key: cord-0040150-9qq2gxa6
authors: Ogra, Pearay L.; Rassin, David K.; Garofalo, Roberto P.
title: Human Milk
date: 2009-05-19
journal: Infectious Diseases of the Fetus and Newborn Infant
DOI: 10.1016/b0-72-160537-0/50007-4
sha: 1dce62b8c8e1acbd7fd97c6df2d23b4da9039984
doc_id: 40150
cord_uid: 9qq2gxa6

nan

Mother's milk delivered naturally through breast-feeding has been the sole source of infant nutrition in mammalian species for millions of years. Since human beings learned to domesticate cattle about 10,000 years ago, nonhuman mammalian milk also has been used to supplement or replace maternal milk in the human infant. The development and widespread use of commercially prepared infant formula products have been phenomena of the 20th century and notably of the past 6 decades. Additional information acquired during the last several years has reinforced existing concepts on the role of breast-feeding in protecting the infant against infections, in providing an ideal source of infant nutrition, in modulating infectious immune responses, and in suppressing the evolution of neoplasms and autoimmune disease later in life. ' Over the past few decades, the immune responses on intestinal and respiratory mucosal surfaces to local infections have been intensely studied. These investigations have led to the development of concepts of immunity on mucosal surfaces of gastrointestinal, respiratory, and genitourinary tracts and identification of mucosa-associated lymphoid tissue (MALT) and local mechanisms of defense that are distinct from the internal (systemic) immune system.

This chapter reviews existing information on major aspects of the physiologic, nutritional, immunologic, and anti-infective components of the products of lactation. Also discussed is the most recent evidence on the contribution of human milk to the development of immunologic integrity in the infant and its influence on the outcome of infections and other host-antigen interactions in the neonate.

'This chapter is dedicated to Lars A. Hansen, MD, PhD, the discoverer of sIgA in human milk, the father of modem "mother's milk feeding practices," and a remarkable human being.

The rudimentary mammary tissue undergoes several developmental changes during morphogenesis and lactogenesis: In the 4-mm human embryo, the breast tissue appears as a tiny mammary band on the chest by the 7-mm embryonic stage, the mammary band develops into the mammary line, along which eventually develops the true mammary anlage; by the 12-mm stage, a primitive epithelial nodule develops; by the 30-mm stage, the primitive mammary bud appears. These initial phases of development take place in both genders (Table 5 -1). Further development in the male, however, appears to be limited by androgenic or other maleassociated substan~es.4'~ Castration in male rat embryos early in gestation leads to female breast development, whereas ovariectomy in the female does not alter the course of development of the mammary anlage. Toward the end of pregnancy, initial phases of fetal mammary differentiation seem to occur under the influence of placental and transplacentally acquired maternal hormones, with transient development of the excretory and lactiferous ductular systems. Such growth, differentiation, and secretory activities are transient and regress soon after birth. 536 At thelarche, and later on at menarche, true mammary growth and development begin in association with rapidly increasing levels of estrogens, progesterone, growth hormone, insulin, adrenocorticosteroids, and pr~lactin.~,' Estrogens appear to be important for the growth and development of the ductular system, and progestins, for lobuloalveolar development (see Table 5 -1). Final differentiation of the breast associated with growth and proliferation of the acinar lobes and alveoli continues to be influenced by the levels of estrogen and progesterone. Other peptide hormones, such as prolactin, insulin, and placental chorionic somatomammotropin, appear to be far more important for the subsequent induction and maintenance of lactation (see Table 5 -1).

It appears that prolactin secretion from the pituitary gland is under neural control and that the increasing innervation of the breast observed throughout pregnancy is regulated by estrogens.' Intense neural input in virgin and parturient but not in currently pregnant mammals has been shown to result in lactation. For example, lactation in goats can be induced by milking maneuvers. Adoptive breast-feeding also is well documented in primitive human societies. Sudden and permanent cessation of suckling can result in the termination of milk secretion and involution of the breast to the prepregnant state as the concentrations of prolactin decline. Estrogen and progesterone also may amplify the direct effects of prolactin or may induce additional receptors for this peptide hormone on appropriate target tissues in the breast.

Breast tissue is responsive to hormones, even as a rudimentary structure, as illustrated by the secretion of "witch's milk" by both male and female newborns in response to exposure to maternal secretion of placental lactogen, estrogens, and proge~terone.~ The secretion of this early milk ceases after exposure to maternal hormones has waned. Sexual differentiation, marked by puberty, is the next major stage in mammary development. As pointed out earlier, androgens inhibit the development of mammary tissue in the male, whereas the development of mammary tissue in the female is dependent on estrogen, progesterone, and pituitary hormones.8 The postpubertal mammary gland undergoes cyclical changes in response to the release of hormones that takes place during the menstrual cycle. The last stage of development occurs during menopause, when the decline in estrogen secretion results in some atrophy of mammary tissue.

During the menstrual cycle, the mammary gland responds to the sequential release of estrogen and progesterone with a hyperplasia of the ductal system that continues through the secretory phase and declines with the onset of menstruation. The concentration of prolactin modestly increases during the follicular stage of the menstrual cycle but remains constant during the secretory phase.g Prolactin secretion appears to be held in readiness for the induction and maintenance of lactation. Initiation and Maintenance of Lactation. Pregnancy is marked by profound hormonal changes reflecting major secretory contributions from the placenta, the hypothalamus, and the pituitary gland, with contributions from a number of other endocrine glands (e.g., the pancreas, thyroid, and parathyroid). Increased estrogen and progesterone levels during pregnancy stimulate secretion of prolactin from the pituitary, whereas placental lactogen appears to inhibit the release of a prolactin-inhibiting factor from the hypothalamus. Prolactin, lactogen, estrogen, and progesterone all aid in preparing the mammary gland for lactation. Initially in gestation, an increased growth of ductule and alveolobular tissue occurs in response to estrogen and progesterone. In the beginning of the second trimester, secretory material begins to appear in the luminal cells. By the middle of the second trimester, mammary development has proceeded sufficiently to permit lactation to occur should parturition take place.

Once the infant is delivered, a major regulatory factor, the placenta, is lost, and new regulatory factors including the maternal-infant interaction and neuroendocrine regulation are gained for control of lactation. Loss of placental hormone secretion results in an endocrine hypothalamic stimulation of prolactin release from the anterior pituitary gland, as well as neural stimulation of oxytocin from the posterior pituitary. The stimulation of the nipple by suckling activates a neural pathway that results in release of both prolactin and oxytocin. Prolactin is responsible for stimulating milk production, whereas oxytocin stimulates milk ejection (the combination is known as the let-down reflex). Oxytocin also stimulates uterine contractions, which the mother may feel while she is breast-feeding; this response helps to restore the uterus to prepregnancy tone.

Milk production and ejection are thus dependent on the complex interaction of stimulation by the infant's suckling, neural reflex of the hypothalamus to such stimulation, release of hormones from the anterior and posterior pituitary, and response of the mammary gland to these hormones to complete the cycle.

Mik Secretion. Milk is produced as the result of synthetic mechanisms within the mammary gland, as well as the transport of components from blood. Milk-specific proteins are synthesized in the mammary secretory cells, packaged in secretory vesicles, and exocytosed into the alveolar lumen. Lactose is secreted into the milk in a similar manner, whereas many monovalent ions, such as sodium, potassium, and chloride, are dependent on active transport systems based on sodium-potassium adenosine triphosphatases (Na', K'-ATPases). In some situations, the mammary epithelium, which may behave as a "mammary barrier" between interstitial fluid derived from blood and the milk because of the lack of space between these cells, may "leak," permitting direct diffusion of components into the milk. This barrier results in the formation of different pools or compartments of milk components within the mammary gland and is responsible for maintaining gradients of these components from the blood to the milk.

Lipid droplets can be observed within the secretory cells of the mammary gland and are surrounded by a milk fat globule membrane. These fat droplets appear to fuse with the apical membrane of the secretory cells and then to be either exocytosed or "pinched o f f into the milk.8 Some whole cells also are found in milk, including leukocytes, macrophages, lymphocytes, and broken or shed mammary epithelial cells. The mechanisms by which these cells enter the milk are complex and include, among others, specific cellular receptor-mediated homing of antigen sensitized lymphocytes.

As the structure of the mammary gland is compartmentalized, so is that of the milk. The gross composition of milk consists of cytoplasm encased by cellular membranes in milk fat globule membranes (fat compartments made up of fat droplets), a soluble compartment containing water-soluble constituents, a casein-micelle compartment containing acidprecipitable proteins with calcium and lactose, and a cellular compartment. The relative amounts of these components change during the course of lactation, generally with less fat and more protein in early lactation than in late lactation. Thus, the infant consumes a dynamic complex solution that has physical properties permitting unique separation of different functional constituents from one another, presumably in forms that best support growth and development.

Lactation Performance. Successful lactation performance depends on continued effective contributions from the neural, endocrine, and maternal-infant interactions that were initiated at the time of delivery. The part of this complex behavior most liable to inhibition is the mother-child interaction. An early attachment of the infant to the breast is mandatory to begin stimulation of the neural pathways essential to maintaining prolactin and oxytocin release.

A healthy newborn infant placed between the mother's breasts will locate a nipple and begin to suck spontaneously within the first hour of birth." This rapid attachment to the mother may reflect olfactory stimuli from the breast received by the infant at birth." Frequent feedings are necessary for the mother to maintain an appropriate level of milk production for the infant's proper growth and development. Programs to support lactation performance must emphasize proper maternal-infant bonding, relaxation of the mother, support for the mother, technical assistance to initiate breast-feeding properly and to cope with problems, and reduction of environmental hindrances. Such hindrances may include lack of rooming-in in the hospital, use of extra formula feeds, and lack of convenient day care for working mothers.

Lactation ceases when suckling stops; therefore, any behavior that reduces the amount of suckling by the infant initiates weaning or the end of lactation. Introduction of water in bottles or of 1 or 2 bottles of formula a day may begin the weaning process regardless of the time after parturition but can be most damaging to the process when the mother-infant dyad is first establishing lactation.

Colostrum and milk contain a rich diversity of nutrients, including electrolytes, vitamins, minerals, and trace metals; nitrogenous products; enzymes; and immunologically specific cellular and soluble products. The distribution and relative content of various nutritional substances found in human milk are presented in Table 5 -2. The chemical composition often exhibits considerable variation among lactating women and in the same woman at different times Table 5-2 Distribution of Secretory Products  in Human Colostrum and Milk" chemical components, especially nitrogen, calcium, and sodium, decrease significantly as the duration of lactation increase^.'^^'^ Several components, however, have been found to change in concentration as a function of water content, because their total daily output appears to be remarkably constant, at least during the first 8 weeks of lactation.20321 Milk production progresses through three distinct phases, characterized by the secretion of colostrum, transitional (early) milk, and mature milk. Colostrum comprises lactational products detected just before and for the first 3 to 4 days of lactation. It consists of yellowish, thick fluid, with a mean energy value of greater than 66 kilocalories (kcal)/dL, and contains high concentrations of immunoglobulin, protein, fat, fat-soluble vitamins, and ash. Transitional milk usually is observed between days 5 and 14 of lactation, and mature milk is found thereafter. The concentrations of many nutritional components decline as milk production progresses to synthesis of mature milk. The content of fat-soluble vitamins and proteins decreases as the water content of milk increases. Conversely, levels of lactose, fat, and water-soluble vitamins and total caloric content have been shown to increase as lactation mature^.^^"^ As the result of several manufacturing errors, the nutrient composition of infant formulas has been l e g i~l a t e d ,~~ resulting in the paradoxical situation that human milk may not always meet the recommended standards for some nutrients, whereas infant formulas may exceed the recommendation. Human milk nutrient composition varies with time of lactation (colostrum versus early milk versus mature milk) and, to some extent, maternal nutritional status. The appropriate amounts of each nutrient must be considered within these constraints.

