key: cord-0040101-zy10cxvm authors: Chheda, Sadhana; Keeney, Susan E.; Goldman, Armond S. title: Immunology of Human Milk and Host Immunity date: 2013-09-18 journal: Fetal and Neonatal Physiology DOI: 10.1016/b978-0-7216-9654-6.50166-1 sha: 7e9be8ea0a11448758a8bbeb795e133da555bd43 doc_id: 40101 cord_uid: zy10cxvm nan Paul Ehrlich reported in 1891 the first evidence that immunity could be transmitted through breast-feeding in experimental animals. 1, 2 Few organized studies of that possibility in humans were reported, however, until the 1920s, when Woodbury 3 and Grulee and colleagues 4, 5 in separate studies found that the incidence and severity of diarrheal diseases were much lower in breast-fed than cow's milk-fed infants.Those observations were confirmed repeatedly in developing and industrialized countries. [6] [7] [8] [9] [10] [11] [12] [13] [14] Furthermore, it was found that the specificity of the protection provided by breast-feeding encompassed bacterial and viral enteric infections due to pathogens such as Shigella species, [8] [9] [10] Salmonella species, 9 Escherichia coli, 9 Vibrio cholerae, 11 rotavirus, [12] [13] [14] and poliovirus. 15 The following explanations for the protection provided by breast-feeding were advanced: 1. Because human milk was less contaminated with pathogenic microorganisms than formula feedings, fewer infections would be transmitted to the breast-fed infant. 2. Because of the increased spacing of births in lactating women due to contraceptive effects of lactation, the density of children susceptible to common contagious agents would be lower in families where breast-feeding was practiced. 16 3. In addition, infants who were breast-fed would be less likely to be in group-care facilities and thus would be less exposed to children harboring microbial pathogens. These propositions were reasonable, but they did not completely explain the protection provided by breast-feeding. In that respect, Wyatt and Mata from Guatemala found that manifestations of infection in breast-fed infants were low even when bacterial enteropathogens such as Shigella were recovered from the nipples and areola of the breast of the mother. 6 Furthermore, some evidence emerged that breast-fed infants may be more resistant to certain common respiratory infections. [17] [18] [19] [20] Despite those earlier studies, the concept, characteristics, and many of the components of the immune system in human milk were not revealed until the last half of the 20th century. 21 By 1973, the following general features of the antimicrobial agents of the immune system in human milk were evident: 22 1. Certain postnatal developmental delays in the immune system are replaced by those same agents in human milk. 2. Other postnatal delays in the immune system are offset by dissimilar agents in human milk. 3. Agents in human milk initiate or augment functions that are otherwise poorly expressed in the infant. 4. Agents in human milk alter the physiologic and biochemical states of the alimentary tract from one suited for fetal life to one that is appropriate for extrauterine life. 5. Defense agents in human milk protect without provoking inflammation, and some agents in human milk inhibit inflammation. 6. Defense agents in human milk have an enhanced survival in the gastrointestinal tract of the recipient infant. 7. Growth factors in human milk augment the proliferation of a commensal enteric bacterial flora. The realization of many of those evolutionary outcomes came about as a consequence of the discovery of an expanded immune system in human milk that consisted of not only antimicrobial agents but also of anti-inflammatory 25, 26 and immunomodulating agents. 26 The nature and functions of these agents are described in following sections of this chapter. The physical features, functions, and quantities of antimicrobial agents in human milk are summarized in Table 163 -1 and are discussed in the following sections. The principal proteins in human milk that are antimicrobial are secretory immunoglobulin A (IgA) antibodies, other immunoglobulins, lactoferrin, lysozyme, mucins, and lactadhedrin.These proteins, except for immunoglobulins other than secretory IgA, are better represented in human milk than other mammalian milks used in human infant nutrition. The concentrations of IgM are much lower in human milk than in serum. 27 IgM molecules in blood and milk are pentamers. However, unlike serum IgM, some human milk IgM is complexed 163 / Immunology of Human Milk and Host Immunity 1611 to secretory component, and the antibody specificities of human milk IgM may be similar to those of secretory IgA in human milk (see later discussion). IgG is also present in human milk, albeit in modest amounts. 27 All IgG subclasses are represented in human milk, 28 but the relative proportion of IgG4 is higher in human milk than serum. 28 Very little IgD is present in human milk. 29 IgE, the immunoglobulin responsible for immediate hypersensitivity reactions, is essentially absent in human milk. 30 Secretory IgA comprises more than 95% of the immunoglobulins in human milk. 27 This type of IgA consists of two identical IgA monomers united by a 15-kD polypeptide called the joining chain and complexed to a 75-kD glycopeptide, the secretory component. 31, 32 Secretory IgA is assembled when dimeric IgA produced by plasma cells in the stroma of the mammary gland binds to the first domain of polymeric immunoglobulin receptors on the basolateral surface of epithelial cells. 33 Investigations of the unusual specificities of antibodies in human milk were spurred by epidemiologic evidence that human milk protects against common enteric and respiratory infectious pathogens and the discovery of secretory IgA in human milk by Lars Å Hanson. 34, 35 This led to studies of the origins of B cells that are responsible for the production of the immunoglobulin part of those antibodies and mechanism of the assembly of the final molecule, secretory IgA. The specificities of many antibodies were found to be due to immunogen-triggered events in the intestinal tract. 36 It was later ascertained that antigen-stimulated B cells from Peyer patches of the lower small intestinal tract migrated to the mammary gland and that the process was under hormonal control. 37, 38 In addition, a B-cell pathway between lymphoid tissues in the bronchi and the mammary gland was discovered. 39 This process may be controlled by a mucosal adhesion-cell adhesion system (e.g., mucosal addressin cell adhesion molecule, or MAdCAM 40 ), and its counterstructure, α4β 7 integrin, 41 and certain cytokines. During mucosal antigenic stimulation, cytokines released from mononuclear cells in Peyer patches induce local B cells to switch from IgM + to IgA + . [42] [43] [44] [45] These isotype-switched B cells then migrate sequentially into local intestinal lymphatic channels and lymph nodes, the thoracic duct, and the vascular circulation. Because of lactogenic hormones and other influences that are poorly understood, the cells move from the vascular compartment to the lactating mammary gland. These IgA + B cells differentiate to IgA producing-secreting plasma cells that remain in the lamina propria of the mammary gland. In keeping with other mucosal lymphoid tissues, IgA dimers produced by plasma cells in the mammary gland principally contain λ-light chains, whereas κ-light chains predominate in immunoglobulins in human sera. 46 IgA dimers produced by those plasma cells bind to polymeric immunoglobulin receptors on the basolateral external membranes of mammary gland epithelial cells. 31, 32, 47, 48 The resultant receptor-dimeric IgA complex is transported to the apical side of and Entameoba histolytica the cell where the original intracytoplasmic portion of the receptor is cleaved away. The remaining molecule, secretory IgA, is secreted into milk. Thus, enteromammary and bronchomammary pathways protect the immunologically immature infant against the pathogens in the environment of the dyad (Table 163-2) .This is important given that secretory IgA antibodies and the antigenbinding repertoire of immunoglobulin molecules are not optimally produced during early infancy. 