, which is interspersed in some regions by a specialized epithelial cell type known as the M cell. The brush border glycocalyx that characterizes villus enterocytes is absent from the apical surface of the M cell. It is replaced by microfolds (hence 'M cell') that are more accessible to luminal antigens. M cells use transepithelial vesicular transport to carry microbes to antigen-presenting cells (APCs) in the underlying gut-associated lymphoid tissue (GALT) (Krahenbuhl & Neutra, 2000). The GALT is divided into discrete inductive and effector sites. M cells are contained within inductive sites in the GALT, known as Peyer's patches. Peyer's patches are aggregations of lymphoid follicles found primarily in the distal ileum of the small intestine (Nagler-Andersson, 2001). Located within the dome-like structure of the follicle-associated epithelium, M cells have long been thought to act as 'gateways to the mucosal immune system', delivering antigens to

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the APCs in the Peyer's patch subepithelial dome. It turns out, however, that M cells are not the only cell types capable of transporting antigen across the epithelial barrier. A recent report indicates that one type of professional APC, the dendritic cell (DC), might extend its dendritic-like processes through epithelial tight junctions and sample luminal antigen directly (Rescigno et al., 2001) (Fig. 7. la). The integrity of the barrier would be maintained during the process because tight junctions are reformed by proteins that are expressed on both enterocytes and DCs.

Fig. 7.1 Gut-associated lymphoid tissue, (a) FAE: follicle-associated epithelium; SED: subepithelial dome; DC: dendritic cell, (b) An intestinal villi. IEL: intraepithelial lymphocytes; LP: lamina propria; T: T cells; B: IgA-secreting B; DC: dendritic cell.

The aggregations of lymphoid follicles that form the Peyer's patches are the most discernible of the follicles dispersed throughout the small and large intestine. Like lymph nodes Peyer's patches have B cell follicles and germinal centres that are surrounded by areas that contain T cells. As seen in Fig. 7.1b, the gut contains loosely organized effector sites, primarily within the lamina propria of the intestinal villi. The lymphocytes found in the

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lamina propria are largely immunoglobulin A (IgA)-secreting plasma cells and memory T effector cells (Farstad et al., 2000). The role of secretory Ig A in excluding antigen from entering the epithelium has long been appreciated. It has recently been suggested that IgA also runs a shuttle service, using the plgR to actively transport antigen out of the lamina propria to the apical surface of the enterocyte (Robinson et al., 2001). These observations indicate a twofold barrier function for secretory IgA, both in guarding the epithelium from microbial entry and as a lamina propria 'pump', that excrets IgA-bounded antigens or microbes that penetrate the barrier back into the lumen. Immunoglobulin production typically requires T cell help. CD4+ helper cells (Th) are divided into two functional subsets on the basis of their pattern of cytokine secretion. In general, Thl cells produce interferon y (IFNy), which is important for cell-mediated immune responses and inflammation Th2 cells secrete interleukins (IL-4, IL-5 and IL-13), and induce B cell activation and differentiation. Unlike other immunoglobulin isotypes, signals through transforming growth factor P (TGF-P)/TGF-|3 receptor type II on B cells are vital for class switching to IgA. Th2 cytokines therefore control B cell differentiation into IgA-secreting plasma cells. TGF-(3 is abundantly expressed in the GALT and is central to two of the most distinctive functions of GALT: secretion of IgA and the generation of regulatory T cells (Weiner, 2001). Besides, the GALT contains some unusual T cells: the intestinal ephitelium that might be unique in its ability to function as an important site for extrathymic maturation of a substantial population of T cells, the thymus-independent development of intra-epithelial lymphocytes (lELs), a subpopulation of T cells which resides between the epithelial cells, above the basement membrane. The enterocytes themselves produce IL-7, which is important for the development of these lELs. Expression of CDS aa homodimers, rather than the CD 8 ap heterodimer that is expressed by CD8+ T cells in other sites, identifies extrathymically derived lELs. However, no evidence has been obtained for a functional role for the CDS aa chain in these lELs, which include y5 TCR+ lELs and many ap+ TCR lELs that develop without passing through the thymus. Infection by a large variety of pathogens results in the activation of lELs, but, so far, no defensive role has been ascribed to the extrathymically derived CDS aa+ subset (Marquez et al., 2000). Moreover, the ability of the GALT to respond quickly and effectively to repeated assaults by enteric pathogens is enhanced by the accumulation at these sites of memory T effector cells. Many pathogens are transported into the Peyer's patches through M cells. Once the epithelial barrier is breached, a rapid host response is required to confine the invaders to the mucosa. Microbes initiate a host-protective response by activating an evolutionarily conserved, primitive pattern-recognition system (Medzhistov & Janeway, 1997). One class of pattern-recognition receptors for these pathogen-associated molecular patterns (PAMPs) was originally identified as toll receptors in Drosophila. Some of their mammalian counterparts, the toll-like receptors (TLRs), have recently been described (Akira et al., 2001). Macrophages and DCs waiting just below the epithelial dome are well positioned to detect microbial entry into the Peyer's patch. Signalling by TLRs would then induce the upregulation of the co-stimulatory molecules that provide the requisite 'second signal' for the activation of naive T cells. However, the essential microbial structure elements that are recognized by these TLRs are common to both commensal flora and pathogens.

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The commensal bacteria that reside in the luminal flora activate the innate immune system by signalling through TLRs. These commensal antigens gain access to the GALT through DCs that are recruited into the epithelium and take them up as well as pathogenic and non-pathogenic bacteria (Rescigno et al., 2001). However, DCs carrying non-invasive bacteria are not observed deeper in the intestinal villi, indicating that they remain in the lamina propria. Perhaps pathogenic bacteria might induce maturation and migration of DCs, whereas commensal bacteria do not. The ability of DCs to discriminate between pathogens and commensals is related in part to differential signalling by TLRs or groups of TLRs. TLRs have a crucial role in the detection of microbial infection in mammals and insects. In mammals, these receptors have evolved to recognize conserved products unique to microbial metabolism. This specificity allows the toll proteins to detect the presence of infection and to induce activation of inflammatory and antimicrobial innate immune responses. Recognition of microbial products by TLRs expressed on DCs triggers functional maturation of dendritic cells and leads to initiation of antigenspecific adaptive immune responses (Medzhistov, 2001) as described in Fig. 7.2.

Fig. 7.2

Initiation of antigen-specific responses.

Perhaps commensals bear as yet unidentified PAMP that elicits an anti-inflammatory cytokine programme, or lack a PAMP that is related to invasiveness and that induces inflammatory cytokine production. In the intestinal mucosa, professional APCs in the Peyer's patches and lamina propria are not the only cells that express TLRs. Intestinal intraepithelial cells also posses both TLRs and the capacity of secreting a wide variety of cytokines and chemokines. Bacterial lipopolysaccharide (LPS) can activate the nuclear transcription factor B (NF-kB) that switches the cytokine secretion programme that characterizes an inflammatory response (Nagler-Andersson, 2001). The epithelial surface, acting as a physical barrier, excludes most commensals, but some

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are able to make it through. It has been described that binding of non-pathogenic bacteria to the epithelium suppressed the transcription of proinflammatory cytokines, blocking the NFkB/inhibitory kB (I-kB) pathway. Therefore, signalling by commensals blocks I-kB degradation and prevents the release of NF-kB and its translocation into the nucleus. Pathogenic recognition turns on this pathway. Also, the mucosa, especially in the gastrointestinal tract, is constantly exposed to foreign substances, and secretes IgA antibodies into the lumen, providing an immunological barrier to limit the penetration of antigens into the mucous membrane (Lamm, 1997). Several studies have allowed Robinson et al. (2001) to propose that mucosal antigens in the lamina propria provide an internal barrier beneath the epithelium that can trap antigens missed by the initial IgA barrier in the lumen. During infection of the mucosae, antigens will be released into the lamina propria from microbial pathogens. The authors think that foreign antigens are a regular presence in mucosal lamina propria. Regardless of the origins of particulate antigens at those sites, IgA antibodies are in a position to bind and efficiently transport antigens out of the body and into the lumen and even excrete particles as large as intact viruses.

Tolerance to food Apart from commensal bacteria, the other main source of potential antigenic stimulation for the GALT comes from food proteins. A large body of experimental evidence has shown that oral administration of soluble antigens induces systemic non-responsiveness to peripheral antigen challenge. Typically known as oral tolerance, the induction of non-responsiveness to dietary antigen is likely to have a vital physiological role in preventing hypersensitivity reactions to food. Often described as systemic non-responsiveness accompanied by local, mucosal (IgA) immunity, new evidence indicates that the predominant mucosal response to non-pathogenic luminal antigens (dietary or commensal) is also one of tolerance. Oral tolerance has been defined as an antigen-specific hyporesponsiveness after prior oral encounter of the antigen. This theme has been previously described (Roux et al., 2000; Nagler-Andersson, 2001).

Effect of nutritional factors on the microenvironment of mucosal immune system Many nutritional factors play important roles in key cellular and metabolic processes. Specific micronutrient deficiencies, concomitant with protein-energy malnutrition, alter immune responses. The severity of an immunological impairment depends upon the severity of the nutrient imbalance, its interactions with other essential nutrients, age of the subject and presence of concomitant infection. However, excessive intake of nutrients may also exert deleterious effects (Chandra, 1997). Protein deficiency alters the kinetics of cellular proliferation in lymphoid organs, such as the thymus (Pallaro et al., 2001), the GALT (Lopez & Roux, 1989) and the BALT (Marquez et al., 1997).

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Protein deprivation at weaning, as shown in an experimental model, provokes an arrest in thymus maturation and a severe impairment in B and T cell differentiation in the GALT (Lopez & Roux, 1989; Pallaro et al., 2001). Moreover, the administration of a low quality dietary protein (maize flour diet), during either long or short periods of time, provokes severe atrophy of the thymus and important changes in GALT, expressed as an arrest in cellular proliferation and differentiation. These observations indicate a synergistic action between low protein quality and low dietary protein concentration, probably due to a low complete protein intake that leads to a lower intake of essential amino acids, especially the limiting amino acid lysine (Pallaro et al., 1996, 1997). Recent studies show that, in addition to low protein concentration, low quality dietary protein is a limiting factor at certain steps of the cellular intrathymic pathways, probably related to the requirement of specific amino acids for an optimal immune response (Pallaro et al., 2001). Isolated deficiencies of micronutrients are rare, with the exception of iron, vitamin B and zinc. The relationship within nutritional factors and immune responses has been intensely reviewed (Scrimshaw & Sangiovani, 1997; Pallaro & Slobodianik, 1999).

Nutrition and immunity Malnutrition has an adverse impact on cell-mediated, secretory and humoral immunity, as well as on non-specific host defences. Although most clinical observations concerning this problem have been made in patients with generalized malnutrition, deficiencies or excess of single nutrients can also cause acquired functional derangements. Acquired anergy can generally be reversed when malnutrition is corrected. Further, secondary nutritional derangements tend to be less severe than primary congenital defects of an immune or non-specific defence component. Unlike the relative resistance to infection seen in patients with uncomplicated starvation, cachexia associated with severe disease, malignancy, or trauma is typically accompanied by superimposed infections, especially due to opportunistic microorganisms that are normally of low pathogenicity. The hypermetabolism of most severe diseases is accompanied by complex biochemical responses, increased energy generation, and an inability to conserve amino acid stores. Secondary derangements in the function of defensive mechanisms and immune responses may then emerge rapidly. Further, loss of body nutrients during primary infection increases the susceptibility of a patient to secondary infections. Chronically malnourished patients often exhibit an infection or infestation as a coexisting problem. Moreover, Solomons (2000) has pointed to the existence of a complex interaction between malnutrition and infection, which was first delineated 40 years ago by Scrimshaw and Sangiovani (1997). Events in the past decade suggest that this interaction must be investigated. Nutritional effects on immune mechanisms Although generalized malnutrition can affect all aspects of host immunity, the impact if greatest on T cell functions and cell-mediated immunity and smallest on B cell functions and humoral immunity. Effects on secretory immunity fall in between.

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Slight leukopenia, together with relative lymphocytosis was found in patients suffering from eating disorders (Marcos et al., 1993). In fact, plasma concentrations of IgM, IgG, IgA, IgD, and IgE may all be greater than normal in malnourished infants, and blocking antibodies and antibodies against food antigens may increase. The humoral immunity, judged by serum IgA concentration, was different in adolescent females suffering from obesity or anorexia nervosa; obese patients showed the highest values (Varela et al., 1997). Apparently paradoxical increases in plasma Ig values during malnutrition have not been explained with certainty, but may involve the presence of infectious or parasitic diseases. Children of developing countries tend to experience a continuing heavy exposure to multiple antigens. Contsondis (1995) has shown that vitamin A supplementation results in an increased IgG response to the wild measles virus. On the other hand, Semba et al. (1992) have shown an increased IgG response to tetanus toxoid, after vitamin A supplementation. During severe generalized malnutrition, lymphoid tissue atrophy develops primarily in the T cell areas and circulating T cell numbers decline. Loss of delayed dermal hypersensitivity to previously encountered antigens is typical and de novo dermal sensitization responses are impaired. In vitro responsiveness to mitogens and antigens is impaired in T cells from patients with generalized protein-energy malnutrition. Host-versus-graft reactions are delayed. Experimental animal models in rats have been well described (Pallaro et al., 1996, 2001; Marquez et al., 1997). Protein undernutrition has been shown to suppress significantly the secretion of lysozyme into tears. Lysozyme levels in tears were significantly lower (50% less) in moderately malnourished grade II children than in normal children. The synthesis and secretion into tears of other locally produced proteins, slgA and amylase were also impaired. Decreased activity of lysozyme has also been observed in leukocytes of children with protein-calorie malnutrition (PCM). Reduced concentrations of slgA and, to lesser extent, lysozyme, are potentially important in the defence of mucosal surfaces. The lower levels differ significantly from those serum proteins, such as total protein, IgG, aminopeptidase, and albumin, levels of which in tears were not influenced by nutritional status. This indicates that the reduced levels of slgA, amylase and lysozyme were not due to reduced volume of secretion in malnourished children, as has been suggested by experimental studies on saliva in malnourished rats. It appears that the mechanism involves an impairment in the local synthesis and/or secretion of these proteins. These observations have been confirmed by studying parotid saliva during the renutrition of children with kwashiorkor or marasmic malnutrition. Saliva flow rate (millimetre per minute) increased about 50% during a 4-week renutrition with a highprotein diet. Therefore, the non-specific immunity due to the washing effects of secretions was significantly increased. Decreased total salivary IgA levels in children with acquired immune deficiency syndrome (AIDS), with a compromised nutritional status were reported by Slobodianik et al. (1996). Essential ammo acid deficiency and immune response Although experimentation in protein malnutrition is easier to conduct, specific essential amino acid deficiency may be of more importance and significance to human conditions of

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malnutrition. Is has long been realized that many proteins, especially of plant origin, are deficient in certain essential amino acids. For example, wheat gluten and maize protein are limited in lysine, and soya protein is limited in methionine. Malnutrition often occurs when these foods are consumed as sole sources of protein in the diet. Pallaro et al. (1996) have shown that the administration of a low-quality dietary protein (cooked maize flour) from weaning onwards provokes damage in the rat mesenteric lymph node due to the lack of B—T cell cooperation. This diet would produce a disturbance in zinc metabolism concomitant to, or as a consequence of, the state induced by the administration of this type of protein. Begum et al. (1970) have analysed two basic diets consumed by the majority of Indian preschool children. The wheat-based diet was limited in lysine, methionine, threonine and isoleucine but adequate in other essential amino acids and nutrients. The rice-based diet was limited in methionine and threonine. Furthermore, it is theoretically possible to acquire essential amino acid malnutrition even when good quality protein is consumed in limited quality. The level of each essential amino acid needed for optimal growth of mice has been determined (John & Bell, 1976). For example, mice fed an 8% casein diet consume inadequate levels of methionine, but the levels of other essential amino acids were considered adequate. Early work by Schaedler and Dubos (1956) had indicated that the dietary level of protein and quality of the dietary protein are both important with respect to the resistance of infection. Mice fed with 20% gluten diet were more susceptible to infection than mice fed with 20% casein diet. In another such investigation, Robertson and Doyle fed four-week-old rats a wheat gluten (lysine limited), soybean protein (methionine limited), or casein-based diet (Robertson & Doyle, 1936). The animals fed the casein diet were less susceptible to infection with Salmonella enteritidis. Gray (1963) showed that animals fed a lysine-deficient diet were more susceptible to Bacillus anthracis, the causative agent of anthrax. The decreased resistance remained even after supplementation of lysine in the diet. More recently, it has been shown that CF1 and Swiss Webster mice fed diets limited in leucine, isoleucine, valine, or lysine had greater susceptibility to S. typhimurium, but CD1 mice fed these diets were not more susceptible compared with well-fed mice (Petro & Bhattacharjee, 1980, 1981). An increased susceptibility to S. typhimurium has been demonstrated in mice fed an hystidine- or threonine-limited diet (Bitar & Bhattacharjee, 1982). However, in these studies the resistance to L. monocytogenes was only impaired in mice fed a methionine- or threonine-limited diet. Both of these studies indicate that the increased susceptibility to S. typhimurium may be the result of a delay in synthesizing adequate antibodies after exposure to the bacterial antigens. However, by day 8 after exposure there was no significant difference among all the dietary groups with respect to their antibodymediated resistance. Attempts to nutritionally rehabilitate mice fed from weaning diets limited in histidine or threonine for two weeks, but not three weeks, were successful. Nutritional deficiences and mucosal immune defence systems in animals Marginal protein malnutrition in a nutritionally well-defined animal model system confirmed the observations made in humans that protein maltnutrition suppressed slgA. slgA was significantly lower in the tears and vaginal secretions of marginally malnourished guinea pigs fed an 8% protein diet. IgG, which was the predominant immunoglobulin in vaginal secretions, was not affected by nutritional stress. It would be expected that mechanisms of

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host defence against a microbe infecting a mucosal surface should be related to levels of secretory antibodies. A significant depression of slgA levels was found in both tears and genital secretions of guinea pigs suffering marginal and severe protein deprivation. slgA in both tear and genital secretions of animals fed a 9% protein diet (marginally malnourished) were significantly lower than control levels both before and after infection with guinea pig inclusions conjunctivitis (GPIC). Post-infection slgA levels in tears of these marginally malnourished animals reached only 69% of post-infection control levels, and the genital secretion slgA levels averaged only 61% of controls. In the severely malnourished group, slgA reached only 60% of control levels in tears and 59% of controls in genital secretions. In addition, malnourished groups and their controls showed significant increases in slgA for both tears and genital secretions after GPIC infection, suggesting that the organism did elicit a secretory antibody response in all animals studied. slgA anti-GPIC antibody was produced in tears of all but the most severely malnourished guinea pigs after infection with GPIC. As in human secretions, the levels of IgG were not affected in genital secretions, in which IgG is the principal immunoglobulin. The gradual increase in slgA levels in the weaning animals with increasing age suggests that a slgA development process may be affected by nutritional stress. Total protein in the secretions of both severely malnourished groups and their controls increased significantly after infection, possibly reflecting the IgA response, since IgG levels in genital secretions and aminopeptidase in both tears and genital secretions did not increase after infection. IgG should play a significant role in mucosal immunity, as it is the second secretory immunoglubulin by concentration in most secretions. The effects of protein malnutrition in its concentration in saliva confirm the study of IgG in vaginal secretions and human secretions. Salivary IgG levels in moderately protein-malnourished rats did not decrease, but their amylase levels did. Amylase is largely locally synthesized, but much of IgG comes from the serum into saliva by transudation. Serum IgG levels are generally not suppressed by protein malnutrition. These animal and human studies strongly suggest that severe protein or calorie deficiencies do not significantly alter total IgG in secretions. In trying to understand how protein deficiency suppresses slgA, they tested the hypothesis that T cell functions were suppressed. As the thymus is known to be suppressed by protein deficiency, they treated protein-malnourished young mice with a thymus hormone preparation, thymosin fraction V. However they found that there was no apparent effect on the quantity of slgA in the intestinal secretions of these mice (Bell et al., 1976; Watson et al., 1977; Watson & McMurray, 1979; Lim et al., 1981). Also, the effect of severe protein deficiency in Wistar rats at weaning has been studied in bone marrow, which is a primary lymphoid organ, and which some authors consider to be associated with the mucosal immune system (Olmos et al., 2001). Cytogenetic studies in bone marrow cells from Wistar rats with protein malnutrition have principally shown: (a) decreased number of viable bone marrow cells; (b) diminished percentage of mitosis; and (c) severe alterations in the percentage of 3,11 and 12 chromosome pairs bearing nucleolar organizing regions (NORs), that reflects a poor ribosomal gene activity showing that RNA transcription is affected but (d) a 20% casein diet administered for five days reversed this situation. However, in a rat model of immunodeficiency, permanent alterations in the thymus and in the GALT were seen. An increase in the number of yS+ T cells in the gut lamina propria and in the number of CDS aoc+, CD25 + , Y$+ sub-populations of intestinal intraepithelial

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lymphocytes (ilEL) was shown by immunohistochemistry. After isolating rat ilEL, by flow cytometry Marquez et al. (2000) concluded that there exists an 'in situ origin and extrathymic maturation of the CD8 OCOC+ ilEL in the intestinal intraephitelium. The increase of TCR y8 + cells may be triggered by the carbohydrate dextrin, to provide immune protection and inflammation at the intestinal level. Recent data indicate that y8 + T cells play an important role by influencing local cellular traffic, promoting the influx of lymphocytes and monocytes, and limiting the access of inflammatory cells that do not contribute to protection but may cause tissue damage. The important role of 78 lymphocytes in surveillance and repair of damaged tissues is wellknown, and studies by others indicate that some y8 + T cells recognize non-specific protein ligands. Besides, cell culture and animal studies have identified a role for y8 IEL in the maintenance of intestinal cell homeostasis. Therefore, we may assume that the yS+ ilEL population increases to provide immune protection and control of inflammation at the intestinal level (Marquez et al., 2000). It is well-known that yS T cells can be stimulated by LPS from Escherichia coli or other bacterial infections. Nutritional deficits, excesses, or imbalances influence host resistance in a diverse manner. Although it is traditionally thought that malnutrition impairs host resistance, such an assumption is not always true. Hundreds of reported studies of infection in malnourished human or animal hosts were evaluated and interactions in which the presence of malnutrition made the infection seem more severe were classified as antagonistic. In virtually all studies in humans, malnutrition either contributed to a synergistic increase in disease severity or had no demonstrable effect. The same synergistic trend was evident in most experimental bacterial infections in animals. In contrast, experimental viral infections showed an antagonistic interaction about as frequently as a synergistic one, and parasitic infestations fell about midway between bacterial and viral infections. Although the fundamental mechanisms that could account for synergistic or antagonistic interactions have not been identified with certainty, the nutritional status of the host can influence the metabolic processes of an invading microorganism as well as those of the host. Because most bacteria possess their own replicating machinery and have relatively simple nutritional needs, bacteria can generally replicate within the intra- or extracellular body fluids of even a malnourished host. Viral replication, on the other hand, is an intracellular event requiring an usurpation of molecular mechanisms, biochemical pathways, and substrate molecules already present within a host cell. If malnutrition causes functional derangements of host cell metabolism, viral replication may not flourish. Malnutrition could thus ameliorate the severity of some viral diseases and thereby appear antagonistic. Antiviral drugs that interfere with a key metabolic process have an analogous action in their ability to inhibit viral replication. Although parasites possess the sophisticated molecular machinery necessary for maturation and replicative cycles, their relatively large size and complex nutritional requirements may cause them to compete with host cells for available key nutrients and substrate molecules. Parasites are occasionally more successful than the host in that competition as illustrated by the development of megaloblastic anaemia in some patients with fish tapewarm infestation. Uptake of vitamin K by these parasites causes an overt deficiency in the host.

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Malnutrition affects the epithelium that lines the vast mucosal surfaces of the gastrointestinal tract and enhances the possibility of infection. The existence of the M cell in the intestinal follicle-associated epithelium allows transport of enteric microorganisms (Fig. 7.3), therefore several pathogenic bacteria exploit the M cell transport mechanism to infect mucosal tissues and/or spread systemically before they can be halted by an immune response.

Fig. 7.3 Diagram of an M cell that allows the transport of the antigen across the epithelial barrier. Neutra et al. (1996) have described very well the interactions of microorganisms with M cells, especially Vibrio cholerae, Salmonella typbimurium, Shigella flexneri and reovirus. The nutritional status of the host can have important effects on the final adequacy of host defensive measures and the outcome of an infection.

Probiotics in malnutrition Introduction Since the digestive tracts of animals are far from sterile, nature has evolved an ingenious internal ecosystem that controls the pathogens with a beneficial microfiora such as lactic acid bacteria, bifidobacteria and others. These microorganisms are present in some natural foods (yoghurt, kefir) or concentrated supplements called probiotics (Fuller, 1989), as discussed in other chapters. Probiotic microorganisms are not nutrients in themselves, but they play a vital symbiotic role in digestion because they can resist digestive acids, adhere to intestinal walls, compete with or destroy disease-causing microorganisms and enhance immunological functions. Normally these helpful microbes reside in the gastrointestinal tract to act as a living barrier to harmful infections, but if this natural balance in disrupted by the widespread use of antibiotics, stress, immunosuppressive therapies or, immunodeficiences such as malnutrition, probiotic administration can play an important role in reconstituting the microfiora environment (Venturi et al., 1999). Despite all the positive reports on probiotics, much more comprehensive clinical research and trials are needed before live culture biotherapy becomes common place. Different microbial strains have different effects and standards of dosage and

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timing may affect the response, while commercial products vary widely in content and quality. Even though probiotics represent a safe non-invasive, and well-tolerated dietary intervention, more research must be done to predict which probiotics are most effective in the treatment of specific diseases or as vaccine vectors to improve the function of the immune system, or for inclusion in nutritional programmes. Efficacy of probiotics has been shown in a wide range of gastrointestinal diseases. Lactobacillus GG alone (Vanderhoof eta/., 1999) or in combination with Bifidobacterium bifidum and S. thermophilus, has been successful in the treatment of Clostridium difficile and reduces the frequency and severity of infections causing acute diarrhoea in children. Similarly antibiotic-induced diarrhoea can be treated by the administration of Saaharomyces boulardii (Madsen, 2001). Probiotic preparations have shown promise in preventing remission in ulcerative colitis as well as reducing the incidence of postoperative Crohn's disease (Faubion & Sandborn, 2000; Guslandi et al., 2000). The mechanisms of action of probiotics may include receptor competition, effects on mucin secretion or probiotic immunomodulation of GALT. Oral administration of viable probiotic compounds has been demonstrated to be safe, but one of the major problems is ensuring the survival of the microbe during the passage from the mouth to the colon. Consequently, microbial strains used as probiotics must be both acid- and bile-resistant. For probiotic bacteria to colonize the colon permanently, continuous consumption must be practised to ensure beneficial effects. In addition better designed and properly conducted clinical trials are necessary to carefully explore and characterize the therapeutic applications of probiotics. Yoghurt and other fermented milks offer tremendous potential for promoting health, having the capacity to improve nutrition, and at the same time reducing the risk of infection. Fermented milks show particular promise in reducing the incidence of malnutrition, lactose intolerance, and diarrhoea. In conventional yoghurt, milk is fermented with starter cultures of L. delbrueckii spp. bulgaricus and S. thermophilus, although other LAB or bifidobacteria may also be used in other forms of fermented milks. The health benefits associated with consumption of yoghurt are well documented (Savaiano et al., 1984; Hitchins et al., 1985; Puri et al., 1996; Meydani & Ha, 2000). The beneficial effects of yoghurt can be divided into nutritional, physiological, antimicrobial and antitumour. These two last properties of yoghurt are intimately related to activation of the immune system. The interaction of nutrient deficiencies and immune status has been the focus of increasing research in the last two decades. Nutrients derived from proteins, vitamins, fats, micronutrients and minerals interact with the immune cells, especially those associated with the gut. Nutritional deficiencies are caused by inadequate intake or reduced bioavailability defined as the percentage of nutrient that is absorbed and available for use by the body. Yoghurt and fermented milks in general are an important source of nutrients and minerals with high bioavailability.