Minerals.The mineral content of human milk is low relative to that of infant formulas and very low compared with that of cow's milk, from which most formulas are prepared, so that although human milk is sufficient to support growth and development, it also represents a fairly low solute load to the developing kidney. The levels of major minerals tend to decline during lactation, with the exception of that of magnesium, but with considerable variability among women tested.25 Sodium, potassium, chloride, calcium, zinc, and phosphorus all appear to be more bioavailable in human milk than in infant formulas, reflecting their lower concentrations in human milk. Iron is readily bioavailable to the infant from human milk but may have to be supplemented later in Preterm infants fed human milk may need supplements of calcium and sodium.28

Vitamins. Human milk contains sufficient vitamins to maintain infant growth and development, with the caveat that water-soluble vitamins are particularly dependent on maternal intake of these nutrients.29 The preterm infant may require supplements of vitamins D, E, and K when fed human milk.30.3' The low content of vitamin D in human milk has been related to the development of rickets in a few breast-fed infants, as discussed later.32 Vitamin D deficiency may be a particular problem in breast-fed infants who are not exposed to at least 30 minutes of sunshine per week." Carbohydrates and Energy. Lactose is the primary sugar found in human milk and usually is the carbohydrate chosen for the preparation of commercial formulas. Lactose supplies approximately half the energy (of a total 67 kcal/dL) taken in by the infant from human milk. Lactose (a disaccharide of glucose and galactose) also may be important to the neonate as a carrier of galactose, which may be more readily incorporated into gangliosides in the central nervous system than galactose derived from glucose in the neonate.34 Also, glycogen may be synthesized more efficiently from galactose than from glucose in the neonate because of the relatively low activity of glucokinase in early de~elopment.~~ Human milk also contains other sugars, including glucose and galactose and more than 100 different oligosa~charides.~~ These oligosaccharides may have protective functions for the infant, especially with respect to their ability to bind to gastrointestinal pathogens.37

Lipids. Fats provide almost half of the calories in human milk, primarily in the form of triacylglycerols (trigly~erides).~~ These lipids are supplied in the form of fat globules enclosed in plasma membranes derived from the mammary epithelial cells.39 The essential fatty acid, linoleic acid, supplies about 10% of the calories derived from the lipid fraction. The triacylglycerols serve as precursors for prostaglandins, steroids, and phospholipids and as carriers for fat-soluble vitamins. The lipid profiles of human milk differ dramatically from those of commercial formulas, and despite considerable adaptation of such formulas, human milk lipids are absorbed more efficiently by the infant.

Cholesterol, an important lipid constituent of human milk (12 mg/dL), usually is found in only trace amounts in commercial formulas. It has been suggested that cholesterol may be an essential nutrient for the neonate.40 A lack of cholesterol in early development may result in turning on of cholesterol-synthetic mechanisms that are difficult to turn off later in life, influencing induction of hypercholesterolemia.4' Some studies suggest that breast-feeding of the neonate is associated with lowered adult serum cholesterol levels and reduced deaths from ischemic heart disease>* Recently, interest has increased in the role that long-chain polyunsaturated fatty acids (LC-PUFAs) may play in human milk, especially docosahexaenoic acid (DHA) and arachidonic acid (AA). These LC-PUFAs are not found in unsupplemented infant formulas but are present in human milk. They are structural components of brain and retinal membranes and thus may be important for both cognitive and visual development. In addition, they may have a role in preventing a t~p y .~~ Numerous studies have found that infants fed formula without DHA or AA have reduced red blood cell amounts of these fatty however, findings in visual and cognitive functional studies in term infants have been i n c o n~i s t e n t .~~'~~ These studies have been complicated by the finding of slower growth in some preterm infants fed LC-PUFA-supplemented formulas.48 The inconsistent findings in supplemented formula-fed babies may reflect the difficulty in determining the optimal amounts of DHA to AA, and their precursors linoleic and linolenic acids, in such supplemented formulas. Thus, these lipids appear to be best delivered from human milk.

Protein and Nonprotein Nitrogen. The exact protein content of mature human milk is variable but falls close to l.og/dL, in contrast with that of infant formulas, which usually contain 1.5 g/dL; the milk from mothers who deliver preterm infants may have slightly more protein. 49 The nutritionally available protein may be even less than 1.0 g/dL-as low as 0.8 g/dL-as a result of the proportion of proteins that is utilized for non-nutritional purposes. In addition, human milk contains a considerably greater percentage of nonprotein nitrogen (25% of the total nitrogen) when compared with formulas (5% of the total nitr~gen).~' Human milk protein is primarily whey predominant (acidsoluble protein), whereas formulas prepared from bovine milk classically reflect the 18% whey-82% casein protein composition in that species. The whey-to-casein protein ratio in humans may change during lactation, with the whey component ranging from 90% (early milk) to 60% (mature milk) to 50% (late rnilk)?l Formulas for preterm infants have been reconstituted from bovine milk to provide 60% whey and 40% casein proteins; all of the major formulas for term infants in the United States are now bovine whey protein-predominant preparations, in an attempt to make them closer to human milk in composition. These differences in protein quality are reflected by differences in the plasma and urine amino acid responses of infants fed human milk or formulas that are casein protein predominant or whey protein p r e d~m i n a n t .~~'~~ In general, however, term infants do not respond with the dramatic differences seen in preterm infants when fed formulas with different protein q~a l i t y .~~-~'

The nonprotein nitrogen component of human milk contains a variety of compounds that may be of importance to the development of the neonate: polyamines, nucleotides, creatinine, urea, free amino acids, carnitine, and t a~r i n e .~~ The significance of the presence of these components is not always clear, but when they are not fed, as in the case of infant formulas that contain little taurine5' or of soy formulas that contain little carnitine,m apparent deficiencies that may influence the development of the infant occur. Taurine is important for bile salt conjugation, as well as for support of appropriate development of the brain and retina:' whereas carnitine appears to be important for appropriate fatty acid metabolism.6' Nucleotides, in particular, appear to bridge the gap between the nutritional and the immunologic roles of human milk components. Human milk contains a majority of these compounds in the form of polymeric nucleotides or nucleic whereas formulas contain nucleotides (when they are supplemented) only in the monomeric forms (Table 5-3) . Nucleotides appear to enhance intestinal development, promote iron absorption, and modify lipid metabolism in their nutritional role.64 On the other hand, these compounds perform an immunologic function by promoting killer cell cytotoxicity and interleukin-2 (IL-2) production by stimulated mononuclear cells from infants either breast-fed or fed nucleotide-supplemented f0rrnulas.6~ Nucleotide supplementation also has been reported to reduce the number of episodes of infant diarrhea in a group of lower-socioeconomic-status infants in Chile, in a manner analogous to that for protection afforded by human milk.* In 1998 it was reported that nucleotide-supplemented formulas promote the immune response of infants to Haemophilus influenzae type b polysaccharide immunization at 7 months of age, and a similar response was observed for diphtheria imm~nization.6~ Infants fed human milk for more than 6 months demonstrated a similar response and also exhibited an enhanced titer response to oral polio vaccine; this latter response was not observed in the nucleotide-supplemented formula-fed Thus, nucleotides are emerging as both nutritional and immunologic components of human milk.

Nutritional Proteins. As noted, the nutritional proteins in human milk are classified as either whey (acid-soluble) or casein (acid-precipitable). Within these two classes of proteins, several specific proteins are responsible for supporting the nutritional needs of the infant.

Human casein is made up primarily of pand K-casein, although the actual distribution of these two proteins is not clear.68 By contrast, bovine milk contains asland a,,-casein (neither of which is found in human milk), in addition to Pand ~-casein.6~ These two human milk casein proteins appear to account for approximately 30% of the protein found in human milk, in contrast with the earlier calculation of 40% (the amount commonly used to prepare reconstituted, so-called humanized formulas from bovine milk, which normally contains 82% casein proteins).

The whey protein fraction contains all of the proposed functional proteins in human milk (immunoglobulins, lysozyme, lactoferrin, enzymes, cytokines, peptide hormones), in addition to the major nutritional protein, a-lactalbumin. The whey proteins make up approximately 70% of human milk proteins, in contrast with 18% in bovine milk. Whereas a-lactalbumin is the major whey protein in human milk, P-lactoglobulin is the major whey protein in bovine milk (and is not found in human milk).50 A consistent fraction of human milk whey protein is made up of serum albumin. Its source remains unclear; some evidence indicates that it may be synthesized in the mammary gland.70 Most of the serum albumin, however, probably is synthesized outside the mammary gland.

Thus, milk proteins are characterized by their site of synthesis, as well as being species specific. Therefore, proteins such as a-lactalbumin and P-lactoglobulin are species and organ specific, whereas proteins such as serum albumin are species specific but not organ ~pecific.~' The net result of these differences in proteins utilized for nutrition by the neonate is that different amounts of amino acids are ingested by the neonate, depending on the source of milk, even reconstitution of the whey and casein classes of proteins from one species in a ratio similar to that of another species does not result in an identical amino acid intake. These differences are reflected in plasma amino acid profiles of infants fed commercial milks versus human milk, regardless of the ratios of re~onstitution.5~ Bioactive Proteins and Peptides. Whereas a major proportion of human milk protein is composed of the nutritional proteins just described, a significant number of the remaining proteins subserve a variety of functions, either other than or in addition to the nutritional support of the neonate. These proteins include carrier proteins, enzymes, hormones, growth factors, immunoglobulins, and cytokines (the latter two are discussed later under "Resistance to Infection"). Whether these proteins are still functional once they have been ingested by the neonate has not always been established, but it is clear that human milk supplies a mixture that is potentially far more complex than just nutritional substrate.

Currier Proteins. A number of nutrients are supplied to the neonate bound to proteins found in human milk. This binding may play an important role in making these nutrients bioavailable. Lactoferrin is an iron-binding protein (a property that also may play a role in its bacteriostatic action) that is apparently absorbed intact by the infant. 72 Lactoferrin may be important in the improved absorption of iron by the infant from human mitk compared with that from cow's milk preparations, which contain little lactoferrin.26 Lactoferrin also may bind other minerals, including zinc and manganese, although the preferred mineral form appears to be the ferric ion.

A number of other proteins appear to be important as carriers of vitamins and hormones. Folate-binding, vitamin B,,-binding, and vitamin D-binding proteins all have been identified in human milk. These proteins appear to have some resistance to proteolysis, especially when they are saturated with the appropriate vitamin l i g a~~d .~~ Serum albumin acts as a carrier of a number of ligands, whereas a-lactalbumin acts as a carrier for calcium. Finally, proteins that bind thyroid hormone and corticosteroids have been reported to be present in human although serum albumin may in part fulfill this function.

Enzymes. The activity of more than 30 enzymes has been detected in human milk.76 Most of these enzymes appear to originate from the blood, with a few originating from secretory epithelial cells of the mammary gland. Little is known about the role of these enzymes, other than lysozyme and the lipases, in human milk. The enzymes found in human milk range from ATPases to antioxidant enzymes, such as catalase, to phosphatases and glycolytic enzymes. Although these enzymes have important roles in normal body metabolism, it is not clear how many of them either function in the milk itself or survive ingestion by the infant to function in the neonate.