49 Furthermore, some secretory IgA molecules in human milk are antiidiotypic antibodies and therefore may operate as immunizing agents. 50 The quantity of secretory IgA declines as lactation proceeds, but a considerable amount of secretory IgA is transmitted to the recipient infant throughout breast-feeding. [51] [52] [53] [54] The concentrations of secretory IgA in human milk are highest in colostrum 51 and then gradually decline to a plateau of about 1 mg/mL. 52 The approximate mean intake of secretory IgA per day in healthy fullterm breast-fed infants is approximately 125 mg/kg per day at 1 month and approximately 75 mg/kg per day by 4 months. 54 Secretory IgA is resistant to intestinal proteases such as pancreatic trypsin. 55 Although the first IgA subclass, IgA1, is susceptible to bacterial proteases that attack the hinge region of the molecule, 56 the second subclass, IgA2, is resistant to those proteases and is disproportionally increased in human milk. 27 Furthermore, secretory IgA antibodies against these bacterial IgA proteases are found in human milk. 56 In keeping with those observations, the amount of secretory IgA excreted in the stools of low birth weight infants fed human milk was about 30 times that in infants fed a cow's milk formula. 57 In addition, the urinary excretion of secretory IgA antibodies in the recipients increased as a result of human milk feedings. 58, 59 The origin of secretory IgA antibodies in the urine of infants fed human milk is undetermined. It is improbable that they are from human milk because there is no known mechanism for the transport of the entire molecule from the gastrointestinal tract to the blood or from blood to urine. Lactoferrin is a single-chain glycoprotein with two globular lobes, each of which displays a site that binds ferric iron. 60 In over 90% of lactoferrin in human milk, 61 iron-binding sites are available to compete with siderophilic bacteria and fungal enterochelin for ferric iron. [62] [63] [64] [65] The chelation of iron disrupts the proliferation of those microbial pathogens. In addition, the chelation is enhanced by bicarbonate, the principal buffer in human milk. Lactoferrin also kills some bacteria 66 and fungi, 67 and the responsible part of the molecule (lactoferricin) 67, 68 acts by damaging outer membranes of pathogens. 68 The action is dependent on Ca 2+ , Mg 2+ , or Fe 3+ but not on the ability to chelate Fe 3+ . 68 There is also evidence that lactoferrin inhibits certain viruses in a manner that is independent of iron chelation. [69] [70] [71] [72] The mean concentration of lactoferrin in human colostrum is between 5 and 6 mg/mL. 51 As the volume of milk production increases, the concentration falls to about 1 mg/ml at 2 to 3 months of lactation. 52 The mean intake of milk lactoferrin in healthy breast-fed full-term infants is about 260 mg/kg per day at 1 month and 125 mg/kg per day by 4 months. 54 Because of resistance of lactoferrin to proteolysis, 73 the excretion of lactoferrin in the stools is higher in infants fed human milk than in those fed a cow's milk formula. 57, 74, 75 The quantity of lactoferrin excreted in stools of low birth weight infants fed a human milk preparation is approximately 185 times that excreted by infants fed a cow's milk formula. 57 That estimate, however, may be too high because of the presence of immunoreactive fragments of lactoferrin in the stools of human milk-fed infants. 76 There is also a significant increment in the urinary excretion of intact and fragmented lactoferrin as a result of human milk feedings. 57, 76 Stable isotope studies suggest that those increments in urinary lactoferrin and its fragments originate from ingested human milk lactoferrin. 77 Lysozyme, a 15-kD single chain protein, lyses susceptible bacteria by hydrolyzing β-1,4 linkages between N-acetylmuramic acid and 2-acetylamino-2-deoxy-D-glucose residues in cell walls. 78 High concentrations of lysozyme are present in human milk during all stages of lactation, 51-54 but longitudinal changes in quantities of lysozyme during lactation are unlike most other immune factors in human milk. The mean concentration of lysozyme is about 70 µg/ml in colostrum, 51 20 µg/ml at 1 month, and 250 µg/ml by 6 months of lactation. 52 The approximate mean daily intake of milk lysozyme in healthy full-term, completely breast-fed infants is 3 to 4 mg/kg per day at 1 month and 6 mg/kg per day by 4 months of age. 54 The high content of lysozyme in human milk and its in vitro resistance to proteolysis are in keeping with an eightfold increase in the amount of lysozyme excreted in the stools of low birth weight infants fed human milk compared with findings in infants fed a cow's milk formula. 57 However, in contrast to secretory IgA and lactoferrin, the urinary excretion of this protein is not increased in infants fed human milk. 59 The lysozyme C gene gave rise some 300 to 400 million years ago to a gene that codes for α-lactalbumin, a protein expressed only in the lactating mammary gland.The protein is a component of lactose synthetase. It is of interest that three domains of this evolutionary descendant of lysozyme are antibacterial. 79 Furthermore, multimeric α-lactalbumin may be antineoplastic. 80 Fibronectin, a high molecular weight protein that facilitates the uptake of many types of particulates by mononuclear phagocytes, is present in human milk (mean concentration in colostrum, 13 µg/ml). 81 The in vivo effects of this broadspectrum opsonin in human milk are not known. All components of the classical and alternative pathways of complement are in human milk, but the concentrations of these components, except for C3, are low. 82, 83 Milk mucins are high molecular weight proteins that are greatly glycosylated. 84 About two-thirds of the mucin in human milk is membrane bound.The concentration of mucin in human milk is between 50 and 90 mg/ml. A number of milk mucins have been identified.The most prominent one is MUC1. MUC1 has molecular weights between 250 and 450 kDa and is primarily bound to membranes of milk fat globules. In that respect, human milk fat globules and mucin from their membranes inhibit the binding of S-fimbriated E. coli to human epithelial cells. 85 The in vivo fate of ingested MUC1 has been investigated. It has been found to be resistant to intragastric digestion in preterm infants. 86 Major fragments of MUC1 are detected in feces of breastfed infants. 87 Furthermore, mucins from such feces are more able to inhibit bacterial adhesion than feces from formula-fed infants. 88 It was originally reported that human milk mucin defended against rotavirus, the most common cause of infectious enteritis in human infants, in an experimental murine model. 89 Rotavirus bound not only to the milk-mucin complex, but also to a 49-kDa component of the complex. The active component was later found to be a separate glycoprotein that was designated as lactadherin. 90 Like human MUC1, lactadhedrin is resistant to intragastric digestion. 91 Oligosaccharides in human milk are produced by glycosyltransferases in the mammary gland. Some of these abundant compounds are receptor analogues that inhibit the binding of certain enteric or respiratory bacterial pathogens and their toxins to epithelial cells. [92] [93] [94] [95] Many types of oligosaccharides have been identified in human milk, and new types are still being recognized. 