Protein malnutrition and immune response One of the key functions of the intestine is to prevent lumen bacteria and endotoxins from reaching organs and tissues. Failure of this intestinal barrier function results in the spread of

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bacteria from the gut to systemic organs, a process that is called bacterial translocation. Protein malnutrition disrupts the normal ecology of the microflora (Welsh et al., 1998), affecting strict anaerobes (Poxton et al., 1997), which may disrupt the normal microflora producing overgrowth of some indigenous flora (Bengmark, 1998). The importance of gastrointestinal defences, in particular mucosal immune system function, against pathogens, microbes and toxins is well-known due to the increasing recognition that the intestinal tract is an important immunological organ. Protein-energy malnutrition (PEM) results in an increased risk of gastrointestinal infection, which is attributable in part to an impaired immune response (Chandra, 1996). The relationship between nutrition and immunity is well demonstrated, thus the function of many cells in the immune system relies on metabolic pathways that in turn require various nutrients as critical cofactors which affect bodily defence mechanisms (MacDermott, 1993). In developing countries malnutrition is the commonest cause of immunodeficiency, with an impaired systemic and mucosal immunity. In PEM most of the host defence mechanisms are breached depending on the severity of protein deficiency and enhanced susceptibility to infection. Protein malnutrition leads to mucosal villous atrophy, abnormal mucin formation, thymus involution, and impairment in the secretion of slgA (Sullivan et al., 1993). For details about PEM and the immune system see Chandra's chapter in this book. The detrimental effect of severe malnutrition on the mucosal immune system can be reversed with adequate renutrition (Castillo et al., 1991), which can restore all gastrointestinal functions, regenerate mucosa, mucus production and reduce the load on the local immune system. During the renutrition process, both the composition of the diet and the administration route have a profound influence on the intestinal morphology and function, so enteral nutrition seems to be superior to parenteral nutrition. A high protein enteral diet improves systemic immunity with a reduction in the incidence of infection (McDonald et al., 1991). The presence of a number of growth factors and hormones in the milk of various species, including human and bovine, have a beneficial effect on the host and suggest a potential role in the renutrition process (Meisel & Bockelmann, 1999). Fermented milk could also be useful in this renutrition process.

Effects of probiotic addition to a renutrition diet There is no doubt about the impairment of immunity in PEM, however it is possible to restore all gastrointestinal functions by appropriate renutrition. The beneficial effect of yoghurt and LAB probiotics on the healthy and the immunodeficient host has been extensively demonstrated (Bengmark & Gianott, 1995). Mucosal studies have demonstrated that the ingestion of fermented products enhances innate and acquired immunity. In addition to being a nutrient-dense calorie source containing high quality protein, absorbable calcium, phosphorous and vitamin A, such products can be considered a functional food that supply minor components that have been shown to play a role in decreasing the risk of certain diseases, particularly gastrointestinal disorders. Probiotics and fermented milks are useful dietary supplements for aiding recovery from malnutrition. Similarly the addition of bacterial supplements, such as selected LAB or fermented milks to an enteral feeding formula may improve not only the nutritional state but also the intestinal microflora and the

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Fig. 7.4 Number of IgA and IgG secreting cells present in the small intestine of malnourished + L. casei (a) and renourished + L. easel (b) mice treated with different doses and periods of L. casei. The secreting cells were determined by immunofluorescent tests performed on histological slices. Values are means ± SD of n = 5. W-N: well-nourished control; M-N: malnourished control; Re7d: renourished control.

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Fig. 7.5 Photograph of haematoxylin-eosin stained histological samples of thymus. (a) Well-nourished control ( x 40 magnification); (b) malnourished control (x 40 magnification); (c) renourished mice treated with diluted yoghurt (½ Yo) for five consecutive days (x 40 magnification).

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cont.

immune system, as well as eliminating toxins and assisting in the regulation of mucus production (Isolauri et al., 1991; Rowland, 1992; Allori et al., 2000). In previous studies performed in an experimental model in mice we demonstrated that L. casei induced an increase in the synthesis of secretory IgA and that previous feeding with this lactobacillus affords protection against infection with Salmonella typhimurium (Perdigon et al., 2001; Vintini et al., 2000). We also demonstrated that yoghurt increased the gut mucosal immunity (Perdigon et al., 1994). However, using the same experimental model of severe malnutrition, we showed that yoghurt and L. casei were not able to increase the protective intestinal mechanisms to the same levels as those found in the well-nourished host (Perdigon et al., 1995; Aguero et al., 1996; Perdigon & Oliver, 2000), when they are administered before a renutrition diet. This may be due to an overstimulation of the immune system in damaged intestinal tissue, as a consequence of severe malnutrition. Thus we suggested that the addition of probiotics for immunodeficiency caused by malnutrition would be advisable after mucosal recovery by adequate refeeding to avoid a harmful effect on the atrophied mucosa by malnutrition. Taking into account the above results we tested the effects of different doses of L. casei or yoghurt added after a renutrition milk diet in a non-severe malnutrition experimental model. Animals were malnourished on weaning at 21 days by feeding with a protein-free diet supplemented with vitamins, minerals and essential fatty acids in order to fulfil nutritional requirements. At the end of this process malnourished animals were renourished with 10% non-fat milk (NFM) for seven days, after which groups of mice were given suspension of L. casei at a concentration of 10 , 107 and 108 cfu/day/animal for two, five or seven consecutive days. Other groups of animals after renutrition with milk received an addition to the diet of either undiluted yoghurt (Yo) or diluted (1:1) yoghurt (^ Yo) for two, five or seven consecutive days. We determined the effect of different doses and periods of L. casei or yoghurt feeding on the body weight, haematological and serum total protein values, the number of

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IgA B lymphocytes and T cells associated with the gut mucosa. We also studied the recovery of the intestinal tissue by histological and ultrastructural microscopy observations of the small intestine. Thymus recovery was also demonstrated by histological studies. We observed that the optimal dose for each of the probiotics was 107 cfu/day/mouse for L. casei administered for five days and yoghurt diluted ½ for five days, to obtain recovery of the intestinal functions (Gauffin Cano et al., 2002a, b). In our model the mucosa impaired by malnutrition recovered with the optimal dose of L. case! administered after a period of seven days of milk renutrition. An increase in the number of IgA but not IgG was also seen (Fig. 7.4a, b). It may be hypothesized that IgA antibodies contribute to local homeostasis not only by performing immune exclusion on the mucosal surface but also by trapping the antigens in the lamina propria (Brandtzaeg, 1998). We showed that the immune increase in the number of IgA+ cells is dose-dependent. This IgA could provide a better intestinal barrier against enteropathogens. In spite of these results, we concluded that the dose of a single strain of bacterium, such as in our experiments with L. casei, must be carefully controlled to avoid side effects by overstimulation to the damaged tissue, which can promote gut barrier compromise. The increase of values determined for IgG secreting cells with some doses would indicate an inflammatory response. IgG antibodies activate complement and may thereby impair the surface epithelium with an enhancement of intestinal permeability. These values and the recovery of the intestine determined by histological and ultrastructural studies were the parameters chosen to select the optimal dose (Gauffin Cano et al., 2002a). When we determined the effect of yoghurt consumption in our model of malnutrition after milk renutrition for seven days, we showed that the diet of renutrition supplemented with yoghurt is effective in improving nutritional, histological and immunological parameters. With probiotics at the optimal dose (½ Yo for five days) we also observed an important thymus weight recovery with an improvement in the histological structure of this tissue. This effect was not observed with L. casei (Fig. 7.5a—c). In the experiments performed with yoghurt, we used both undiluted and diluted yoghurt to avoid overstimulation as was observed with L. casei. We demonstrated that five days of diluted yoghurt feeding was the optimal dose for improving the intestinal barrier and the mucosal immune system. However, high doses of yoghurt (seven days) was also effective. This conclusion for the optimal dose was reached mainly on the basis of changes in the number of IgA and histological studies of the small intestine (Gauffin Cano et al., 2002b). The results obtained for the optimal dose of yoghurt on the B and T cell population are shown in Fig. 7.6. We observed that yoghurt might provide a better intestinal barrier by increasing the numbers of IgA cells in the lamina propria and subsequently enhancing the production of secretory IgA. The increase in the CD4+ T population means that yoghurt is able to restore most of the immunological functions, by the multiple roles of the T cells in the activation of the immune system. The low values obtained for CD8+ T cells responsible for cytotoxic activity means that yoghurt could be modulating the inflammatory immune response. In conclusion, these studies show that the impaired gut barrier and mucosal immune system function produced by malnutrition can be reversed by addition of probiotics to the renutrition diet. L. casei could be used as an oral adjuvant if the dose is carefully chosen. However, our results were better with yoghurt than with L. casei and they suggested strongly

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Fig. 7.6 Number of IgA secreting cells, CD4+, and CD8+ T cells. The immune cells were determined by immunofluorescenttestson histological slices of small intestine from wellnourished, malnourished, and renourished mice treated with diluted yoghurt for five consecutive days. Values are means ± SD of n = 5.

that the addition of a renutrition diet with yoghurt is good practice. This probiotic does not induce any harmful effects. The thymus and intestinal recovery was also better using yoghurt. These results are not surprising considering the important nutritional values of yoghurt and the improved bioavailability of enzymes, and macronutrients. Although our results were obtained in an animal model, and the effect of probiotics in humans have not yet been well established, we suggest that the consumption of yoghurt by malnourished children after enteral renutrition, could accelerate the recovery of immunological intestinal function.

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Nagler-Andersson, C. (2001) Man the barrier! Strategic defenses in the intestinal mucosa. Nature Reviews Immunology, 1, 59-67. Neutra, M.R., Frey, A. & Kraehenbuhl, J.P. (1996) Epithelial M cells: gateways for mucosal infection and immunization. Cell, 86, 345-8. Neutra, M.R., Mantis, NJ. & Krahenbuhl, J.P. (2001) Collaboration of epithelial cells with organized mucosal lymphoid tissues. Nature Immunology, 2, 1004—9. Olmos, S., Garcia Reinoso, M.F., Marquez, M.G. & Roux, M.E. (2001) Cytogenetic studies in bone marrow cells from Wistar rats in protein malnutrition. Metabolism, 50, 1825-9. Pallaro, A.N. & Slobodianik, N.H. (1999) Dietary protein quality and zinc levels in growing rats. Nutrition Research, 19, 1089-95. Pallaro, A.N., Fernandez, I., Rio, M.E., Roux, M.E. & Slobodianik, N.H. (1996) Low-quality dietary protein: its impact on Wistar rats mesenteric lymph nodes. Journal of Nutrition Immunology, 4, 67—74. Pallaro, A.N., Rio, M.E., Roux, M.E. & Slobodianik, N.H. (1997) Amino acid supplementation: Its effect on the thymus in growing tats. Journal of Nutrition Immunology, 5, 29— 37 Pallaro, A.N., Roux, M.E. & Slobodianik, N.H. (2001) Nutrition disorders and immunelogic parameters: study of the thymus in growing rats. Nutrition, 17, 724—8. Perdigon, G. & Oliver, G. (2000) Modulation of the immune response of the immunesuppressed host by probiotics. In: Probiotics 3: Immunomodulation by the gut microflora and probiotics (eds. R. Fuller & G. Perdigon). Kluwer Academic Publisher, London, pp. 148— 75. Perdigon, G., Rachid, M., De Budeguer, M.V. & Valdez, J.C. (1994) Effect of yogurt feeding on the small and large intestine associated lymphoid cells in mice. Journal of Dairy Research, 61 (4), 553-62. Perdigon, G., Aguero, G., Alvarez, S., de Allori, C. & Pesce de Ruiz Holgado, A. (1995) Effect of viable Lactobacillus casei feeding on immunity of the mucosae and intestinal microflora in malnourished mice. Milchwissenschaft, 50, 125-45. Perdigon, G., Fuller, R. & Raya, R. (2001) Lactic acid bacteria and their effect on the immune system. Current Issues in Intestinal Microbiology, 2 (1), 27—42. Petro, T.M. & Bhattacharjee, J.K. (1980) Effect of dietary essential amino acid limitations upon native levels of murine serum immunoglobulins, transferrin and complement. Infection and Immunity, 27, 513. Petro, T.M. & Bhattacharjee, J.K. (1981) Effect of dietary essential amino acid limitations upon the susceptibility to Salmonella typhimurium and the effect upon humoral and cellular immune responses in mice. Infection and Immunity, 32, 251. Poxton, I.R., Brown, R., Sawyerr, A. & Ferguson, A. (1997) The mucosal anaerobic gramnegative bacteria of the human colon. Clinical Infection Disease, 25 (Suppl. 2), S1ll—13. Puri, P., Rattan, A., Bijlani, R.L., Mahapatra, S.C. & Nath, I. (1996) Splenic and intestinal lymphocyte proliferation response in mice fed milk or yoghurt and challenged with Salmonella typhimurium. International Journal of Food Science Nutrition, 9, 23-8. Rescigno, M. et al. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunology, 2, 361—7. Robertson, E.G. & Doyle, M.E. (1936) Higher resistance of rats fed casein than those fed vegetables proteins. Proceedings of the Society of Experimental Biology and Medicine, 35, 374.

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Robinson, J.K., Blanchard, T.G., Levine, A.D., Emancipator, S.N. & Lamm M.E. (2001) Mucosal IgA mediated excretory immune system — in vivo. Journal of Immunology, 166, 3688-92. Roux, M.E., Lopez, M., Del, C. & Florin, C.A. (2000) Mucosal immunity. In: Probiotics 3. Immunomodulation by the gut microflora andprobiotics (eds. R. Fuller & G. Perdigon), pp. 12— 28. Kluwer Academic Publishers, The Netherlands. Rowland, I.R. (1992) Metabolic interactions in the gut. In: Probiotics: the scientific basis (ed. R. Fuller). Chapman and Hall, London, pp. 29—53. Savaiano, D.A., Elanouar, A.A., Smith, D.E. & Levitt, M.D. (1984) Lactose malabsortion from yoghurt, pasteurized yoghurt, sweet acidophilus milk, and cultured milk in lactosedeficient individuals. American Journal of Clinical Nutrition, 40, 1219—23. Schaedler, R.W. & Dubos, R.J. (1956) Reversible changes in the susceptibility of mice to bacterial infections II. Changes brought about by nutritional disturbances. Journal of Experimental Medicine, 104, 67. Scrimshaw, N.S. & Sangiovani, J.P. (1997) Synergism of nutrition, infection and immunity: an overview. American Journal of Clinical Nutrition, 66, 4645—775. Semba, R.D., Muhilal, S.A.L., Natadisastra, G. et al. (1992) Depressed immune response to tetanus in children with vitamin A deficiency. Journal of Nutrition, 122, 101-7. Slobodianik, N., Pallaro, A., Rio, M.E. et al. (1996) Prealbumin, retinol-binding protein and total salivary IgA in children with AIDS. Clinical Chemistry, 42(3), 471—2. Solomons, N.W. (2000) Miconutrients and infection. Nutrition, 16, 1093-5. Sullivan, D.A., Vaerman, J.P. & Soo, C. (1993) Influence of severe protein malnutrition on rat lacrimal, salivary and gastrointestinal immune expression during development, adulthod and ageing. Immunology, 78 (2), 308-17. Vanderhoof, J.A., Whitney, D.B., Antonson, D.L., Manner, T.L., Lupo, J.V. & Young, RJ. (1999) Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children. Journal of Pediatrics, 135 (5), 564-8. Varela, P., Slobodianik, N., Pallaro, A. et al. (1997) Some nutritional parameters in adolescent females suffering from obesity or anorexia nervosa. A comparative study. World Review Nutrition Diet, 82, 168-74. Venturi, A., Gionchetti, P., Rizzello, F. et al. (1999) Impact on the composition maintenance treatment of patients with ulcerative colitis. Alimentary Pharmacology and Therapy, 13 (8), 1103-8. Vintini, E., Alvarez, S., Medina, M., Medici, M., de Budeguer, M.V. & Perdigon, G. (2000) Gut mucosal immunostimulation by lactic acid bacteria. Biocell, 24 (3), 223-32. Watson, R.R. & McMurray, D.N. (1979) The effects of malnutrition on the secretory and cellular immune processes. CRC Critical Review of Food and Nutrition, 113-59Watson, R.R., Horton, R.G. & Clinton, J.M. (1977) Suppression of secretory IgA antibodies in protein malnourished guinea pigs following chlamidial eye and vaginal infection. Fed. Proceedings, 36, 1251. Weiner, H. (2001) The mucosal milieu creates tolerogenic dendritic cells and TH1 and TH3 regulatory cells. Nature Immunology, 2, 671—2. Welsh, F.K., Farmery, S.M., MacLennan, K. et al. (1998) Gut barrier function in malnourished patients. Gut, 42(3),396-401.

Immune Activation Versus Hyporesponsiveness and Tolerance in the Gut

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J. Chin1 & A. Mullbacher2 1

Immunology & Microbiology, Elizabeth Macarthur Agricultural Institute, NSW Department of Agriculture, PMB 8, Camden, Sydney, NSW 2570, Australia, and 2 Department of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia

Introduction At birth, the mammalian neonate is transferred from the pathogen protective confines of the uterus to an opportunistic and antigenic challenging environment. Although armed with a functional immune system, it is generally considered that both innate and adaptive immunity is relatively immature and easily subjected to modulation by external stimuli. These include a plethora of hormones, cytokines and chemokines contained in breast milk as well as maternal immune cells such as macrophages, lymphocytes and neutrophils, that can potentially shape and condition the growth of the gastrointestinal tract (GIT) and the complex array of immune cells that service this organ. At the same time, the GIT of the newborn is being rapidly colonized by microorganisms that are mostly of maternal origin as well as those from the environment including food sources. Some of these microorganisms will colonize various geographical compartments of the GIT and in particular niches within each compartment to become autochthonous bacteria characteristic of the individual. Often, the neonate is confronted by pathogenic bacteria that it must distinguish from the commensal population of innocuous, harmless and even beneficial bacteria. Understanding how the immune system mobilizes against 'foe' and not against 'friend' is a vital aspect of defining a role for probiotic bacteria as functional foods capable of promoting a healthy outcome the consumer.

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Innate and adaptive immunity Immune responses have traditionally been divided functionally into innate and adaptive categories. Innate immune responses are already functionally present prior to encounter with antigen. Once confronted by 'foreign' or 'non-self antigens emanating from alien pathogens, innate responses result in the mobilization of phagocytic cells, complement activation and the production of antimicrobial peptides. Initiation of innate immunity is triggered when an antigen docks or interacts with a restricted set of highly conserved receptors known as pattern-recognition receptors (PRRs). PRRs recognize pathogen-associated molecular patterns (PAMPs) or antigenic signatures that are commonly associated with classes of microbial pathogens, lipopolysaccharides (LPS) being a classic example (Medzhitov & Janeway, 1997). PRRs are not restricted to cells of the innate immune system and can be found on immune effector cells involved in the presentation of antigen including macrophages, dendritic cells and B lymphocytes. PRRs possess characteristic protein structure consisting of calcium-dependent lectin domains, scavenger-receptor protein domains and leucine-rich repeat domains (Fearon & Locksley, 1997). As part of the acute phase response, the liver synthesizes and secretes mannan-binding lectins that function as opsonins by binding to the carbohydrates associated with bacteria, viruses and fungi. Pathogens coated with mannan or mannose-binding lectins are immediately flagged for recognition by the complement system and targeted for phagocytosis. Special endocytic PRRs receptors on the surface of phagocytes, e.g. macrophage mannose receptor, when engaged with the corresponding PAMP, immediately facilitate the uptake and delivery of the pathogen into lysosomes where they are destroyed. Toll-like receptors (TLRs) are a recent inclusion in the family of PRRs. These cell surface receptors, through their large extracellular leucine-rich repeat domain, have the capacity to recognize molecular signatures associated with Gram-negative and Gram-positive pathogens. When engaged, transmembrane TLRs commute information intracellularly by signal transduction pathways that activate transcription factors of the nuclear factor-KB and AP-1 family (Anderson, 2000). There have been as many as 10 TLRs discovered to date (Imler & Hoffmann, 2001). One of the earliest TLR - TLR4 - was identified as being defective in C3H/HeJ mice that were non-responsive to LPS and highly susceptible to Gram-negative infections. TLR4 is now known to interact with both CD 14 and MD-2 on membrane surfaces for the binding of LPS. LPS in the blood circulation is usually associated with a serum protein called lipopolysaccharide-binding protein (LBP), which transfers LPS to the CD 14 receptor anchored in the cell membrane via a glycosylphosphoinositol tail. Since CD 14 does not possess an intracellular signalling domain, further activation is reliant upon the formation of a complex between CD14-LPS and TLR-4 and MD-2. TLR4 therefore functions as a co-receptor in transducing the LPS signal intracellularly. While LPS is the PAMP of Gram-negative bacteria, lipotechoic acid (LTA) and peptidoglycan (PGN) are the PAMP signatures associated with Gram-positive bacteria. LTA is anchored in the bacteria membrane by glycolipids and PGN consists of alternating (1,4) linked N-acetylmuramyl and Nacetylglucosaminyl glycan cross-linked by short peptides. Both LTA and PG also exploit the CD 14 receptor for binding but intracellular signalling is mediated by a different coreceptor - TLR2 (Schwandner et al., 1999). In other situations, the toll receptor TLR5 is

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capable of directly recognizing bacterial flagellin (Gewirtz et al., 2001; Hayashi et al., 2001). While the innate immune response is dictated by a select number of PRRs (estimated to be about 102—103) that have been evolutionarily conserved and inheritable, adaptive immune responses involving the B cell receptor (BcR) and T cell receptor (TcR), depend upon the generation of an infinite number of receptors by somatic random processes in the course of their development and selection. The maintenance of immunological memory of antigen reactive B and T cells (with a repertoire of as many as 10 and 10 possible combinations, respectively) is therefore entirely dependent upon selection pressure in the course of an individual's antigenic experience and such information cannot be inherited (Medzhitov & Janeway, 2000). Innate immunity is reliant upon immediate and direct recognition of PAMPs by PRRs. Such recognition is invariably associated with transmembrane signalling and gene activation as well as antigen assimilation. In the case of internalized protein antigens, intracellular processing occurs in the lysosome of antigen-presenting cells (APCs) and results in the generation of peptides that are bound in the groove of major histocompatibility complex (MHC) class II molecules or in the case of cross-priming, in the groove of class I MHC (Carbone et al., 1998) for re-presentation on the cell surface. T cells involved in adaptive immune responses cannot recognize antigen directly. Instead, T cells recognize protein antigens via their TcR—CD 3 complex only if the processed peptides are presented as a peptide—MHC—class I or class II complex (pMHCI or pMHCII) on the APC's cell surface. Acquired immunity therefore occurs temporally only after innate immune responses have been initiated.

Antigen and the T cell response Encounter of antigen by naive T cells can lead to priming or tolerance. Priming occurs when TcR-pMHCI or TcR-pMHCII antigen engagement is accompanied by co-stimulation involving a second signal interaction between another set of cell surface ligand receptors, such as that between CD40L (CD40 ligand) on the T cell and CD40 on the APC; in this case a dentritic cell (DC) is obligatory (Lanzaveccia & Sallusto, 2001). This requirement for costimulation is necessary for the induction of cytotoxic T cell responses (CTL) under noninflammatory conditions (Schoenberger et al., 1998). CD28 is another class of constitutively expressed second signal receptor on the T cell surface. Activation of APCs by way of toll receptors also induces the expression of second signal ligands such as B7.1 (CD80) and B7.2 (CD86). CD28-B7.1 or CD28-B7.2 engagement provides the necessary co-stimulatory second signal that accompanies TcR-pMHCII interactions to initiate proliferation, cytokine production and resistance to apoptosis of naive T cells (Lenschow eta!., 1996). To ensure that T cells do not always become activated by B7 interactions with CD28, an alternative set of surface receptor and member of the immunoglobulin (Ig) superfamily - CD 152 (also known as CTLA-4) — plays a major role in competing for binding with B7. B7-CTLA-4 interactions have the opposite effect to B7-CD28 engagement and result in down-regulation of T cell activation by facilitating antigen (Ag)-specific apoptosis and suppression of cytokine synthesis (Liu et al., 2001).

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Antigen-presenting cells in the GIT Macrophages are strategically positioned in the subepithelial mucosa and represent a frontline defence barrier against invading pathogens and their products. It has been assumed that these macrophages were developmentally similar and possibly derived from CD 14+ monocytes and should share surface expression of TLRs and CD 14 for the detection of LPS and LPS-binding protein. However, investigations by Smith et al. (2001) have shown clearly that lamina propria macrophages lack CD 14 expression as well as CD89, the receptor for IgA (FcaR) even though they expressed both TLR2 and TLR4. Accordingly, these macrophages were markedly less responsive to LPS-induced cytokine production and phagocytosis of IgA opsonized particles. These findings indicate that intestinal macrophages may have originated from CDl4- monocytes and down-regulation of LPS and IgA-mediated functions is probably an important feature to maintain homeostasis and to reduce inflammatory responses in the gut. Exposure to a variety of antigens derived from organisms living in the gut can have a profound effect on the polarization of intestinal DCs to commit to the DC1 or DC2 phenotype. Depending on the initial antigenic encounter of the DC in the intestinal microenvironment, DCs mature as APCs capable of directing T cell differentiation (Lanzavecchia & Sallusto, 2001) with three potential signals. Once matured, DCs cannot be repolarized by exposure to new microbial stimuli or cytokines (De Jong et al., 2002). The ramification of this scenario is that the vast majority of DCs in the gut would be DC2, committed to a Th2 phenotype by virtue of the preponderance of Th2 biased antigens such as LPS. Therefore, it was surprising that Viney et al. (1998) found that expansion of DCs in the GIT with the haemopoietic growth factor Flt3 ligand (Flt3L) induced increased systemic tolerance after feeding soluble antigen instead of increasing immune responsiveness. Flow cytometry confirmed that DCs expanded by Flt3L displayed low levels of both second signal receptors CD80 and CD86. Whether the Flt3L-expanded DC subset is identical to a similar tolerogenic DC population reported to be responsible for tolerance induction in the lamina propria (Harper et al., 1996) remains to be elucidated.