Lysozyme appears to have a part in the antibacterial function of human milk, whereas the lipases have a more nutrient-related role in modulating fat metabolism for the neonate. Two lipases have been identified in human milk, a lipoprotein lipase and a bile salt-stimulated l i p a~e .~~ Lipoprotein lipase appears to be involved in determining the pattern of lipids found in human milk by regulating uptake into milk at the level of the mammary gland. Human milk bile salt-stimulated lipase is an acid-stable protein that compensates for the low activity of lipases secreted into the digestive tract during early devel~pment.'~ Thus, these two enzymes regulate both the amount and the pattern of lipid that appears in milk as well as the extremely efficient absorption of lipid by the infant. Human milk lipid is absorbed much more readily than lipid from commercial milk formulas despite the many adaptations that have been made to improve absorption, illustrating the effective mechanisms supported by the lipases.

Hormones and Growth Factors. Both peptide and steroid hormones, as well as growth factors, have been identified in trace amounts in human milk, although as with most of the enzymes, it is not clear to what degree they function in the neonate to whom they have been supplied. As discussed previously, binding proteins for corticosteroids and thyroxine have been identified in milk and, by extrapolation from observations of other milk components, may play a role in making these bioactive compounds more readily available to the infant.

Among the hormones identified in human milk are insulin, oxytocin, calcitonin, and prolactin. Most of these hormones appear to be absorbed by the infant, but their role in in vivo function remains unclear.79 Breast-fed infants appear to have a different endocrine response from that of formulafed infants, presumably reflecting the intake of hormones from human milk. 80 The advantages or disadvantages to the infant of these responses, however, are unknown.

Human milk also contains a rich mixture of growth factors, including epidermal growth factor (EGF) and nerve growth factor." In addition, a variety of gastrointestinal peptides have been identified in human milk. Presumably, the supply of these various factors to the infant through the milk compensates for their possible deficiency in the infant during early development.

The composition of human milk provides a complex and complete nutritional substrate to the neonate. Human milk supplies not only individual nutrients but also enzymes involved in metabolism, carriers to improve absorption, and hormones that may regulate metabolic rates. Commercial formulas have not yet been developed to the point that they can provide an analogous complete nutritional system.

Fresh human milk contains a wealth of components that provide specific, as well as nonspecific, defenses against infectious agents and environmental macromolecules (Table 5-4). These component factors include cells, such as T and B lymphocytes, polymorphonuclear neutrophils (PMNs) (i.e., polymorphonuclear leukocytes), and macrophages; soluble products, especially immunoglobulins; secretory immunoglobulin A (sIgA); immunomodulatory cytokines and cytokine receptors; components of the complement system; several carrier proteins; enzymes; and a number of endocrine hormones or hormone-like substances. Additional soluble factors that are active against streptococci, staphylococci, and tumor viruses also have been identified.22 Other soluble milk factors with potential implications in host defense include the bifidus factor, which promotes growth of bifidobacteria, and an EGF, which promotes growth of mucosal epithelium and maturation of intestinal brush border. The developmental characteristics of sIgA have been studied more extensively than those of other component~.8~-~~

On the basis of available information, it is clear that a majority of IgA-producing cells observed in milk have their origin in the precursor immunocompetent cells in the gutassociated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT). Exposure of IgA precursor B lymphocytes in the GALT or BALT to microbial and dietary antigens in the mucosal lumen is an important prerequisite for their initial activation and proliferation. Such antigensensitized cells eventually are transported through the systemic circulation to other mucosal surfaces, including the mammary glands and, as plasma cells, initiate the synthesis of immunoglobulin against specific antigens previously experienced in the mucosa of the respiratory or alimentary t r a~t .~' ,~~ It has been proposed that T cells observed in the milk also may be derived from GALT and BALT in a manner similar to that of IgA-producing cells. Little or no information is available regarding the site of origin of other cellular or soluble immunologic components normally present in human milk. Specific antibody and cellular immune reactivity against many respiratory and enteric bacterial and viral pathogens and ingested food proteins also are present in human breast milk ( 

IgA antibodies found in milk possess specificity for Immunoglobulin A. As observed in other peripheral mucosal sites, the major class of immunoglobulin in human colostrum and milk is the 1 1 S sIgA. Other isotypes-namely, 7s IgA, IgG, IgM, I@, and IgE-also are present. The IgA exists as a dimer of two 7s IgA molecules linked together by a polypeptide chain, the J-chain, and is associated with a nonimmunoglobulin protein referred to as the secretory component. The sIgA protein constitutes about 75% of the total nitrogen content of human milk. The IgA dimers produced by plasma cells at the basal surface of the mammary epithelium are transported to specialized columnar epithelial cells, where they acquire the secretory component before their discharge into the alveolar Sequential quantitation of class-specific immunoglobulin in human colostrum and milk has demonstrated that the highest levels of sIgA and IgM are present during the first few days of lactation ( Fig. 5-1) . Levels of IgA are 4 to 5 times greater than those of IgM, 20 to 30 times greater than those of IgG, and 5 to 6 times greater than those of serum As lactation progresses, IgA declines to levels that range from 20 to 27 mg per gram of protein, and IgM levels decline to 3.5 to 4.1 mg/g. IgG levels do not show any significant change during early and late lactation and usually are maintained in the range of 1.4 to 4.9 mg/g (see Fig. 5-1) . Although a dramatic and rapid decline in milk IgA and IgM occurs during the first week of life, this decrease is more than balanced by an increase in the volume of milk produced as the process of lactation becomes established (see Fig. 5 infesious agents endemic to or pathogenic for-the intestinal and respiratory tracts (see Table 5 -4). These antibodies may be present in the milk in the absence of specific circulating IgA. In a study in which pregnant women were given oral feedings of Escherichia coli 083, development of IgA antibody in human milk was evident in the absence of detectable serum antibody-specific response^.^' In another study, investigators have observed similar responses in animal models using intrabronchial immunization with Streptococcus pneumoniae. These and other s t~d i e s~~-~~ have strongly supported the concept of a bronchomammary, as well as an enteromammary, axis of immunologic reactivity in the breast.

Despite the elegance of studies that have defined the mechanisms of IgA cell trafficking from GALT and BALT to the mammary glands, it is clear that the actual number of B cells or IgA plasma cells in the mammary glands is sparse. At the same time, colostrum and milk may contain large amounts of IgA (as much as 11 g in colostrum and as much as 1 to 3 g per day in later milk), as shown in Table 5 -6. The reasons underlying the apparent disparity between the content of immunoglobulin-producing cells and concentrations of immunoglobulin are not known. It may be related to the unique hormonal environment of the mammary glands. The hormones that have been consistently observed in human milk are listed in Table 5 -4.

The effect of pregnancy-and lactation-related hormones on regulation of immunologic reactivity present in the resting and lactating breast has been examined.g0 In a study on immunoglobulin production in the nonlactating human breast, several interesting findings were noted." Few mononuclear cells were present in the nonlactating breast of nulliparous and of parous women, although IgA-containing cells predominated. Synthesis of IgA appeared to be slightly increased in the parous women. IgA was found in the mammary tissues during the proliferative stage of the menstrual cycle in the nulliparous women and during the luted phase in the parous women. The number of IgA-producing cells in the nonlactating breast was observed to increase with parity. These findings suggest that the immunologic makeup of the nonlactating, as well as the lactating, breast may be significantly influenced by the hormonal milieu. In another study of virgin mice given exogenously administered hormones?2 an extended exposure to estrogen, progesterone, and prolactin was necessary for maximal increments in IgA-producing plasma cells in the breast. Similarly, castrated males exposed to these hormones became moderately receptive to mammary gland homing of cells specific for IgA synthesis. As would be expected, testosterone eliminated female breast receptivity to these cells. These studies suggest the existence of a hormonally determined homing mechanism in the mammary gland for class-specific, immunoglobulin-producing cells.

More recent studies have proposed another possible influence of lactational hormones on immunocompetent cells. In limited observations, combinations of prolactin with estrogen and progesterone (in concentrations observed normally at the beginning of parturition) appeared to have an amplifjmg effect on the synthesis and secretion of IgA from peripheral blood lymphocyte^.^^ This observation raises the possibility that the high levels of sIgA observed in colostrum and milk may be the result of selective, hormonally mediated proliferation of antigen-sensitized IgA cells in the peripheral blood. The immunoglobulin could acquire secretory component during its passage through the mammary epithelium and eventually appear in the colostrum or milk as mature sIgA. Although the appearance of sIga antibody in milk characteristically follows antigenic exposure in the GALT or BALT, the precise nature of the IgA content in milk appears to be determined by a variety of other factors operating in the mucosal lymphoid tissue. These include the regulatory T cell network in the GALT and possibly in the BALT,94 the nature of antigens (soluble proteins versus particulate microbial and the route of primary versus secondary antigenic exposure?

It has been estimated that the breast-fed infant may consistently receive an amount of about 1 g of IgA each day.

Approximately Yloo of this amount each day is IgM and IgG.97,98 The estimates of lactational immunoglobulin delivered to the breast-fed infant at different periods of lactation are presented in Table 5 -6. Most ingested IgA is eliminated in the feces, although up to 10% may be absorbed from the intestine into the circulation within the first 18 to 24 hours after birth. Approximately 70% to 75% of ingested milk IgA survives passage through the gut and is excreted in the fe~es.9~ Feces of breast-fed infants contain functional antibodies present in the ingested mi".'"'' Other studies also support the finding of prolonged survival of milk IgA in the gastrointestinal tract. Infants fed human milk have demonstrated the presence of all immunoglobulin classes in the feces. Fecal IgA content was three to four times greater than that of IgM after human milk feeding. Comparative studies on survival of human milk IgA and bovine IgG in the neonatal intestinal tract have suggested that the fecal content of IgA may be 14 to 20 times greater after human milk feeding than that of bovine IgG after feeding of bovine immune globulin.'" Direct information about the role of milk IgA in antimicrobial defense is available in several studies. Secretory IgA interferes with bacterial adherence to cell surfaces. lo* Colostrum and milk can inhibit the activity of E. coli and Vibrio cholerue enterotoxins in experimental setting~.''~ The antitoxic activity of human milk appears to correlate well with its IgA content but not with its IgM and IgG content.