96, 97 Oligosaccharides in human milk are different than those found in commercial milk formulas. Although the quantities of total gangliosides in human and bovine milk are similar, the relative frequencies of each type of ganglioside in milk from these two species are distinct. For example, much more monosialoganglioside 3 and GM 1 are found in human than bovine milk. [97] [98] [99] The chemistry of these compounds dictates the specificity of their binding to the adherence structures of bacterial pathogens. For example, GM 1 gangliosides are receptor analogues for toxins produced by V. cholerae and E. coli, 93 whereas the globotriaosylceramide Gb3 binds to the β subunits of Shigatoxin. 100 A fucosyloligosaccharide inhibits the stable toxin of E. coli, 94 whereas a different one inhibits Campylobacter jejuni. 101 Oligosaccharides in human milk also interfere with the attachment of Haemophilus influenzae and Streptococcus pneumoniae. 95 In that regard, G1cNAc(β1-3) Gal-disaccharide subunits block the attachment of S. pneumoniae to respiratory epithelium. In vivo animal experiments also suggest that oligosaccharides and glycoconjugates in human milk protect against certain enteric bacterial infections. 102 In that regard, certain human milk oligosaccharides survive passage through the alimentary tract 103 and some of the absorbed carbohydrate is then excreted into the urinary tract. 104 Sugars that are present in several glycoconjugates including mucins, lactadherin, and secretory IgA also interfere with the binding of bacterial pathogens to epithelial cells. 105 In addition to the direct antibacterial effects of the carbohydrates in human milk, nitrogen-containing oligosaccharides, glycoproteins, and glycopeptides in human milk are growth promoters for Lactobacilli and Bifidobacilli. 106, 107 For example, the growth-promoter activity associated with caseins may reside in the oligosaccharide moiety of those complex molecules. 107 These factors are responsible to a great extent for the predominance of Lactobacilli and Bifidobacilli in the bacterial flora of the large intestine of breast-fed infants found in most studies. The bacteria produce large amounts of acetic acid, which aids in suppressing multiplication of enteropathogens. It has also been reported that Lactobacilli strain GG aids in the recovery from acute rotavirus infections 108 and may enhance the formation of specific IgG, IgA, and IgM antibodies. 109 In addition, enteric commensal bacteria may stimulate the production of low molecular weight, antibacterial peptides, such as defensins. 101 These types of defense mechanisms may contribute to the comparative 163 / Immunology of Human Milk and Host Immunity 1613 paucity in stools of breast-fed infants of bacterial pathogens most often found in urinary tract infections (P-fimbriated E. coli). 111 Fatty acids and monoglycerides generated by the enzymatic digestion of lipid substrates in human milk disrupt enveloped viruses. [112] [113] [114] These antiviral lipids may aid to prevent coronavirus infections of the intestinal tract 115 and defend against intestinal parasites such as Giardia lamblia and Entameoba histolytica. 116, 117 Monoglycerides from milk lipid hydrolysis also inactivate certain gram-positive and gram-negative bacteria. 118 The in vivo hydrolysis of ingested milk lipids in early infancy occurs because of two enzymatic mechanisms.The first is due to the action of lingual lipase and the second is due to the activation of human milk bile-salt stimulated lipase in the duodenum. Thus, it is likely that the products of lipid digestion contribute to the defense of the breast-fed infant against enteric infections. Living leukocytes are found in human milk. 119 In contrast to B cells that transform into plasma cells that remain sessile in the mammary gland, other leukocytes attracted to the site traverse the mammary epithelium and become part of the milk secretions. The highest concentrations of leukocytes in human milk occur in the first few days of lactation (1-3 × 10 6 /ml). 120 The several types of leukocytes and their major features follow. The relative frequencies of T cells and B cells among lymphocytes in early human milk secretions are 83% and 6%, respectively. 121 The small number of natural killer (NK) cells in human milk 121 is in keeping with the low cytotoxic activity of human milk leukocytes. 122 The small number of B cells is a reflection that most B cells that enter the lamina propria of the mammary gland transform into sessile plasma cells. Both CD4 + (helper) and CD8 + (cytotoxic/suppressor) T-cell subpopulations are present in human milk, 121, 123 but compared with human blood T cells, the proportion of cytotoxic/suppressor T cells (CD8 + ) in human milk is increased. 121 Virtually all CD4 + and CD8 + T cells in human milk bear the CD45 isoform, CD45RO, that is indicative of cellular activation. 121, 124 In addition, an increased proportion of the T cells displays other phenotypic markers of activation. 121, 124 T cells in human milk produce certain cytokines such as interferon-γ, 124 macrophage migration inhibitory factor, 120 and a monocyte chemotactic factor. 120 The production of interferon-γ is consistent with the CD45RO phenotype of T cells in human milk 121, 123 and the finding that CD45RO + T cells are the major source of that cytokine. 121 Additional cytokines are produced by human milk leukocytes, 124 but the extents of their production and secretion have not been determined. Neutrophils and macrophages in human milk are laden with milk fat globules and perhaps with other membranes that have been phagocytized. Because of these intracytoplasmic bodies, the cells are difficult to identify by common staining methods.They can be identified however by their content of myeloperoxidase (in the case of neutrophils), 120 nonspecific esterase (in the case of macrophages), 120 or by the surface expression of CD14 (in the case of macrophages). 125 Both types of cells in human milk are phagocytic.There is some evidence that the respiratory burst occurs in milk macrophages after stimulation, 126 but their intracellular killing activities appear to be reduced.The macrophages have also been found to process and present antigens to T cells. 127 After exposure to chemoattractants, human milk neutrophils (compared with blood neutrophils) do not increase their adherence, polarity, directed migration, 128 or deformability. 129 Some of those features appear to be due to agents in human milk. For example, the decreased calcium influx by human milk neutrophils has been duplicated by incubating blood neutrophils in human milk. 130 Unlike human milk neutrophils, the motility of macrophages in human milk is increased compared with their counterparts in blood. 131 These features of neutrophils and macrophages in human milk appear to be due to cellular activation, because these cells display phenotypic markers of activation including an increased expression of CD11b/CD18 and a decreased expression of CD62L (L-selectin). 125 The in vivo fate and role of human milk leukocytes in defense of the infant are not well understood.The area about the upper alimentary and respiratory tracts seems to provide potential sites for human milk leukocytes to enter. It is of considerable interest that small numbers of memory T cells are detected in blood in infancy. 132 Thus, it may be possible that maternal memory T cells in milk compensate for the developmental delay in their production in the infant. There is evidence from experimental animal studies that milk lymphocytes enter tissues of the neonate, 120 but that has not been demonstrated in humans.There are also reports of transfer of cellular immunity by breastfeeding. 133 It will be important to ascertain whether those reports will be verified by testing for cellular immunity against many different antigens in young infants who have or have not been breast-fed. Inflammatory agents and systems that give rise to them are poorly represented in human milk. 25 These include (1) the coagulation system, (2) the kallikrein-kininogen system, (3) major components of the complement system, (4) IgE, (5) basophils, mast cells, eosinophils, and (6) cytotoxic lymphocytes. Certain proinflammatory cytokines (see subsequent discussion) are found in human milk, but there is no clinical evidence that they generate inflammatory processes in the recipient. In contrast to the paucity of inflammatory agents, human milk contains a host of anti-inflammatory agents. 