T cell function in the GIT - the CD4+ population Mucosal T cells are present as intraepithelial lymphocytes in the gut wall of the GIT. They are strategically positioned to encounter antigen presented on the basolateral surface of lECs and also by a range of APCs including DCs, monocytes, macrophages and polymorphonucleocytes (PMN) that transit or are mobilized to the submucosa. However, in the absence of an appropriate second co-stimulatory signal from the APC, engagement of the TcR/CD3 complex by MHC II peptide constitutes a one-signal activation and does not lead to activation of the T cell. These hyporesponsive T cells do not produce cytokines or undergo cell division. They represent a form of tolerance referred to as anergy (Bretscher, 1992). In contrast, a number of inflammatory conditions of the gut with suspected immune aetiopathology arise from overactivity of the second signalling process. This may involve a subpopulation of memory T cells that appear to have the ability to expand by co-stimulation alone without the need of antigen to be presented (Flynn & Miillbacher, 1997). Examples of

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major forms of inflammatory bowel disease include Crohn's disease and ulcerative colitis. Using an adoptive transfer model of murine colitis, Liu et al. (2001) showed that transfer of CD45RB high CD4 T cells from syngeneic wild type mice induced transmural inflammation in SCID recipients. The same subset of effector T cells obtained from mice with a CD28 background (i.e. no co-stimulation receptor) failed to elicit any clinical symptoms. This emphasized the importance of second signalling in exacerbating an inflammatory response. Blockage of B7-CD28 interactions with anti B7.1 but not B7.2 antibodies also suppressed the inflammatory response. Another subset of lymphocytes from CD28-/- mice with the CD25 + CD4 + phenotype successfully prevented inflammation when co-administered with CD45RBhighCD4+ T cells. However, this ameliorating influence of CD25 + CD4 + T cells could be blocked with anti-CTLA-4 antibodies, thereby confirming that CD25 CD4 T cells mediate their anti-inflammatory properties via competitive CTLA-4-CD28 binding. In recent years, experimental evidence has shown that a unique lineage of thymus derived CD4 T cells can also play a major role in the induction of tolerance (Sakaguchi, 2000). This population is characterized by the presence of both CD25 and CD4 surface markers. A significant reduction in numbers of these regulatory CD2 5+ CD4+ T cells (Tr) in peripheral lymphoid tissues appears to be correlated with the appearance of autoimmunity in mice thymectomized three days after birth. Although little is known about the activation of CD25+ CD4+ T cells, they appear to be unlike anergic T cells that have been activated by a single signal (antigen alone in the absence of co-stimulation), and function primarily as noncytotoxic suppressor T cells. The possibility that such a population may play a role in peripheral tolerance induction following systemic or oral exposure to antigen was investigated by Thorstenson and Khoruts (2001). Using an adoptive transfer model of RAG-2 deficient DO 11.10 TCT transgenic donor mice, they were able to show the appearance of CD25 + CD4 + T cells following intravenous injection of ovalbumin peptide or oral administration of ovalbumin. This population appeared only under tolerogenic conditions at low dose antigen exposure such that cell progression was limited. These CD25+ CD4 T cells did not produce interleukin 2 (IL-2) and expressed high levels of CTLA-4 and were thus capable of blocking second signal activation. Clearly it is possible to identify the two separate routes to tolerance induction. High dose antigen exposure in absence of co-stimulation results in tolerance due to induction of anergic T cells. Low dose antigen exposure associated with CTLA-4 suppression of second signal activation results in tolerance due to suppressor T cells. Violation of these conditions by co-administering low dose antigen in the presence of adjuvant or an inflammatory signal inhibits the development of the suppressor CD25 + CD4 + T cell population. T cells in the intestinal wall have been recruited from peripheral circulation and are believed to be activated because of surface expression of CD 152. However, expression of CTLA-4 on the cell surface also attenuates their function and they retain a suppressive phenotype. T cells expressing CTLA-4 have the capacity to block immune responsiveness in the GIT and this can affect the clinical course of disease. Several investigators have reported that blockade of CTLA-4 with anti-CTLA-4 monoclonal antibodies can improve immune function and promote rapid and protective primary responses against the intestinal nematode Nippostrongylus brasiliensis (McCoy et al., 1997) and an increase in the rejection rate of pre-established colon carcinomas (Leach et al., 1996). It is likely that CTLA-4 blockade of T cell function is bypassed in the face of provocation by invasive pathogenic bacteria capable of

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breaching the epithelial barrier. In such a situation, APCs with the propensity to internalize and present antigen may then activate T cells with two signals to override the CTLA-4 blockade. Activated CD4 T helper (Th) cells become polarized effectors by secreting different combinations of cytokines. Thl cells produce beside other cytokines interferon (IFN)-y while Th2 cells secrete IL-4. The manner in which APCs such as DC signal T cells is believed to be critical in the polarizing process. DCs regulate Thl/Th2 switching by deploying a third signal (Kalinski et a!., 1999). Signal 3 is heterogeneous and is mediated by soluble factors such as IL-12 (Kubin et al., 1994), IL-18, IFN-y and membrane-bound molecules like OX40L (Flynn et al., 1998) and ICAM1 (Smits et al., 2002). The type of antigenic encounter appears to be the critical determinant in the control of switching. DCs when exposed to antigens derived from intracellular pathogens such as Mycobacterium tuberculosis or pertussis toxin mature as IL-12 secretors that favour Thl polarization while some helminthic antigens, prostaglandin (PGE2) and cholera toxin favour Th2. Thl and Th2 commitment is controlled by 'master switch' transcription factors T-bet (Szabo et al., 2000) and GAT A-3 (Zheng & Flavell, 1997) activation pathways, respectively. The CDS cytotoxic T cell subpopulation with antibodies against the surface antigen CDS identifies a subpopulation of T cells with predominantly cytotoxic phenotype (CTL). The TcR of CTL recognizes short 8—11 amino acid pMHCI complexes. The development of MHC tetramers, molecules comprised of four identical class I heavy chains, each bound to P2-microglobulin and a single peptide, has enabled the study of CTL activation and identification (Davis et al., 1998). Tetramer binding to T cells has been found to provide very strong one signal activation of naive CTLs without the need for co-stimulation (Wang et al., 2000). Following activation, naive CD8 cells proliferate to become effector CTLs with the capacity to lyse target cells that display the corresponding pMHCI. CTLs play a vital role in eliminating cells that have been infected by intracellular bacteria or viruses. The majority of activated CTLs die by activation-induced cell death (AICD; Russell, 1995). A part of the antigen reactive CTL population differentiates into long lived memory T cells with distinctly different activation requirements to that of naive CTLs (Mullbacher & Flynn, 1996). Surface markers CDlla, CD lib, CD44, CD62L and peanut lectin agglutinin distinguish naive T cells from activated (including memory) T cells but there are no markers that specifically identify the memory population (Miillbacher & Blanden, 2000; Slifka & Whitton, 2000). CTLs can be differentiated into type 1 (Tel) or type 2 (Tc2) based on the secretion of IFN-y and tumour necrosis factor (TNF)-a in the former and IL-4, IL-5, IL-10 and IL-13 in the latter (Dobrzanski et al., 2000).

y5 T cells The major component of T cells found commonly in the intestinal mucosae bear the y5 TcR phenotype. Unlike &P T cells, y5 T cells recognize unique non-peptide antigens (Morita et al., 2000). These T cells utilize V52 and V82 gene segments capable of recognizing microbial antigens that may be intermediates or side products in isoprenoid synthetic pathways (Morita et al., 2001) and include prenyl pyrophosphate, alkylamine antigens and pyrophosphomonoesters. Many of these are also pattern signature molecules of bacterial

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activity. Since antigen recognition is not dependent upon MHC restricted presentation, the activation of 78 T cells resemble that caused by exposure to superantigen or mitogen. Therefore, yS T cells through their pattern recognition TcRs provide an additional strategy in protective surveillance by endowing the adaptive immune system, the capacity to excert effector function similar to that of the innate immune responses. y5 T cells can also recognize peptide antigen. Indeed, the paradigm that peptides are exclusive ligands of T cells is being reassessed, particularly since the discovery that 0$ T cells can be stimulated by lipoarabinomannan and mycolic acids in the context of CDlb (Kaufmann, 1996).

Turnover of T cells The dynamics of T cell turnover is critical to the maintenance of immune homeostasis. Resting CD4 T cells having encountered MHC-peptide antigen embark upon at least seven cycles of cellular division without further need of antigen (Signal 1) or cytokine (Signal 3) (Bajenoff et al., 2002). Initial antigen-driven cell division does not generate effector T cells and appears to fulfil the primary function of clonal expansion of rare antigen-specific T cells. Thereafter, functional commitment is dependent upon repeated contact with antigen-loaded APC and engagement with Signal 2. However, selection of either Thl or Th2 effectors is determined only under the conditions of the appropriate cytokine, e.g. IL-12 or IL-4 produced by DC1 or DC2 and functions as Signal 3, respectively. T cells can be eliminated by apoptosis during the initial antigen-driven cell cycling phase if they do not encounter Signals 2 and 3. Effector Th cells activated by further antigen re-engagement can be eliminated by a process referred to as AICD. AICD is mediated by Fas (CD95, Apo-l)/FasL interactions leading to apoptosis (Dhein et al., 1995). Fas is expressed on all T cells and is upregulated upon their activation. FasL however, is only expressed in TCR-activated T cells. Gld and Ipr mice with homozygous defects in FasL and Fas, respectively, undergo massive lymphoproliferation associated with autoimmune disorders (Gillete-Ferguson & Sidman, 1994). An intrinsic bias towards Th2 phenotype by virtue of the fact that effector Th2 T cells can be maintained simply by antigen/co-stimulator engagement in the presence of IL-4 with no further need for repeated antigen stimulation has been documented. In contrast, Thl commitment requires recurrent TCR engagement by APCs due to a need for sustained activation of the T-bet transcription factor (Bajenoff et al., 2002). CDS T cells on the other hand, undergo a programme of proliferation and differentiation into IFN-y producing cells in the absence of continued antigen re-stimulation (Van Stipdonk et al., 2001) and their turnover is accomplished mainly by TNF/TNFR-mediated apoptosis (Heath et al., 1997).

Intestinal epithelial cells Apart from the skin, no other single layer of cells can perform as complex a series of biological functions as the single layer of intestinal epithelial cells (lECs) that line the gut lumen. lECs can process and present antigen to T cells (Mayer & Shlien, 1987), express cell adhesion molecules (Dippold et al., 1993), secrete cytokines and chemokines (Jung et al., 1995), release eicosanoids (Eckmann et al., 1997), produce nitric oxide (Kolios et al., 1998) and

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express costimulatory molecules. lECs display a number of different receptors on the apical and basal surface and use these as sentinels to relay danger signals from the lumen to the network of mucosal myeloid, lymphoid and mesenchymal cells for the maintenance of intestinal homeostasis. These receptors include toll, peroxisome proliferator-activated receptor (PPAR) and galanin-1 receptor can potentiate and inhibit intestinal inflammation (Hecht et al., 1999; Su et al., 1999). When activated by receptor engagement, a series of protein kinases and phosphatases cascade the information via an inducible transcription factor to the nucleus, eventuating in the upregulation of a set of immediate early (IE) genes. NF-KB is an example of such an inducible transcription factor. NF-KB is a heterodimer composed of RelA (p65) and NF-KB 1 (p50) subunits. NF-KB binds to the promoter/ enhancer region of many IE genes including IL-8, RANTES, IL-2R, Fas, cyclooxygenase-2, complement factors C3 and C4, and MHC class I and II (Jobin & Sartor, 2000). Proinflammatory responses associated with NF-KB induction is normally prevented by the fact that it is complexed with an endogenous cytoplasmic inhibitor known as 1KB of which there are a number of homologous members - iKBa, lKB(3, iKBe, iKBy, BcB, p!05 and pi 00. This family of proteins are characterized by an ankyrin repeat domain involved in protein—protein interaction. NF-KB is inactive when IKBa is avidly bound to its RelA subunit. In response to an appropriate stimulus such as IL-1 or TNF-a, a suite of 1KB kinases (IKK) phosphorylate IKBa on the amino terminus of serine residues 32 and 36. Once phosphorylated, IKBa is immediately ubiquinated and rapidly degraded via a non-lysosomal ATP-dependent 26S proteolytic complex composed of a 700-kDa proteasome, freeing the transcription activator NF-KB. The activity of NF-KB is tightly regulated and it is noteworthy that lECs that line the distal ileum and colon remain non-inflammatory even though they are continuously bathed with lumenal contents rich in EPS (80 |Lig/g faeces), anaerobic (10 ~ /g) and aerobic (10 /g) bacteria. One possible explanation for this may be that the activity of IKK is down-regulated in differentiated or mature lECs compared to undifferentiated crypt cells. It is interesting to note that such down-regulation is also associated with a decreased capability of butyrate-differentiated lECs to bind enteric bacteria in vitro (unpublished observations). Alternatively, commensal bacteria may have evolved strategies to block phosphorylation of 1KB and hence establish a non-inflammatory relationship with the gut lining (Gordon, 2000). Presumably this relationship will be more important in the crypt region where bacteria can attach as compared to villi. Pathogenic bacteria in such an environment would have to compete for binding to enterocytes and probably exploit virulence genes that encode invasion factors to penetrate and trigger an inflammatory response (Elewaut et al., 1999). However, non-invasive bacteria can also activate NF-KB (Eaves-Pyles et al., 1999) but this could be mediated by interaction of alternative virulence gene products that encode haemolysin or cytotoxin capable of provoking the NF-KB pathway.

Peyer's patches - suppression of T cell responsiveness Peyer's patches (PP) fulfil an important immune surveillance role and are considered to be key lymphoid inductive sites for priming T cell responses in the GIT (Mowat & Viney, 1997). In these tissues, naive T cells are drawn from the circulatory system in response to

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chemoattractant signals produced in the form of chemokines (Dieu et al., 1998; Kim et aL, 1998) by stromal cells and endothelial cells of high endothelial venules. Secondary lymphoid tissue chemokine (SLC) is expressed by stromal elements in lymphoid tissues while macrophage-inflammatory protein (MIP) is produced by DCs. Upon extravasation into PP and peripheral lymph node (LN) (Salmi & Jalkanen, 1999), T cells become activated by interaction with APC including DC, macrophages and B cells. This interaction results in the synthesis of adhesion molecules such as LFA-1 and CD 2 that ensure sustained T-APC interactions and subsequent initiation of an immune response. To obtain some insight into T cell activity in different lymphoid organ compartments, Kellermann and McEvoy (2001) employed a number of functional assays to compare the biological activity of CD4 and CDS T cells isolated from PP and peripheral LN. As shown in Table 8.1, T cells from PP were profoundly suppressed in their ability to respond to chemokine-stimulated chemotaxis and actin polymerization. The lack of T cell chemotaxis to SLC, a chemokine with a key role in directing T cells and DC into T cell areas of lymphoid tissues and in promoting T cell activation (Forster et al., 1999), would not lead to activation and the T cells would remain hyporesponsive. Decreased responsiveness to chemokines in T cells and DC was due at least in part, to the down-regulation of mRNA encoding the chemokine receptor CCR7R. In addition, actin polymerization, an early event required to facilitate T cell migration and adhesion, was also compromised as was the T cell response to concanavalin A and phorbol myristate acetate. Since these properties were not mirrored in T cells resident in LN, it is likely that the PP microenvironment imposes hyporesponsiveness.

Table 8.1 Comparison of the biological activity of T lymphocytes isolated from peripheral lymph nodes (LN) and Peyer's patches (PP) (summarized after Kellermann and McEvoy, 2001) Functional assay

LN

PP

Chemotaxis to SLC and MIP-3p

High

Reduced

Chemotaxis to SDF-la and IP-10

High

Reduced

Actin polymerization by staining with BODIPY-phallacidin following activation with SLC or MIP-3p or SDF-la

Rapid-potent

Reduced

CCR7 mRNA by RT-PCR

More abundant

Less abundant

Con A and PMA induced actin polymerization

High

Reduced

Calcium flux assays with fluo-3

High

Decreased

Levels of chemokines - SLC, MIP-3(3MDC, RANTES, Mig and IP-10 determined by quantitative RT-PCR

More abundant

Less abundant

Levels of chemokines - MIP SocTECK and Vic

Less abundant

More abundant

SLC (GCkine), secondary lymphoid-tissue chemokine (CCL21); MIP-3(J, macrophage-inflammatory protein (CCL19); SDF-la, stromal derived factor IP-10 (CXCL10); MDC, monocyte-derived chemokine (CCL22) RANTES (CCL5); Mig, monokine induced by IFN-y (CXCL9); MIP-3oc, (CCL20); TECK, thymus expressed chemokine (CCL25) and Vic (CCL28).

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Additional support for this idea was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) analysis of B cell depleted PP and LN which revealed higher levels of IL-4 and IL-lOmRNA in the latter, and higher IL-12 and IFN-y in the former. PPs therefore appear to predispose towards a Th2 milieu while LNs provide an environment favouring Thl maturation. This is in line with the continuous exposure of mucosal lymphoid tissues to externally derived antigens in contrast to peripheral LN. The microenvironment of PP mandates a suppressed response that is attained in part by attenuating T cell and APC interactions.

Probiotic bacteria - a biological role as live or dead organisms The literature is awash with reports (references cited in Kailasapathy & Chin, 2000) illustrating the health benefits that have ensued as a consequence of purposefully modifying the relationship with our immediate enteric microbial environment through the consumption of probiotic bacteria (after Saavedra, 1999). Without analysis of the underlying mechanisms in almost all clinical trials, it can be argued that these benefits have occurred because the live microbial supplement (probiotic) has improved intestinal microbial balance (Fuller, 1991). This effect may result from a direct antagonistic action of the probiotic agent against the pathogen in a clinical disease setting. However, in practical terms, often regular administration of large numbers of probiotic bacteria is required to attain numerical superiority. One would suspect that repeated high dose administration of live probiotic bacteria would not be required if these organisms were capable of efficient colonization and proliferation. Secondly, it is likely that not all benefits accruing from probiotic feeding are due to microbial antagonism or competitive superiority. Many reports have documented improvement of clinical symptoms caused by viral gastroenteritis following probiotic feeding. Other mechanisms promoted by bacteria feeding such as their impact on immune activation are most likely responsible for shortened duration and viral shedding of rotavirus after treatment with Lactobacillus GG and Bifidobacterium. Based on current knowledge on interactions between bacteria and intestinal epithelial cells and immune cells in the GIT, activation of the immune system is dependent upon signalling events between antigen (as whole bacteria) with innate receptors, and between antigen processed by APC and cells of the adaptive immune response. While colonization and antagonistic activity requires live probiotic bacteria, immune activation can most likely proceed without the need for them to be alive. In addition to the already wide range of biological molecules of bacterial origin with 'danger signalling' properties, the genetic material of dead bacteria also contains a preponderance of danger CpG motifs that have the capacity to activate Thl immune responses (Klinman et al., 1996). Even if the large doses of probiotic bacteria administered failed to survive, it is conceivable that their DNA should be able to impact upon the immune system to favour a Thl response.

Immune activation by probiotic bacteria feeding Previously, we have documented in vivo immune activation by probiotic bacteria in mice by using a co-fed marker antigen - ovalbumin (Chin et al., 2000; Matsuzaki & Chin, 2000;

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Chin, 2001). However, the design of these experiments were focused upon T and B cell responses against the marker antigen — ovalbumin. The ability of probiotic feeding to modulate T and B cell function may be studied directly by their activation state postfeeding. The underlying principle behind these experiments is based on the premise that (1) (2) (3) (4)

non-pathogenic probiotic bacteria and their antigens can activate innate responses to facilitate antigen uptake and presentation by APCs T cells exposed to Signal 1 and 2 and/or 3 engagement, upon departure from the GIT would transit to draining mesenteric lymph nodes and the spleen expansion of activated T cells should result in an increased responsiveness to polyclonal T cell mitogens activated and committed B cells would become less responsive to further activation by LPS, a known B cell mitogen.

Figure 8.1 summarizes the feeding protocol used to analyse the effect of oral administration of bacteria on T and B cell responses in female 12-week-old BALB/c mice. This study was extended to include bacteria species not normally considered to be probiotic strains. Bacteria used included Streptococcus bovis (Sb — a haemolytic enteric isolate from cattle faeces); Lactobacillus fermentum (Lf); Streptococcus thermophilus (St); Lactobacillus casei (Lc — Shirota strain); Bifidobacter bifidum (Bb) and Pediococcus pentaceous (Pp - a strain normally used in salami starter cultures). A dose of 108CFU in lOOu.1 was administered via a gavage tube on alternate days, three times a week for three weeks. Mice from each treatment group, including negative controls, were euthanased seven days after the last feed. Blood was collected by cardiac puncture and the spleen removed. Erythrocytes were removed by hypotonic lysis and remaining splenocytes were cultured in microtitre plates in the presence of phytohaemmagglutinin (PHA), concanavalin A (conA) or LPS at the indicated concentrations per well. The results in Fig. 8.2a show that bacteria feeding generally increased the capacity of splenocytes to respond to PHA compared to those from unfed (no bacteria feeding) controls (UC).

Day 1 to Day 21 - Probiotic feeding (C57/B1 & BALB/c) 10 mg per dose per mouse, 3 times a week for three weeks Day 21 - Euthanase BALB/c mice for post-feeding analysis Vaccinate C57/B1 subset intradermally with SAMA4-adjuvanted Ovalbumin (100 ng in 100 nl) Day 28 - Boost C57/B1 intradermally with SAMA4-adjuvanted Ovalbumin (100 jig in 100 ul) Day 35 - Euthanase C57/B1 Remove spleen for cytotoxicity and lymphoproliferation assay Collect blood for ELISA antibody assays Fig. 8.1 Schedule for feeding probiotic bacteria, intradermal ovalbumin vaccination and collection of tissues and blood for immunological analyses.

Immune Action vs.

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Fig. 8.2 Lymphoproliterative response to polyclonal mitogens in spleen cells obtained from mice after bacteria feeding. Polyclonal mitogens used include (a) PHA (phytohaemagglutinin A); (b) Con A (concanavalin A); and (c) IPS (lipopolysaccharide). Bacteria used for feeding were Streptococcus bovis (Sb - a haemolytic enteric isolate from cattle faeces); Lactobacillus fermentum (Lf); Streptococcus thermophilus (St); Lactobacillus easel (Lc - Shirota strain); Bifidobacter bifidum (Bb) and Pediococcus pentaceous (Pp - a strain normally used in salami starter cultures). A dose of 108CFU in 100|al was administered via a gavage tube on alternate days, three times a week for three weeks.

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The best responses were elicited by prior feeding with Lc > Sb > Lf. Although there was a change in the response profile to ConA (viz. Lf > Lc > Sb), prior feeding with these three species also promoted a better response than St, Bb and Pp (Fig. 8.2b). In contrast to the response observed with T cell mitogens, probiotic feeding generally decreased B cell responsiveness to LPS in all treatment groups compared to unfed controls. The most significant suppression in proliferation was observed in mice given Sb, Lf and St (Fig. 8.2c). These results confirm that T cells have been activated in vivo and expanded as a consequence of feeding different Gram-positive bacteria while B cell responsiveness to LPS has been attenuated. A possible explanation for this may be the recent finding that LTA from lactic acid bacteria (LAB) can temper LPS-associated inflammatory responses through competitive engagement of soluble CD 14 (Vidal et al., 2002). This would result in decreased immunomodulation from endogenous LPS. Based on these observations, we believe that the judicious use of probiotic consortiums may generate a health benefit by tolerizing the LPSresponsiveness of host cells in the GIT. In vitro support for this concept has come recently from work carried out in Michalek's laboratory where endotoxin tolerance was induced differentially by sequential stimulation of the human macrophage cell line THP-1 with LPS from E. coli and Porpbyromonas gingivalis (Martin et al., 2001). An important question arises from these experiments: will modulation of T and B cell responsiveness by probiotic feeding affect the animal's ability to respond to further antigenic challenge? To investigate this facet, experiments with C57/B1 mice were carried out with oral feeding of probiotic bacteria in exactly the same manner as the experiment shown in Fig. 8.2, before intradermal vaccination with SAMA4-adjuvanted ovalbumin (Chin & San Gil, 1998) at seven-day intervals. Spleen cells from mice euthanased seven days after the last vaccination were assayed for cytotoxic T cell responses against ovalbumin using a standard Cr51 release assay against MHC-compatible targets pulsed with the ovalbumin peptide - SIINFEKL (Chin et al., 2000). The results in Fig. 8.3 show that probiotic feeding significantly reduced the cytolytic potential of splenocytes to lyse the appropriate target cells compared to unfed/ vaccinated mice (Pos - positive control). Similar experiments carried out previously by co-feeding ovalbumin with probiotic bacteria (Chin et al., 2000) have clearly demonstrated, tolerance induction against ovalbumin (complete elimination of a cytotoxic response against targets expressing ovalbumin peptide) and failure of probiotic feeding to influence such tolerance induction. The present observation of partial suppression of the cytotoxic response by probiotic feeding is not typical of tolerance induction and probably represents a form of restricted expansion of ovalbuminspecific CD8 T cells, most likely attributable to immune deviation. Probiotic feeding with Sb, Lf, St, Bb and Pp also increased the T cell proliferative response to ovalbumin (Fig. 8.4). Further analysis of the Ig isotype response against ovalbumin by enzyme-linked immunosorbent assay (ELISA) revealed that the antibody response was predominantly IgGl with little or no detectable IgG2a or IgG2b antibodies against ovalbumin (Fig. 8.5). These results clearly show that the modulation of immune responses by probiotic feeding with the selected test species, has not altered the intrinsic bias towards a Th2 response.

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Fig. 8.3 Effect of oral bacteria feeding on the cytotoxic T cell response of spleen cells against syngeneic targets expressing the ovalbumin peptide SIINFEKL post-vaccination. Treatment groups represent mice fed Streptococcus bovis (Sb); Lactobacillus fermentum (Li); Streptococcus thermophilus (Si); Lactobacillus casei (Lc - Shirota strain); Bifidobacter bifidum (Bb) and Pediococcus pentaceous (Pp), respectively. Individual animal responses (n = 3 per panel) are represented by open (assayed against RMA targets/ peptide) or closed (assayed against targets only) symbols O, n, and A.

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Fig. 8.4 Lymphoproliferative response to ovalbumin in spleen cells obtained from mice after bacteria feeding and vaccination. Treatment groups represent mice fed Streptococcus bovis (Sb); Lactobacillus fermentum (Lf); Streptococcus thermophilus (Si); Lactobacillus easel (Lc - Shirota strain); Bifidobacter bifidum (Bb) and Pediococcus pentaceous (Pp), respectively.

Probiosis - is it Gram-positive versus Gram-negative bacteria or it is live versus dead bacteria? Almost all pathogenic Gram-positive or Gram-negative bacteria exemplified by Staphylococcus aureus and Bordetella pertussis, respectively, have the ability to synthesize molecules that interfere or modulate the host immune response. Pathogenic Gram-positive bacteria produce superantigens capable of activating large numbers of T cells via V[3 elements of their TcR without the constraints of MHC restriction (Blackman & Woodland, 1995). Pathogenic Gram-negative bacteria release large amounts of lipooligosaccharides (LOS) or LPS. While different species of Gram-negative bacteria may differ immensely in their oligosaccharide sequence (O-antigen), most of the biological activity resides in lipid A. Lipid A is attached to the polysaccharide through a specific sugar — 2-keto-3-deoxyoctulosonic acid (KDO). Lipid A itself consists of a phosphorylated p-l,6-linked glucosamine disaccharide to which long fatty acid chains are attached. The biological activity of LPS differs between different Gramnegative bacteria and may be due primarily to differences in the three-dimensional shape of lipid A (Netea et al., 2002). Lipid A from E. coli is conical, binds avidly to TLR4 and acts as a very potent inducer of cytokine production. Porphyromonas gingivalis lipid A is more cylindrical, binds to TLR2 and appears to be more selective in the cytokines it can induce (Martin et al., 2001).

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Fig. 8.5 Antibody isotype response of mice to ovalbumin after bacteria feeding and vaccination. Treatment groups represent mice fed Streptococcus bovis (Sb); Lactobacillus fermentum (Lf); Streptococcus thermophilus (St); Lactobacillus casei (Lc - Shirota strain); Bifidobacter bifidum (Bb) and Pediococcus pentaceous (Pp), respectively, (a) IgGl response; (b) lgG2a response; and (c) lgG2b response.