Precoating of V cholerue with specific sIgA protects infant mice from disease. ' @' Similar results have been obtained by using specific purified milk sIgA in preventing E. coli-and Shigella dysenteriue-induced disease in rabbit^.''^ Less definite, but suggestive, is a study conducted with human milk feeding relative to the intestinal implantation of orally administered live poliovirus vaccine. '06 This study found that breast-feeding may reduce the degree of seroconversion for poliovirus antibody in the vaccinated infants. Because antipolio IgA is present in human milk and colostrum, the investigators concluded that specific IgA may bind poliovirus and influence viral replication in the intestinal mucosa. Extensive experience with oral polio immunization worldwide, however, has not found an association between breastfeeding and live vaccine failures. Other studies have clearly shown that the magnitude of poliovirus replication in the intestine is determined by the presence and level of preexisting sIgA antibody. With high levels of intestinal IgA antibody, little or no replication of vaccine virus was observed in the gut. With lower levels, varying degrees of viral replication could be dem~nstrated."~ Indirect evidence, obtained from a more clinical perspective, suggests a protective role for milk against a variety of mucosal infections. Breast-feeding has been strongly implicated in supporting gastrointestinal homeostasis in the neonate and in establishing normal gut flora. Observations have shown the absence of diarrheal disease in breast-fed infants, even in the face of contamination of the fed milk with E. coli and Shigella species.'08 A preventive and therapeutic role for breast-feeding also has been suggested in nursery outbreaks of such disease due to enteropathogenic strains of E. coli1O9 and diarrhea associated with rotavirus."' Breast-feeding plays an inhibitory role in the appearance of E. coli 0 8 3 agglutinins found in the feces of colonized infants. A decrease in the incidence of neonatal sepsis, specifically that associated with gram-negative bacilli and E. coli K1 serotypes, also has been linked to breast-feeding. ' ' 'J l2 Milk IgA, possibly by limiting ingestion of foreign antigens by the neonate, or by binding of foreign proteins with specific antibodies to prevent absorption, or by both processes, may decrease the incidence of atopic-allergic diseases."3-' l5 The frequency of IgE skin test-positive results has been described as being lower among breast-fed infants, possibly because of decreased exposure to cow's milk proteins or presence of maternal blocking antibodies.' l6 Indirect epidemiologic data suggest that breast-feeding is protective against certain respiratory bacterial and viral infection~."~~"~ Whereas the epidemiologic studies strongly support a protective role of breast-feeding, it is not possible in these studies to dissect the relative contribution of sIgA from that of other soluble or cellular components present in colostrum and milk. 1  7  3  80  80  120  1 1,000  3  10  45  45  50  40  2000  7 1-2 4 20 25 10 1000 7-28 1-2 2 10-15 10 10 1000 c50

1-2 0.5-1 10-15 10 10 1000

aEstirnates based on the available data for total immunoglobulin and daily protein synthesis (see references 6, 83, 84).

Immunoglobulin G and Immunoglobulin M. Normal neonates exhibit characteristic paucity or lack of IgA during the first 7 to 10 days after birth. At that time, the presence of IgM and IgG in milk may be important to compensate for immunologic functions not present in the mucosal sites. For example, both IgG and IgM participate in complement fixation and specific bactericidal activity, functions not associated with IgA. Studies carried out after oral feeding of immune serum globulin (mostly IgG) have suggested that IgG may survive in the gastrointestinal tract of low-birthweight infants.'" Thus, other immunoglobulin isotypes in milk also may be able to serve as effective substitutes for IgA in the neonates of IgA-deficient mothers in prevention of infection with enteric or respiratory pathogens. in release of IgA.I3' In other studies, milk macrophages have been found to be efficient in release of superoxide anions after in vitro stimulation with phorbol myristate a~e t a t e . '~~"~~ Milk macrophages have the capacity to be primed by appropriate stimulation for greater release of superoxide anions.'33 It has been shown that milk macrophages obtained from preterm-delivered lactating mothers have a significantly higher phagocytic index than that for the macrophages in term milk. The bactericidal activity appears to be similar in pre-and full-term milk macrophages, however.13* In neutrophils, mdk macrophages appear to be activated, as demonstrated by the increased expression of CD1 Ib and decreased expression of L-se1e~tin.I~~ The precise functions of macrophages in colostrum or milk have not been fully explored. These cells have been suggested as potential transport vehicles for IgA.1283'29 Milk macrophages possess phagocytic activity against Staphylococcus aureus, E. coli, and Candida albicans, with possible cytocidal activity against the first two organism^.'^^ Milk macrophages participate in antibody-dependent, cell-mediated cytotoxicity for herpes simplex virus type 1-infected cells.'35 Infection of milk macrophages by respiratory syncytial virus results in the production of the pro-inflammatory cytokines IL-lP, IL-6, and tumor necrosis factor-a (TNF-cx).'~~ These cells also are involved in a variety of other biosynthetic and excretory activities, including production of lactoferrin, ly~ozyme,'~' components of c~rnplernent,'~~ properdin factor B, epithelial growth factor( s), and T lymphocyte-suppressive factor(s)." Milk macrophages also have been suggested to be important in regulation of T cell f u r~c t i o n . '~~' '~~ -

Human colostrum and milk contain lymphocytes, monocytesmacrophages, neutrophils, and epithelial cell^."^ Early colostrum contains the highest concentration of cells, approximately 1 x lo6 to 3 x lo6 cells per mL. By the end of the first week of lactation, cell concentration is of the order of lo5 cells per mL. Total cell numbers delivered to the newborn throughout lactation may, however, remain constant when adjustments are made for the increase in volume of milk produced.'24 The two major cell populations in human milk are difficult to distinguish by common staining methods because of the large number of intracytoplasmic inclusions, neutrophils, and macrophages. More accurate estimates made by flow cytometry analysis suggest that the relative percentages of neutrophils, macrophages, and lymphocytes in early milk samples are approximately 80%, 15%, and 4%, re~pectively.'~~*'~~ The remaining cells are present in smaller amounts, especially in the absence of active suckling, engorgement, or local breast infection. Colostral cells also elicit antibody-and lectin-dependent cellular cytotoxic responses. The NK, as well as the antibody-and lectin-dependent, responses in colostral cells, however, have been observed to be significantly lower than those of autologous peripheral blood cells.

Reduced cellular cytotoxicity of colostral cells also has been observed against virus-infected targets and certain bacteria. In fact, with several specific virus-infected targets, colostrum and mdk cells conspicuously lack cellular cytotoxicity when compared with autologous peripheral blood cells. There is also an apparent exclusion of cytolytic T cells in the milk that are specific for certain human leukocyte antigen (HLA) phenotypes.'423143 A majority of T lymphocytes in colostrum and milk are mature CD3' cells. Both CD4' (helper) and CD8' (cytotoxic and suppressor) populations are present in human milk, with a proportion of CD8' T cells higher than that found in human blood (Table 5-7) . The CD4'lCD8+ ratio in milk is significantly lower than that observed in peripheral blood and is not due to an increase of CD8' cells in the peripheral blood of women during the postpartum period. Colostral and milk T lymphocytes manifest in vitro proliferative responses on stimulation with a number of mitogens and antigens. Several studies have shown a selectivity in lymphocyte stimulation responses in colostral and milk lymphocytes to various antigens when compared with peripheral blood lymphocyte Antigens such as rubella virus stimulate T lymphocytes in secretory sites and milk, as well as in systemic sites.IZ4 By contrast, E. coli K1 antigen whose exposure is limited to mucosal sites produces stimulation of lymphoproliferative responses only in milk lymphocytes. These findings support the concept of select T cell populations in the mammary gland.

In addition to antigen selectivity, a general hyporesponsiveness to mitogenic stimulation of milk lymphocytes relative to peripheral blood lymphocytes has been o b~e r v e d . '~~' '~~ The decreased reactivity of milk lymphocytes to phytohemagglutinin (PHA) may be partly the result of a relative deficiency of certain populations of T cells in milk. Macrophage-T cell interactions also have been postulated as being responsible for this relative hyporesponsi~eness,~~ although it is not known whether the effects are the result of decreased helper or increased suppressor function. Recent studies have shown that milk lymphocytes exhibit reduced responses to allogeneic cells but display good ability to stimulate al10reactivity.I~~ Treatment of milk lymphocytes with monoclonal antibodies cytotoxic for T lymphocytes or with anti-HLA class I1 antigen-specific monoclonal antibodies has resulted in a substantial reduction in in vitro proliferative responses to bacterial antigens. It appears that, in general, the T cell proliferative responses to PHA and tetanus toxoid in breast-fed infants are significantly higher than those in bottle-fed infants, possibly secondary to the presence of maternally derived cell growth factors and other lymphokines present in human milk.1403145

Virtually all CD4' and CD8' T cells in milk bear the CD45 isoform CD45RO that is associated with immunologic m e m~r y . '~~' '~~ In addition, the proportion of T cells that display other phenotypic markers of activation, including CD25 (IL-2R) and HLA-DR, is much greater than that in b l~o d . "~, '~~ Consistent with their memory phenotype, T cells in human milk produce interferon-y (IFN-Y).'~~ Furthermore, a significantly greater number of CD4' T cells in colostrum express the CD40 ligand (CD40L) compared with autologous or heterologous blood T cells.I4' Cognate interaction between the CD40L on T cells and the CD40 on B cells is a necessary step for antibody production in vivo and is congenitally deficient in the newborn. The function of these memory T cells in the recipient human infant is currently unknown, however. Mucous membrane sites in the alimentary or respiratory tract, or both, of the recipient infant would seem to be potential entry sites for human milk leukocytes. Of considerable interest, very small numbers of memory T cells are detected in blood in infancy.'49 Thus, it is possible that maternal memory T cells in milk compensate for the developmental delay in their production in the infant. In this regard, the proportion of T lymphocytes bearing the T cell receptor-yG (TCR-yG) is approximately two times greater in colostrum than in b l~o d . '~~, '~'

Human TCR-yG' cells populate organized lymphoid tissues and represent half of the intraepithelial lymphocytes in the Thus, the intestinal epithelia may have a selective affinity for TCR-yG' cells and provide a favorable environment for maternal T cells in milk to be transferred to the breast-fed infant. Evidence from experimental animal studies indicates that milk lymphocytes enter tissues of the n e~n a t e , '~~. '~~ but this has not been demonstrated in humans. In addition, the possible transfer of histocompatibility antigens and T cells to the neonate through breast-feeding has been examined by determining the fate of skin grafts in suckling rats fed by allogeneic mothers.'57 Such foster feeding of milk may result not only in increased allogeneic graft survival but also in development of "runting" syndrome, possibly as a result of a graft-versus-host-type reaction in the breast-fed animal. Effects of the transfer may be related to dosage of ingested allogeneic cells, in that increasing cell numbers transferred may prolong skin graft survival but may also increase the likelihood of a graft-versus-host reaction. Of note, the suckling rat gut has a higher degree of permeability to whole proteins than that characteristic of the human intestine. Furthermore, clinical experience in immunodeficient neonates has never supported the development of graftversus-host reaction-like disease in the breast-fed human infant. In humans, possible transfer of maternal T cell reactivity to tuberculin protein from the mother to the neonate throu h the process of breast-feeding has been that maternal cellular products or soluble mediators of cellular reactivity may be transferred passively to the neonate through breast-feeding. Admittedly, however, the occurrence of such phenomena in humans has not been studied carefully. Thus, it must be emphasized that at present, evidence to suggest any T cell-mediated immunologic risks associated with breast-feeding in humans is lacking. On the other hand, it is still unknown whether milk T cells, either TCR-aV or TCR-yF, play a role in the transfer of adoptive immunoprotection to the recipient infant. Although the absolute counts in actively nursing mothers exhibit considerable variability between different samples, highest numbers are generally observed during the first 3 to 4 days of lactation. The numbers of neutrophils decrease significantly after 3 to 4 weeks of lactation, and only rare neutrophils are observed in samples collected after 60 to 80 days post partum. Leukocytes in human milk appear to be metabolically activated. Indeed, although the neutrophils are phagocytic and produce toxic oxygen radicals, they do not respond well to chemoattractants by increasing their adherence, polarity, or directed migration in in vitro systems.Im This diminished response was found to be due to prior activation in that the neutrophils in milk displayed a phenotypic pattern that is typical of activated neutrophils.