25 They include (1) factors that promote the growth of epithelium and thus strengthen mucosal barriers, (2) antioxidants, (3) agents such as lactoferrin that interfere with certain complement components, 25, 134 (4) enzymes that degrade mediators of inflammation, (5) protease inhibitors, 135 (6) agents that bind to substrates such as lysozyme to elastin, 136 (7) cytoprotective agents such as prostaglandins E 1 , E 2 , and F 2α , 137, 138 and (8) agents that inhibit the functions of inflammatory leukocytes (Table 163-3) . 25 Like the antimicrobial factors, many of these factors are adapted to operate in the hostile environment of the alimentary tract. The main antioxidants in human milk include an ascorbate-like compound, 139 uric acid, 139 α-tocopherol 140,141 and β-carotene. 140, 141 In fact, blood levels of α-tocopherol and β-carotene are higher in breast-fed than formula-fed infants not supplemented with those agents. 141 Mucosal growth factors in human milk include epithelial growth factor, 142 lactoferrin, 143 cortisol, 144 and polyamines. 145, 146 Other hormones and growth factors in human milk 147 may also affect the growth, differentiation, and turnover of epithelial cells. These agents may therefore limit the penetration of free antigens and pathogenic microorganisms and affect other barrier functions of the intestinal tract. In keeping with that notion, there are significant differences between the biophysical and biochemical organization and functions of mucosal barriers in adults and 1614 XX / Developmental Immunobiology neonates. 148, 149 Furthermore, maturation of those functions may be accelerated by human milk. 150, 151 Enzymes in human milk degrade inflammatory mediators that may damage the gastrointestinal tract. In that respect, plateletactivating factor (PAF) plays a role in an intestinal injury in rats induced by endotoxin and hypoxia. 152 Furthermore, an acetylhydrolase that degrades PAF is present in human milk, 153 and the production of human PAF-acetylhydrolase is developmentally delayed. 154 Published results of investigations also indicate that human milk feedings lessen intestinal permeability in young infants. [155] [156] [157] Three sets of observations provide the basis of the concept of immunomodulating agents in human milk: 1. Epidemiologic investigations suggest that older children who were breast-fed during infancy may be at less risk for developing certain chronic diseases that are mediated by immunologic, inflammatory, or oncogenic mechanisms.The diseases in question are type 1 diabetes mellitus, 158 lymphomas, 159 acute lymphocytic leukemia, 160 and Crohn's disease. 161 Although preventing or lessening infections by antimicrobial agents or by anti-inflammatory agents in human milk may have long-term consequences, agents that influence the development of systemic or mucosal defenses of the infant may also be responsible for those possible long-term effects. infants cannot be accounted for by passive transfer of those substances from human milk. Breast-feeding primes the recipient to produce higher blood levels of interferon-α in response to respiratory syncytial virus infections. 162 In addition, increments in blood levels of fibronectin achieved by breast-feeding cannot be accounted for by the amounts of that protein in human milk. Moreover, breast-feeding leads to a more rapid development of systemic 163 and secretory 163, 164 antibody responses and of secretory IgA in external secretions [57] [58] [59] including urine, 58, 59 which is far removed from the route of ingestion. Therefore, those increments are not due to absorption of those same factors from human milk. 3. The third line of evidence is the discovery that all leukocytes in human milk are activated (see previous section on leukocytes). Investigations revealed that human milk enhances the movement of blood monocytes in vitro. In addition, much of that motility was abrogated by antibodies to tumor necrosis factor-α (TNF-α). 165 Subsequently,TNF-α in human milk was detected immunochemically. 166 Many other cytokines have been found in human milk. They include Th1 cytokines such as interferon-γ, 167 interleukin (IL)-12, 168 and IL-18 169 ; proinflammatory cytokines including IL-1β) 170 and IL-6 171, 172 ; chemotaxins including IL-8, 173 regulated on activation, normal T expressed and secreted (RANTES), 174 and eotaxin 174 ; antiinflammatory agents such as transforming growth factor-β (TGF-β) 173, 175 and IL-10 176 ; and the cellular growth factors EGF, 142 granulocyte colony-stimulating factor (G-CSF), 177 macrophage-CSF, 178 hepatic growth factor, 179 and erythropoietin 180 (Table 163-4 ).There are controversies concerning the quantities of some of these agents in human milk. The discrepancies between the results of some of the studies may depend on differences in storage conditions of the specimens and the types of immunoassays. The sites and extents of their effects on the recipient infant are not determined. Several other immunomodulating agents are in human milk including β-casomorphins, 181 prolactin, 182, 183 antiidiotypic antibodies, 50 α-tocopherol 140, 141 and a host of nucleotides that enhance NK-cell, macrophage, and Th1-cell activities. [184] [185] [186] As previously mentioned, seven somewhat overlapping evolutionary outcomes concerning the relationships between the immune status of infants and defense agents in human milk have been recognized. 50, 51 In respect to the first evolutionary outcome, many aspects of the human immune system are incompletely developed at birth, and the immaturity is most marked in very low birth weight infants. These developmental delays include (1) the mobilization and function of neutrophils, 187 (2) the production of lysozyme 188 and secretory IgA 189, 190 at mucosal sites, (3) memory T cells that bear CD45RO, 135 (4) the complete expression of the antibody repertoire, 191 and (5) the production of certain cytokines including TNF-α, 192, 194 interferonγ, 194, 193 G-CSF, 196 GM-CSF, 197 and IL-3. 196 Many of those developmentally delayed defense factors are well represented in human milk (Table 163-5) . For example, secretory IgA antibodies in human milk compensate for the low production of secretory IgA at mucosal sites during early infancy. It is also important that the antibody response achieved through this pathway is polyclonal and is directed against not only protein, but also polysaccharide antigens, because infants display a more restricted clonality 198 and do not mount an IgG antibody response to polysaccharide antigens. 199 The problem has been modified by the introduction of conjugate vaccines. Even so, the 163 / Immunology of Human Milk and Host Immunity 1615 antibody response to conjugate vaccines is higher in breast-fed than cow's milk-fed infants. 200 An additional example is the interrelationship between the amount of lysozyme produced by the infant and the quantity secreted into milk. Indeed, the necessity of high lysozyme levels in human milk is coupled to the low production of the protein by mucosal cells during infancy. 188 It is likely that the attainment of normal intraluminal concentrations of lysozyme in infancy is dependent on breast-feeding.This is in keeping with the finding of higher lysozyme activities in stools of breast-fed than in nonbreast-fed infants. 57 The potential in vivo effects of immune factors in human milk in the recipient infant depend on the survival of those agents. Although it may be argued that defense agents in human milk would be destroyed by the digestive processes in the gastrointestinal tract, many of these agents may be bioactive in the alimentary and respiratory tracts for the following reasons: 1. Protein components may affect the epithelium, leukocytes, or other cells of proximal parts of the alimentary or respiratory tracts where proteolytic enzymes are not produced. 