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Although most probiotic bacteria are Gram-positive LAB, little is known about the immunomodulatory molecules they produce or are associated with their structure. The use of non-pathogenic enteric Gram-negative bacteria such as E. coli as probiotics, can competitively exclude pathogenic E. coli but may not be capable of subverting the host immune system, unless possession of LPS presents the only danger signal. In the absence of other clear danger signals such as toxins, non-pathogenic probiotic bacteria may also share common determinants such as adhesion and colonization factors. If biological activity of probiotic bacteria is reliant upon specific molecules, then delivery of large numbers of dead probiotic bacteria can provide the necessary molecular mass to confer an immunomodulatory response via the enterocytes or when absorbed. However, probiotic bacteria may only produce specific molecules when they have successfully colonized and viability becomes an important prerequisite for the production of both constitutive and induced signalling molecules. An interesting example is the production of quorum-quenching N-Acyl homoserine lactonase by Bacillus species (Dong et al., 2002). Many other Gram-negative bacteria within the GIT communicate and signal vital processes through the production of the quorum-sensing factor N-Acyl homoserine lactone (AHL). Destruction of AHL by the quorum-quenching factor (QQF) can interfere with survival processes and greatly disrupt the balance of microbial diversity. This may be one reason why Bacillus spores have been used effectively as probiotics in animals. It is not known at present whether QQF can act on y-butyrolactones that are used as signalling molecules by Gram-positive bacteria. To date, there have been very few reports demonstrating that individual strains of probiotic bacteria can alter the Th2 imbalance and promote instead, a Thl response. Maassen et al. (2000) showed that only oral in vivo feeding of BALB/c mice with live L. brevis and L. reuteri compared to L. murinus, L. casei, L. gasseri, L. plantarum and L. fermentum, were able to modulate immune responses towards the production of Th2 cytokines and to initiate a switch towards IgG2a production. Judicious use of an appropriate probiotic species or strain, can provide a practical approach to convert the Th2 bias of the GIT in an attempt to reduce the incidence of allergic responses (Matsuzaki & Chin, 2000). According to the 'hygeine' hypothesis (Chin, 2002), epidemiological evidence suggests that a lack of infection experience in a clinical setting has increased the population predisposition to allergic disorders in developed countries (Wohlleben & Erb, 2001). Atopic individuals are therefore more prone to allergies due to the imbalance towards a Th2 mucosal immune response or conversely, a lack of prior experience of Thl engaging experiences. In accordance with current views on subverting the Th2 bias with ancillary 'danger' signals, oral administration of a commercial preparation of killed respiratory pathogenic bacteria (OM-85) consisting of Haemophilus influenzae, Streptococcus pneumonias, Streptococcus pyogenes, Streptococcus viridans, Klebsiella pneumoniae, Klebsiella ozaenae, Staphylococcus aureus and Moraxella catarrhalis to rats, was effective in up-regulating both primary and secondary IgG2b responses. This was accompanied by increased IFN-y interferon and decreased IL-4 production and an increased capacity for development of Thl-dependent delayed hypersensitivity to a challenge antigen (Bowman & Holt, 2001).

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Summary and perspective Both the innate and adaptive arms of the immune system have evolved the ability to recognize and respond to danger signals associated with potential pathogens. So great is this capacity to mount an immune response that there is always a constant danger that an overreactive immune system may do more harm than good in its efforts to protect the host. Therefore, a complex set of regulatory mechanisms has been set in place to attenuate and/or enhance these responses. The most important issues confronting researchers in the field of probiotics are: (1) (2)

(3) (4) (5) (6)

Characterization of the molecules associated with, or produced by probiotic bacteria that are capable of affecting other bacteria, host enterocytes and immune cells. Development of in vitro assays to study molecular signalling events in target cells (particularly differential gene induction using proteomic technology) mediated by interaction with or attachment by live and/or dead probiotic bacteria. Testing the signalling interactions of probiotic bacteria in vivo. Unravelling the interactions between probiotic bacteria and other commensals and/or pathogens in the GIT. Characterizing the expression of new genes in probiotic bacteria in the host intestinal environment that is initiated by the process of colonization. Dissecting the interactions between probiotic bacteria and their antigens on signalling events between enterocytes and other immune cells in the submucosae.

Until more information is obtained, we remain greatly constrained in our efforts to develop designer probiotic formulations and delivery mechanisms capable of sustaining the appropriate immune response and achieving predictable and reliable clinical outcomes from therapeutic probiotic usage. Nonetheless, it is clear from ongoing research, that immune responses can be modulated by probiotic feeding. However, and more importantly, we may have to modify our current ideas about what constitutes a probiotic. In view of new knowledge on some of the mechanisms involved in mucosal activation by lumen bacteria, it might perhaps be more appropriate to achieve a probiosismediated health outcome based on the delivery of live commensal bacteria that are not exclusively LAB, and enhancing their efficacy with the inclusion of killed pathogenic bacteria capable of imparting non-pathological but still functional danger signals to educate the mucosal immune system.

Acknowledgements The authors acknowledge the contributions of Bernadette Turner for conducting the lymphoproliferation and cytotoxic assays, Sameer Dixit for preparing the probiotic bacteria and Ron Thla Ha for assisting in the cytotoxic T cell assays.

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McCoy, K., Camberis, M. & Le Gros, G. (1997) Protective immunity to nematode infection is induced by CTLA-4 blockade. Journal of Experimental Medicine, 186, 183—7. Medzhitov, R. & Janeway, C.A. Jr (2000) Innate immunity. New England Journal of Medicine, 9, 338-44. Morita, C.T., Mariuzza, R.A. & Brenner, M.B. (2000) Antigen recognition by human y8 T cells: pattern recognition by the adaptive immune system. Springer Seminars in Immunopathology, 22, 191-217. Morita, C.T., Lee, H.K., Wang, H., Li, H., Mariuzza, R.A. & Tanaka, Y. (2001) Structure features of nonpeptide prenylpyrophosphates that determine their antigenicity for human jo T cells. Journal of Immunology, 167, 36—41. Mowat, A.M. & Viney. J.L. (1997) The anatomical basis of intestinal immunity. Immunology Review, 156, 145-66. Miillbacher, A. & Flynn, K. (1996). Aspects of memory cytotoxic T cells. Immunology Review, 150, 113-127. Miillbacher, A. & Blanden, R.V. (2000) Cytotoxic T cell memory. In: Cytotoxic Cells: Basic Mechanisms and Medical Applications (ed. M.V. Sitkovsky, & P. Henkard). LippincottWilliams & Wilkins, Philadelphia, pp. 317-25. Netea, M.G., van Deuren, M., Kullberg, B., Cavaillon, J.-M. & Van der Meer, J.W. (2002) Does the shape of lipid A determine the interaction of LPS with Toll-like receptors. Trends in Immunology, 23, 135-9Russell, J.H. (1995) Activation-induced death of mature T cells in the regulation of immune responses. Current Opinion in Immunology, 7, 382—8. Saavedra, J.M. (1999) Probiotics plus antibiotics: Regulating our bacterial environment. Journal of Pediatrics, 135, 535-7. Sakaguchi, S. (2000) Regulatory T cells: key controllers of immunologic self-tolerance. Cell, 101, 455-8. Salmi, M. & Jalkanen, S. (1999) Molecules controlling lymphocyte migration to the gut. Gut, 45, 148-53. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M. & Kirschning, C.J. (1999) Peptidoglycan and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. Journal of Biological Chemistry, 274, 17406—9. Slifka, M.K. & Whitton, J.L. (2000) Activated and memory CD8+ T cells can be distinguished by their cytokine profiles and phenotypic markers. Journal of Immunology, 164, 208-16. Smith, P., Smythies, L., Mosteller-Barnum, M. et al. (2001) Intestinal macrophages lack CD 14 and CD89 and consequently are down-regulated for LPS and IgA-mediated activities. Journal of Immunology, 167, 2651-6. Smits, H.H., de Jong, E., Schuitemaker, N. (2002) Intracellular adhesion molecule-1/LFA-1 ligation favours human Thl development. Journal of Immunology, 168, 1710—16. Su, C, Wen, X., Bailey, S.T. et al. (1999) A novel therapy for colitis utilizing PPAR-y ligands to inhibit the epithelial inflammatory response. Journal of Clinical Investigation, 104, 383-9Szabo, S.J., Sean, T., Costa, G., Zhang, X., Fathman, C. & Glimcher, L. (2000) A novel transcription factor, T-bet, directs Thl lineage commitment. Cell, 100, 655-69. Thorstenson, K.M. & Khoruts, A. (2001) Generation of anergic and potentially

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immunoregulatory CD25 CD4 T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen. Journal of Immunology, 167, 188-95. Van Stipdone, M., Lemmens, E. & Schoenberger, S.P. (2001) Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nature Immunology, 2, 243—6. Vidal, K., Donnet-Hughes, A. & Granato, D. (2002) Lipoteichoic acids from Lactobacillus johmonii and Lactobacillus acidophilus antagonize the responsiveness of human intestinal epithelial HT29 cells to LPS and Gram-negative bacteria. Infection and Immunity, 70, 2057-64. Viney, J., Mowat, A., O'Malley, J., Williamson, E. & Fanger, N.A. (1998) Expanding dendritic cells in vivo enhances the induction of oral tolerance. Journal of Immunology, 160, 5815-25. Wang, B., Maile, R., Greenwood, R., Collins, E.J. & Frelinger, J.A. (2000) Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells. Journal of Immunology, 164, 1216-22. Wohlleben, G. & Erb, K.J. (2001) Atopic disorders: a vaccine around the corner? Trends in Immunology, 22, 618-26. Zheng, W. & R.A. Flavell (1997) The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell, 89, 587-96.

Food Hypersensitivity and Allergic Diseases

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R.K. Chandra Memorial University of Newfoundland, Janeway Child Health Centre, Suite 2J740 St John's, Newfoundland A1B 3V6, Canada

Introduction Adverse reactions to foods have been recorded in historical accounts for 3000 years. Most individuals can tolerate most foods most of the time. However, as many as one-third of individuals experience some side effect to ingestion of foods at least once during their lives; only a small proportion of these are truly due to 'allergy'. Food allergy is defined as a reaction that has a demonstrable immunological reaction. Allergic disease is a common cause of morbidity, particularly in young children. The prevalence of allergic disease has increased in the last 20 years in most countries. The sequential order of occurrence of allergy is food hypersensitivity, gastrointestinal manifestations, atopic eczema, asthma, and hay fever. This implies that the prevention of food hypersensitivity in the first few months of life may well reduce the chances of the occurrence of other allergic disorders. And the beneficial effect may last well beyond childhood. A variety of factors increase the risk of allergic disease, for example hereditary predisposition, exposure to 'allergenic' foods and environmental triggers such as house dust mites and tobacco in early life. Prolonged breast feeding, the use of a partial whey hydrolysed formula, delayed introduction of certain 'allergenic foods', and avoidance of inhalant allergens reduces the incidence of eczema and asthma, especially in high risk infants. These preventive measures are extremely cost-effective and should be adopted widely at the community level. The subject of food hypersensitivity and allergic disease has been reviewed by several authors (Chandra 1988, 1997b, 2002a; Estaban 1992; Walker et al. 1993; Chandra et al. 1995; Samartin et al. 2001; Buttriss 2002; Thomson & Chandra 2002) and a selective review is presented here.

Immunological mechanisms Four types of processes may underlie sensitization to foods and the subsequent manifestation of clinical syndromes attributable to it (Chandra 1988; Chandra etal. 1995). These are: type

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I or immunoglobulin E (IgE) mediated hypersensitivity that manifests as urticaria, angioedema and anaphylaxis. In type II, the antigen binds to the cell surface and the presence of antibodies disrupts the membrane leading to cell death. Neutropenia and thrombocytopenia are examples. In type III, antigen-antibody-complement immune complexes get trapped in small blood vessels or the glomerulus, resulting in vasculitis and nephropathy, respectively. Type IV is mediated by T lymphocytes and this delayed hypersensitivity can show as a variety of clinical syndromes. In many patients, more than one process may be working at the same time. It follows that unless relevant tests are done to identify all these four types of reactions, the diagnosis of allergy may be missed. There is much recent knowledge about the role of cell-mediated immunity and of cytokines in the pathogenesis of food allergy (Samartin et al. 2001). It is believed that there are two functional subsets of T helper (TH) cells. TH1 cells are primarily involved in mounting a protective response to invading microorganisms including bacteria and viruses. These cells predominantly produce interleukin-2 and interferon-gamma. TH2 cells are responsible for allergic responses and produce interleukin-4 and other cytokines. A shift in TH1/TH2 ratio to the latter may well predispose to the clinical development of atopic disorders including eczema and asthma. It is now possible to set up experiments in which purified antigens or whole extracts of foods are used to stimulate T cells in vitro and depending upon the cytokine profile, e.g. interleukin 4/interferon gamma, one can predict the occurrence of allergic responses. There is preliminary information to suggest that these changes may correlate with persistence of food hypersensitivity.

Food allergens In general, food antigens are polypeptides or proteins (Buttriss 2002). Occasionally, a small sized food molecule may piggy back on a protein or polypeptide, i.e. act as a hapten, and elicit an immune response. Thus, it is possible to visualize a reaction to the digested end products of foods. For reasons that are not well understood, certain foods are more likely to cause allergic reactions than other foods (Table 9.1). This knowledge can be used to plan 'hypoallergenic' diets for diagnosis and for management. However, any food can cause serious reactions in a person. Most food antigens that cause reactions are in the molecular weight range of 14 000 and 70 000. Many are glycoproteins.

Clinical manifestations Any food can produce any symptom (Chandra 1988; Estaban 1992). All organs and systems can be affected by allergic disease. These include gastrointestinal symptoms (nausea, vomiting, colic, diarrhoea, malabsorption, blood in stools), skin symptoms (rashes of various types, eczema, urticaria, angioedema), respiratory symptoms (wheezing, asthma, rhinitis), neurological symptoms (migraine), joint symptoms (arthralgia, arthritis), psychological symptoms (behaviour problems, hyperactivity, reduced attention span, tics) and miscellaneous symptoms (anaphylaxis) (Thompson & Chandra 2002).

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Table 9.1

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Allergenic foods

Foods that cause allergic reactions frequently Milk and other dairy products Eggs Nuts, especially peanuts Fish and shellfish Wheat, soy Pork, beef Tomato, carrot, celery, mushrooms, corn Chicken Orange, pineapple, kiwi Garlic, basil, savory, paprika, mustard Yeast Food colours and preservatives Foods that cause allergic reactions less often Sugar Rice, oats, barley Apricot, cranberry, grape, peach, pear, plum Sweet potato, tapioca Asparagus, lettuce, cauliflower, spinach, squash, broccoli, artichoke Tea, coffee Lamb, turkey Olive oil, safflower oil

The consumption of alcohol prior to or within two hours of the ingestion of food allergen worsens the severity of clinical manifestations. Similarly, modest or exhaustive exercise worsens symptoms and may precipitate anaphylaxis. Several deaths have been reported where an individual with modest allergy to food engaged in exercise within a few hours of the ingestion of the involved allergen. In true food allergy, more than one organ system may be affected in the same individual. On the other hand, the reported occurrence of several symptoms in different organs, for example a combination of diarrhoea, headache, fatigue, rashes, abdominal 'swelling' and cramps, visual problems, respiratory distress and others, might be indicative of a psychosomatic problem: in this scenario, a systematic psychological assessment for depression is warranted. Food allergy is generally not permanent. In infants with cow's milk allergy, tolerance to milk often occurs within one year. Peanut allergy is more persistent (Table 9-2). If food allergy occurs for the first time in adult life, it is more likely to persist for many years and even be for life. There is preliminary evidence to suggest that tests of lymphocyte stimulation in the presence of the food allergen and the pattern of cytokine production may predict the duration of persistence of allergic symptoms.

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Table 9.2

Prevalence of food allergy by age

Allergen

Number examined in 1st yr

Number positive in 1st yr

Number positive at 2yr

Number positive at Syr

Number positive at 5 yr

Milk Egg Peanut

480 480 480

186 236 120

64 128 95

18 45 83

6 19 68

Identification of high risk infants for prevention Attempts at prevention of allergic disease and assurance of parental motivation should first identify those newborns that are more likely to develop allergies later in childhood. A number of clinical and laboratory tests have been assessed for these objectives. These include positive parental history of atopy particularly if the same clinical syndrome is present in both parents, elevated neonatal IgE, reduced number of CD8+ gamma-delta receptor positive lymphocytes, reduced serum mannan-binding protein, and altered cord blood alphalinoleinic acid levels. If one combines all these factors, it is possible to predict with more than 95% certainty the chances of development of allergic disease in children (Estaban 1992; Chandra 2000, 2002a; Wahn & von Mutius 2001). However, steps to reduce the incidence of allergy should be carried out in all infants with more vigorous efforts being made in those with biparental history of atopy and elevated neonatal blood IgE concentration.

Preventive measures Breast feeding There is no doubt that exclusive breast feeding for four months or longer is associated with reduced occurrence of proven allergic disease (Table 9.3), such as eczema and asthma. Longterm studies are available to indicate that the benefits are for at least 18 years of age (Chandra 1979, 1997a, 2002b; Saarinen & Kajosaari 1995). The addition of a standard cow's milk formula or soy formula reduces the benefits of Table 9.3 Influence of exclusive breast feeding on cumulative incidence of allergic disease. Follow-up for 18 years Group Breast-fed < 4 months > 4 months

Low risk

High risk

5.8% 3.6% 8.6%

33.4% 24.3% 56.7%

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breast feeding. Therefore, exclusivity of breast feeding is important. The benefits are enhanced if the mother restricts common allergenic foods from her own diet during lactation (Chandra et al. 1989). These include dairy products, egg, fish, nuts and soy. Even though this approach might seem very restrictive, mothers who have had a previous child with allergy would go to any length to reduce the risk of the disease in a subsequent child. For this reason, the acceptance rate of the restricted diet by mothers who have had a previous child with allergy is more than 95%. Of course, breast feeding should be promoted for other reasons as well. It promotes optimum physical and intellectual growth. It reduces the risk of necrotizing enterocolitis, diabetes mellitus type I and type II, lymphoma, and infections in general (Chandra 2000). Despite the availability of modern infant formulae, exclusive breast-fed infants are healthier than formula-fed infants in all countries, even industrialized ones. These data have been reviewed elsewhere (Saarinen et al. 1995; Chandra 2000). Hydrolysed formulas Hydrolysis reduces the 'allergenecity' of milk proteins. There are many arguments in favour of using a partial hydrolysed formula (pHF) rather than an extensively hydrolysed formula (eHF) (Chandra 1997; Baumgartner et al. 1998). The data have been subjected to metaanalysis. For treatment of proven cow's milk allergy, an extensive hydrolysis is more beneficial, even though the taste of the milk is bad and the product costs are high. However, for prevention even partial hydrolysis is adequate. The molecular size is less than 5000 daltons, the majority of the molecules being < 1000 daltons. These recommendations have been endorsed by the European Society of Pediatric Gastroenterology, Hepatology and Nutrition, and the European Community (see Chandra 2002b). Cost considerations are also important (Chandra 2002a,b). It cannot be emphasized too strongly that the design and execution of the trials are very important and critical elements that would determine the validity of the results (Hattewig et al., 1989; Chandra et al., 1995; Kleinman et al., 1991). For studies of reduced exposure to food allergens during pregnancy, it is important to start the precautions prior to the 10th week of pregnancy, a time threshold that allows for the fetus to become capable of mounting an immune response. Many long-term studies with follow up of at least 60 months have concluded that feeding pHF from birth decreases the cumulative incidence as well as the period prevalence of atopic disease, particularly in those with a strong family history of allergy. To illustrate this point, the results of our study are described. 216 high risk infants whose mothers had elected not to breast feed were randomized to receive exclusively a partial whey hydrolysed formula (Good Start or NAN.HA), a conventional standard cow milk formula (Similac), or a soy formula (Isomil). Follow up until the age of five years showed a significant lowering in the cumulative incidence of atopic disease, especially eczema and asthma (Chandra 1997). The many prospective controlled studies in this field have been discussed and analysed recently (Baumgartner et al., 1998). A total of 15 out of 25 published studies that examined the impact of early exclusive feeding on atopic disease and that met the strict inclusion and exclusion criteria for meta-analysis were included. All studies were conducted in industrialized countries, viz. Canada, Europe. Exclusive feeding of the assigned formula was for a minimum of three months. Oral challenges to document cow's milk and other food allergies

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were conducted in some studies. The Fixed Effect Model and the Random Effect Model of statistical evaluation were conducted. A total of 576 infants satisfied all the criteria for evaluation by meta-analysis. At all examination points of 6, 12, 18, 24, 36, 48 and 60 months, pHF fed infants had a lower incidence of allergic disease. For the age period four to six months, the percentage of pHF fed infants who showed allergy was about one-quarter that of the control group (odds ratio 0.25, 95% confidence intervals 0.18—0.36). This trend continued until age five years. Should pHF/eHF be recommended for all infants, not just for those with parental history? There are some preliminary recent studies in unselected populations that conclude that indeed all infants would benefit from this approach. Other measures There are few controlled studies on other measures that might also help in reducing the incidence of allergic disease. These include delayed introduction of selected 'allergenic' foods particularly egg, fish and nuts, reduced exposure to tobacco smoke and house dust mites.

Management of food allergy The common sense approach to the treatment of food allergy has been known for decades. Firstly, the diagnosis should be confirmed without any doubt. Many individuals are put on restrictive diets or even pharmacological products that have side effects without the benefit of a final diagnosis. Secondly, the foods proven to be involved should be avoided strictly. Occasionally, antihistamines and even steroids might be needed for short-term therapy. Those who have experienced anaphylactic reactions to foods must carry self-administered adrenaline with them all the time (Thompson & Chandra 2002). The injection of adrenaline can be taught even to young children from the age of four years. The injection can be administered in the thigh and through clothes. The needle should be kept inside the muscle for at least 10 seconds. There are no significant side effects except mild tachycardia and palpitation. A second dose can be repeated after 1520 minutes if there is no significant relief of symptoms. With the first injection of adrenaline administered within 5—10 minutes of the onset of symptoms, one can virtually eliminate the possibility of a fatal outcome.

References Arshad, S.H., Matthews, S., Grant, C. & Hide, D.W. (1992) Effect of allergen avoidance on development of allergic disorders in infancy. Lancet, 339, 1493—7. Baumgartner, M., Brown, C.A., Exl, B.-M., Secretin, M.-C, van't Hoft, M. & Haschke, F. (1998) Controlled trials investigating the use of one partially hydrolyzed whey formula for dietary prevention of atopic manifestations until 60 months of age; an overview using meta-analytic techniques. Nutrition Research, 18, 1425—42. Buttriss, J. (ed.) (2002) Adverse Reactions to Food. Blackwell Science, Oxford.

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Chandra, R.K. (1979) Prospective studies on the effect of breast feeding on incidence of infection and allergy. Acta Pediatrica Scandinavica, 68, 691—4. Chandra, R.K. (ed.) (1988) Food Allergy. Nutrition Research Education Foundation, St John's, Newfoundland, Canada. Chandra, R.K. (1997a) Five-year follow-up of high-risk infants with family history of allergy who were exclusively breast-fed or fed partial whey hydrolysate, soy, and conventional cow's milk formulas. Journal of Pediatric Gastroenterology and Nutrition, 24, 380—8. Chandra, R.K. (1997b) Food hypersensitivity and allergic disease;a selective review. American Journal of Clinical Nutrition, 66, 526S—9S. Chandra, R.K. (2000) Food allergy and nutrition in early life. Proceedings of the Nutrition Society, 59, 1-6. Chandra, R.K. (2002a) Food allergy. Indian Journal of Pediatrics, 69, 251-5. Chandra, R.K. (2002b) Food hypersensitivity and allergic diseases. European Journal of Clinical Nutrition, 56, Suppl. 3, SI—3. Chandra, R.K. (2002c) Breast feeding, hydrolysate formulas and delayed introduction of selected foods in the prevention of food hypersensitivity and allergic disease. Nutrition Research, 22, 125-35. Chandra, R.K. (2002d) Preventive nutrition; consideration of cost-benefit and cost-effective ratios. Nutrition Research, 22, 1-3. Chandra, R.K. & Baker, M. (1983) Numerical and functional deficiency of suppressor T cells precedes development of atopic eczema. Lancet, ii, 1393-4. Chandra, R.K., Puri, S. & Hamed, A. (1989) Influence of maternal diet during lactation and use of formula feeds on development of atopic eczema in high risk infants. British Medical Journal, 299, 228-30. Chandra, R.K., Gill, B. & Kumari, S. (1995) Food allergic and atopic disease. Critical Review of Allergy Immunology, 13, 293—314. Estaban, M.M. (ed.) (1992) Adverse reactions to foods in infancy and childhood. Journal Pediatric, 121, S1-S126. Hattevig, G., Kjellman, B., Sigurs, N.& Kjellman, N.-I. (1989) The maternal avoidance of eggs, cow's milk and fish during lactation upon allergic manifestations in infants. Clinical Allergy, 19, 27-32. Kleinman, R.E., Bahna, S., Powell, G.F. & Sampson, H.A. (1991) Use of infant formulas in infants with cow milk allergy. A review and recommendations. Pediatric Allergy Immunology, 2, 146—55. Koopman, L.P. (2002) Risk factors for the development of atopic disease in infancy and early childhood. Doctoral thesis, Erasmus Universiteit Rotterdam. Saarinen, U.M. & Kajosaari, M. (1995) Breast feeding as prophylaxis against atopic disease; prospective follow-up study until 17 y old. Lancet, 346, 1065—9. Samartin, S., Marcos, A. & Chandra, R.K. (2001) Food hypersensitivity. Nutrition Research,

21,473-97. Thompson, K. & Chandra, R.K. (2002) The management and prevention of food prophylaxis. Nutrition Research, 22, 89-110. Wahn, U. & von Mutius, E. (2001) Childhood risk factors for atopy and the importance of early intervention. Journal of Allergy and Clinical Immunology, 107, 567—74. Walker, W.A., Harmatz, P.R. & Wershil, B.K. (eds) (1993) Immunophysiology of the Gut. Academic Press, New York.