The expression of CD1 lb, the a chain subunit of Mac-1, was increased, and the expression of L-selectin was decreased.125

Epithelial Cells. On the basis of their anatomic distribution, epithelial cells in the human mammary gland can be classified into two main types: myoepithelial and luminal. Epithelial cells of both types, however, appear to be more heterogeneous on histologic and physicochemical testing. 142,161,162 They include secretory cells, which contain abundant rough endoplasmic reticulum, lipid droplets, and Golgi apparatus. The secretory cells appear to produce casein micelle. The squamous epithelial cells usually are seen in the regions of the cutaneous junction of the nipples, especially near the galactophores. The ductal or luminal cells, which exist in clusters, have many short microvilli, tight junctions, and remnants of desmosomes.'6's'62 Studies using monoclonal antibodies have shown that in rodents, as many as 10 different types of epithelial cells in the adult mammary glands may exist. These cell types probably represent various stages of differentiation of mammary gland epithelium. These include, in the mammary end buds, the distinct cell types of the tip and the main compartment peripheral cell types I and I1 and, in alveoli as well as in the ducts of the mammary glands, the luminal cell types I and I1 and myoepithelial cells.161 It is not known, however, whether similar epithelial cell differentiation occurs in the human mammary gland.

In human milk, relatively few epithelial cells are observed in the early phases of lactation. Most epithelial cells appear after 2 to 3 weeks and are seen in appreciable numbers, even as long as 180 to 200 days after the onset of lactation. With the possible exception of the synthesis of secretory component and casein and possibly other products, with which secretory epithelial cells have been associated in the stroma of the mammary gland, the role of epithelial cells in the milk remains to be defined.

Possible Functional Roles for Cellular Elements. The information reviewed thus far provides strong evidence for the existence of a number of dynamic cellular reactions in the mammary gland, colostrum, and milk. Unfortunately, the specific functional role, collectively or individually, for the epithelial cells, monocytes, neutrophils, or lymphocytes in the mammary gland or the milk remains to be defined. In view of the high degree of selectivity and the differences in the quantitative and functional distribution of cellular elements, it is suggested that the mammary gland, like mucosal surfaces, may function somewhat partitioned from the cellular elements in peripheral blood, in a manner similar to that for other peripheral sites (such as the genital tract) of the common mucosal system. It is, however, not known whether the characteristic proportions of macrophages, T lymphocytes, other cytotoxic cells, or epithelial cells are designed for any specific functions localized to the mammary gland in the lactating mother or to epithelium or lumen of the intestinal or respiratory mucosa of the breast-feeding infant, or both. The observations on the transfer of delayed hypersensitivity reactions in human neonates and of graftversus-host reactivity in the rat raise the possibility that milk cells may function as important vehicles in transfer of maternal immunity to neonates. The potential beneficial and harmful roles of such cell-mediated transfer through the mucosal routes, however, need to be investigated further. The paucity of NK and other cytotoxic cells in the colostrum may have a role for the breast-feeding neonate, especially in influencing the antigen processing and uptake of replicating microorganisms and their immune response at systemic or mucosal levels or both. Although colostral cells await further elucidation of their function in the mammary glands and the suckling neonate, it is likely that their presence in the milk represents a highly selective phenomenon and not a mere contamination with peripheral blood cells.

Human colostrum and milk contain all components of the complement system. Active production of C3 has been reported in vitro in breast milk cell culture^.'^^"^ Interf e r~n , '~~ migration inhibition factor,15" and a-fet~protein'~" also are present in human milk, although their roles have not yet been fully elucidated (see Table 5 -4).

Iron-binding proteins present in colostrum and milk, such as lactoferrin,'66 have bacteriostatic activity in vitro against E. coli, S. aureus, and C. a l b i c~n s . '~~ Some evidence suggests enhanced bactericidal activity of lactoferrin in association with IgA. Lysozyme and bifidus factor (a collection of glycosamides that promote growth of Lactobacillus and bifidobacterial species, whose growth in turn inhibits growth of enteric gram-negative aerobic bacilli) may function as ancillary inhibitors of gut and skin pathogens. Antistaphylococcal factors appear to be active against experimental staphylococcal infections and may be important for local mammary gland pr~tection.'""~~ Of particular interest is the demonstration of certain oligosaccharides that prevent attachment of S. pneurnoniae to human epithelial cells'68 and of high-molecular-weight substances that inhibit virulence of enterotoxins of Enterobacteriaceae organisms (see Table 5 -4).

Nonimmunoglobulin antiviral factors have been demonstrated in lipid and aqueous phases of human milk. These factors have shown activity against influenza A and B viruses, herpes simplex virus, Semliki Forest virus, Japanese B encephalitis virus, rubella virus, rhinovirus, and rotavirus (see Table 5 Several recent studies have generated interest in the potential role of nonantibody proteins, bile salt lipases, whey proteins, and trace metals present in human milk in the control of enteric infection^.'^'-'^^ Several species of grampositive and gram-negative bacteria frequently can be killed by incubation with human milk whey but not commercial infant f~rrnula."~ The mechanisms responsible for such antibacterial activity are not known. The synergistic inter-action among IgA, lactoferrin, and iron has been suggested to play a role in such d e f e n~e . '~' -'~~ Concentrations of free fatty acid and possibly monoglycerides seem to increase during storage of milk because of spontaneous lipolysis generated by lipoprotein l i p a~e . '~~. '~~ Antibody-independent antiparasitic effect of stored, but not fresh, human milk against Giardia lamblia or Entamoeba histolytica has been attributed to such free fatty acids. '77 In additional studies conducted in vitro, bile salt-stimulated lipase, the major lipase in human milk, has been found to cause hydrolysis of milk triglycerides. It remains to be seen whether free fatty acids induce significant in vivo protection in the intestine against intestinal parasites. On the other hand, bile salts themselves may stimulate the growth of G. 1arr1blia.I~~ Nonantibody proteins, several carrier proteins, and cellular enzyme proteins are present in milk in high concentrations. Concentrations of lysozyme range from 30 to 50 mg/100 mL in early colostrum to 5 to 10 mg/100 mL in late milk. The susceptibility of an organism to lysozyme depends on the availability of the peptidoglycan substrate. In certain situations in which the peptidoglycan may be blocked by lipoproteins, the organisms are relatively resistant to l y~o z y m e s . '~~~'~~

General Features. The defense agents in human milk, although biochemically diverse, share certain features: ( 1 ) They usually are common to mucosal sites. (2) They are adapted to resist digestion in the gastrointestinal tract of the recipient infant. (3) They protect by noninflammatory mechanisms. (4) They act synergistically with each other or with factors produced by the infant. (5) Most components of the immune system in human milk are produced throughout lactation and during gradual weaning, but ( 6 ) there is often an inverse relationship between the production of these factors in the mammary gland and their production by the infant during the same time frames of lactation and postnatal development. Indeed, as lactation proceeds, the concentration of many factors in human milk declines. Concomitantly, the mucosal production of these factors rises in the developing infant. It is unclear whether the inverse relationship between these processes is due to feedback mechanisms or whether the processes are independent.

Carbohydrate Components. Human milk contains several oligosaccharides and glycoconjugates, including monosialogangliosides that are receptor analogues for heat-labile toxins produced by V: cholerae and E. ~o l i '~' ;

fucosecontaining oligosaccharides that inhibit the hemagglutinin activity of the classic strain of V: ~holerae'~'; hcosylated oligosaccharides that protect against heat-stable enterotoxin of E. coli''o; mannose-containing high-molecular-weight glycoproteins that block the binding of the El Tor strain of V ~holerae'~'; and glycoproteins and glycolipids that interfere with the binding of colonization factor (CFNII) h b r i a e on enterotoxigenic E. coli. ' 81 The inhibition of toxin binding is associated with acidic glycolipids containing sialic acid (gangliosides). Although the quantities of total gangliosides in human and in bovine milk are similar, the relative frequencies of each type of ganglioside in milk from these two species are distinct. More than 50 types of monosialylated oligosaccharides have been identified in human milk, and new types are still being recognized.IS2 Monosialoganglioside 3 constitutes about 74% of total gangliosides in human milk, but the percentage is much lower in bovine milk.ls3*"4 Also, the level of the enterotoxin receptor ganglioside G,, is 10 times greater in human than in bovine milk.Is4 This difference may be of clinical importance because GM, inhibits enterotoxins of E. coli and V: ch~lerae.''~ It also is of interest that intact human milk fat globules, as well as the mucin from the membranes of these structures, inhibit the binding of S-fimbriated E. coli to human buccal epithelial cells.'s6

Oligosaccharides in human milk also interfere with the attachment of H. influenzae and S. pne~moniae."~ In this regard, N-acetylglucosamine (GlcNAc) ( 1 -3)Gal-disaccharide subunits block the attachment of S. pneumoniae to respiratory epithelium. Moreover, recent evidence indicates that human milk interferes with the binding of human immunodeficiency virus (HIV) envelope antigen gp120 to CD4 molecules on T cells."' Some evidence from animal models suggests that the oligosaccharides and glycoconjugates in human milk protect in but relevant clinical data are scarce .192 In addition to the direct antimicrobial effects of the carbohydrates in human milk, nitrogen-containing oligosaccharides in human milk are growth promoters for Lactobacillus btj5dus var. pennsylvani~us,''~ glycoproteins, and glyc~peptides.'~~~''~ The bifidus growth promoter activity associated with caseins may reside in the oligosaccharide moiety of those complex molecules.196 It appears that these factors are responsible to a great extent for the predominance of Lactobacillus species in the bacterial flora of the large intestine of the breast-fed infant. These bacteria produce large amounts of acetic acid, which aids in suppressing the multiplication of enteropathogens. It also has been reported that Lactobacillus species strain GG aids in the recovery from acute rotavirus infection^''^ and may enhance the formation of circulating cells that produce specific antibodies of the IgG, IgA, and Igh4 isotypes, as well as serum levels of those antibodie~.'~'

Substrata in Human Milk. Human milk supplies defense agents from fat as it is partially digested in the recipient's alimentary tract. Fatty acids and monoglycerides produced from milk fats by bile salt-stimulated lipase or lipoprotein lipase in human milk,"' lingual/gastric lipase from the recipient from birth,2"" or pancreatic lipase after a few weeks of age are able to disrupt enveloped viruses.zo1-205 These antiviral lipids may aid in preventing coronavirus infections of the intestinal traci? and also may defend against intestinal parasites such as G.

Proteins. The principal proteins in human milk that have direct antimicrobial properties include the following.

a-lactalbumin. a-Lactalbumin is a major component of the milk proteins and may possess some important functions of immunologic defense. This protein appears as large complexes of several a-lactalbumin molecules, which can induce apoptosis in transformed embryonic and lymphoid cell lines. A lower number of such aggregated a-lactalbumin molecules binding oleic acid as a co-factor can induce cytolysis of several types of malignant cells. Such preparations of human a-lactalbumin made lethal to tumor cells (HAMLET) are highly effective in inducing apoptosis. The antitumor cytolytic activity with HAMLET also has been observed against large numbers of human tumors.