2. Ingested proteins may escape intragastric-intraduodenal digestion because of developmental delays in the production of gastric HCl and pancreatic proteases. 201 This resistance to digestion may be augmented by the protection provided by the buffering capacity of human milk that shields some acid-labile components of milk, antiproteases in human milk, 135 inherent resistance of many defense agents in human milk to digestive processes, and the protection against digestion of some defense agents in human milk because they are compartmentalized. 166, 172 In that respect, much of the TNF-α in human milk is bound to soluble receptors. 202 This thesis is borne out as previously discussed by an increased survival of certain human milk defense agents in the alimentary tract of the recipient infant. Maturational delays of the immune system are generally more profound in premature infants. Furthermore, the potential immunologic problems are compounded by the shortened duration of placental transfer of IgG to the fetus. 203 That predisposes premature infants to certain opportunistic infections. Moreover, major medical problems during the newborn period including pulmonary diseases, 204 nutritional imbalances, and invasive clinical procedures increase the risks of premature infants to infections. Interferon-␥ T-helper 1 cytokine-macrophage activator Interleukin-1␤ Activates T cells and macrophages Interleukin-6 Enhances IgA production Interleukin-8 Chemotaxin for neutrophils and CD8 + T cells Interleukin-10 Th2 cytokine Inhibits production of many pro inflammatory cytokines Interleukin-12 Th1 cytokine Enhances production of interferon-␥ TNF-␣ Enhances production of polymeric Ig receptors TGF-␤ Enhances isotype switching to IgA + B cells G-CSF Increases granulocyte (neutrophil) production M-CSF Increases monocyte production G-CSF = granulocyte colony stimulating factor; M-CSF = monocyte colony stimulating factor; TGF-␤ = transforming growth factor-␤; TNF-␣ = tumor necrosis factor-␣. PAF-AH = platelet activating factor-acetylhydrolase;TNF-␣ = tumor necrosis factor-␣ Milk from women who have delivered prematurely contains many of the same antimicrobial factors that are found in milk from women who have delivered after a full-term pregnancy. 205 These include secretory IgA, lactoferrin, and lysozyme.The concentrations of those defense agents are higher in preterm than term milk.Those higher concentrations may be in large part due to a lower volume of milk produced by women who have delivered prematurely.That may not be the total explanation for the higher concentrations in that the patterns of the concentrations of some of the antimicrobial factors in preterm and term milk are not exactly the same. 205 Moreover, the concentrations of most anti-inflammatory and immunomodulating factors in preterm milk have not been established. In addition to the protection against enteric infections and respiratory infections such as otitis media, there are several indications that human milk feedings protect premature infants against systemic infections that are more prone to occur in immature infants. Winberg and his colleagues in Sweden 206 reported that the risk of bacterial sepsis was less in premature newborn infants who were fed human milk. These observations were confirmed by Yu and co-workers in Australia 207 and Nayaryanan and her associates in India, 208 who found that supplemental feedings of expressed human milk were associated with a reduced frequency of infections in low birth weight infants. Human milk also protects against many cases of necrotizing enterocolitis (NEC). 209 The factors in human milk that are responsible for this protection remain to be elucidated, but evidence from human and experimental animal studies suggests that IgA, 210 erythropoietin, 211,212 PAF-acetylhydrolase, 153 and IL-10 213 are likely possibilities. In each case, there is a developmental delay in the production of the suspected factor, and the agent in question is well-represented in human milk. Two contrasting experimental animal models of cytokine gene deficiency suggest that anti-inflammatory cytokines in human milk may prevent disorders due to inflammatory processes. Mice homozygous for the TGF-β1 null gene display spontaneous, infiltrations of macrophages and T cells in many organ sites; the lungs, heart, and salivary glands are most prominently involved. [214] [215] [216] Furthermore, there is experimental evidence that the effects of the TGF-β1 deficiency are mitigated by the ingestion of that cytokine in murine milk. 216 In the second animal model, a targeted IL-10 gene deletion was engineered in mice. In those IL-10-deficient animals, a fatal enterocolitis began directly after weaning, and it was dependent on establishment of an enteric bacterial flora. 213 The enterocolitis had some features of Crohn's disease and NEC. Much of the enterocolitis in those animals was prevented by intraperitoneal injections of IL-10 given at the start of weaning. 217 Although it has not been established whether human milk feedings protect against the pulmonary and vascular effects of hyperoxia, some experimental evidence suggests that one of the anti-inflammatory components of human milk, α 1 -antitrypsin, prevents many of those features in hyperoxic neonatal rats including elevations in pulmonary elastolytic activity. 218 The possible effects of human milk upon the development of atopic diseases have been investigated by many groups, but there is no consensus whether breast-feeding protects against those disorders, 219 except for atopic dermatitis 220 or when food allergens are avoided by complete breast-feeding. Much of the disagreement is probably due to confounding variables including variations in the genetic predisposition to atopic disorders, the sufficiency of breast-feeding, dietary exposures not appreciated by the parents, and exposures to inhalant allergens or irritants that might lead to lung damage. Furthermore, there is evidence that increased exposures to infectious diseases facilitate Th1 responses that lead to the development of cellular immunity, whereas much lower exposures engender Th2 responses that lead to antibody formation and hence to possible IgE-mediated hypersensitivity.Thus, the effect of breast-feeding on the risk of atopic diseases may well depend on a multiplicity of factors that are not equally represented in all investigated populations. Moreover, the question is complicated by the transmission of foreign food antigens in human milk 221 and the triggering of allergic reactions by those antigens in some recipient infants. 222 Why only a subpopulation of breast-fed infants develops atopic diseases is unknown. To establish whether a breast-fed infant is reacting to a foreign food antigen in human milk, it is necessary to conduct trials of dietary elimination and oral challenge with the food in question in the mother while she is breast-feeding. 223 If those trials suggest that the infant is reacting to a foreign food antigen in human milk, then the problem may be avoided by eliminating the food allergen from the maternal diet. If the food allergen is a basic food such as cow's milk, the woman must have a diet that supplies the correct types and quantities of nutrients to meet the needs of lactation. 20 If long-term elimination is impractical, then breast-feeding may be stopped and the infant tried on a hypoallergenic formula. In addition, the development of allergic disease in breast-fed infants may be due to alterations in the types of fatty acids found in milks produced by mothers of the allergic infants. 224, 225 The influence of human milk feedings upon the rate of rehospitalizations of premature infants was examined in the 1988 National Maternal and Infant Health Survey conducted by The National Institutes of Child Health and Human Development. 