Nutritional and Microbial Modulation of Carcinogenesis

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R. Hughes & I. Rowland Northern Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 ISA

Introduction It is generally accepted that diet plays a major role in the aetiology of certain cancers of the gastrointestinal tract particularly the stomach, colon and rectum. The effect of diet on cancer at various sites has been the subject of many reviews (WCRF, 1997; Cummings & Bingham, 1998). Evidence for an effect of diet appears to be strongest for colorectal cancer. Colon cancer is the fourth most common cancer worldwide (Boyle & Langman, 2000). International incidence rates vary approximately twenty-fold with Westernised countries showing highest rates (Parkin et al., 1992). Genetic factors account for 5—10% of cases as evidenced by the existence of genetic syndromes which carry an increased risk. The commonest hereditary syndromes are familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC). The remaining cases have been attributed to environmental factors, particularly diet, making this cancer a potentially avoidable disease. Epidemiological cross sectional comparisons, case—control studies and trends in food intake show high rates of colorectal cancer in populations consuming diets high in red meat (especially processed meat) and fat and low in starch, non-starch polysaccharides (NSP) and vegetables (WCRF, 1997; Potter, 1999). Although evidence from prospective studies supports these findings, estimates of relative risk are not high. Indeed recent prospective trials have shown no protective effect of vegetables and fibre (Fuchs et al., 1999; Michels et al., 2000) and no increased risk with red meat (Key et al., 1998). Recently, limitations of prospective studies have been noted in terms of inaccurate dietary intake data due to measurement error, nutrient interactions and individual variations in genetic polymorphisms. Mechanisms by which diet is associated with colon cancer causation or prevention are largely unknown and the subject of much research. Carcinogenic agents may be present in

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the diet or formed in vivo during digestion. Mechanisms currently under investigation are listed in Table 10.1. Many of these mechanisms involve the metabolic activities of the bacterial flora normally resident in the human colon. The colonic lumen harbours over 10 bacteria per gram of contents (Cummings & MacFarlane, 1991). It is not surprising therefore that the activities of this population have an important impact on the health of the host. This chapter discusses the detrimental and beneficial consequences of bacterial activity in relation to carcinogenesis. As most intestinal bacteria are found in the large intestine in humans, most of the chapter will refer to carcinogenic effects in the colon. Table 10.1 Summary of the proposed mechanisms whereby diet may increase or reduce colon cancer risk Mechanism

Food/nutrient

Mechanisms associated with increased risk Genotoxin formation during cooking Formation of toxic products during fermentation Induction of oxidative DMA damage Increased secondary bile acid formation Increased growth factor formation

Red meat Red meat protein Iron (red meat) and alcohol Saturated fat Refined carbohydrate

Protective mechanisms Maintenance of DNA integrity Carcinogen binding Reduced DNA damage Formation of protective fermentation products

Induction of Phase II detoxification enzymes Decreased mutagen formation

Folate Calcium and probiotics Antioxidants in fruit and vegetables Fibre/non-starch polysaccharides Non-digestible oligosaccharides Resistant starch Glucosinolates/Brass/ca vegetables Fibre/non-starch polysaccharides Probiotics

Bacteria and cancer overview Bacteria have been linked to cancer by two mechanisms, induction of chronic inflammation following bacterial infection and production of toxic bacterial metabolites. Helicobacter pylori infection is known to increase risk of adenocarcinoma of the distal stomach. H. pylori is the first bacterium to be termed a definitive cause of cancer by the International Agency for Research into Cancer (IARC). The inflammatory effects of H. pylori infection have been related to cancer due to increased cell proliferation and production of mutagenic free radicals and N-nitrosocompounds (NOC) (Moss, 1999). In addition H. pylori infection was associated with changes in oncogene and tumour suppressor gene expression as shown by increased ras p21 expression and p53 mutation in H. pylori positive cases of gastric cancer (Wang et al., 2002). Dietary vitamin C has been associated with reduced gastric cancer risk (Correa et al.,

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1998). Although this may be related to its ability to scavenge reactive oxygen species and inhibit NOC formation, high dose vitamin C has also been shown to inhibit H. pylori growth and colonisation (Zhang et al., 1997) and at physiological concentrations it induced H. pylori associated apoptosis and cell cycle arrest in vitro (Zhang et al., 2002). The prevalence of H. pylori infection is falling in developed countries and this has been linked to changes in the epidemiology of gastrointestinal diseases, in particular reduced incidence of gastric cancers in Western countries (Blaser, 1998; Logan & Walker, 2001). Improved nutrition, water supplies and reduced family sizes have been associated with reduced H. pylori colonisation (Blaser, 1998). H. pylori status has also been positively associated with colorectal adenoma risk in humans (Breuer-Katschinski et al., 1999). Bacterial involvement in colorectal cancer has been widely studied with most information being derived from animal work and some human studies. Increased Bacteroides numbers has been associated with increased colon cancer risk in humans (Hill et al., 1971; Moore & Moore, 1995). In another study lecithinase-negative Clostridium and Lactobacillus were more abundant in colon cancer patients (Kanazawa et al., 1996). Some Lactobacillus species and Eubacteruium aerofaciens have been associated with reduced risk (Moore & Moore, 1995). In animals, colonic tumour formation is dependent upon the presence of intestinal flora (Reddy et al., 1974). A high incidence of spontaneous colorectal cancer has been demonstrated in the T-cell receptor P chain and p53 double knockout mice. Further work on this model showed that adenocarcinoma of the colon did not occur in germ free TCRp~~p53 mice but adenocarcinomas were detected in 70% of the conventionalised animals showing a major role for intestinal flora (Kado et al., 2001). Streptococcus bovis has been implicated in colonic neoplasia. Supplements of Streptococcus bovis and antigens extracted from the bacterial cell wall were shown to induce formation of hyperproliferative aberrant colonic crypts and increase expression of proliferation markers in carcinogen treated rats (Ellmerich et al., 2000). The effect of individual bacteria on cancer risk varies. Mice mono-associated with Mitsuokella multiacida, Clostridium butyricum or Bifidobacterium longum had a higher incidence of colonic adenoma (68% in each case) as compared with those associated with Lactobacillus acidophilus (30%) (Horie et al., 1999). Bacterial colonisation was associated with lower faecal pH (L. acidophilus) and bile acid deconjugation (Clostridia). Bacterial strains belonging to the lactic acid producing bacteria, Lactobacillus and Bifidobacterium have been associated with a number of health effects including possible cancer protective effects as discussed later. These bacteria are termed 'probiotics'. The generally accepted definition for probiotics is that they are live microbial food ingredients which beneficially affect the host by improving its intestinal flora. In the colon, cancer protective effects of certain probiotic bacteria have been associated with cellular effects including suppressed ornithine decarboxylase activity and ras-p21 expression (Singh et al., 1997) and reduced O -methylguanine adduct DNA levels (Arimochi et al., 1997). Bacteria may influence carcinogenesis via several mechanisms. They may have a direct effect by binding potential mutagens thus reducing exposure to the host (Morotomi & Mutai, 1986; Orrhage et al., 1994). Certain strains resident in the normal gut flora, are also known to produce and release toxins which can bind specific cell surface receptors and affect intracellular signal transduction. This interaction has been reviewed by Fasano (1999). Cytotoxic effects of toxins A and B (Clostridium difficile toxins) and enterotoxin (Clostridium perfringens toxin) have been reported in intestinal cells cultured in vitro (Fiorentini et al., 1989,

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1992; McClane, 1996). Toxins A and B have been shown to induce apoptosis in vitro (Mahida et al., 1996; Fiorentini et al., 1998a). In contrast, the toxin produced by Escherichia coli (CNF1) inhibits apoptosis possibly by increasing expression of BCL2 and BCLXl (Fiorentini et al., 1998b). Cytolethal distending toxins (CDTs) found in E. coli, Shigella dysenteriae, Campylobacter spp. and Haemophilus ducreyi, irreversibly inhibit cell division at the G2/M stage (Comayras et al., 1997). Biological activities of the major toxins produced by bacteria have been extensively reviewed (Donnelli et al., 2000). To date, the vast majority of mechanisms whereby bacteria are involved in carcinogenesis involve toxic or protective products of bacterial metabolism. Such metabolic activities include numerous enzymic reactions and degradation of undigested residue for energy. Diet can substantially modulate these activities by providing a vast array of substrates. Toxic and protective consequences of bacterial metabolism are outlined below.

Bacterial metabolites Enzyme activities The enzymic activities of the gut microflora towards ingested foreign compounds such as nitro-aromatics, azo compounds, and nitrate can lead to the generation of genotoxic and carcinogenic products. Enzymes commonly implicated include P-glucuronidase, (3-glycosidase, azoreductase, nitroreductase, nitrate reductase, the conversion of pre-carcinogen 2amino-3-methyl-7H-imidazo[4,5-f]quinoline (IQ) to 7-hydroxy-2-amino-3,6-dihydro-3methyl-7H-imidazo[4,5-f]quinoline-7-one (7OHIQ). Bacterial enzyme activity is affected by age, antibiotic use and diet. In particular, high beef, high fat and low fibre diets have been shown to increase caecal nitrate reductase and P-glucuronidase activities in rat caecal contents (Rumney et al., 1993a; Hambly et al., 1997). In humans, faecal P-glucuronidase, Pglucosidase and sulphatase activities decreased following conversion from an omnivorous diet to a lactovegetarian diet (Johansson et al., 1990). Apple pectin, wheat bran and oat bran have also been shown to suppress faecal enzyme activities (P-glucuronidase, P-glycosidase, nitroreductase and 7a-dehydoxylase) in humans (Mallett et al., 1988; Reddy 1990). Dietary sources of certain non-digestible oligosaccharides (galactooligosacharides and fructooligosaccharides), termed prebiotics, have been shown to suppress faecal P-glucuronidase and nitrate reductase activities in humans (Buddington et al., 1996) and animals (Rowland & Tanaka, 1993; Kikuchi et al., 1996; Rowland et al., 1998). Some of these studies reported a concomitant increase in probiotic bacterial numbers (Rowland & Tanaka, 1993; Kikuchi et al., 1996). Many probiotic strains have low activities of enzymes involved in the formation of genotoxic agents as compared to other major anaerobes in the gut (Saito et al., 1992) suggesting that prebiotics affect bacterial enzyme activities by influencing gut flora composition. $-glucuronidase and $-glycosidase Bacterial P-glucuronidase deconjugates glucuronide compounds. Substrates include xenobiotic compounds previously conjugated in the liver to reduce endogenous exposure to these toxic agents. Deconjugation in the colon thus releases the parent compound, or its hepatic

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metabolite for ultimate absorption back into the enterohepatic circulation. Escherichia coli and Clostridium species have the highest (3-glucuronidase activity (Rowland et al., 1985). Colon carcinogens such as 1,2-dimethylhydrazine (DMH), azoxymethane (AOM) and methylazoxymethanol (MAM) require deconjugation in order to induce carcinogenesis. Indeed, faecal [3-glucuronidase activity has been positively associated (significant) with AOM induced preneoplastic aberrant crypt foci formation (Arimouchi et al., 1999). In humans, faecal (3-glucuronidase activity was shown to be 1.7 times (12.1 times after sonication) higher in colorectal cancer patients as compared to healthy controls suggesting a role for this enzyme in carcinogenesis (Kim & Jin, 2001). Bacterial (3-glycosidases hydolyse the sugar moiety of plant glycosides to obtain energy. Dietary sources of plant glycosides include fruits, vegetables and beverages, such as tea and wine, derived from plants. Plant glycosides are relatively harmless; however, upon hydrolysis they release aglycones which are known to exert toxic, mutagenic and carcinogenic effects (Rowland et al., 1985; Goldin, 1986). In contrast, aglycones released during hydrolysis of flavonoids have anticarcinogenic and antimutagenic properties (as discussed below). Therefore hydrolysis of plant glycosides in the gut can lead, potentially, to both adverse and beneficial consequences for man. Enzymic hydrolysis of plant flavonoids Phytoestrogens are a group of diphenolic compounds found in plants. Two classes of phytoestrogens, lignans and isoflavones are structurally similar to mammalian oestradiol (Setchell & Aldercreutz, 1988). Both have been shown to have a range of health effects including anticancer properties such as modulation of steroid hormone metabolism and reduced proliferation of hormone dependent cancer cells (Bingham et al., 1998). Isoflavonoids and lignans undergo extensive metabolism in the human body, with the intestinal flora being the major site of biotransformation. The glycosides of isoflavonoids are rapidly hydrolysed by gut bacteria to release the aglycones genistein and daidzein which are further metabolised by colonic bacteria to equol, desmethylangolensin and /7-ethylphenol (Franke & Custer, 1996). Plant lignans are converted to enterolactone and enterodiol by hydrolysis, dehydroxylation, demethylation, and oxidation reactions catalysed by the facultative anaerobes of the intestinal tract (Setchell & Aldercreutz, 1988). Microbial involvement in phytoestrogen metabolism has been demonstrated in rats fed phytoestrogens. In this study, the isoflavone metabolites, equol and desmethylangolensin and the lignan metabolites, enterolactone and enterodiol, were detected in urine from human flora associated (HFA) rats and not from germ free rats (Rowland et al., 1999). Some authors suggest that individual variations in gut flora composition account for the considerable interindividual variation in plasma and urinary concentrations of isoflavonoids and lignans and their metabolites in humans (Xu et al., 1995, 1998; Lampe et al., 1998; King & Bursill, 1998; Rowland et al., 2000). In one study, the interindividual variation in isoflavone metabolites was highly significant and in the case of equol, related to diet. Those who excreted high levels of equol had a significantly lower low fat intake as compared to poor excretors (Rowland et al., 2000). The authors concluded that dietary effects on gut microflora may account for interindividual variations in phytoestrogen metabolism. This may have important implications for risk of certain cancers. Epidemiological studies provide good evidence for a protective role of soy phytoestrogens against hormonal cancers such as breast and prostate cancer (Aldercreutz, 1993). In terms of

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colon cancer the evidence stems mainly from case—control studies and is as yet unclear as approximately equal numbers of studies show protective, causative or no effect (Bingham et al., 1998). Other work has however shown that lignans present in flax/linseed or rye bran suppress colonic aberrant crypt foci incidence in carcinogen treated animals (Serraino & Thompson, 1992; Steele et al., 1995; Jenab & Thompson, 1996; Davies et al., 1999). A similar effect of the soy isoflavone genistein was shown in some (Steele et al., 1995) but not all of the above studies (Davies et al., 1999). In vitro work has shown that genistein suppressed proliferation of the colon cancer cells HT29 and CACO-2 in a dose dependent manner (Kuo, 1996). Although large scale epidemiological evidence is lacking, emerging in vitro and animal work suggests that bacterial metabolites of dietary phytoestrogens may exert effects at the cellular level which is relevant to carcinogenesis. Further work is needed in this area to determine the importance of these metabolites to the host. Nitroreductase and nitrate reductase Nitrocompounds and nitrates are reduced by nitro and nitrate reductases, respectively, in the human colon. Aromatic and heterocyclic nitro compounds are extensively used in industrial processes and found in diesel exhaust, cigarette smoke and airborne particulates. Both are reduced by bacterial nitroreductases to potentially toxic N-nitroso and N-hydroxy compounds before conversion to aromatic amines. Nitrate is a common contaminant of food (particularly vegetables) and drinking water. Nitrate is readily converted to nitrite in the human colon via bacterial nitrate reductase activity. This is the most important route of nitrate dissimilation (Allison & MacFarlane, 1988). Nitrate and nitrite reduction may contribute to endogenous N-nitrosation in the large intestine, as oxides of nitrogen can react with nitrogenous compounds such as amines, amides and methylureas to form NOC. This has potentially toxic consequences for the host as many NOC are metabolised to form potent DNA alkylating agents (Mirvish, 1995; Tricker, 1997). Bacterial strains belonging to Escherichia, Pseudomonas, Proteus, Klebsiella and Neissera have been shown to be N-nitrosate nitrogenous precursors in vitro and N-nitrosation activity is dependent upon the presence of nitrate and nitrite reductase genes (Calmels et al., 1985, 1988, 1996). Endogenous large intestinal NOC formation has been demonstrated in rats and shown to be dependent upon the presence of intestinal flora (Massey et al., 1988). More recently NOC have been detected in human faecal samples at levels ranging from 82 to 1010 J-ig/kg and excretion is positively related to dietary nitrate (Rowland et al., 1991) and red meat (Bingham et al., 1996; Silvester et al., 1997; Hughes et al., 2001). Each of these studies reported a high interindividual variation in faecal NOC excretion which may reflect individual variations in bacterial nitrate and nitrite reducing enzyme activities. Endogenous NOC formation may be one mechanism to explain the role of meat in colon cancer risk. However, NOC detected in these studies were determined using a group selective approach so the nature of the NOC measured is unknown. The risk, therefore, associated with increased faecal NOC excretion has yet to be determined and is an ongoing area of research. Metabolism of heterocyclic amines IQ is one of several heterocyclic aromatic amines (HAA) formed in small quantities when foods, particularly meat and fish, are cooked at high temperatures. HAAs readily undergo

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hepatic N-oxidation by P4501A2 enzymes and subsequent N-glucuronidation. The conjugated N-hydroxy metabolites are transported to the colon where they are deconjugated by bacterial p-glucuronidases and reabsorbed. The remaining N-hydroxy derivatives are substrates for 0-acetylation by colonic N-acetyltransferases (NATs) producing Nacetoxyarylamines which readily form DNA adducts (Turesky et al., 1991). Animal studies have shown increased colon cancer in animals fed well cooked meat containing high levels of HAA (Layton et al., 1995; Pence et al., 1998) and IQ is one carcinogen commonly used to induce cancer in animal models of colon cancer (Reddy & Rivenson, 1993). In humans, epidemiological studies have now suggested that the toxic effects of HAA depend upon the individual's ability to metabolise them. Individuals who were phenotyped or genotyped as fast acetylators (i.e. possess high NAT activity) were shown to have a higher risk of colon cancer with increased meat intake (Roberts-Thomson et al., 1996; Welfare et al., 1997). Animal work has suggested that the intestinal microflora plays an equal or possibly a more important role in conversion of HAAs to genotoxic metabolites as compared with mammalian enzymes. In one study, DNA damage was shown to be over 65% greater in colonocytes and hepatocytes from IQ treated HFA rats as compared to germ free rats (Kassie et al., 1999). The carcinogenic effect of IQ was initially attributed to the generation of the toxic metabolite 7-OHIQ during bacterial metabolism. 7-OHIQ has been found in faeces of individuals consuming a fried meat rich diet, indicating that the formation of 7-OHIQ can occur in vivo in man (Carman et al., 1988). This metabolite was shown to be a direct-acting and potent mutagen in Salmonella typhimurium and an inducer of DNA damage in colon cells in vitro (Ohgaki et al., 1991; Rumney et al., 1993b). Subsequent work however has failed to confirm 7-OHIQ toxic effects in mammalian cells and animals. However bacterial metabolism of HAA is still thought to be an important source of genotoxic agents. In addition, probiotics may prevent HAA carcinogenicity. 100% and 80% suppression of IQ-induced colon and liver tumours, respectively, were noted in rats fed supplements of Bifidobacterium longum (Reddy & Rivenson, 1993). Suggested protective effects include direct binding to the carcinogen or changes in bacterial enzyme activities such as P-glucuronidase. The impact of intestinal flora on carcinogenic effects of HAAs has been reviewed elsewhere (Knasmiiller et al., 2001). Metabolism of sulphur-containing compounds Sources of sulphur in the large intestine include sulphate and sulphites from processed foods and beverages, sulphur-containing amino acids from protein-rich foods and endogenous sources including sulphated polysaccharides such as mucin and chondroitin sulphate. Sulphates, sulphites and sulphur-containing amino acids are metabolised by sulphatereducing bacteria and amino acid fermenting bacteria, respectively, producing hydrogen sulphide (H2S) (MacFarlane et al., 1992a). H2S has been implicated in the aetiology of ulcerative colitis as numerous toxic effects have been shown in experimental studies. Human studies have shown that faecal concentrations of H2S are higher in patients with untreated ulcerative colitis as compared to healthy controls (Pitcher et al., 2000). In the same study patients with active ulcerative colitis had significantly higher faecal sulphate-reducing bacteria counts as compared to those in remission. The toxic consequences of H2S production

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may be mitigated by the presence of certain intestinal enzymes. Rhodanese is located in the submucosa and crypts of the colon and has recently been shown to be the principal enzyme involved in H2S detoxification (Picton et al., 2002). In vitro and animal studies have shown that sulphide induces apoptosis, increases goblet cell depletion and increases proliferation (Aslam et al., 1992; Christl et al., 1994). In addition sulphide inhibits butyrate metabolism in vitro (Moore et al., 1997). Butyrate is an important energy source for colonocytes. Doses of sulphide used in these studies ranged from 0.2 to 2 mmol/L corresponding to levels detected in human faeces (0.2—3.4 mmol/day) (Magee et al., 2000). In healthy human subjects faecal sulphide concentrations increased in response to increased doses of red meat, a rich source of sulphur amino acids (Magee et al., 2000). The effect of other dietary sources of sulphur on faecal sulphide levels are as yet unknown; however, evidence suggests that any dietary effects occur via changes in bacterial metabolism. Secondary bile acids The bile acids cholic and chenodeoxycholic acids are synthesised from cholesterol in the liver, secreted in the bile and metabolised by colonic bacteria. Bacterial cholylglycine hydrolase (CGH) deconjugates the conjugated acids. The unconjugated form is then dehydroxylated by 7a-dehydroxylase (70C-DH) to form the secondary bile acids - deoxycholic acid (DCA) and lithocholic acid (LCA). Regional differences in bile acid metabolism occur as shown by differences in caecal and faecal CGH and 70C-DH levels (Thomas et al., 2001). Most metabolism occurs in proximal regions. Secondary bile acids, which comprise over 80% of faecal bile acids, are postulated to play an important role in the aetiology of colon cancer by acting as promoters of the tumorigenic process. In experimental animal models they have been shown to increase colonic aberant crypt foci formation (Sutherland & Bird, 1994) and disrupt colonic mucosal cell membrane integrity leading to a compensatory increase in mucosal proliferation (Nagengast et al., 1995). Toxic effects in vitro include DNA damage (Pool-Zobel & Leucht, 1997), proliferation and polyamine production (Milovic et al., 2000), induction of the activator protein-1 (AP-1) transcription factor (Matheson et al., 1996). Changes in AP-1 activation will affect gene expression and has been associated with neoplastic transformation (Angel & Karin, 1991). AP-1 dependent gene transcription by DCA was later shown to be correlated with induction of cell proliferation (Glinghammer et al., 1999). Interestingly, this paper also showed that lipid components of human faecal water which is in direct contact with the colon epithelium, also activate AP-1. Secondary bile acids are now thought to influence cell growth by interacting with two important cell signalling systems, i.e. prostaglandin E2 and protein kinase C. Both systems are known to be regulators of cell growth, differentiation and apoptosis (Pongracz et al., 1995; Radley et al., 1996). Most studies focus on a promotional role for bile acids in carcinogenesis. Recently, however a role in later stages of carcinogenesis was suggested. LCA in particular was shown to increase invasion of colon cancer cells (CACO2) in vitro and this was associated with increased matrix metalloproteinase 2 (MMP2) secretion (Halvorsen et al., 2000). Enhanced MMP activity has been implicated in metastasis (Chambres & Matrisian, 1997). In human studies, the role of bile acids as colon carcinogens is less clear as several casecontrol studies have reported no difference in faecal bile acid excretion between colorectal adenoma or cancer patients as compared to controls (Nagengest, 1988). However, other

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studies reported elevated faecal secondary bile acid levels as compared to healthy controls (Kishidaet al., 1997; Owen, 1997). Comparison of such studies is difficult as some measured bile acids in faecal samples while others measured bile acids in faecal water samples. It is now accepted that the aqueous phase of faecal samples is more indicative of material in contact with the colonic mucosa. Concentrations of the secondary bile acids, DCA and LCA, have been shown to be significantly higher in faecal water from patients with colonic polyps or cancer as compared to controls with normal colons (Stadler et al., 1988). Other studies have shown no differences in faecal water bile acid concentrations (de Kok et al., 1999). Bile acid concentration in faecal water is influenced by diet. Bile acid concentrations, particularly DCA, were found to be significantly lower in vegetarian subjects as compared to omnivores in a human trial (van Faassen et al., 1993). In another study, a high fat, low calcium and low fibre diet significantly increased faecal water bile acid concentration and cytotoxicity as compared to a low fat, high fibre and high calcium diet (Rafter et al., 1987). Calcium and fibre have been shown to reduce bile acid exposure by binding the acids and reducing transit time, respectively (Marteau et al., 1994; Lupton et al., 1996). Other factors affecting faecal bile acid profiles include transit time, age, hepatic function and factors affecting colonic flora metabolism.

Fecapentaenes Fecapentaenes 12 and 14 (FP12 and FP14) have been identified and consist of a glyceryl ether compound containing a pentaene moiety with a chain length of 12 or 14. The gut microflora, particularly species of Bacteroides, have been implicated in FP formation as FP were produced in vitro by faecal suspensions under anaerobic conditions. Synthesis was inhibited by antibiotics and heat sterilization (Hirai et al., 1982). FP have been detected in faecal samples from humans consuming Western meat-containing diets and are thought to contribute to the mutagenicity of these samples. Average FP concentration in human faecal samples is approximately 500ng/g dry weight although concentrations up to 10000ng/g have been detected. In vitro work has shown that FPs are potent direct-acting mutagens. At low concentrations (0.6-10 U,g/mL), FP12 induces single strand DNA breaks, gene mutations, chromosome aberrations, sister chromatid exchanges and unscheduled DNA synthesis in human fibroblasts in vitro (Plummer et al., 1986). Mechanisms by which FPs induce DNA damage and carcinogenesis have been reviewed previously (Povey et al., 1991). Animal studies have provided conflicting results for FP induced carcinogenicity. Rodent bioassays have indicated that FP12 does not have carcinogenic or tumour-initiating activity (Ward et al., 1988; Weisburger et al., 1990; Shamsuddin et al., 1991). However, Zarkovic et al. (1993) showed that FPs may possess tumour-promoting activity in a rat colon carcinogenesis model using N-methyl-N-nitrosurea (MNU) as an initiating agent. In another study, proliferative effects of FP12 were demonstrated in rat colon epithelia (Hinzman et al., 1987). Human studies have shown that FP excretion is influenced by the contents of the bowel including the presence of FP binding and solubilising factors. Dietary fibre adsorbs FPs increasing their excretion. Certain bile acids (cholic and deoxylcholic acids) increase FP solubility thus reducing their excretion, this effect is reversed by calcium (De Kok et al., 1993). This dietary effect may explain results from a human study which

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showed higher faecal FP excretion in vegetarians (a low colon cancer risk group) as compared to omnivores (De Kok et a/., 1992). Indeed, lower faecal FP levels have been detected in colorectal cancer patients than in controls (Schiffman et al., 1989)- Although FP are known faecal mutagens their role as colon carcinogens is as yet unclear and may be dependent upon the endogenous environment.

Reduced exposure to toxic compounds Certain metabolic activities of the gut microflora may reduce human exposure to toxic compounds and certain dietary carcinogens. Microbial involvement in phytoestrogen metabolism has already been mentioned. Studies have reported the ability of freeze dried preparations of intestinal bacteria, especially lactic acid bacteria, to bind dietary carcinogens including heterocyclic amines, the fungal toxin aflatoxin Bl (AFB1) and the food contaminant AF2 (Morotomi & Mutai, 1986). In some studies, binding was associated with a concomitant decrease in mutagenic activity in vitro (Morotomi & Mutai, 1986; Orrhage et al., 1994). In theory, carcinogen binding should decrease the bioavailability of ingested carcinogens in the gut, reducing their capacity to damage the intestinal mucosa. However a recent study showed that lactic acid bacteria administered to rats, had no effect on absorption or genotoxic activity in vivo of carcinogens (Bolognani et al., 1997). Conditions required for carcinogen binding in vivo may differ from those showing successful binding in vitro. In addition, the toxicological significance of carcinogen binding remains to be established. An additional beneficial effect of the colonic microflora is the breakdown of glucosinolates. Glucosinolates are found in brassica vegetables and their hydrolysis products in particular isothiocyanates are known to influence enzymes involved in carcinogen detoxification (phase II enzyme metabolism) (Zhang et al., 1992). Microbial involvement in glucosinolate breakdown was recently shown following incubation of watercress with a human faecal suspension. Eighteen per cent of the glucosinolates present were hydrolysed to isothiocyanates within a twohour period (Getahun & Chung, 1999).

Formation of toxic and protective agents during fermentation The main function of the colonic flora is to salvage energy from undigested material for growth and hence increase biomass. Figure 10.1 summarises the fermentative activities of the colonic flora. Carbohydrate provides the main fermentable substrates in the human large intestine. These substrates include non-absorbable sugars such as dietary fibre/NSP, resistant starch, non-digestible oligosaccharides (NDO) and certain bulk sweeteners. Other fermentable substrates include nitrogenous material such as proteins and amino acids, which are typically fermented upon exhaustion of carbohydrate sources. Indeed, levels of protein fermentation products increase progressively from the left to right colon (MacFarlane et al., 1992b). The nature and extent of fermentation depends upon the characteristics of the bacterial flora, colonic transit time and the availability of nutrients.

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Fig. 10.1

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Overview of fermentation in the colon.