Lactofem'n. Lactoferrin, the dominant whey protein in human milk, is a single-chain glycoprotein with two globular lobes, both of which display a site that binds ferric iron.209 More than 90% of the lactoferrin in human milk is in the form of apolactoferrin (i.e., it does not contain ferric iron)?" which competes with siderophilic bacteria and fungi for ferric iron2"-2'5 and thus disrupts the proliferation of these microbial pathogens. The epithelial growth-promoting activities of lactoferrin in human milk also may aid in the defense of the recipient infant. 216 The mean concentration of lactoferrin in human colostrum is between 5 and 6 mg/mL?17 As the volume of milk production increases, the concentration falls to about 1 mg/mL at 2 to 3 months of l a~t a t i o n . "~~~'~ Because of its resistance to proteolysis,220-222 the excretion of lactoferrin in stool is hi her in human milk-fed than in cow's milk-fed infant^. '''-^^^ The mean intake of milk lactoferrin per day in healthy breast-fed, full-term infants is about 260 mg/kg at 1 month of lactation and 125 mg/kg by 4 month^."^ The quantity of lactoferrin excreted in the stools of low-birth-weight infants fed human milk is approximately 185 times that in stools of infants fed a cow's milk formula. 226 That estimate, however, may be too high because of the presence of immunoreactive fragments of lactoferrin in the stools of human milk-fed infants. 227 In addition, a significant increment in the urinary excretion of intact and fragmented lactoferrin occurs as a result of human mdk fee ding^.'^^.^^^ Recent stable isotope studies suggest that the increments in urinary lactoferrin and its fragments are principally from ingested human milk la~toferrin.~~' Lysozyme. Relatively high concentrations of lysozyme single-chain protein are present in human milk.218,219,231-235 This 15-kDa agent lyses susceptible bacteria by hydrolyzing p-1,4 linkages between N-acetylmuramic acid and 2-acetylamino-2-deoxyy-~-glucose residues in cell Lysozyme is relatively resistant to digestion by trypsin or denaturation due to acid. The mean concentration of lysozyme is about 70 pg/mL in colostrum,218 about 20 pg/mL at 1 month of lactation, and 250 pg/mL by 6 The approximate mean daily intake of milk lysozyme in healthy, full-term, completely breast-fed infants is 3 to 4mg/kg at 1 month of lactation and 6 mg/kg by 4 months. 223 Few studies have been conducted to examine the fate of human milk lysozyme ingested by the infant. The amount of lysozyme excreted in the stools of low-birth-weight infants fed human milk is approximately eight times that found in the stools of infants fed a cow's milk formula,226 but the urinary excretion of this protein does not increase as a result of human milk feedings.

Fibronectin. Fibronectin, a high-molecular-weight protein that facilitates the uptake of many types of particulates by mononuclear phagocytic cells, is present in human milk (mean concentration in colostrum, 13.4 mg/L).237 The in vivo effects and fate of this broad-spectrum opsonin in human milk are not known. Complement Components. The components of the classical and alternative pathways of complement are present in human milk, but the concentrations of these components, except C3, are exceptionally i0w.163,164

Although a direct anti-inflammatory effect of human milk has not been demonstrated in vivo, a number of clinical observations suggest that breast-feeding protects the recipient infant from injury to the intestinal or respiratory r n u c~s a . '~~~~~~ This protection may be due in part to the more rapid elimination or neutralization of microbial pathogens in the lumen of the gastrointestinal tract by specific or broad-spectrum defense agents from human milk, but other features of human milk suggest that this is not the sole explanation. Phlogistic agents and the systems that give rise to them are poorly represented in human milk.240 By contrast, human milk contains a host of antiinflammatory agents,24' including a heterogeneous group of growth factors with cytoprotective and trophic activity for the mucosal epithelium, antioxidants, antiproteases, cytokines and cytokine receptors and antagonists, and other bioactive agents that inhibit inflammatory mediators or block the selected activation of leukocytes. Like the antimicrobial factors, some of these factors are well adapted to operate in the hostile environment of the recipient's alimentary tract.

Growth factors in human milk include EGF,'69,242 the transforming growth factors TGF-cx'~~ and TGF-P,'" lactof e r k 2 I 6 mammary gland-derived growth factor:45 and p~l y a m i n e s . '~*~~~ These and a host of including insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), growth hormone-releasing factor (GHF), hepatocyte growth factor (HGF), prolactin, leptin, and may affect the growth and maturation of epithelial barriers, limit the penetration of pathogenic microorganisms and free antigens, and prevent allergic sensitization. Corticosterone, a glucocorticoid that is present in high concentrations in rat milk, speeds gut closure in the neonatal rat.250 Although macromolecular absorption does not appear to be as marked in the human n e~n a t e ?~' .~~~ the function of the mucosal barrier system in early infancy is important to host defense, and this system may be affected by factors in human milk. In this regard, the maturation of the intestinal tract as measured by mucosal mass, DNA, and protein content of the small intestinal tract appears to be influenced by milk, particularly early milk secretions.254

Antioxidant activity in colostrum has been shown to be associated with an ascorbate compound and uric acid?55 In addition, two other antioxidants present in human milk, c i -t o~o p h e r o l~~~~~~~ and p-~arotene,'~~ are absorbed into the circulation by the recipient gastrointestinal mucosa. Serum vitamin E concentrations rise in breast-fed infants from a mean of 0.3 mg/mL at birth to approximately 0.9 mg/mL on the fourth day of life. 256 The pleiotropic cytokine IL-10, a potent suppresser of macrophage, T cell, and NK cell function, has been demonstrated at very high concentrations in samples of human milk collected during the first 80 hours of lactation.258 IL-10 is present not only in the aqueous phase of the milk but also in the lipid layer. Its bioactive properties were confirmed by the finding that human milk samples inhibited blood lymphocyte proliferation and that this property was greatly reduced by treatment with anti-IL-10 antibody. Of interest, mice with a targeted disruption in the IL-10 gene, when raised under conventional housing conditions, spontaneously develop a generalized enterocolitis that becomes apparent at the age of 4 to 8 weeks (time of weaning).25y These observations suggest that IL-10 in human milk may play a critical role in the homeostasis of the immature intestinal barrier by regulating aberrant immune responses to foreign antigens. Soluble receptors and cytokine receptor antagonists also are potent anti-inflammatory agents. Human colostrum and mature milk have been shown to contain biologically active levels of IL-1 receptor antagonist (IL-1Ra) and soluble TNF-a receptors I and I1 (sTNF-aRI and sTNF-~RII).~~' The in vivo relevance of these observations also has been confirmed in a chemically induced colitis model of rats. Animals with colitis fed human milk had significantly lower neutrophilic inflammation than animals fed either chow or infant formula.26' Similar "protective" effects were seen in rats with colitis fed an infant formula supplemented with IL-1Ra,261 suggesting that this anti-inflammatory agent present in milk may contribute to the broad protection against different injuries provided by human milk feeding.

The presence in human milk of platelet-activating factor acetylhydrolase (PAF-AH), the enzyme that catalyzes the degradation and inactivation of PAF, is intriguing.262 Indeed, elevated serum concentrations of PAF have been found in rat and human neonates with necrotizing enterocolitis (NEC), whereas the concentrations of PAF-AH were found to be significantly lower than in control (unaffected) neonates.2633264 It also is of interest that serum concentrations of PAF-AH at birth are below those in adults and then gradually rise. 265 The enzyme is actively transferred from the mucosal to the serosal fluid in intestine of neonatal rats, particularly in the earliest postnatal period.266 Other antiinflammatory factors present in human milk include an IgE-binding factor, related antigenically to the Fc&RII (the lower-affinity receptor for I@), that suppresses the in vitro synthesis of human IgE,267 and the glycophosphoinositolcontaining molecule protectin (CD59) that inhibits insertion of the complement membrane attack complex (MAC) to cell targets. 268 The in vivo fate and effects of these antiinflammatory factors in human milk are still poorly understood.

Several seemingly unrelated types of observations suggest that breast-feeding modulates the development of the immune system of the recipient infant:

Both prospective and retrospective epidemiologic studies have shown that breast-fed infants are at less risk for development of certain chronic immunologically mediated disorders later in childhood, including allergic diseases,269 Crohn's disease,270 ulcerative insulin-dependent diabetes m e l l i t~s ,~~~ and some lymphomas. In regard to cell-mediated immunity, breast-fed infants given bacille Calmette-Gukrin (BCG) vaccine either at birth or later show a significantly higher lymphocyte transformation response to purified protein derivative (PPD) than that in infants who were never breast-fed?" Moreover, maternal renal allografts survive better in persons who were breast-fed than in those who were n~t .~~' -~' ' In this respect, the in vitro allogeneic responses between the blood lymphocytes of mothers (stimulating cells) and their infants (responding cells), as measured by an analysis of the frequencies of cytotoxic T lymphocyte (CTL) precursors directed against HLA alloantigens (CTL allorepertoire), are low in breast-fed infants2'' Increased levels of certain immune factors in breast-fed infants, which could not be explained simply by passive transfer of those substances, also suggest an immunomodulatory activity of human milk. Breast-fed infants produce higher blood levels of interferon in response to respiratory syncytial virus infection.282 It also was found that the increments in blood levels of fibronectin that were achieved by breast-feeding could not be due to the amounts of that protein in human milk.237 In addition, it was found that human milk feeding led to a more rapid development in the appearance of sIgA in These and other observations suggest that the ability of human milk to modulate the development of the infant's own mucosal and systemic immune systems may be associated with immunoregulatory factors present in colostrum and in more mature milk. Several different types of immunomodulatory agents can be identified in human milk.241 Among the numerous substances with proven or potential ability to modulate the infant immune response are pr~lactin?'~ a-to~opherol?~~ lact~ferrin:~~ nucleotides,6' anti-idiotypic s I~A ? '~ and cytokine~.~'~ It is evident that many of these factors in milk have other primary biologic functions, as in the case of hormones or growth factors, and that their potential as immune regulatory agents overlaps with their antimicrobial or anti-inflammatory proper tie^.'^' Cytokines in Human Milk In the 1990s, several cytokines, chemokines, and growth factors that mediate the effector phases of natural and specific immunity were discovered in human milk. These include IL-lp, IL-6, IL-7, IL-10, IL-12, IL-8, growth-related peptide-a (GRO-a), monocyte chemotactic protein-1 (MCP-1 ), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and TGF-P (Table 5-8) . Human milk displays a number of cytokine-characteristic biologic activities, including the stimulation of growth, differentiation of immunoglobulin production by B cells?88-2yo enhancement of thymocyte proliferation,2y' inhibition of IL-2 production by T cells?92 and suppression of IgE production.267 IL-lp2y3 and TNF-a 294 were the first two cytokines quantified in human milk. In colostrum, TNF-a is present mainly in fractions of molecular weight between 80 and 195 kDa, probably bound to its soluble receptors?60 Milk TNF-a is secreted both by milk macro phage^^^^*^^^ and by the mammary epithelium.296 IL-6 was first demonstrated in human milk by a specific bioassay. 297 In this study, anti-11-6-neutralizing antibodies Eotaxina ~~ G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRO-a, growth-related peptide-a; IL, interleukin; MCP-1, monocyte chemotactic protein-1; M-CSF, monocyte colony-stimulating factor; RANTES, regulated upon activation, normal T cell expressed and secreted; TGF-B, transforming growth factor+; TNF-a, tumor necrosis factor-a.

inhibited IgA production by colostrum mononuclear cells, suggesting that IL-6 may be involved in the production of IgA in the mammary gland. The presence of IL-6 in milk also has been demonstrated by immunoassays.294~296~298. 299 In like manner, IL-6 is localized in high-molecular-weight fractions of human milk. 298 The association of IL-6 with its own receptor has not been studied in milk, although the expression of IL-6 receptor by the mammary epithelium2% and in secreted form in the milk260 may explain the high molecular weight of this cytokine in human milk. The expression of IL-6 messenger ribonucleic acid (mRNA) and protein in milk cells and in the mammary gland epithelium suggests that both milk mononuclear cells and the mammary gland are likely major sources of this cytokine.2953296'300

The presence of IFN-y in human milk also has been reported, 151,296,299 although some investigators have found significant levels of IFN-y only in milk samples obtained from mothers whose infants had been delivered by cesarean section. The significance of this observation is not clear at present. IFN-y bioactivity as well as its association with specific subsets of milk T cells also remains to be deterrnined.l5' (The presence and possible function of IL-10 in human milk are discussed in the section "Anti-inflammatory Agents.") Chemokines are a novel class of small cytokines with discrete target cell selectivity that are able to recruit and activate different populations of le~kocytes.~~' Two major subfamilies, the CXC and the CC chemokines, are defined by the splicing of the conserved cysteine residues, which are separated by either one amino acid (CXC chemokines) or adjacent amino acids (CC chemokines). IL-8 and GRO-a belong to the CXC family and are mainly chemotactic factors for neutrophils. On the other hand, CC chemokines, which include MCP-1, macrophage inflammatory proteinla (MIP-la), and RANTES (regulated upon activation, normal T cell expressed and secreted), are chemotactic factors for monocytes, basophils and eosinophils, and T lymphocytes. 302 The presence of both CXC and CC chemokines has been described in human milk (see Table 5 -8).