226 Although a cause-effect relationship could not be definitively established, the feeding of human milk was an independent predictor of decreased risk for rehospitalization. Thus, human milk feeding may have beneficial effects on the premature infant that extend beyond the initial hospitalization. Human milk contains an array of host resistance factors that are antimicrobial, anti-inflammatory, or immunomodulating. This immune system is adapted to function at mucosal sites and to protect the recipient against a host of infectious and inflammatory processes that are common in the developing infant. In addition, there may be long-term health benefits to the recipient by human milk feedings that apparently are due to alterations in the immune system. The precise ways in which the immunologic agents in human milk protect the child and how those agents interact with the developing immune system of the recipient are not well understood.These research issues will require the coordinated efforts of neonatologists, immunologists, molecular biologists, and other clinical and basic scientists. The lungs are unique internal organs, situated within the body yet interposed between the host and its environment. The development of pulmonary host defense mechanisms capable of restricting the growth of environmental pathogens was therefore an essential step in the evolution of air-breathing animals. To effect gas exchange with the environment, the lungs must be able to buffer the potentially injurious effects to airways and alveoli of multiple substances, including pathogenic organisms, which may be present in the air stream. In a 3.5-kg neonate, with a typical minute ventilation ranging from 100 to 150 ml/(kg • min), this requires the lungs to filter approximately 30 L of inhaled air hourly; a problematic task in that the alveolar surface area requiring protection is 20 times the average neonatal body surface area. 1 Mechanisms must also exist to prevent, or contain, effects of potential pathogens delivered by aspiration of oropharyngeal secretions. Concomitantly, the lungs as reticuloendothelial structures are also responsible for filtering all blood returning to the left atrium via the pulmonary circulation. Thus, the extensive alveolar-capillary membrane, composed of both immune and nonimmune cells, may encounter pathogens by hematogenous routes, as well as by inhalation. Beyond this significant environmental exposure, host defense of the lung presents other unique challenges. 2 Individual alveoli are exposed to the environment in parallel, and thus must be somewhat self-sufficient in initial antigen response. Furthermore, the alveolar-capillary interface, teleologically evolved for gas exchange, offers little barrier to pathogen movement in either direction. Finally, even mild inflammation in this critical location can significantly impair gas exchange, and threaten host survival. Because airways in the lower respiratory tract normally contain few colonies of essentially commensal organisms, evolved pulmonary mechanisms of pathogen containment and clearance, both immunologic and nonimmunologic, are clearly effective. Available pulmonary host defenses can be broadly categorized as either mechanical or immunologic. Examples of mechanical defenses include the larynx and epiglottis (which are anatomi-cally situated to minimize aspiration of oropharyngeal material) airway angulation, mucus secretion, and mucociliary clearance mechanisms, including the cough reflex. These mechanisms result in progressive filtering of about 99% of inhaled particles as they pass through the conducting airways, so that overall level of antigen exposure at a given site is inversely related to its depth within the respiratory tree. Mechanical barrier components of host defense thus minimize "bulk" exposure to pathogens, and antigens, minimizing the frequency of host immune response activation. Available immunologic mechanisms are by recent convention broadly categorized as either innate, or adaptive. Innate immune responses are nonspecific, relying on host recognition of "pathogen-associated molecular patterns," such as peptidoglycans, endotoxin, or fungal mannans. Such foreign patterns are recognized, and ligated, either by soluble bioactive substances within the airway (defensins, collectins) or by pattern recognition receptors on macrophages. Subsequent generation of "early response cytokines" (tumor necrosis factor, interleukin [IL]-1) and chemotaxins (leukotrienes, chemokines, split components of complement) leads to recruitment of additional cellular elements of innate immunity, granulocytes and natural killer cells. By means of concurrent cytokine networking with other cells in the alveolar milieu (including epithelia and fibroblasts), alveolar macrophages activate antigen-presenting cells, and lymphocytes move into the alveolar compartment; recruitment of the specialized lymphocytes, T cells and B cells, heralds the onset of the adaptive immune response. These cells manifest specific receptors somatically generated in response to specific antigens, facilitating immunologic "memory" and long-term cell-mediated and humoral immunity. Adaptive immunity thus initiates a targeted response aimed at containment and clearance of a specific antigen, allowing titration of nonspecific, and potentially host injurious, alveolar inflammation. Because of the anatomic location of elements of pulmonary innate immunity, these responses typically precede those of adaptive immunity. However, complex Experimentelle Untersuchangen über Immunität. I. ueber ricin Experimentelle Untersuchangen über Immunität. II. Üeber abrin The relation between breast and artificial feeding and infant mortality Breast and artificially-fed infants. Influence on morbidity and mortality of twenty thousand infants Breast and artificially-fed infants. A study of the age incidence in the morbidity and mortality in twenty thousand cases Bacteria in colostrum and milk of Guatemalan Indian women Diarrhoeal disease in a cohort of Guatemalan village children observed from birth to age two years Shigella infection in breast-fed Guatemalan Indian neonates The protective effect of human milk against diarrhea: a review of studies from Bangladesh Breast-feeding as a determinant of severity in shigellosis: evidence for protection throughout the first three years of life in Bangladeshi children Protection against Cholera in breastfed children by antibodies in breast milk Rotavirus infection in a maternity unit Effects of antibodies, trypsin and trypsin inhibitors on susceptibility of neonates to rotavirus infections Modulation of rotavirus enteritis during breastfeeding Antipoliomyelitic activity of human and bovine colostrum and milk Breast feeding, birth spacing and their effects on birth survival Breast-feeding protects against respiratory syncytial virus infections Breast-feeding and respiratory syncytial virus infection Protective effect of breastfeeding against infection Infant outcomes. Nutrition During Lactation. P 1953-1956 AS: The immunological system in human milk: the past-a pathway to the future Host resistance factors in human milk Evolution of immunological functions of the mammary gland and the postnatal development of immunity Modulation of the gastrointestinal tract of infants by human milk. Interfaces and interactions. An evolutionary perspective Anti-inflammatory properties of human milk Expression of functional immunomodulatory and antiinflammatory factors in human milk Protein and Non-Protein Nitrogen in Human Milk Local production of IgG4 in human colostrum IgD-a mucosal immunoglobulin? The relative paucity of IgE in human milk Polymeric IgA is complexed with secretory component (SC) on the surface of human intestinal epithelial cells A transmembrane precursor of secretory component. The receptor for transcellular transport of polymeric immunoglobulins Characterization of a critical binding site for human polymeric