Carbohydrate fermentation Carbohydrate fermentation results in production of short-chain fatty acids (SCFA), hydrogen, carbon dioxide and stimulation of bacterial growth. Bacterial biomass is the major constituent of stools from humans eating a Western diet (Stephen & Cummings, 1980). Increasing dietary non-digestible carbohydrate increases bacterial biomass and thus stool weight and reduces transit time (Bingham, 1990). This is often considered to be a beneficial effect as increased stool bulk and decreased transit time should reduce exposure of the colonic mucosa to potentially toxic agents in the faecal stream. On a population basis, increased stool weight has been associated with reduced colorectal cancer risk (Cummings et a/., 1992). Non-digestible carbohydrate supplements have also been shown to reduce faecal excretion of potentially toxic products of protein fermentation in healthy humans (Kelsay et al., 1978), probably reflecting reduced need for protein as an energy source in the large intestine. Short chain fatty acids Acetate, proprionate and butyrate are the principal SCFAs produced during fermentation. SCFA concentrations vary regionally in the large intestine with concentrations being highest in the proximal colon (MacFarlane & MacFarlane, 1997). SCFAs are important energy sources for cells lining the colon thus influencing colonocyte growth, differentiation and cell transport. They are rapidly absorbed from the large intestine and metabo-

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lised at various sites providing energy to muscle, kidney, heart and brain. Metabolic effects of SCFAs have been reviewed elsewhere (Cummings & MacFarlane, 1997; D'Argenio & Mazzacca, 1999). Of particular interest in the cancer field is the cellular effects of butyrate. In vitro studies have shown that butyrate inhibits hydrogen peroxide induced DNA damage (Rosignoli et al., 2001), improves epithelial barrier function (Mariadason et al., 1997) and reduces cell invasion (Emenaker & Basson, 1998) in colon cancer cells. Physiological doses of butyrate (2—4 mM) have been shown to induce differentiation and apoptosis in colon adenoma and cancer cells (Hague & Paraskeva, 1995). In the colonic crypt, apoptosis maintains the balance in cell number between newly generated and surviving cells and at the luminal surface where differentiated epithelial cells are exfoliated (Potten, 1992). The apoptotic inducing effect of butyrate has been related to its ability to inhibit the enzyme histone deacetylase. Inhibition of histone deacetylase activity increases the accessibility of DNA to a variety of transcription factors (Dangond & Gullans, 1998). A lack of butyrate has been linked to mucosal atrophy, inflammation and gastrointestinal disorders such as ulcerative colitis (Harig et al., 1989). Increased colonic butyrate formation has often been proposed as one of the protective mechanisms of high fibre diets. The amount and type of SCFA produced varies depending on the fermentation substrate. Cummings and MacFarlane (1997) summarised SCFA yields from a number of in vitro fermentation studies. Starch provided the best source of butyrate followed by oat bran and wheat bran. SCFAs also reduce the pH of the colonic lumen thereby preventing growth of pathogenic bacteria. A low pH has also been associated with decreased colon cancer risk in humans (Segal et al., 1995).

Products of colonic protein fermentation On average, 12 g of proteinaceous material or 0.5—4 g total nitrogen enters the large intestine each day in the form of protein (48—51%) and peptides (20-30%) (Cummings & MacFarlane, 1991). Sources of protein in the colon include dietary residues, pancreatic enzymes, mucus and exfoliated epithelial cells. Gut flora are responsible for most of the proteolytic activity in human faecal samples, with Bacteroides spp. being the major contributors (MacFarlane et al., 1986, 1988). Analysis of gut contents from sudden death victims showed that proteolysis and amino acid fermentation are restricted to caecal contents (MacFarlane et al., 1986). Products of protein fermentation include SCFAs, hydrogen, carbon dioxide, branched-chain fatty acids (BCFA) such as isobutyrate, isovalerate and 2methylbutyrate and ammonia, amines, phenols and indoles. The initial step in bacterial proteolysis involves hydrolysis of the polypeptide chains producing peptides and amino acids, which can be utilised for assimilation. Potentially toxic products of protein metabolism include ammonia, phenols, sulphides and certain amines. In humans, the amount of protein entering the colon is largely determined by dietary protein consumption (Silvester & Cummings, 1995). Increased protein intake has also been associated with increased faecal excretion of protein metabolites such as ammonia (Cummings et al., 1979; Geypens et al., 1997; Silvester et al., 1997); NOCs (Bingham et al., 1996; Silvester et al., 1997; Hughes et al., 2001); sulphides (Magee et al., 2000) and urinary phenol excretion (Cummings et al., 1979; Geypens et al., 1997). Dietary protein intake is therefore a major influence on bacterial proteolysis.

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Amines formed in the large intestine include methylamine, pyrrolidine, butylamine, putrescine, histidine and taurine (Drasar & Hill, 1974). Species belonging to the genera Clostridium, Bifidobacterium and Bacteroides have been shown to form substantial quantities of amines (Allison & MacFarlane, 1989). The physiological significance of amine formation to the host is largely unknown. However cellular effects exist as putrescine is known to regulate cell growth and differentiation in the gastrointestinal epithelium (Seidel et al., 1984). A possible toxic effect may also be evident as most amines undergo N-nitrosation reactions in the presence of potent nitrosating agents forming potentially toxic NOC (Mirvish, 1995). The concentration of ammonia in human faecal samples ranges from 12 to 30 mM and is influenced by diet. While protein increases faecal ammonia excretion, fermentable carbohydrates reduce faecal ammonia excretion during periods of low protein intake (Cummings et al., 1979). Bacteria assimilate ammonia to form bacterial protein during carbohydrate fermentation. Ammonia concentrations as low as 5—lOmM have been shown to alter the morphology and intermediary metabolism of intestinal cells, affect DNA synthesis and reduce the lifespan of cells (Visek, 1978). Such effects suggest a role in tumour promotion via increased mucosal cell turnover and hence the likelihood of multiplication of damaged cells. Indeed, uterosigmoidoscopy patients have luminal ammonia concentrations as high as lOOmM and are at an increased risk of developing tumours distal to the site of the ureteric implantation (McConnel et al., 1979). Phenols and indoles are formed following bacterial degradation of aromatic amino acids. Intestinal bacteria involved in these processes include Clostridia (Elsden et al., 1976), Bacteroides (Chung et al., 1975), Enterobacteria (Botsford & Desmoss, 1972), Biftdobacteria (Aragozzini et al., 1979) and Lactobacillus (Yokoyama & Carlson, 1974). Phenolic compounds are absorbed in the colon, detoxified by the liver and excreted in urine (phenol, ^-cresol and 4-ethylphenol) (Tamm & Villako, 1971). Physiological levels of phenolic compounds in colonic contents are normally low (Bassert et al., 1986) making the relation of these compounds to colorectal mucosal damage unclear. In vitro work, however has shown that phenol may enhance N-nitrosation of dimethylamine by nitrite and the reaction between phenol and nitrite produces the mutagen diazoquinone (Kikugawa & Kato, 1986).

Probiotics, prebiotics and cancer Experimental work has shown that pro- and prebiotics have favourable effects on various markers of colon cancer risk. Anticancer properties have been mentioned throughout this chapter and are summarised in Table 10.2. To date, evidence for cancer protective effects is derived from in vitro studies and from animal models of colon cancer. Epidemiological evidence is lacking while evidence from human intervention trials is limited; however, results are beginning to emerge in the literature. Despite this, there is epidemiological work showing that dietary sources of pro- and prebiotics reduce colon cancer risk. For example, soya and garlic are good sources of the prebiotic inulin and anticancer effects of soya and garlic are well established in the literature (Bingham et al., 1998; Fournier et al., 1998; Bianchini & Vainio, 2001; Fleischauer & Arab, 2001). Prebiotic non-digestible carbohydrates are also found in chicory, asparagus, artichoke, onions, and leeks. Inulin is a fructan consisting mainly as (3-(2-l) fructosyl-fructose

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Table 10.2

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Effects of probiotics in animal models of colon cancer

Effect

Probiotic

References

Reduced colon tumour incidence

L. acidophilus B. longum Lactobacillus GG B. longum L. acidophilus Lactobacillus supplement produced from L. case/ L. acidophilus L. fermentum L. rhamnosus

Goldin & Gorbach (1980) Reddy et al. (1993) Goldin et al. (1996) Singh et al. (1997) Mclntosh et al. (1999) Fukui et al. (2001)

Reduced ACF formation

B. longum B. longum Bifidobactehum spp. B. longum

Kulkarni et al. (1994) Challa et al. (1997) Abdelali et al. (1995) Rowland et al. (1998)

Suppression of bacterial enzyme activities associated with carcinogen activation

L. acodphilus L. acidophilus B. longum

Goldin & Gorbach (1976) Cole et al. (1989) Rowland et al. (1998)

Reduced DNA damage

L. case/ L. acidophilus, L. gasseri, L. confusus, B. longum, B. breve and L. acidophilus L. bulgaricus 191R St. thermophilus

Pool-Zobel et al. (1993)

Pool-Zobel et al. (1996) Wollowski et al. (1999)

ACF, aberrant crypt foci.

links (Roberfroid & Delzenne, 1998). Inulin-type fructans including natural inulin, enyzmatically-hydrolysed inulin (i.e. oligofructose) and synthetic fructooligosacharrides are the most widely studied prebiotic sources. Commercial sources of probiotics include capsules, powders, enriched yoghurts and fermented milks. Epidemiological evidence for anticancer effects of fermented milk products and yoghurt also exist but the studies are less conclusive with some showing protective and others no effects (Malhotra, 1977; Young & Wolf, 1988; Peters et al., 1992; Boutron et al., 1996). In France consumption of yoghurt was associated with reduced risk of large adenomas in a human case-control study (Boutron et al., 1996). The authors also showed a lack of effect of calcium on colorectal cancer risk and suggested that lactic acid bacteria are the most likely active components in yoghurt. Experimental work in humans has shown that milk fermented with probiotics suppresses urinary mutagen excretion (Lidbeck et al., 1992; Hayatsu et al., 1993) and faecal (3-glucuronidase activity (Bouhnik et al., 1996). Several reviews have discussed the anticancer effects of probiotics and their

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dietary precursors (Brady et al., 2000; Hirayama & Rafter, 2000; Wollowski et al., 2001). Not all studies reported protective effects of lactic acid bacteria. Probiotic anticancer effects tend to be strain specific. Administration of several probiotic strains together however may be more successful. In mice, administration of a mixed lactic acid bacteria culture as a dietary supplement inhibited early adenoma development and ultimate tumour yield (Fukui et al., 2001). Several anticancer effects of probiotics have been demonstrated in humans as summarised in Table 10.3. Organisms must exhibit several characteristics for successful use as probiotics. The organism should maintain viability and activity in the carrier food before consumption, survive transit through the upper gastrointestinal tract, survive and grow in the intestine producing beneficial effects with no toxic consequence to the host (Kim, 1988). In order to study cancer protective effects it is important that human trials also demonstrate successful transit and colonisation of the probiotic strain. Overall, experimental and animal work show promising anticancer effects of several probiotic strains which deserve confirmation in well designed human trials. As for probiotics the possible anticancer effects of prebiotics originate from experimental studies. Studies have shown that prebiotics affect tissue and physiological markers of colon carcinogenesis. Antigenotoxic effects have been reported in the colonic mucosa of carcinogen induced animals. For example, lactulose reduced DNA damage (Rowland et al., 1996), increased colonic glutathione-S-transferase activity and suppressed aberrant crypt foci formation (Challa et al., 1997). Inulin suppressed preneoplastic aberrant crypt foci formation (Rao et al., 1998; Reddy, 1998; Rowland et al., 1998) and induced apoptosis (Hughes & Rowland, 2001) at dietary concentrations of 5—10%. Interestingly, two of these studies showed a concomitant suppression of caecal (3-glucuronidase activity formation (Reddy, 1998; Rowland et al., 1998). Modulatory effects of prebiotics on caecal and faecal bacterial enzyme activities have also been demonstrated in humans and associated with increased faecal count of bifidobacteria and decreases in clostridia and enterobacteria (Terada et al., 1992; Buddington et al., 1996). Stimulation of probiotic numbers in the colonic lumen is one mechanism whereby prebiotics exert anticancer effects. However, as well as modulating gut flora composition, prebiotics may exert cancer protective effects via their fermentation products. In humans, inulin and oligofructose intake increased stool output (Gibson et al., 1995; Den Hond et al., 2000) reflecting increased bacterial biomass. Fermentation studies have shown that butyrate is produced during bacterial fermentation of inulin and oligofructose in vitro (Wang & Gibson, 1993; Gibson & Roberfroid, 1995) and in animals (Campbell et al., 1997; Djouzi et al., 1997) at levels comparable to that produced from many NSP (Cummings & MacFarlane, 1997). The cellular effects of butyrate have already been discussed. Recent studies have reported more potent anticancer effects of pro- and prebiotics when administered together. Combinations of pro- and prebiotics are termed synbiotics. Administration of bifidobacteria with oligofructose, inulin or lactulose significantly suppressed aberrant crypt foci formation in rat colon and for each study the effect was greater as compared to single doses of the pro- and prebiotic (Challa et al., 1997; Rowland et al., 1998; Gallaher & Khil, 1999). No consistent effects were evident when the bifidobacteria were administered with oligosaccharides other than the fructan oligo-

Table 10.3

Anticancer effects of probiotics in humans

Effect

Study group

Organism

References

Reduced tumour recurrence

Bladder cancer patients

L case/

Aso et al. (1992, 1995)

Reduced mucosal cell proliferation and decreased faecal pH

Colon adenoma patients

L. acidophilus and B. bifidum

Biasco et al. (1991)

Reduced urinary mutagenicity

Healthy Healthy

Milk fermented with L. acidophilus or L. case/

Lidbeck et al. (1992) Hayatsu et al. (1993)

z r+ rt-'

o'

Reduced faecal enzyme activity (3-glucuronidase 1 Nitroreductase > Choloylglycine hydrolase J (3-glucuronidase (3-glucuronidase 1 (3-glucosidase J

3

Q)_

CD 3 Q. S

o' O CT

91

Healthy

L. rhamnosus GG

Bouhnik et al. (1996)

s o

Healthy

Bifidobacter fermented milk

Bouhnik et al. (1996)

» o

Healthy

L. case/ fermented milk

Spanhaak et al. (1998)

ao

Q.

c_ 3

CD O

o ou CD

O C/5 <S>

ro 10 (O

224

Gut Flora, Nutrition, Immunity and Health

saccharides (Gallaher & Khil, 1999). Administration of the probiotic together with its nutritional precursor may aid survival of the bacteria during intestinal transit as its specific substrate is readily available for fermentation. These anticancer effects should now be confirmed in human studies.

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The Role of Nutrition in Immunity of the Aged

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S. Walrand,1 M.-P. Vasson1 & B. Lesourd2 1

Laboratoire de Biochimie, Biologie Moleculaire et Nutrition, EA 2416, Centre de Recherche en Nutrition Humaine, Faculte de Pharmacie, 28 place Henri-Dunant, BP 38, 63001, France, and 2 Nutrition Geriatrique, Departement de Gerontologie Clinique, Hopital Nord, CHU de Clermont-Ferrand, route de Chateaugay, BP 56, 63118 Cebazat, France

Introduction Improvements in health care and nutrition in the last century have increased most people's life expectancy. One result is that the elderly are now the fastest-growing segment of the population in developed countries; every day more than 5500 American citizens turn 65. For many of these people, the years ahead will bring significant changes: changes in their social roles, in their family life, but also in their health concerns and in their nutritional needs and priorities. As the human body enters its senior years, its ability to fight off infection and other health problems, such as neoplastic diseases, diminishes significantly. Many factors can contribute to this decline, such as the presence of comorbid conditions, environmental factors, and the senescence of normal defence mechanisms in elderly subjects, termed 'immuno-senescence'. The immune system, which is responsible for fighting most diseases, simply does not function as efficiently in older adults as in younger people. Two complementary systems of immunity rid humans of pathogens and cancer cells: innate immunity and acquired immunity. Innate immunity provides a rapid but incomplete defence against threatening agents until the slower, more definitive adaptative immune response develops. The age-related changes in the acquired immune system, supported by T and B lymphocytes, are well documented, whereas innate immunity has not yet received due attention in the elderly. Among all the factors affecting the defence mechanisms, it has been proved that nutritional status plays a major role in a healthy immune system. In both healthy and nutritionally deficient elderly persons, dietary supplements, such as vitamins and minerals, have been found to enhance the response of the immune system, resulting in some cases in fewer days of infectious illnesses.

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This chapter highlights the changes in immune status and responses that develop with aging and examines the role of nutritional factors in maintaining or improving immune competence in older adults.

Aging and immune function Innate immune system The immune system is the most diverse, ubiquitous, and important system in the body, acting as it does to protect us from a hostile environment. Innate immunity involves all the areas that come into contact with the outside (skin, mucous membranes, lung lining and the whole intestinal system), plus the cells that non-specifically recognize and kill foreign agents (neutrophils, macrophages and natural killer cells) (Ginaldi et al., 1999c). Age-related changes in skin and mucous barriers Aging is associated with thinning and drying of the skin and a decline in its blood flow. Similarly, the mucous membranes become drier and more susceptible to injury, and the synthesis of proteins associated with keratinization may be slowed in elderly individuals (Lavker et al., 1987). These changes in the integrity of membranes can enable easier attachment and allow invasion of bacteria. In the gastrointestinal mucosa, for example, a thinning of the cells that line the gut can result in leakage of toxins into the bloodstream (Arranz & Ferguson, 1992; Schmucker et al., 2001). Any crossing of the natural barrier of the body by what is considered foreign by the immune system will result in activation of the system and heightened risk of infectious disease. Age-related changes in phagocytic cells After an organism has gained entry, the first line of defence is the body's non-specific effector mechanisms, in particular the phagocytic cells such as the polymorphonuclear neutrophils (PMNs) and macrophages. Phagocytic defensive functions consist of a sequence of events including migration, adhesion, phagocytosis, and the release of compounds able to destroy microbial agents, such as reactive oxygen species (ROS). Curiously, investigators of the aging immune system have mainly concentrated on lymphocytes; less attention has been paid to the function of the innate system in the elderly, even though phagocytes are known to play a major role in primary host defence against infections (Hellewell & Williams, 1994). Some phagocytic activities have been shown to be impaired in aged people, thus aggravating susceptibility to infectious agents and contributing to infection-induced morbidity and mortality (Makinodan et al., 1984). PM.N function

Studies on PMN function in older persons have yielded conflicting results, but it is generally agreed that just as in other immune system components, there is a general decrease in functional activities (Ginaldi et al., 1999c; Lord et al., 2001). Neutrophils from aged subjects often exhibit a diminished chemotactic activity against complement-derived chemotactic factors (Polignano et al., 1994; Wikby et al., 1994) or the peptide fMET-LEU-PHE (fMLP;

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Corberand et al., 1981; Antonaci et al., 1984; Niwa et al., 1989; Ortega et al., 1993) even when the population meets the health admission criteria of the SENIEUR protocol (Fig. 11.1; Ligthart et al., 1984). The chemoattractant fMLP binds to a specific cell surface receptor (Williams et al., 1977) and activates PMN migration by a mechanism triggered by intracellular calcium (Naccache et al., 1977) and inducing cell cytoskeleton modification (Rao & Varani, 1982; Rao & Cohen, 1990). Rao et al. (1992) showed that the expression of the chemotactic peptide receptor is lower in elderly subjects than in young adults. FMLP-induced actin polymerization is also decreased in PMNs from elderly people (Rao et al., 1992). The defect in actin polymerization may reflect alteration in PMN cytoskeleton response to stimuli and explain the defective chemotaxis noted in elderly individuals. In addition, exposure of the PMN to fMLP is also associated with an instantaneous elevation of the intracellular cytosolic calcium concentration that clearly precedes the start of cell migration (Korchak et al., 1984a,b; Khalfi et al., 1996). Lipschitz et al. (1988) demonstrated that aging results in alterations in neutrophil calcium homeostasis that may play a role in the age-related decline in neutrophil chemotaxis. In this study, cytosolic calcium remained significantly lower in fMLP-stimulated PMNs from old persons compared with young, whereas permeability to extracellular calcium and efflux of calcium from the cell were also significantly diminished (Lipschitz et al., 1988). These results are strong evidence for a causal connection between altered calcium homeostasis and diminution in response to fMLP activation in the aged. Nevertheless, in vivo evaluation of PMN migration using the skin window technique was quantitatively in the normal range in elderly subjects, indicating that neither endogenous generation of chemotactic compounds,

Image Not Available

Fig. 11.1 Chemotactic activity of PMN against casein and fMLP during aging (Corberand et al., 1981; Niwa et al., 1989). ND: not determined, a, b: P= 0.05.

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nor PMN movement machineries are affected by the aging process (Biasi et al., 1996). These latter results are at variance with other literature data (Corberand et al., 1981; Antonaci et al., 1984;Niwatf */., 1989; Ortega et al., 1993; Polignanotf */., 1994; Wikby et al., 1994). However, PMN chemotaxis is a very complex phenomenon, based on the integrity of cell membranes and receptors, cytoskeleton dynamics and the action of chemical substances. Unlike in vitro techniques, the skin windows method used by Biasi makes it possible to evaluate the PMN migration in a global way. It is conceivable that in vitro evaluation of chemotaxis, carried out under specific conditions, might unmask specific defects that are not relevant for in vivo migration. Most studies (Antonaci etal., 1984; Damtew #
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PMNs and other immune cells. In resting cells, NADPH oxidase is dissociated and thus dormant, and consists of one membrane-bound component and four components stored in cytosolic granules (Babior, 1984; Dahlgren & Karlsson, 1999). Upon PMN activation, the cytosolic proteins translocate to the membrane component and a functional electron-transfer system is formed leading to O2' generation. O2'~ is then rapidly converted, through enzyme activities (superoxide dismutase, catalase, myeloperoxidase) into other ROS that include hydrogen peroxide (H2O2), hydroxyl radical ('OH) and hypochlorous acid (HOCl) (Casimir & Teahan, 1994). The importance of O2' and H2O2 production for PMN bactericidal activity is well known, a lack of production of these species in patients with chronic granulomatous disease resulting in an increased susceptibility to bacterial infection (Smith & Curnutte, 1991). A significant reduction of ROS production by stimulated PMNs has been found in healthy old donors by different groups using different technique and types of stimulating agents (Table 11.1). A decrease in O?' generation by PMNs after phorbol-12-acetatel3-myristate (PMA) activation was observed in elderly subjects (Braga et al., 1998a, b). Other results (Tortorella et al., 1993; Biasi et al., 1996) showed that PMNs in suspension from aged persons displayed a PMA-triggered O2' responsiveness overlapping that seen in younger counterparts, while a

Table 11.1 ROS production by stimulated PMNs in healthy elderly and young adults according to the stimulant used Unit

Stimulants

Adult

Elderly

10 ng/ml 10 ng/ml 2.5.10'6 M 10~6M

39.7 ± 7.3 21.5 ±6.2 202.7 + 31.1 207.2 ± 26.6

40.1 ± 6.8 21.2 ±6. 7 116.5 + 23.2* 180.3 + 17.6

nM/106 cells nM/106 cells mV mV

17.3 ±4.2 9.3 + 3.3 76.8 ± 32.3 2.0 ±0.2

9.1 ±3.2* 3.6 ±2. 7* 24.6 ± 14.8* 1.7 ±0.1

nM/106 cells nM/106 cells mV arbitrary unit

8.2 ± 3.2 72.8 ± 33.7 1.54 ± 0.41 1.10 ±0.12

8.4 ±6.1 58.6 ± 14.8 1.51 ± 0.40 0.90 ± 0.18

5.4 ± 1.0

3. 2 ±0.3*

nM/106 cells mV nM/minlO6 cells activated/ non-activated arbitrary unit

PMA1

Tortorella eta/. (1993) Biasi et al. (1996) Braga et al. (1998a) Walrand et al. (2001b) FMLP2

Tortorella et al. (1993) Biasi et al. (1996) Braga et al. (1998a, b) Walrand (unpublished)

10-7M 10-7M 5.10'7 M 10"5M

Zymosan 3 Biasi et al. (1996) Braga et al. (1998) Niwa et al. (1989) Kuriowa et al. (1989)

0.1 mg/ml 1 mg/ml 1 mg/ml 2 mg/ml

Walrand (unpublished)

1 mg/ml

1

Phorbol 12-myristate 13-acetate. Formyl-methionyl-leucyl-phenylalanine. 3 0psonized Zymosan. * P < 0.05 compared with adult subjects. 2

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significant decrease in respiratory burst was observed in the presence of fMLP in the same populations. The discrepancies observed between PMN burst oxidative response in elderly persons according to the stimulant used may be related to the different biochemical pathways activated by these two commonly used soluble stimulating agents. PMA is a lipophilic chemical that lacks specific cell surface receptors on PMNs. This compound directly activates protein kinase C (Casimir & Teahan, 1994), which is involved in the assembly and activation of NADH,H oxidase, without the complication of receptor regulation and independently of the calcium system (Casimir & Teahan, 1994; Robinson & Badwey, 1995). FMLP, a formylated oligopeptide structurally similar to bacteria-derived peptides, is a ligand that binds to a specific fMLP receptor on the cell surface and acts by means of a mechanism that is dependent on both intra- and extracellular calcium and on a process that includes phospholipase activation (Casimir & Teahan, 1994; Braga et al., 1998a). Other studies (Kuroiwa et al., 1989; Niwa et al., 1989; Fietta et al., 1994; Krause et al., 1999) report that ROS generation induced by particulate opsonized agents, such as Candida albicans and zymosan, was not different in young and old individuals. Therefore, the final release of ROS seems to be dependent on the type of stimulant used and the type of activated receptor in elderly subjects. Very interesting work carried out by Braga et al. (1998b) used luminol-amplified chemiluminescence, a technique that specifically identifies ROS, and five different stimulants (two particulate, Candida albicans and zymosan, and three soluble ones, fMLP, PMA and polyanetholesulfonate) to evaluate the oxidative burst activity of PMNs in relation to age. They observed a dichotomy between the effects of Candida albicans and zymosan (particles), which were not significantly different in the elderly subjects compared with the young controls, and those of soluble agents, which showed a significant reduction in the chemiluminescence signal in the elderly group. Considering the different results obtained with the various stimulants adopted in this study, it may be postulated that aging can influence the different transductional pathways in different ways. For example, PMNs from healthy elderly persons generated significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, after stimulation by fMLP, resulting in a decrease in O2' production (Lipschitz et al., 1991). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in fMLP-induced O2° generation was corrected if the intervening signal transduction steps were bypassed by ionophore elevation of cytosolic calcium (Lipschitz et al., 1991). Thus aging seems to be associated with a reduced ability to produce key second messengers, impairing microbicidal function. This latter observation was confirmed by Corberand et al. (1981) who showed that Candida-\d\\mg activity was significantly decreased in subjects over 80 years old. Macrophage function

Because of their tissue locations, studies of macrophage function in aged humans are scarce and yield conflicting results compared with investigations using animal models. Some authors (Gardner et al., 1981; Nielsen et al., 1984) found no difference between blood monocyte functions from young and old humans in their ability to kill bacteria or generate cytokines, although others observed that the capacity of tissue macrophages from rodents to present antigen and produce cytokines declined (Davila et al., 1990; Ding et al., 1994) or increased (Ershler, 1993; Lesourd & Mazari, 1999) with age. For example, the ability of glycogen-elicited peritoneal macrophages triggered by Staphylococcus epidermidis to secrete