IL-8 concentration was first determined in a small group of milk samples by Basolo and colleagues.296 These investigators identified the expression and secretion of IL-8 by mammary epithelial cells, although milk cells also appear to produce this ~h e m o k i n e .~~~.~" Another member of the CXC chemokine found in human milk is GRO-a, along with the two CC chemokines MCP-1 and RANTES.300 Expression of MCP-1 and, to a lesser extent, RANTES mRNA was confirmed in studies of milk cells. 300 Recently, high levels of the CC chemokine eotaxin, a potent and specific chemotactic factor for eosinophils and subtype 2 helper T cells (TH2), also have been demonstrated in human milk. 303 Colony-stimulating factors-highly specific protein factors that regulate cell proliferation and differentiation in the process of hematopoiesis-were discovered relatively recently in human milk. Although colony-stimulating activity was demonstrated in milk in 1983:04 G-CSF, M-CSF, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were not specifically identified and measured in human milk until the 1990s.151,305-307 The concentrations of M-CSF in particular appear to be 10-to 100-fold those in serum, and M-CSF evidently is produced by epithelial cells of the ducts and alveoli of the mammary gland under the regulatory activity of female sex hormones. 306 Although it is tempting to speculate that cytokines present in milk may be able to interact with mucosal tissues in the respiratory and alimentary tracts of the recipient infant, the functional expression of specific receptors for cytokines on epithelial or lymphoid cells in the airway and gastrointestinal mucosa has not been fully explored.24' A receptor-independent mechanism of cytokine uptake by the gastrointestinal mucosa during the neonatal period has not been demonstrated to date.

Several investigators have examined the effects of prematurity, early weaning, galactorrhea, and maternal malnutrition on the process of lactation. The immunologic aspects of these studies have focused largely on evaluation of the total content of sIgA and specific antibody activity. As described previously, the mammary secretions of nonlactating breast contain sIgA, although the amount appears to be much lower than in the lactating breast.308 Mammary secretions of patients with galactorrhea appear to contain sIgA in concentrations similar to those of normal postpartum colostrum.309 Although malnutrition has been associated with reduced secretory antibody response in other external secretions, maternal malnutrition does not seem to affect the total sIgA concentration or antimicrobial-specific antibody activity in the m~tk.~'O The nutritional as well as immunologic composition of milk from mothers of premature infants appears to be significantly different from that of milk from mothers of infants born at term.14,219,311,312 Comparative studies conducted during the first 12 weeks of lactation suggest that the mean concentrations of lactoferrin and lysozyme are higher in preterm than in term milk. Secretory IgA is the predominant immunoglobulin in preterm as well as in term milk, although the sIgA concentration appears to be significantly higher in preterm milk collected during the first 8 to 12 weeks of lactation. Secretory IgA antibody activity against certain organisms (E. coli somatic antigen) in the preterm milk was observed to be somewhat less than, or at best similar to, that found in term milk. In addition, the number of lymphocytes and macrophages in milk appears to be lower at 2 weeks but significantly higher at 12 weeks in milk from mothers with preterm (born at 34 to 38 weeks of gestational age) infants than in milk from those with fullterm infants.311 The authors of these investigations have proposed that some of the observed changes may reflect the lower volume of milk produced by mothers delivered of preterm infants. The possibility remains that changes in the immunologic profile of preterm milk may be a consequence of inadequate stimulation by the preterm infant, alterations in the maternal hormonal milieu, or other factors underlying premature delivery itself.

Development of mucosal integrity in the gut appears to depend on maturation of the mucosal tissue itself and the establishment of a normal gut flora. The former represents an anatomic and enzymatic blockage to invasion of microorganisms and antigens, and the latter, an inhibition of colonization by pathogenic bacteria. Although permeability of the neonatal gut to immunoglobulin is rather short-lived or incompletely developed, unprotected or damaged neonatal gut is permeable to a host of other proteins and macromolecules for several weeks or longer. Large milk protein peptides and bovine serum albumin have been shown to enter the circulation and produce a circulating antibody response. The inflamed or ischemic gut is even more porous to both antigens and pathogens. A variety of proven and presumed mechanisms for the role of both IgA and the normal flora have been proposed to compensate for these temporary inadequacies. Evidence for gut-trophic substances in humans is still preliminary. Ample epidemiologic evidence exists for a positive effect of breast-feeding in establishing the normal gut flora. Most compelling are the observations in rural Guatemala of gross contamination of milk by potentially pathogenic aerobic, gram-negative bacilli, including E. coli and Shigella species, with an absence both of diarrheal illness and of significant numbers of these organisms in the feces of infants during the period of lactation. In addition, the diverse serotypes of aerobic, gramnegative bacilli present in the oropharynx and the gastrointestinal tract of the neonate may serve as a source of antigen to boost the presensitized mammary glands, leading to a further modulation of specific bacterial growth in the muc0sa.3~~ The precise role of antibody that blocks adherence of these pathogens to the gut and the effects of other factors, such as lactoperoxidase, lactoferrin, lysozyme, and Bifidobacterium bifidum, in those situations are undetermined.

Extensive epidemiologic evidence supports the "prophylactic value" of breast-feeding in the prevention or amelioration of diarrheal disease and is summarized in several revieWS.22,82,314,3 15 Ample experimental animal data on the value of specific colostral antibody in preventing diarrheal illness are available from studies of colostral deprivation.

These include colibacteriosis associated with E. coli K88 in swine; rotaviral gastroenteritis in cattle, swine, and sheep; and diarrheal illness associated with transmissible gastroenteritis of swine. 316 In humans, cholera is rare in infancy, especially in endemic areas where the prevalence of breast-feeding is high. The experience with an outbreak of cholera in the Persian Gulf lends support to the possibility that the absence of breast-feeding is an important variable in increasing the risk of cholera in infancy.

A few reports have claimed that nursery outbreaks of diarrhea associated with enteropathogenic strains of E. coli can be interrupted by use of breast milk. Conflicting data exist regarding prevention of human rotaviral infection and disease. Evaluation of nursery outbreaks of rotaviral disease has suggested that the incidence both of infection and of illness was lower in breast-fed infants, but the incidence of symptoms in formula-fed infants also was very low. Studies carried out in Japan have noted a fivefold decrease in incidence of rotaviral infection among breast-fed infants younger than 6 months of age. It must be emphasized that most rotavirus infections in neonates are asymptomatic, regardless of breast-or b~ttle-feeding."~-~~' On the basis of careful clinical observations, Bishop and c o -~o r k e r s~~~ in Australia first questioned the positive effects of breastfeeding in rotavirus infection. More recent case-control studies of enteric viral infections in breast-fed infants have suggested that breast-feeding may protect infants from hospitalization rather than from infection itself.323s3"' Longitudinal follow-up of a large cohort of infants during a community outbreak of rotavirus has shown that attack rates of rotavirus infection were similar in breast-fed and in bottle-fed infants. The frequency of clinical disease with diarrhea, however, appeared to be significantly lower in breast-fed infants. Of interest, the protection observed in these patients was more a reflection of altered microbial flora from breast-feeding than of specific immunologic protection against rotavirus. Thus, it appears that breastfeeding provides significant protection against diarrheal disease, although the mechanism of such protection remains to be defined.323x324

NEC is a complex illness of the stressed premature infant, often associated with hypoxia, gut mucosal ischemia, and necrolysis and death.325,326 Clinical manifestations have, on a few occasions, been associated with bacteremia and invasion by gram-negative bacilli, particularly Klebsiella pneumoniae, into the intestinal submucosa. Clinical manifestations include abdominal distention, gastric retention, and bloody diarrhea. Classic radiographic findings include air in the bowel wall (pneumatosis intestinalis), air in the portal system, and free infradiaphragmatic air (signifying perforation). Treatment involves decompression, systemic antibiotics, and, often, ~u r g e r y .~~~"~~ A number of studies have suggested a beneficial role of breast milk in preventing or modifying the development of NEC in high-risk human infants. Some pediatric centers have claimed virtual absence of NEC in breast-fed infants; however, many instances of the failure of milk feeding to prevent human NEC also have been reported. In fact, outbreaks of NEC related to Klebsiella and Salmonella species secondary to banked human milk feedings have been docu-mented. 127.330.331 In an asphyxiated neonatal rat model of NEC, the entire syndrome could be prevented with feeding of maternal milk. The crucial factor in the milk appeared to be the cells, probably the macro phage^.'^^ It also is possible that antibody and nonspecific factors play a role, as does establishment of a gut flora. Prophylactic oral administration of immunoglobulin has been found to have a profound influence on the outcome of NEC in well-controlled studies.331 Penetration of the gut by pathogens and antigens is increased with ischemic damage, and noncellular elements of milk may aid in blockage of this transit. 332 The role of enteric anaerobic organisms has been seriously considered in the pathogenesis of NEC. Cytolytic toxins of Clostridium dificile and other clostridial species have been demonstrated in infants with NEC, often significantly more frequently than in normal Clearly, NEC is a complex disease entity whose pathogenesis and cause remain to be defined. Although breastfeeding may be protective, a number of other factors are clearly related to the mechanism of mucosal injury and the pathogenesis of this syndrome.

The incidence of bacteremia among premature infants fed breast milk has been suggested to be significantly lower than that among those receiving formula feedings or no feeding.337"39 It has been shown that a high percentage of cases of neonatal bacteremia and meningitis caused by 

One of the most challenging developments in human milk research has been the demonstration in breast-fed infants of a reduced incidence of diseases with auto-or dysregulated immunity, long after the termination of brea~t-feeding.'~~-~~~ Since the first report in 1936,343 numerous published studies have addressed the effect of infant feeding on the development of atopic disease and asthma. Beneficial results of breast-feeding as prophylaxis against atopy have been observed in most of the studies; in others, however, beneficial effects were reported only in infants with a genetically determined risk for atopic disease. Finally, no beneficial effect at all or even an increased risk has been suggested in some breast-fed infants. Kramer, in an extensive meta-analysis of 50 studies published before 1986 that focused on infant feeding and atopic disease, has attempted to shed some light on the c o n t r o~e r s y .~~ Seven of the 13 studies on asthma included in this analysis claimed a protective effect of breast-feeding, whereas 6 claimed no protection. Several serious methodologic drawbacks, however, have been noted in this analysis. In a number of the studies analyzed, early infant feeding history was obtained months or years after the feeding period, ascertainment of the infant feeding history was obtained by interviewers who were aware of the disease outcome, or insufficient duration and exclusivity of breast-feeding were documented; all were confounding variables that considered inappropriate "exposure standards." Nonblind ascertainment of disease outcome was found to be the most common violation of the "outcome standards."