Ig on secretory component Comparative immunological studies of the immune globulins of human milk and blood serum Immunological characterization of chromatographically separated protein fractions from human colostrum Antibody forming cells in human colostrum after oral immunisation Origin of IgA secretory plasma cells in the mammary gland Hormonal induction of the secretory immune system in the mammary gland Broncho-mammary axis in the immune response to respiratory syncytial virus A tissue-specific endothelial cell molecule involved in lymphocyte homing Expression and function of the MAdCAM-1 receptor, integrin α 4β7, on human leukocytes Mucosal homeostasis: role of interleukins, isotype-specific factors and contrasuppression in the IgA response Control of isotype switching by T cells and cytokines Cytokine regulation of localized inflammation. Induction of activated B cells and IL-6 mediated polyclonal IgG and IgA synthesis in inflamed human gingiva Ig isotype switching in B lymphocytes. The effect of T-cell derived interleukins, cytokines, cholera toxin, and antigen on isotype switch frequency of a cloned B cell lymphoma Sequential assay of human milk immunoglobulins show a predominance of lambda chains Secretory component on epithelial cells is a surface receptor for polymeric immunoglobulins Studies on translocation of immunoglobulins across intestinal epithelium. II. Immunoelectron microscopic localization of immunoglobulins and secretory component in human intestinal mucosa Development of the human antibody repertoire Anti-idiotypic antibodies to polio virus in commercial immunoglobulin preparations, human serum, and milk Human milk banking II. Relative stability of immunologic factors in stored colostrum Immunologic factors in human milk during the first year of lactation Immunologic components in human milk during the second year of lactation Daily ingestion of immunologic components in human milk during the first four months of life Increased resistance of immunoglobulin dimers to proteolytic degradation after binding of secretory component Inhibition of bacterial IgA proteases by human secretory IgA and serum Enhanced fecal excretion of selected immune factors in very low birth weight infants fed fortified human milk Breast feeding increases concentrations of IgA in infants' urine Human milk feeding enhances the urinary excretion of immunologic factors in low birth weight infants Structure of human lactoferrin at 3.1-Å resolution Iron in human milk Iron-binding proteins in milk and resistance of Escherichia coli infection in infants Bacteriostasis of a milk-sensitive strain of Escherichia coli by immunoglobulins and iron-binding proteins in association Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactoferrin and secretory immunoglobulin A isolated from human milk Kinetic effect of human lactoferrin on the growth of Escherichia coli A bactericidial effect of lactoferrin Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment Multiple molecular forms of human lactoferrin. Identification of a class of lactoferrin that possesses ribonuclease activity and lacks iron binding capacity Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts A milk protein lactoferrin enhances human T cell leukemia virus type I and suppresses HIV-1 infection Antiadenovirus activity of milk proteins: lactoferrin prevents viral infection The effect of trypsin and chymotrypsin on the in vitro antimicrobial and iron-binding properties of lactoferrin in human milk and bovine colostrum Characterization and properties of the human and bovine lactotransferrins extracted from the feces of newborn infants The persistence of human milk proteins in the breast-fed infant Molecular forms of lactoferrin in stool and urine from infants fed human milk Origin of intact lactoferrin and its DNAbinding fragments found in the urine of human milk-fed preterm infants. Evaluation of stable isotopic enrichment Mechanism of lysozyme action Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule Multimeric α-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei Plasma fibronectin concentrations in breast fed and formula fed neonates Developmental aspects of complement components in the newborn.The presence of complement components and C3 proactivator (properdin factor B) in human colostrum Complement system in human colostrum: presence of nine complement components and factors of alternative pathway in human colostrum Chemistry of milk mucins and their anti-microbial action Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants Detection of large fragments of human milk mucin MUC1 in feces of breast-fed infants Inhibition of adhesion of S-fimbriated E coli to epithelial cells by meconium, stool of breast-fed and formula-fed infants-mucins are the major inhibitory component Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis Role of human-milk lactadhedrin in protection against symptomatic rotavirus infection Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants Inhibition of bacterial adhesion and toxin binding by glycoconjugate and oligosaccharide receptor analogues in human milk Trace amounts of ganglioside GM1 in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli Fucosylated oligosaccharides of human milk protect suckling mice from heat-stable enterotoxin of Escherichia coli Inhibition of attachment of Streptococcus pneumoniae and Haemophilus influenzae by human milk and receptor oligosaccharides Oligosaccharides from human milk as revealed by matrix-associated laser desorption/ionization mass spectrometry Carbohydrates in milk: analysis, quantities, and significance Human and bovine milk: comparison of ganglioside composition and enterotoxin-inhibitory activity Oligosaccharides and glycoconjugates in human milk Human milk contains the Shiga toxin and Shiga-like toxin receptor glycolipid Gb 3 Human milk glycoconjugates that inhibit pathogens Do the binding properties of oligosaccharides in human milk protect human infants from gastrointestinal bacteria? Survival of human milk oligosaccharides in the intestine of infants Urinary excretion of lactose and complex oligosaccharides in preterm infants XX / Developmental Immunobiology Nutrient Regulation During Pregnancy, Lactation, and Infant Growth Secretory IgA carries oligosaccharides for Escherichia coli type 1 fimbrial lectins Undialyzable growth factors for Lactobacillus bifidus var. Pennsylvanicus Bifidobacterium bifidus var. Pennsylvanicus growth promoting activity of human milk casein and its derivates A human Lactobacillus strain (Lactobacillus GG) promotes recovery from acute diarrhea in children Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain Inducible expression of human β defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier Adhesion and entry of uropathogenic Escherichia coli Effect of antiviral lipids, heat, and freezing on the activity of viruses in human milk Membrane-disruptive effect of human milk: Inactivation of enveloped viruses Inactivation of enveloped viruses and killing of cells by fatty acids and monoglycerides Isolation and propagation of a human enteric coronavirus Human milk kills parasitic protozoa Cholate-dependent killing of Giardia lamblia by human milk Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk The cells of human colostrum. I. In vitro studies of morphology and functions Transfer of maternal leukocytes to the infant by human milk Activated-memory T cells in human milk Human colostral cytotoxicity. II. Relative defects in colostral leukocyte cytotoxicity and inhibition of peripheral blood leukocyte cytotoxicity by colostrum Human breast milk T cells display the phenotype and functional characteristics of memory T cells Lymphokine production by human milk lymphocytes Increased expression of CD11b and decreased expression of L-selectin Oxygen metabolism of human colostral macrophages Cellular immunity in human milk Decreased response of human milk leukocytes to