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both tumour necrosis factor-OC (TNFa) and interleukin-1 (IL1) was decreased in aged rats (Bradley et al., 1989). In addition, aging resulted in a significant reduction in the capacity of resident peritoneal macrophages from old mice to respond to high doses of interferon-y (IFNy) and lipopolysaccharide (LPS) with increased tumor cytolysis (Lu et al., 1999). Davila et al. (1990) also observed that the production of O2'~ by macrophages in the presence of opsonized zymosan and recombinant INFy was 75% lower in 23-month-old than in threemonth-old rats. Furthermore, the secretion of TNFa in response to INFy and LPS was almost absent in macrophages from aged rats (Davila et al., 1990). Ding et al. (1994) reported that in vitro INF-, PMA-, or opsonized-induced release of H2O2 and nitric oxide by peritoneal macrophages was 50% lower in old than in adult mice. As the ability of macrophages to secrete ROS, reactive nitrogen intermediate and cytokines correlates closely with their ability to perform two critical effector functions, namely intracellular killing of microorganism and lysis of tumour cells, the diminished response of macrophages to activating signals may be one aspect of the impaired immune response in advanced age. Nevertheless, other authors (Nafziger et al., 1993; Lesourd & Mazari, 1999) report that macrophage functions were preserved or even enhanced with aging. In these studies, IL1 production by stimulated macrophages was sustained in old mice, while IL6 generation was increased (Ershler, 1993). The greater secretion of macrophage cytokines may lead to higher and longer-lasting body metabolic changes in old individuals, since monokines play a central role in controlling body metabolism. For example, proinflammatory cytokines induced muscle protein breakdown, which is particularly damaging in old individuals, since the aging process/w se is responsible for sarcopenia (Beaufrere & Boirie, 1998). Thus any disease may induce higher muscle loss in aged subjects than in adults; the muscle destroyed being not fully rebuilt after recovery. When disease follows disease, body protein decrease may then push the elderly into progressive and gradually increasing protein-energy malnutrition (PEM) and, therefore, into progressive and gradually reduced immune response (Ershler, 1993; Lesourd & Mazuri, 1999). Age-related changes in natural killer cells Human natural killer (NK) cells are a population of large granular lymphocytes defined by the CD3 , CD16+, CD56+ surface phenotype. NK cells are involved in the recognition and lysis of tumour and virally infected cells (Solana et al., 1999). Reports on NK cell cytotoxicity in healthy elderly populations were conflicting, indicating either a decrease or no modification of NK cell number, phenotype and function. A significant increase of mature and immature NK cells with age was found by most authors in both absolute count and proportion (Krishnaraj & Svanborg, 1992; Sansoni et al., 1993; McNerlan et al., 1998). Mature NK, comprising 90—95% of total NK cells, are strongly cytotoxic and presented a low expression of CD56. In contrast, immature NKs are defined by the high surface expression of CD56, are poorly cytotoxic but proliferate strongly in response to IL2 (Solana et al., 1999). The increased percentage of NK cells observed in the elderly is mainly due to the increase in the mature subset, supporting the hypothesis that a phenotypic and functional shift in the maturity status of NK cells occurs during aging (Krishnaraj & Svanborg, 1992). McNerlan et al. (1998) showed that expanded NK cell populations are in an activated state. Although the functions of these subsets remain to be elucidated, their expansion in the elderly may represent a remodelling of the immune system with increasing age, with an increase in non-

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major histocompatibility complex (MHC) restricted cells perhaps compensating for the decline in T or B cell functions in the elderly. Observations (Krishnaraj & Blandford, 1987; Sansoni et al., 1993) in healthy elderly subjects showed that NK cell cytotoxicity was not affected by aging. However, these results indicated that the increase in the number of peripheral NK cells did not correlate with a significant increase in the cytotoxic activity of these cells, suggesting that NK cell cytotoxicity should be impaired when considered on a per-cell basis (Ligthart et al., 1989; Ogata et al., 1997; Solana et al., 1999). Acquired immune system After innate immune cells, the second line of defence to eliminate invading microorganisms is the antigen-specific immune system. The acquired immune system is immature and does not function well in the early stages after birth. Its activity develops quickly by exposure to innumerable antigens, peaks at puberty but starts gradually to decline thereafter. Studies in several long-lived rodent strains and humans indicated that aging is characterized by thymic involution, decline in T cell number, phenotype, proliferative response and function, and a relative maintenance in B cell response. Age-related involution of thymic function Once the essential role of the thymus in immune development and function became clear, the thymic involution that accompanies aging was logically viewed as a clue to the immune dysfunction that characterizes old age. The levels of thymic hormones decline after puberty, but they do not differ significantly between elderly and middle-aged subjects (Steinmann et al., 1985; Ginaldi et al., 1999b). In other words, because this tissue undergoes involution before the onset of age-related changes in immune function, the thymus may be only partly responsible for the T cell dependent changes with age. Nevertheless, physiologically, the main reason why T cells are more susceptible to aging than other immune elements is that the recruitment of T lymphocytes is quite limited, as the thymic capacity to provide T cells to peripheral lymphoid tissues decreases quickly during aging (Hirokawa, 1998). The situation is quite different for other immune cells, which are constantly replenished by the bone marrow throughout life. Given that T cells are mainly produced in the thymus, a causal relationship may thus exist between thymic involution and subsequent age-related decline in T cell functions. In addition, thymus involution is probably of major significance during acute infections, since aged individuals do not replace the destroyed lymphocytes at the efficient rates observed in younger adults in such situations (Lesourd, 1999). Quantitative and qualitative changes in T cell subpopulations in the elderly The number of total lymphocytes in the blood compartment usually decreases in healthy elderly subjects compared with adult ones (Lesourd, 1997; Huppert et al., 1998). This decline represents only 10-15% of lymphocyte counts (Lesourd, 1995) and is partly due to a reduction in the T cell subpopulation. In addition, aged individuals express fewer mature T cells (CD3+) and higher numbers of immature T lymphocytes (CD2+CD3 ), resulting in a reduction in the mature-to-immature T cell ratio (Lesourd & Mazari, 1998). This change, detectable only after age 50 years, slowly increases thereafter, even in the very old (> 90

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years old, Huppert et al., 1998). Furthermore, there are marked modifications in T lymphocyte helper (CD4 ) and cytotoxic (CDS ) subpopulations in aged people. Nevertheless, the alteration in the T helper and cytotoxic mediated components of the immune system are very controversial. T cytotoxic lymphocyte populations have been reported to increase (Stulnig et al., 1995), decrease (Nagel et al., 1981; Sansoni et al., 1993; Sindermann et al., 1993; Huppert et al., 1998) or remain unchanged (Hallgreen et al., 1983; Matour et al., 1989; Arnalichef*/., 1994; Rea «/*/., 1996, 1999; Krausetf */., 1999) in elderly subjects. In addition, most studies (Sansoni et al., 1993; Rea et al., 1996, 1999; Gianni et al., 1997; Huppert et al., 1998; Chen et al., 2002) generally describe a reduction in the T helper subset, whereas some investigators detected no variation (Nagel et al., 1981; Hallgreen et al., 1983; Matour et al., 1989; Sindermann et al., 1993; Stulnig et al., 1995; Krause et al., 1999). The main cause of this marked heterogeneity is the presence of concomitant pathological disorders or complex environmental influences that may themselves affect immune status. To overcome this problem, Ligthart et al. (1984), in the SENIEUR protocol, set strict admission criteria for human immunogerontological studies that include clinical information, laboratory data, and immunopharmacological interferences. A decrease in the CDS subset with no change in the CD4 subpopulation has been described in healthy people unimpaired by any illness and taking no medication liable to exert immunomodulating effects and then meeting the SENIEUR protocol inclusion criteria (Lesourd & Meaume, 1994; Wick & Grubeck-Lowenstein, 1997). In addition, there is recent evidence (Shearer, 1997; Wick & Grubeck-Lowenstein, 1997; Lesourd, 1999; Rea et al., 1999) suggesting that antigen expression on T lymphocyte subsets is subject to qualitative and quantitative modifications with advanced age resulting in T lymphocyte subset equilibrium changes with age. The CD45RA isoform represents primarily naive, i.e. unprimed T lymphocytes that switch to the CD45RO phenotype after antigenic stimulation and result in memory cells (Stulnig et al., 1995). Accordingly, there is a continuous decline in the proportion of CD45RA CD45RO" lymphocytes during aging, parallel to an increase in CD45RA~CD45RO+ cells; a switch is found in both helper and cytotoxic subpopulations (Stulnig et al., 1995; Jakola & Hallgren, 1998; Mazari & Lesourd, 1998; Pawelec et al., 2001). This type of modification, related to antigen exposure, starts in infancy, increases markedly during childhood and early adulthood until age 30 years, and continues thereafter at a far lower rate until death (Cossarizza et al., 1996). Using a simple compartmental model to study the age-dependent kinetics of phenotypic restructuring between naive and memory T cells, Jakola and Hallgren (1998) demonstrated that the average annual turnover rate is the same for CD4 and CDS naive cells, whereas it seems to be 1.5 times higher in CDS memory than in CD4 memory lymphocytes. The annual conversion rate of CD4 naive cells to memory cells is twice the CDS conversion rate. In addition, the transition between naive and memory cells that occurs during the third decade of life (Cossarizza et al., 1996) may be due to these differences in turnover and conversion rates from naive to memory lymphocytes (Jakola & Hallgren, 1998). Furthermore, the CD25 antigen, which is identified as the a chain of the high-affinity IL2 receptor fell with age in both helper and cytotoxic lymphocytes (Rea et al., 1999), making IL2 less effective for immune activation and lymphocyte proliferation (Sindermann et al., 1993; Rea et al., 1996). Conversely, a marked CD3 HLA-DR upregulation was described with increased age, suggesting an increase in antigen presentation by T cells (Stulnig et al.,

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1995; Walrand, 2002). The function of increased HLA-DR expression may be to promote recognition and elimination of damaged or infected cells (Rea et al., 1996) because of compromised gut and epithelial barriers in old persons. Such qualitative and quantitative age changes in antigen expression on T lymphocytes may have a major influence on cell activation and functions during adaptative challenges (e.g. infectious diseases or protein-calorie malnutrition). Age-related changes in T cell function Aging is associated with impairment of T cell functions, most importantly the decline in the ability of T lymphocytes to proliferate and produce IL2 after phytohemagglutinin treatment (Lehtonen et al., 1990; Sindermann, 1993; Rea et al., 1996; Krause et al., 1999; Song et al., 1999; Bruunsgaard, 2000). Age-related decreased proliferation and reduced IL2 secretion are at least partly related to changes in T cell subsets. Increase in memory T lymphocytes may play a significant role in the reduction of lymphocyte proliferation, since memory cells are poor IL2 secretors (Nagelkerken et al., 1991; Hobbs & Ernst, 1997). In addition, immature CD2 CD3 cells, which increase with aging, possess a lower capacity to replicate (Ales-Martinez et al., 1988). Nevertheless, we have reported conflicting results from very carefully selected healthy elderly subjects (Mazari & Lesourd, 1998). In vitro IL2 release and proliferation of stimulated mononuclear cell cultures were not significantly different in 25— 34-year-old and 75—84-year-old healthy elderly subjects selected according to the SENIEUR protocol. In addition, we reported that in this population, a minor micronutrient deficit, such as low folate levels, was associated with decreased lymphocyte IL2 secretion and proliferation (Mazari & Lesourd, 1998). It appears that changes in cellular mediated immunity in aged individuals may be related not only to the aging process per se but also to environmental factors such as health and/or nutritional status. Recent advances in the understanding of the role of cytokines has led to the separation of helper functions between T helper 1 (THl) and T helper 2 (TH2) cells (Mosmann & Coffman, 1989a, b; Shearer, 1997). Both THl and TH2 subsets are activated through different chemokines (Siveke & Hamann, 1998) and express different functions. THl lymphocytes are helper cells for cytotoxic T cells and macrophages and secrete IL2, IL10 and IFNy while TH2 cells secrete IL3, IL4, IL6, IL12 and have helper roles for B lymphocytes and polymorphonuclear eosinophils (Mosmann & Coffman, 1989a, b; Shearer, 1997). Since the early 1990s, immunosenescence has also been associated with a dramatic decrease in the TH1/TH2 cell ratio, due to a reduction in THl function (mainly a decrease in IL2 and INFy release) and increase in TH2 function (IL.4 and IL6 secretion) (Kubo & Cinader, 1990; Ershler et al., 1993; Segal et al., 1997). However, conflicting results have been reported for age-related INFy variations, which have been found to remain constant with age (Chen et al., 1987; Sindermann et al., 1993). IFNy is also secreted by memory T cells, a subset that increases in the elderly, and the apparently contradictory observations may be related to different values for naive/memory T lymphocyte ratio in the populations studied. The agerelated modifications in THl and TH2 function is probably due to cumulative antigenic influences throughout the subject's lifespan, since this has been reported to be associated with a lower TH1/TH2 ratio (Cakman et al., 1996). Taken as a whole, the alteration of THl and TH2 activities may be of major importance in the immune impairment of elderly

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persons, since TH1 mainly induces maturation and activation of the cytotoxic T lymphocytes, which decrease during aging (Bruley-Rosset & Payelle, 1987; Mbawuike et al., 1997), while TH2 induces increased B immunoglobulin production, which increases with advancing age (Batory et al., 1984; Moulias et al., 1984). Changes in B cell number and function in the elderly The main reported finding concerning B cells collected after the revolution caused by the SENIEUR protocol is the paradoxical opposite trend with age of two parameters related to humoral immunity. An increase in most serum immunoglobulin class and subclass levels occurs, concomitant to a decrease in circulating B lymphocytes (Lehtonen et al., 1990; Hulstaert et al., 1994; Stulnig et al., 1995; Rea et al., 1996; Huppert et al., 1998; Ginaldi et al., 1999a; Van Loveren et al., 2001). Both IgA and IgG serum levels increase significantly with age, whereas IgM do not (Lesourd & Mazari, 1999). Among IgG subclasses, only IgGl, IgG2, and IgG3 show a significant increase, whereas IgG4 remains constant or reduced (Paganelli et al., 1992). IgGl and IgG3 are mainly involved in the humoral response to viral and bacterial antigens and IgG2 and IgM in the response to polysaccharides (Ginaldi et al., 1999a). The clinical significance of the increase in immunoglobulin plasma levels in healthy old people remains unclear but it may confer greater protection against viral and bacterial infections. In addition, it is known that IgG4 antibodies are regulated differently and are involved in defence against parasitic infections (Paganelli et al., 1992). The decrease or even absence of IgG4 in plasma of certain elderly subjects, including healthy centenarians, suggests that their role is not essential for healthy survival. The increase in antibody serum concentrations may be a result of decreased catabolism of these proteins, an enhancement of the amount of immunoglobulin secreted per cell, or the number of secreting cells in tissues other than the blood compartment may also be increased (Ligthart et al., 1985). The latter possibility would imply age-related alterations in B lymphocyte homing and recirculation. Findings (Cossarizza et al., 1997; Franceschi et al., 1998; Ginaldi et al., 1999a) suggest that the lymphocyte membrane expression of molecules involved in homing processes, and of cell adhesion molecules, increases with age. Therefore, we cannot dismiss the possibility that peripheral blood does not mirror the real situation of the B cell compartment, and that a normal or even increased number of immunoglobulin-producing cells is present in lymphoid organs, including mucosal tissues (Paganelli et al., 1992). In addition, Lehtonen et al. (1990) observed that in vitro immunoglobulin production is reduced in aged persons in both unstimulated and mitogen-stimulated B cell cultures. IgM production is especially affected, while IgA and IgG secretions are better preserved. The inability to increase the immunoglobulin secretion significantly in vitro suggests an intrinsic defect in the B cell system in the elderly, partly refuting the hypothesis of an increased rate of immunoglobulin production per cell. Finally, the age-related increase in most immunoglobulin classes and subclasses could be the consequence of an increase in autoantibody production. There are two major subsets of B lymphocytes (Bl and B2 cells), which can be distinguished from each other on the basis of their phenotypic characteristics and immunoglobulin secretions. The B1-CD5 lymphocyte subsets appear to be the main source of natural antibodies and autoantibodies, while B2-CD5^ cells produce specific antibodies to foreign antigens. Aging is characterized by an increase in Bl subpopulation through T lymphocyte independent proliferation,

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whereas the ability of bone marrow to generate B2 lymphocytes is impaired due to the decline in T cell cytokine generation (Rosenberg et al., 1983; Ben-Yehuda et al., 1994; Weksler, 1995). The growing number of antibodies produced by Bl lymphocytes with age and the fact that the antibody repertoire of B1 cells is dominated by autoantigen specificities may contribute to the age-associated increase in autoantibody levels described by several authors (Ginaldi et al., 1999a; Lesourd et al., 1998). However, the lack of organ-specific autoantibodies in healthy old subjects and centenarians (Mariotti, 1992, 1998, 1999), plus the fact that most of the classical autoimmune diseases occur in the young (Globerson & Effros, 2000) casts some doubt on the general validity of such a hypothesis. Further studies in humans are needed to clarify this point.

Nutritional regulation of immune function of the aged Effect of protein-energy malnutrition (PEM) on immune responses in aged people Elderly persons are at increased risk of developing PEM because of various social, physical, pathophysiological and psychological factors. PEM exists in apparently healthy aged persons living at home (Steen & Rothenberg, 1998), but this illness is usually predominantly described in elderly people in nursing homes and in hospitalized old patients with medical or surgical disorders (Fiatarone, 1990; Constans et al., 1992; Mowe et al., 1994; Sullivan et al., 1995; Bertozzi et al., 1996; Tierney, 1996; Covinsky et al., 1999). The prevalence of PEM in hospital varies from one study to another according to the indices chosen for the assessment and the arbitrary cut-off points for normal and abnormal anthropometric and biological variables. However, PEM places aged subjects at higher risk of subsequent morbidity, including decreased resistance to infection, slowed wound healing, and increased incidence of disease (Scrimshaw & San Giovanni, 1997). Consequently, PEM is associated with adverse outcomes in older hospitalized patients. Alterations in polymorphonuclear functions during PEM in the elderly The influence of nutritional deficiencies on PMN functions was initially demonstrated in children (Chandra et al., 1976). In this study, the PMN chemotactic index was depressed but correlated more closely with the presence of infection than with nutritional disorders. However, ten years later, Lipschitz et al. (1986) showed that bactericidal activity was altered in undernourished old rats, while bacterial ingestion appeared to be preserved. The decrease in bactericidal ability was related to a reduced O2' production in undernourished animals (Lipschitz & Upuda, 1986). More recently, Cederholm et al. (1999, 2000) reported a lower leukotriene C4 and O2'~ formation by PMNs from chronically ill elderly patients with PEM than by PMNs from age-matched but well-fed controls, when induced by surface receptor dependent stimulus (fMLP). In contrast, the PMN response to PMA, acting directly on enzymes involved in ROS pathways, was intact in the patients. Previous animal studies (Gyllenhammar et al., 1988, 1989; Palmblad et al., 1988) demonstrated that the number and affinity of fMLP receptors were not affected by dietary derangement. Therefore, the definition of the part of the subcellular transduction mechanism for cell activation that is affected by PEM requires further study. In addition, several factors other than PEM may

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affect PMN responses in chronically ill elderly patients, such as medication and concurrent diseases, since for example proinflammatory cytokine release has been shown to interfere strongly with ROS production (Redmond et al., 1991). There is no possible way to separate these factors when studying a population of elderly patients recruited from internal medicine services. Starvation not related to concurrent disease is rare in affluent societies. We recently studied the effect of a controlled fasting period (36 hours) on PMN functions in healthy adult and elderly subjects free of pathological disorders and not undergoing drug treatment, according to the SENIEUR protocol (Walrand, 200la). We showed that acute starvation reduced neutrophil chemoattractant ability toward fMLP in both the adult and elderly subjects (Fig. 11.2).

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Fig. 11.2 PMN chemotactic index (ratio of the number of PMNs that migrated in response to fMLP to the number that migrated spontaneously) in healthy adult and elderly subjects in basal conditions, after 36-hour fasting and 4-hour refeeding periods (Walrand et al., 2001a, b). a,b: P < 0.05. As chemotaxis is a highly glucose-dependent pathway (McMurray et al., 1990; Mowat & Baum, 1971), we hypothesized that the decreased glucose availability during fasting might limit glucose uptake and utilization by PMNs. In addition, fasting clearly induced an increase in O2'~ generation by PMA-stimulated PMNs in adult and elderly healthy people (Walrand et al., 2001a). Some authors (Karasik et al., 1990; Pineiro et al., 1998) have reported that fasting is accompanied by a marked increase in protein kinase C activity, resulting in stimulation of NADPH,H + oxidase enzyme. In summary, we demonstrated that PMN functions, i.e. chemotaxis and ROS production, were not altered by acute starvation in healthy elderly persons in comparison with adult responses, showing that PMN alterations reported in critically ill elderly undernourished patients may be

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partly due to the concomitant pathology and may be independent of the aging process or PEM effects. Alterations in monocyte/macrophage functions during PEM in the elderly Beside this profound impairment of PMNs, monocyte functions are also lowered in malnourished elderly people. The ability of monocytes to release cytokines in LPS- or PHAstimulated in vitro cultures is decreased in elderly subjects with PEM, the same alteration being reported for monocytes from old dietary restricted mice (Arnalich et al., 1994; De la Fuente & Munoz, 1992). For example, III cytokine release is reduced in infected elderly patients with PEM (Nafziger et al., 1993). IL1, in association with other monokines such as TNFa and IL6 represents the central core of the inflammatory response. Therefore, PEM can modify the clinical symptoms of inflammation in undernourished elderly individuals. Furthermore, low production of cytokines is also responsible for decreasing the mobilization of nutritional body reserves, leading to deteriorated defence mechanisms. We also showed that healthy elderly subjects of low body mass index (BMI) produced lower levels of IL1 and IL6 from LPS-stimulated monocytes than similar aged people with higher BMIs (Lesourd et al., 1998). In addition, these subjects had increased serum C-reactive protein and a-1glycoprotein acid levels, indicating a probable in vivo activation of monocytes, since acutephase protein synthesis is boosted by monocyte cytokines. The reduction in in vitro cytokine release that we noted may be explained by a limited capacity of monocyte secretions in aged individuals due to prior in vivo activation. In contrast, previous studies reported that tissue macrophage functions were preserved in PEM animal models (Bradley et al., 1989; McMurray 1998). In addition, dietary caloric restriction from weaning, a regimen that extends lifespan (Masoro, 1998) had no effect on the declining ROS, TNFa and IL6 productions in stimulated macrophages from old mice and rats (Effros et al., 1991; Riley-Roberts et al., 1992). However, we recently showed that TNFoc secretion by peritoneal macrophages was dramatically decreased in adult and old rats after a long-term denutrition period (Walrand et al., 2000a), indicating that in situ responses are altered by PEM. Alterations in T-cell mediated immunity during PEM in the elderly One of the main alterations induced by PEM in the elderly is the dramatic decrease in lymphocyte count (Incalzi et al., 1998). In addition, T lymphocyte distribution, which is characterized by a decrease only in CDS cells in healthy well-nourished old subjects, is associated with a reduction in both CD4 and CDS subsets. Compared with healthy elderly subjects, aged patients with serum albumin lower than 30 g/L often have less than half the level of CD4 counts in peripheral blood (Table 11.2; Lesourd & Mazari, 1997; Lesourd et al., 1998). Decrease in serum albumin levels in the elderly is also associated with lower mature CD 3 T cell subset and higher immature CD2 CD3~ T lymphocyte levels (Lesourd et al., 1998). In addition, proliferation index and release of IL2 by mitogen stimulated lymphocyte cultures is dramatically reduced in these patients (Weindruch et al., 1979; Chandra, 1990a, b; Lesourd & Mazari, 1999). Conflicting results have been published in idiopathic senile anorexia (Arnalich et al., 1994). A significant decrease in CD2 and CD4 lymphocyte subpopulations, but unchanged CD8 subset were observed in senile underweight anorectic

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Table 11.2 Alteration of acquired immune response in PEM elderly subjects (Lesourd, 1995, 1997, 1998)

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subjects. In a short-term fasting model (36 hours), the number and percentage of CDS cell were reduced in healthy adult subjects but did not modify the alteration in T cell distribution (decrease in CD8+ cells) in healthy elderly people (Walrand et al., 2000b, 200la). The lack of response of lymphocyte subpopulation counts to starvation in healthy elderly subjects may be due to low baseline values induced by the aging process. In aged animal models, proteinfree diet or food restriction resulted in a marked thymic involution together with a reduction of splenic T cells, both in number and in antibody response to sheep red blood cells (Konno et al., 1993). This work shows that thymic function is sensitive to protein deficiency and that a well-balanced diet is necessary for preservation of immune functions in old age. Delayed cutaneous hypersensitivity (DCH), a bedside measure of cell-mediated immunity, has been found to be impaired in elderly patients during PEM (Arnalich et al., 1994; Lesourd, 1995; Cederholm et al., 2000). Both the number of positive reactions and the DCH scores were altered by PEM. In these studies, the intensity of the T cell dependent immune deficit has been shown to be proportional to the degree of PEM (Chandra, 1989; Lesourd & Mazari, 1998; Thorslund et al., 1990). Furthermore, in a nine-week study of immune adaptation to marginal protein intakes (0.45 g/kg body weight/day), Castaneda et al. (1995) observed a decrease in DCH response that was similar to reductions seen in elderly persons with PEM. These authors concluded that marginal protein intake alone led to impairment in

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immune function during aging. Nevertheless, neither T cell subsets nor immunoglobulin plasma concentrations were modified by low protein intakes in this study (Castaneda et al., 1995). Alterations in B cell mediated immunity during PEM Antibody reponses are also lowered in the undernourished elderly population. Although the immunoglobulin levels are not altered by undernutrition (Arnalich et al., 1994), seroconversion rates after administration of tetanus toxoid or influenza vaccine are lower in aged people suffering from PEM (Fiilop et al., 1999; Lesourd, 1995; Lesourd & Mazari, 1997). About 75% of vaccine unresponsiveness in hospitalized elderly patients is apparently related to PEM (Table 11.2; Lesourd & Mazari, 1997). After vaccine shots, not only were antibody levels reduced in aged persons presenting malnutrition syndrome, but antibody affinity was also decreased (Chandra, 1989). Therefore, it can be suggested that vaccines in undernourished elderly patients induce a lower level of protection than in well-nourished persons. Immune response during nutritional recovery in undernourished elderly persons The reversibility of immune alterations induced by PEM in chronically ill elderly patients is not clear. Follow-up studies reported that hospitalized elderly subjects that were prescribed nutritional supplementation (Cerderholm et al., 1995a, b) or cyclic enteral nutrition (Hebuterne et al., 1995, 1997) showed a more prominent improvement in their nutritional status than the non-supplemented subjects. In addition, the most pronounced recovery was registered in orosomucoid negative patients showing that inflammatory syndromes may interfere with nutritional state and recovery in elderly subjects. Influence of nutritional recovery on PMN function in old age Nutritional intervention by oral supplementation equivalent to 400 kcal (1.7 mj) and 40 g protein daily induced a weight gain in ill and malnourished elderly subjects that was accompanied by an improvement of O2'~ generating capacities of PMNs in response to PMA or fMLP (Cederholm et al., 1999). Nutritional help may therefore improve the bactericidal function of PMNs of elderly patients during PEM. However, we cannot tell from these data whether the tendency for increased O2* production is conferred by a recovered nutritional status or by some improvement in the underlying disease. In healthy subjects selected according to the SENIEUR protocol, we described a significant lower PMN response to refeeding in fasted healthy elderly than in 36-hour-fasted young adults (Walrand et al., 200la, b). Renutrition led to a recovery of neutrophil migration toward fMLP in the adults, whereas the alteration of chemotaxis induced by fasting was not restored in the old people (Fig. 11.2). In addition, some differences in ROS production, and particularly an elevated H2O2 production, in PMA-activated PMNs between the adult and elderly fasted subjects were also observed during refeeding (Walrand et al., 2001a). As previously described (Antonaci et al., 1991; Niwa et al., 1993; Tortorella et al., 1993), elderly individuals under nutritional stress exhibited metabolic pathway imbalances in H2O2 scavenging enzymes, resulting in a decrease in total cellular myeloperoxidase, catalase, and glutathione peroxidase. The dysregulation of the ROS pathway during aging may thus explain the differences in H2O2 production noted in our study after nutritional fluctuations. Finally, our recent