chemoattractant peptides The effects of colostrum on neutrophil function: decreased deformability with increased cytoskeletal-associated actin Human milk effects on neutrophil calcium metabolism: blockade of calcium influx after agonist stimulation The motility of human milk macrophages in collagen gels Deficient quantitative expression of CD45 isoforms on CD4 + and CD8 + T-cell subpopulations and subsets of CD45RA low CD45RO low T cells in newborn blood Differential modulation of the immune response by breast-or formula-feeding of infants Modulation of classical C3 convertase of complement by tear lactoferrin Protease inhibitors and their relation to protease activity in human milk Lysozyme binds to elastin and protects elastin from elastase-mediated degradation Prostaglandin E 1 , E 2 , and F 2α in human milk and plasma Prostaglandin concentrations in human milk Colostral antioxidants: separation and characterization of two activities in human colostrum Vitamin A and E content of human milk at early stages of lactation Influence of breast-feeding on the restoration of the low serum concentration of vitamin E and β-carotene in the newborn infant Epidermal growth factor is a major growth-promoting agent in human milk Human lactoferrin stimulates thymidine incorporation into DNA of rat crypt cells Changes in the concentration of cortisol in milk during different stages of human lactation Polyamines in human and cow's milk Polyamine concentration in rat milk and food, human milk, and infant formula Hormones and growth factors in human milk Development of the gastrointestinal mucosal barrier. III. Evidence for structural differences in microvillus membranes from newborn and adult rabbits Developmental changes in the activities of sialyl-and fucosyltransferases in the rat intestine Development of the neonatal rat small intestinal barrier to nonspecific macromolecular absorption. II. Role of dietary corticosterone Colostrum-induced enteric mucosal growth in beagle puppies Endotoxin and hypoxia-induced intestinal necrosis in rats: the role of platelet activating factor Presence of platelet-activating factor-acetylhydrolase in milk Serum PAF acetylhydrolase increases during neonatal maturation Early feeding, antenatal glucocorticoids, and human milk decrease intestinal permeability in preterm infants Development of gastrointestinal mucosal barrier. II. The effect of natural versus artificial feeding on intestinal permeability to macro-molecules Intestinal permeability changes during the first month: effect of natural versus artificial feeding A meta-analysis of infant diet and insulin-dependent diabetes mellitus: do biases play a role? Infant feeding in childhood cancer Breast-feeding and risk of childhood acute leukemia Role of infant feeding practices in development of Crohn's disease in childhood Effect of breast feeding on responses of systemic interferon and virus-specific lymphocyte transformation in infants with respiratory syncytial virus infection In-vivo immune responses of breast-and bottle-fed infants to tetanus toxoid antigen and to normal gut flora Development of secretory immunity in breast fed and bottle fed infants Chemokinetic agents for monocytes in human milk: possible role of tumor necrosis factor-alpha Tumor necrosis factor-α in human milk Presence of interferon-gamma and interleukin-6 in colostrum of normal women Interleukin-12 in human milk Interleukin-18 in human milk Interleukin-1β in human colostrum Detection of Il-6 in human milk and its involvement in IgA production Interleukin-6 in human milk Production of interleukin-6 and interleukin-8 by human mammary gland epithelial cells Chemoattractant factors in breast milk from allergic and nonallergic mothers Transforming growth factor-beta (TGF-β) in human milk Interleukin-10 (IL-10) in human milk Human milk contains granulocyte-colony stimulating factor (G-CSF) Identification of macrophage colony-stimulating factor in human milk and mammary epithelial cells Hepatocyte growth factor in human milk and reproductive tract fluids Origin and fate of erythropoietin in human milk Novel opioid peptides derived from human β-casein: human β-casomorphins Bioactive and immunoreactive prolactin variants in human milk Milk-borne prolactin and neonatal development The nucleotide profile of human milk Dietary nucleotide effects upon murine natural killer cell activity and macrophage activation Immunomodulating actions of nucleotides: enhancement of immunoglobulin production by human cord blood lymphocytes Diminished lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 on neonatal neutrophils underlies their impaired CD18-independent adhesion to endothelial cells in vitro Human tracheobronchial secretions: development of mucous glycoprotein and lysozyme-secreting systems Immunology of the neonate.Vienna Development of intestinal mucosal immunity in fetal life and the first postnatal months Development of the human antibody repertoire Neonatal interleukin-1β, interleukin-6, and tumor necrosis factor: cord blood levels and cellular production Decreased interleukin-10 production by neonatal monocytes and T cells: relationship to decreased production and expression of tumour necrosis factor-α and its receptors Cellular and molecular mechanisms for reduced interleukin 4 and interferon-gamma production by neonatal T cells Decreased production of interferongamma by human neonatal cells. Intrinsic and regulatory deficiencies Decreased G-CSF and IL-3 production and gene expression from mononuclear cells of newborn infants Decreased stimulated GM-CSF expression and GM-CSF gene expression but normal numbers of GM-CSF receptors in human term newborns as compared with adults Human cord blood antibody repertoire. Mixed population of V H gene segments and CDR3 distribution in the expression of Cα and Cγ repertoires Prevention of Haemophilus influenzae type b bacterial infections with the capsular polysaccharide vaccine Effect of breast-feeding on antibody response to conjugate vaccine Digestive-absorptive functions in fetuses, infants, and children Soluble tumor necrosis factor-alpha (TNF-alpha) receptors in human colostrum and milk bind to TNF-alpha and neutralize TNF-alpha bioactivity Immunoglobulins in human fetal sera at different stages of gestation Acute respiratory failure and bronchopulmonary dysplasia The effects of prematurity upon the immunologic system in human milk Does breast milk protect against septicaemia in the newborn? Breast milk feeding in very low birthweight infants Partial supplementation with expressed breast-milk for prevention of infection in low-birth-weight infants Breast milk and neonatal necrotising enterocolitis Prevention of necrotizing enterocolitis in low-birth-weight-infants by IgG-IgA feeding Human milk as a potential enteral source of erythropoietin Erythropoietin and the incidence of necrotizing enterocolitis in infants with very low birth weight Interleukin-10-deficient mice develop chronic enterocolitis Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death Maternal rescue of transforming growth factor-β1 null mice Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses Alpha 1-antitrypsin protects neonatal rats from pulmonary vascular and parenchymal effects of oxygen toxicity Maturation of Immunocompetence in breast-fed vs. formula-fed infants Promotion of Breastfeeding Intervention Trial (PROBIT): a randomized trial in the Republic of Belarus The passage of maternal dietary proteins in human breast milk Breast-feeding of allergic infants Association of atopic diseases with breast-feeding: Food allergens, fatty acids, and evolution Breast milk fatty acids in mothers of children with atopic eczema Atopic sensitization during the first year of life in relation to long chain polyunsaturated acids in human milk Predictors of rehospitalization among very low birth weight infants (VLBW) We thank Mrs. Susan C. Kovacevich for her assistance in the preparation of this chapter.