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observations in long-term dietary-restricted old rats revealed that the use of a high protein diet during renutrition resulted in an improvement of H2O2 production by PMA-stimulated PMNs (Walrand et al., 1999, 2000a). Therefore, studies need to be conducted with higherprotein diets providing adequate energy and micronutrient intakes to determine the potential immunological role of such a regimen in malnourished elderly patients. Influence of nutritional recovery on T cell function in old age Eight weeks of enough appropriate nutritional supplements to provide an extra 500 kcal of energy every day, and at least the minimum daily requirement of all essential micronutrients, improved DCH response, increased CD3 , CD4 and CD8 T cell numbers and elevated lymphocyte proliferation response to PHA in a group of undernourished aged subjects (Chandra, 1990a). This improvement in immunological function was associated with evidence of improved nutritional status, mainly in terms of anthropometric indices and levels of albumin, prealbumin, transferrin, retinol-binding protein, zinc and iron (Chandra et al., 1982). Improved NK cell activity, mitogen-induced lymphocyte stimulation response and increased IL2 production by activated lymphocytes were also observed in elderly subjects given nutritional counselling and appropriate supplements for six months to correct diagnosed nutritional deficiencies (Chandra, 1989, 1991). These data indicate that impaired immunocompetence is a correctable abnormality in elderly malnourished patients. However, we noted a lack of response in T lymphocyte subsets (CD3+, CD4+ and CD8+) during the refeeding period in healthy 36-hour-fasted elderly subjects (Walrand et al., 2000b, 2001a). We also investigated two different types of undernourished elderly patients during nutritive therapy: patients with undernutrition in relation with earlier undernourishment and patients with PEM associated with marked acute phase responses in relation with ongoing disease (Lesourd & Mazari, 1997). The classification of the two groups was carried out on acute phase protein levels, i.e. C-reactive protein and oc-l-glycoprotein acid. Refeeding improved immune responses of undernourished elderly subjects, but differently with respect to the levels of acute phase responses (Lesourd & Mazari, 1997). Refeeding induced improved IL2 secretion in PHA-activated lymphocyte cultures, this effect being closely correlated to serum albumin increases. In contrast, immune effect of refeeding needs longer nutritive therapy to be quantifiable in aged patients with major acute phase responses (Table 11.3; Lesourd, 1999). In these subjects, neither IL2 production by stimulated lymphocytes nor serum albumin increased, as long as C-reactive protein remained elevated. This emphasizes the importance of inflammatory processes on immune response in PEM aged patients and the inhibitory impact of these phenomena on nutritional or immune status improvement. Not only does PEM exert a profound impact on the immune responses of aged individuals, but micronutrient deficiencies have the same effects (Licastro et al., 1993; James et al., 1995; Ravaglia et al., 2000). Nutritive therapy in elderly PEM subjects using vitamin Bg (Talbott et al., 1987; Rail & Meydani, 1993) or zinc (Bogden et al., 1987, 1988; Boukai'ba et al., 1993), or a combination of several micronutrients (Penn et al., 1991; Galan et al., 1997; Fortes et al., 1998) induced increased immune responses in aged undernourished patients. For example, Penn et al. (1991) used a mix of antioxidant vitamins (A, E and C) in elderly persons hospitalized in a long-stay unit for 28 days. They found that supplementation improved CD4 T cell numbers and lymphocyte proliferation in treated subjects and

Table 11.3

Changes in immune functions over refeeding in PEM elderly

Period of refeeding (days)

Albumin (g/L)

C-reactive protein (CRP) (mg/L)

111 release (ng/L)

IL6 release (ug/L)

CDS (/mm3)

CD 4 (/mm3)

IL2 release (ug/L)

Subjects with moderate acute acute-phase responses (CRP < 30mg/L at refeeding onset) n = 23 0.87 ± 0.75 0.71 ± 0.38 19.813.7 25.4 ± 4.3 845 + 324 1.72 ± 1.26 1.27 + 0.46 1236 ± 431 29.2 ± 5.1 12. 5 ±3.2 2.91 ± 1.45 1.78 ± 0.47 1461 ± 458 10.4 ± 2.9 31.6 ±6.2 7. 3 ±2.9 3.21 ± 1.34 2.00 + 0.39 1508 ± 417 35.8 ± 4.7

412 787 1236 1278

± 238 ± 358 ± 481 ± 423

0.86 1.19 1.68 2.14

± 0.25 ± 0.33 ± 0.37 + 0.41

Subjects with important acute acute-phase responses (CRP > 50 mg/L at refeeding onset) n = 0.79 ± 0.63 1.01 + 0.57 697 75.6 ± 40.2 25.3 ± 4.8 734 0.87 ± 0.57 0.62 ± 0.34 25.2 ± 6.0 42.1 ±27.3 1.48 ± 1.64 1.15 ± 0.71 17.6 ± 14.4 28.9 ± 6.6 1025 2.38 ± 1.75 1.74 ± 0.63 1384 33.5 ± 6.8 11. 5 ±8.8

462 524 798 1114

± 257 ± 286 ± 346 ± 385

1.06 0.71 1.48 1.89

± 0.32 ±0.29 ± 0.36 ± 0.43

0 21 ±2 42 ±4 93 + 5

0 21 ± 2 42 ±4 93 ±5

24 ± 328 ± 398 ± 402 ± 457

Two groups of profoundly undernourished elderly (albumin < 30g/L) have been followed during three months of refeeding. The first group presented low acute phase responses and had chronic insufficient intakes. It received 37.8 ± 6.4 kcal/kg/day with at least 16% of protein and 50% of carbone hydrates (D 21). The second exhibited high level (CRP > 50 mg/L) for acute phase responses. It was refed with higher levels 44.5 ±7.2 kcal/kg/day with at least 18% of protein and 50% of carbohydrates using either enteral or oral routes (D21). Immune responses were sequentially measured for lymphocytes (CD3 and CD4) absolute counts in peripheral blood, in vivo lymphocyte functions (IL2 releases in PHA-stimulated mononuclear cell cultures) and for monocyte functions (IL1 and IL6 releases in LPS-stimulated monocyte cultures) using previously described techniques (Lesourd & Mazari, 1997, 1999; Lesourd, 1999).

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concluded that antioxidant micronutrient cocktails can improve cell-mediated immune function in elderly long-stay patients (Penn et al., 1991). We conducted a similar study in long-stay undernourished patients using one to three times the recommended dietary allowances (RDA) of micronutrients known to possess immuno-modulating properties (vitamins A, E, and C, zinc, and selenium) (Monget et al., 1996a; b). Supplementation caused trace elements and vitamins blood levels to return to normal in the supplemented elderly group but did not enhance cell-mediated response after six months of treatment. Girodon et al. (1999) also observed that a low-dose supplementation of zinc and selenium for two years provided significant immune improvement in institutionalized elderly patients suffering from nutritional deficiencies by increasing the humoral response after vaccination. The number of patients without respiratory tract infections during the study was higher in groups that received trace elements (Girodon et al., 1999). Such micronutrient supplementations may therefore be of considerable importance to public health by reducing morbidity from infections in elderly hospitalized subjects. Influence of nutritional supplementation in healthy well-nourished elderly subjects Several reports have addressed the question of the influence of micronutrient supplementation in healthy self-sufficient home-living elderly subjects. Of particular interest is the study published by Chandra (1992), which assessed the effect of physiological amounts of vitamins and trace elements on immunocompetence and occurrence of infection-related illness in 96 independently-living healthy elderly individuals. Immunological variables and frequency of illness were recorded at baseline and after 12-month supplementation. Multivitamin and mineral supplementation improved immune response in elderly subjects as demonstrated by increases in peripheral mature CD3 T lymphocytes, mostly due to increased CD4 cell subsets, and NK cell numbers. In addition, PHA-dependent lymphocyte proliferation, IL2 and soluble IL2 receptor release in lymphocyte cultures and NK cell activity were also improved after supplementation (Chandra, 1992). Furthermore, Chandra reported that this type of nutritional strategy may also increase antibody response to influenza vaccines. But the most interesting finding of this work was the reduction of the rate of infection in the supplemented group during the one-year duration of the study (Chandra, 1992). Bogden et al. evaluated the effects of daily supplementation with 15 mg (recommended level), or 100 mg (seven times the RDA) zinc for one year on cellular immune functions in healthy free-living elderly people also consuming a one-a-day type supplement containing physiological amounts of all essential micronutrients except zinc (Bogden et al., 1990, 1994). The latter supplement was administered to ensure that deficiencies of other micronutrients would not limit the ability of the zinc intakes to enhance immune function. Surprisingly, in vitro lymphocyte proliferative capacities and DCH response were higher in the group receiving the multivitamin/multimineral supplement without zinc than in those taking zinc. Furthermore, when subjects discontinued zinc ingestion after one year but continued taking the vitamin and mineral supplement for four months, these lower DCH response rose to the levels found in the group taking micronutrients without zinc (Bogen et al., 1990). The most likely explanation is that this result was due to the immune-enhancing action of the one-a-day type multivitamin/multimineral supplement given to all the study subjects. This nutritional supplement contained a number of micronutrients known to be

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required for optimal cellular immunity (vitamins A, B6, B9, C, E, iron and copper). In addition, high zinc supplementation might have interfered with the beneficial activity of one or more components of the micronutrient supplement. In a similarly designed study (Bogden et al., 1994), the same authors compared DCH responses in a group of healthy elderly persons receiving a vitamin and mineral mix (containing recommended level of zinc) and in a placebo group without micronutrient supplementation. DCH response was increased only in the supplemented group, confirming the positive effect of micronutrient supplementations on immune response in apparently healthy subjects. However, these latter results raise questions about the safety and efficacy of zinc supplementation for improvement of immunocompetence in healthy old subjects. Other authors focused on the effect of fat-soluble vitamin supplementation on immune status of free-living well-nourished elderly people. These latter vitamins are important in the aged population because of their antioxidant protective properties and their ability to maintain an efficient immune system. Meydani et al. (1997) reported that supplementation with vitamin E for four months improved certain clinically relevant indices of cell-mediated immunity in free-living healthy elderly subjects. Subjects consuming 200 mg/day of vitamin E displayed a 65% increase in DCH and a sixfold increase in hepatitis B and tetanus vaccine antibody titres compared with placebo. However, vitamin E supplementation in healthy elderly subjects had no effect on diphtheria antibody titre and did not affect immunoglobulin levels or T and B subpopulation counts (Meydani et al., 1997). In the same way, 100 mg/day vitamin E supplementation caused a beneficial effect on the number of positive DCH reactions and DCH induration diameters in non-institutionalized elderly subjects (Pallast et al., 1999)- In addition, 1 g vitamin C and 200 mg vitamin E administered daily for 16 weeks resulted in a significant increase in lymphoproliferative capacity and PMN functions, i.e. adherence to vascular endothelium, chemotaxis, phagocytosis of latex beads, and O2 production, both in healthy aged women and in women with major depressive disorders and coronary heart disease (De la Fuente et al., 1998). Many theories have been formulated in an attempt to elucidate the immuno-modulatory mechanism of vitamin E during aging (for reviews see Meydani, 1995; Serafini, 2000). Vitamin E is widely recognized as a major lipidsoluble antioxidant in the biological membrane, where it scavenges free radicals, inhibiting the initiation and chain propagation of lipid peroxidation and protecting cellular structures against oxidative stress damage. According to this theory, vitamin E protects the cells of the immune system from the attack of free radicals, which increases with advanced age, preserving membrane fluidity and receptor integrity. Meydani (1995) proposed that this protective effect might delay the immuno-suppressive action of oxidative stress on lymphocyte proliferation and function during aging. However, it has become progressively clear that the effect of vitamin E cannot be ascribed only to its antioxidant capacities, and that some other mechanisms, such as a possible post-translational effect via a vitamin E-mediated reduction of macrophagic mediator production, may be involved (Serafini, 2000). Because these micronutrients possess evident antioxidant properties, the effect of carotenoid supplementation on human lymphocyte function has also been investigated in healthy elderly persons. Santos et al. (1997) reported that short-term (90 mg/day for three weeks) or long-term (50 mg on alternate days for 10-12 years) (3-carotene intakes had no significant effect on DCH response and lymphocyte subset profile (CD3 , CD4 and CDS ) in healthy elderly persons. In addition, there were no differences in production of IL2

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by activated lymphocytes or in lymphocyte proliferation due to PHA as a result of (3carotene supplementation (Santos et al., 1997). These observations have recently been confirmed by Corridan et al. (2001) showing that in well-nourished, free-living, healthy elderly individuals, supplementation with low levels of (3-carotene and lycopene was not associated with either beneficial or detrimental effects on the T cell subset distribution, the expression of functionally associated cell surface molecules (MHC class II, intercellular adhesion molecule type 1 (ICAM1) or lymphocyte function-associated antigen type 3 (LFA3)) and lectin-stimulated lymphocyte proliferation. Finally, we recently reported that three weeks of carotenoid depletion followed by five weeks of (3-carotene, lycopene and lutein repletion had no effect on DCH response during aging (Fig. 11.3; Cardinault et al., 2001). Consistent results of these trials show that carotenoid supplementation has no enhancing effect on acquired immunity in healthy elderly persons.

Image Not Available

Fig. 11.3 Effect of three-week carotenoid depletion followed by five-week carotenoid repletion on DCH response during aging (Cardinault et a/., 2001). a,b: P< 0.05. White = Basal, Grey = Depletion , Black = Repletion.

Nevertheless, in a long-term supplementation study, Santos et al. (1996) reported that |3carotene supplemented elderly subjects had significantly greater NK cell activities than elderly people receiving placebo. The reason for this is unknown. However, it was not due to an increase in the percentage of NK cells, or to an increase in IL2 receptor expression, or to IL2 production (Santos et al., 1996). These authors concluded that p-carotene may act directly on one or more of the lytic stages of NK cell cytotoxicity, or on NK cell activityenhancing cytokines other than IL2, such as IL12. These results mean that long-term (10—12 years) P-carotene supplementation may be beneficial for viral and tumoral surveillance by enhancing NK cell activity in elderly persons.

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Concluding remarks The decline in immune function with age is unanimously recognized and is supported by many epidemiological and clinical observations. These age-related functional changes are responsible for the increased vulnerability to illness of the elderly, which contributes to a higher prevalence of infectious and neoplastic diseases. Furthermore, immune alterations delay recovery after illness in the elderly. Because of the progressive increase in the proportion of elderly people in developed countries, this age-related increase in morbidity will be a major public health concern in the decades to come. Subtle or obvious alterations in immune function may precede the development of disease in elderly subjects. The most consistent feature of the aging immune system is the heterogeneity of the alterations observed. The modifications undergone by each individual component of the immunity have been the subject of much controversy. The main cause of this marked heterogeneity between studies is the presence of concomitant pathological disorders that may themselves affect immune status. To overcome this problem and to describe age-related immune changes, studies have been conducted in populations meeting the inclusion criteria described in the SENIEUR protocol. This tool sets strict admission criteria for human immunogerontological investigations that include clinical information, laboratory data, and immunopharmacological interferences. From these studies, it appears that aging perse affects several innate and acquired immunological indices. The mechanisms of the impairment in the ability of lymphocytes or phagocytes to respond to stimulating signals is still a matter of speculation and may involve either an intrinsic defect in the immune cells that exists before the cells are released into the bloodstream, or an acquired defect related to the environmental components, such as nutritional status. For the past thirty years, many gerontologists have focused on the role of nutrition in the maintenance of the immune system and susceptibility to illness in the elderly. It is now well established that nutritional deficiencies result in an impairment of immune responses in aged people, even in a healthy population. A superimposed infection or inflammation amplifies these alterations and can leave the elderly subject open to infectious diseases. Control studies to examine the nutritional strategies to be employed to improve immunocompetence in both well-nourished and undernourished elderly persons are currently in progress. In these populations, protein-energy supply or immunomodulating nutritional agents, such as vitamins and minerals, could be administered to maintain the immune response or aid the recovery of immune function, and therefore limit the consequences of infectious challenge.

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G. Perdigon1'2 & R. Fuller3 1

Centra de Referenda para Lactobacilos (CERELA), Chacabuco 145 (4000), Tucuman, Argentina 2 Catedra de Immunologia, Fac de Bioquimica, Qufmica y Farmacia, UNT, Tucuman, Argentina 3Russet House, Ryeish Green, Reading, RG7 1ES, UK

The intestinal ecosystem is a complex network of interacting procaryotic (the indigenous gut microflora) and eucaryotic cells. The intestinal flora exerts a strong effect both on gutassociated lymphoid tissue (GALT) activation and development of the regulatory mechanisms (oral tolerance) involved in maintenance of the immune status of the host, and can vary according to the bacterial equilibrium in the gut. Microbial antigens from the digestive flora are the most important antigenic stimulus, and have a considerable influence in the development and functioning of GALT; food antigens also help to maintain these functions. Nutrition is an important factor for the optimal activity of the immune system and both undernutrition or hypernutrition can affect the systemic and mucosal immune response. Thus a delicate balance exists in the intestine among bacterial flora, food antigens and the immune status of the host. Alterations in this dynamic balance, either in the quality of the gut flora the type of food ingested or the intensity of the GALT response effects may have harmful effect on health. The knowledge of the role played by the probiotics and prebiotics in improving the gut flora not only by modifying its composition or its activity but, in the stimulation of the immune system, has stimulated much research in an attempt to develop successful supplements for human use. The different chapters of this book summarize the investigations that have been done on the role of the intestinal microflora, its metabolic activity, its influence on the immune system, the importance of nutrition on the functioning of immune system and how it can be affected by eating disorders or old age. The use of molecular techniques (polymerase chain reaction, PCR, and their modifications) for the identification of bacteria has allowed a better understanding of the composition of the gut microflora than that gained from the traditional techniques using selective bacteriological culture. The use of nucleic acid-based methodologies has considerably improved the identification of the bacterial isolates from faeces. These techniques help us to analyse bacterial communities at the level of genus or species and to understand the way in which they interact. This knowledge also allows us to assess the influence of the gut flora on

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the ontogeny and maturation of the immune system and its role in the incidence of atopic disorders. There is no doubt about the role of the intestinal microflora in physiological and biochemical processes in the gut. The colonic microbiota interacts with the intestine in many ways, it can stimulate peristaltic movement and facilitate the passage of digestive residues through the bowel. Nutritional conditions and stress affect the composition of this intestinal microflora and consequently the metabolic activity of the microorganisms. The most striking effect of diet on bacterial communities in the large intestine can be seen in the microflora of breast- and formula-fed infants, especially in the anaerobic population. Vegetarian diet can affect bacterial metabolism in the large intestine. Intestinal bacteria are a source of vitamin B under some nutritional circumstances, and they participate in the fermentation of complex carbohydrates to short-chain fatty acids (SCFA), protein to peptides and amino acids. If the microbiota is modified, an abnormal colonic fermentation is induced and toxic substances may be produced. The excessive use of antibiotics that modify the composition of gut flora, has led to the use of functional foods such as probiotics (viable microbes) or prebiotics (non viable food components) to improve the intestinal microflora. Now it is known that these functional foods can manage the gut flora and improve resistance to infections, chronic gut disorder, lactose intolerance, food allergy and enhance mineral bioavailability. Both probiotics and prebiotics favour the gut function and induce beneficial effects in the consumer. Not all of these properties attributed to probiotics or prebiotics are supported by good clinical trial evidence but any adverse effects have not been reported for the continuous use of these functional foods. Bacterial carbohydrate fermentation and SCFA production are influenced by the feeding regime. Some probiotic microorganisms such as Propionibacterium can favour the intestinal metabolism of carbohydrates to SCFA, in the large intestine modifying the risk factor for disease and for disease prevention. The metabolic activity of the intestinal flora can also be managed by addition to the food of probiotic microorganisms. Studies on the strong relationship between microflora, nutrition and their influence on the immune state are essential if we are to fully understand their role in disease resistance. The mechanisms for the induction of the innate and adaptative immunity, the kinetic of the immune response, and the immune regulation of this response are described showing the complex network between cytokines, immune cells and the immune response induced. The role of nutrition in the behaviour of the immune system is extremely important. The influence of micronutrients (minerals: zinc, selenium, copper, iron) and vitamins on the immunocompetence are well demonstrated. Vitamin A deficiency has been associated with an increase of diarrhoea and respiratory infections resulting from a decrease of the immune function: vitamin C is one of the food components with high antioxidant properties, thus in eating disorders such as anorexia or bulimia nervosa it can induce a severe malnutrition mainly affecting cellular immunity rather than humoral immunity and the risk of infections is increased. Protein deficiency will also affect immunity, but here, the intestinal mucosal response will be severely affected. Structural changes in the villi and microvilli of the small intestine, in the thymus and in the

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number and activity of immune cells (lymphoid tissues atrophy) are seen. For the recovery of all gut functioning, a renutrition diet with the addition of probiotics could be used. However, only a good knowledge of mucosal immune system functioning will allow us to develop diets for reversing the malnutrition process, ameliorating gut immunity and decreasing the severity of infections. Observations support the idea that the indigenous gut microflora influences the long-term duration of oral tolerance and that Gram-negative bacteria are involved in the maintenance of oral tolerance. The activation or tolerance to an antigen can be induced, depending on the conditions under which it is introduced. In recent years experimental evidence has shown that a T cell CD4+ can play a major role in the induction of tolerance. Other mechanisms have been proposed and the dose of antigens is crucial to produce oral tolerance through the induction of cytokine suppressors. How non-pathogenic probiotic bacteria can influence the activation or down regulation of the immune response is also reviewed, although little is known about the immunomodulatory molecules produced or associated with the cell wall of the probiotic bacteria. Many questions still remain to be answered relating to the mechanisms by which the probiotics work and the signals which modulate the immune system. Over the last twenty years there has been an increase in the incidence of allergic diseases. Environmental changes may play a role, as with disturbances of the intestinal flora due to early dietary diversification. Food allergens are polypeptides, proteins or glycoproteins, thus the reaction is against the digested end product of food able to elicit an immune response. Clinical manifestations include gastrointestinal symptoms. Food allergy is generally not permanent, it can be avoided in infants by exclusive breast feeding or by supplying hydrolysed formula. Even though food allergy is not beneficial for health, the influence of nutrition on it illustrates its affect on the immune system. Bacteria have been linked to cancer by induction of chronic inflammation or by production of toxic bacterial metabolites. Mechanisms by which diet is associated with colon cancer are still not well understood. However, many of these mechanisms involve the metabolic activity of the colonic bacteria. Probiotics and prebiotics can reduce colon cancer risk by modulating gut flora composition or metabolic activity. The combination of probiotic and prebiotic is termed synbiotic and they have been reported to have a more potent anticancer effect, thus the carcinogenesis can be modulated by nutritional and microbial components. Nutritional status plays a major role in a healthy immune system. Many factors can contribute to a decline in immunocompetence such as the failure of defence mechanisms in elderly persons. The immune system does not function efficiently in old people compared with the young. In elderly persons innate and adaptive immunity are affected, dietary supplements (vitamins and minerals) can enhance the immune response but only for few days. The immune function in old age is also due to a thymic involution affecting the activity of T cells. B cells are also affected in the elderly. Elderly persons are at an increased risk of developing protein-energy malnutrition that further impairs the functioning of the immune system. Nutritional strategies are necessary to improve immunocompetence, health and quality of life in the aged. This book focuses on many points of discussion about the role of microflora, its influence in the gut immune system and modulation of the immune response. These factors are also

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important in the prevention of carcinogenesis and in good functioning of the immune system in maintenance of health even in the aged. The role of probiotics and prebiotics in improving health and in the recovery from malnutrition are also discussed. This is a contribution to our understanding of the complex intestinal ecosystem bringing together the three essential aspects: microflora, nutrition and immunity as indicators of good health.

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Index Acetate production 79 Acquired immunity 168, 258 Adhesion molecules 120, 184 Aging and immune function 238 Allergic diseases 201 Allergic to food 201 Amines 37, 213 Amino acid fermentation 35, 219 Animal models 103, 222, 251 Antibody (see Immunoglobulins) Antigen uptake 159 presenting cells 181 Antioxidant vitamins 143, 253 Apoptosis 88, 184, 211 Bacterial IPS 179 B cells 104, 117, 138, 161, 180, 244 Bifidobacteria 4, 28, 54, 91. 167, 188 Body weight 146 Branched chain fatty acids 36 Breast feeding 27, 53, 204 Butyric acid 39, 53, 219 Calcium 69, 168, 179, 209, 239 Cancer 53, 69, 141, 209, 220 Cellular immunity 122, 141, 201, 250 Cholesterol 67, 88 Complement system 12 Cytokines 100,124, 146,158,184,201, 243 Dendritic cells 156, 179 Diarrhoea 27, 86, 167, 203 DNA-RNA hybridisation 4 Electrophoresis 5

Endocytosis 240 Endothelial cells 35, 186, 240 Enteral nutrition 168, 252 Enzyme activities 211, 241 Essential amino acids 162 Faecal flora 40, 84, 217 Fatty acid profiles 27 Fibre diets 32, 219 Folic acid 209 Food antigens 160 Food allergens 202 Functional foods 52 Gastroenteritis 67 Germfree animals 14, 26, 56, 104, 212 Growth factors 168 Gut microflora 55, 210 Glycoprotein 28, 102, 202 Humoral immunity 125, 161, 255 Hydrolases 31, 78 Hypersensitivity response 201 Immune response 14, 67, 140 Immunodeficiency 140, 168 Immunoglobulins 105, 162 Inflammation 102, 138, 181, 219, 258 Innate immune response 100, 159, 179, 238 Interleukins 158 Intestinal epithelial cells 86, 156, 184 Intestinal microflora 8, 26, 35, 83, 210 Iron deficiency 142, 253 Lactic acid bacteria 54, 91, 167, 190, 217 Lactobacillus 9, 28, 54, 90, 167, 187, 210

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Index

Lamina propria 119, 160, 181 Leptin 147 Lipid metabolism 12, 147

Renutrition diet 168 Respiratory burst 240 Ribotyping 2, 6

Macrophages 179, 238, 242 Malnutrition 138, 146, 161, 246 Methanogenic bacteria 30, 79 Micronutrient intakes 139 Mineral absorption 69 Mutagens 29, 210

Secretory IgA (S-IgA) 158 Selectins 101 Selenium 141, 255 Short-chain fatty acids 12, 39, 79, 218 Soybean-oligosaccharides 62 Starch fermentation 42

Nitroreductase 83, 211 Non-digestible carbohydrates 54, 68, 209 Non-starch polysaccharides 55, 78, 209 Nutrients 15, 138, 161

Targeted prebiotics 65 T cells 104, 109, 141, 161, 180, 202, 244 Thymic function 115, 182, 244 Tolerance 89, 131, 160, 182 Toll-like receptors 13, 156, 179 Toxins 36, 56, 167, 171, 190, 210, 238 Translocation 168

Oligosaccharides 27, 54, 59, 78, 192, 221 Oligonucleotide probes 2, 58 Oral tolerance 160 Peptides 31, 179, 219 Peyer's patches 9, 119, 157, 185 Phagocytosis 39, 138, 240 Phenols 36, 60, 78, 219 Polysaccharides 40 Prebiotics 52, 59, 82, 220 Probiotics 6, 52, 55, 89, 166, 187, 220 Propionate 12, 39, 53, 83 Protein energy malnutrition 160, 243 Protein kinase C 249 Proteolytic microorganisms 53

Urinary sulphate 30 Ulcerative colitis 53, 167, 182, 219 Uronic acids 83 Vitamins 30, 138, 162, 255 Western diet 9, 29, 61, 218 Xylose 32 Yoghurt 56,90, 167, Zinc 139, 163, 253

Quorum sensing 15, 194