Kramer's analysis also found that failure to control for confounding variables was a common violation in "statistical analysis standards" identified in several studies. Indeed, the effect of infant feeding on subsequent asthma may be confounded by other variables that are associated both with infant feeding and with unique investigational conditions. Factors that seem to have the greatest potential for confounding effects include the family history of atopic disease, socioeconomic status, and parental cigarette smoking. Only 1 of 13 studies on asthma included in the meta-analysis adequately controlled for these confounding factors. Moreover, 3 of the studies that did not demonstrate a protective effect of breast-feeding on asthma had inadequate statistical power. The effect of infant feeding on the severity of outcome and on the age at onset of the disease was virtually ignored in most of the Although this extensive meta-analysis may suggest some uncertainty about the prophylactic benefit of breast-feeding, two recent studies strongly support a positive effect of breast-feeding on the development of atopic disease and asthma. The first s t u d y consisted of prospective, longterm evaluation from infancy until the age of 17 years; the prevalence of atopy was significantly higher in those infants with short-duration (less than 1 month) or no breast-feeding, which increased to a demonstrable difference by the age of 17 years, than in the infants with intermediate-duration (1 to 6 months) or prolonged (longer than 6 months) breastfeeding. The differences in the prevalence of atopy persisted when the groups were divided according to positive or negative atopic heredity. Furthermore, the atopy manifestations in the different infant feeding groups did not remain constant with age. In particular, respiratory allergy, including asthma, increased greatly in prevalence up to the age of 17 years, with a prevalence as high as 64% in the group with short-duration or no brea~t-feeding.~~~ In the second study, a prospective, longitudinal study of the prevalence and risk factors for acute and chronic respiratory illness in childhood, the investigators examined the relationship of infant feeding to recurrent wheezing at age 6 years and the association with lower respiratory tract illnesses associated with wheezing early in life?45 Children who were never breastfed had significantly higher rates of recurrent wheezing at 6 years of age. Increasing duration of breast-feeding beyond 1 month was not associated with significantly lower rates of recurrent wheezing. The effect of breast-feeding was apparent for children both with and without wheezing lower respiratory tract illnesses in the first 6 months of life. In contrast with the findings of the first study, however, the effect of breast-feeding was significant only among nonatopic children. 345 The exact mechanisms by which breast-feeding seems to confer long-lasting protection against allergic sensitization are poorly understood. It is likely, however, that multiple synergistic mechanisms may be responsible for this effect, including ( 1) maturation of the recipient gastrointestinal and airway mucosa, promoted by growth factors present in human milk242-244; (2) inhibition of antigen absorption by milk s I~A~~~; (3) reduced incidence of mucosal infections and consequent sensitization to bystander antigens347; (4) changes in the microbial flora of the intestine of breast-fed infants325; and (5) direct immunomodulatory activity of human milk components on the recipient A number of earlier and more recent studies have greatly contributed to the understanding of macromolecular transport across the immature gut and its consequences in terms of the generation of circulating antibody or immune complexes, the processes that are blocked predominantly by sIgA, the glycocalyx, and the intestinal enzymes. These mucosal immunologic events have been the basis for the concept of immune exclusion. Immune exclusion is not absolute, however, because uptake of some antigens across the gut may be enhanced rather than blocked by interaction with antibody at the mucosal surface. Beginning with the observations of IgA-deficient patients, it has become clear that the absence of the IgA barrier in the gut is associated with both an increased incidence of circulating antibodies directed against many food antigens and an increased occurrence of atopicallergic diseases.346 Some studies have noted complement activation in serum after feeding of bovine milk to children with cow's milk allergy. The neonate is similar in some respects to the @-deficient patient,348 and increased transintestinal uptake of food antigen with consequent circulating antibody formation in the premature infant has been reported. 349 Other studies have suggested that early breastfeeding, even of short duration, is associated with a decreased serum antibody response to cow's milk proteins.253 Prolonged breast-feeding not only may partially exclude foreign antigens through immune exclusion but may also, because the mother's milk is the infant's sole food, prevent their ingestion.350 It must, however, be emphasized that intact bovine milk proteins and other food antigens and antibodies have been observed in samples of colostrum and milk.6

As described previously, epidemiologic evidence suggests that bacterial and viral respiratory infections are less frequent and less severe among breast-fed infants in a variety of cultures and socioeconomic settings. Antibodies and immunologic reactivity directed against herpes simplex virus, respiratory syncytial virus, and other infectious ave been quantitated in colostrum and milk. Adoptive experiments in suckling ferrets have shown that protection of the young against respiratory syncytial virus can be transferred in colostrum containing specific antibody. The neonatal ferret gut, however, is quite permeable to macromolecules and permits passage of large quantities of virus-specific IgG. In the absence of either documented antibody or cellular transfer in the human neonate across the mucosa, any mechanisms of protection against respiratory syncytial virus and other respiratory pathogens remain obscure.

Data are lacking in humans regarding passive protection on other mucosal surfaces, such as the eye, ear, or genitourinary tract. Some epidemiologic evidence suggests that recurrence of otitis media with effusion is strongly associated with early bottle-feeding and that breast-feeding may confer protection against otitis media with effusion for the first 3 years of life.353 Foster feeding-acquired antibody to herpes simplex virus has been found to result in agents86,95,1 18,351,352 h significant protection against reinfection challenge in experimental animal studies. 351 A number of other benefits have been associated with breast-feeding, including natural contraception during active nursing354 and protection against sudden infant death syndr0me,3~~ diabetes,356 obesity,357 and high cholesterol level and ischemic heart disease later in life.42 Of particular recent interest has been the association of breast-feeding with improved intellectual performance in older children. Several studies have demonstrated enhanced cognitive outcome in breast-fed children, although controversy exists regarding the mechanisms by which such improved performance may occur.358-360 Health benefits for the mother also may be associated with breast-feeding: A reduced incidence of breast cancer has been noted in women who have lactated.361

Several potentially harmful effects have been associated with breast-feeding. Some provocative data suggest that nonautologous human milk may, under certain conditions, be nutritionally inadequate for the premature infant.22323 The concentration of anti-Rhesus factor (anti-Rh) antibodies in milk appears to be too low to pose any threat to the incompatible neonate. Variable concentrations of medicinal products and their metabolites are excreted in colostrum and milk (Table 5-9) . Environmental contaminants such as dichlorodiphenyl trichloroethane (DDT), polychlorinated biphenyls (PCBs), and mercury have been demonstrated in high concentrations in human milk.22, 362 The failure to initiate lactation properly during early breast-feeding may present a risk of dehydration to the infant, because insufficient fluids may be ingested. Inappropriate introduction of bottles and pacifiers also may interfere with proper induction of lactation. Later in lactation, introduction of bottles may induce premature weaning as the result of a reduction in the milk supply.

Although human milk is the optimal form of nutrition for most healthy term infants, some circumstances have been identified in which breast-feeding is contraindicated and some in which continued breast-feeding should be conducted with caution to protect the infant. Infants with inherited metabolic diseases may be best nourished by treatment with alternative forms of nutrition. In particular, neonates with diagnosed galactosemia need to have galactose removed from their diet; in other words, they need to be switched to a milk containing lactose-free carbohydrate (because lactose is a glucose-galactose disaccharide). Infants diagnosed with phenylketonuria may receive some human milk to support their requirement for phenylalanine but often may be better managed by use of specially prepared commercial milks.

Management of hyperbilirubinemia associated with breastfeeding, so-called breast milk jaundice, has been an area of some controversy. The mechanism responsible for this form of jaundice is unknown but has been suggested to reflect inhibitors of glucuronidation, deficiency of related enzymes, excessive lipid breakdown, and insufficient milk intake.363.364 Recent recommendations suggest that a more laissez-faire approach to this problem is appropriate.365 Increasing milk volume by increasing the number of feedings may be the most appropriate approach to breast milk jaundice; however, severe cases may necessitate phototherapy. Increased intake of fluids in breast-feeding infants appears to be effective in many cases. 366 Several instances of specific nutrient deficiencies in breast-fed infants have been described, specifically related to lack of vitamin K, vitamin D, vitamin B,,, folk acid, vitamin C, and carnitine. In each of these instances, several case reports have appeared warning against deficiencies that have resulted in clinical consequences to the neonate. For example, hemorrhagic disease reported in a few breast-fed infants was successfully treated with vitamin K.367 These infants did not receive vitamin K at birth. Mothers who practice unusual dietary habits, such as strict vegetarianism, may have reduced levels of vitamin B,, and folic acid in their milk, and deficiencies in breast-fed infants of such mothers have been reported.3689369 Cases of rickets in breast-fed infants have been reported, particularly during winter among infants not exposed to the sun.32337o Deficiency of carnitine, a nutrient responsible for modulating fat absorption, also has been reported to result in clinical symptoms in breast-fed infants in mothers ingesting unusual d i e t~. h ' -~~'

These various clinical expressions of nutrient deficiency in milk are of concern, but they also should be put in the context of nutrient deficiencies observed in formula-fed infants. Clearly, millions of infants in developing countries are at severe risk of malnutrition when they are formula-fed because of the economic stress of supplying sufficient formula. Even in developed countries, large numbers of nutrient deficiencies and associated clinical symptoms have occurred as a result of accidents in the manufacture of formulas.372 The most notable of these accidents have taught us the effects of early vitamin B, deficiency, folic acid deficiency, and chloride deficiency. Formula feeding also has been associated with an increased incidence of diabete~?'~ Thus, some situations arise in which breast-feeding must be carefully considered as an appropriate feeding modality for the infant. Commercial formulas also represent risks, however. The infant is best served by observant pediatricians and mothers who promptly respond to any clinical signs in the neonate.

The presence of microbial contamination in milk is of serious concern. Contaminated milk has been implicated in neonatal infection with S. uureus, group B hemolytic streptococci, mycobacteria, and, possibly, Salmonella species (Table 5-10). Mastitis and breast abscess have been associated with the presence of bacterial pathogens in human milk. Such inflammation of the breast will often resolve even with continued breast-feeding. Resolution of the inflammation may be related to the presence of antisecretory factor (AF), a factor induced in the milk by enterotoxin-producing bacteria that appears to promote recovery from acute bacterial mastititis. In general, feeding an infant from a breast affected by an abscess is not recommended.' Infant feeding on the affected breast may be resumed, however, once the mother has received adequate treatment. Furthermore, breastfeeding may continue on the unaffected breast. Mothers with active tuberculosis should refrain from breast-feeding for at least 2 weeks or longer after institution of appropriate treatment if they are considered contagious.' Viral contaminants of maternal origin in the milk include rubella virus, herpes simplex virus, hepatitis B virus (HBV), cytomegalovirus (CMV), HIV-1, human T-lymphotropic virus type 1 (HTLV-l), and, possibly, HTLV-2 (see Table 5 -10). For most viruses, although transmission has been documented as evidenced by seroconversion, no serious illness in the neonate, with the possible exception of CMV infection-related illness secondary to breast-feeding, has been rep~rted.''~~'~ Occasional reports of possible neonatal herpes simplex virus infection associated with presence of the virus in the mother's milk may just as easily have been caused by an infant-to-mammary gland rather than a mammary gland-to-infant route of in~culation.~'~ Both the RNA-dependent DNA polymerase and structural proteins of C-type tumor viruses, possibly related to mouse mammary tumor and Mason-Pfizer viruses, have been identified in human breast tissue and products of lactation.*' An association between breast-feeding of female infants and the development of breast cancer has been hypothesized in families with a strong history of carcinoma of the breast. Epidemiologic evidence to support such an association is lacking, however. Breast-feeding may in fact be a protective factor relative to maternal risk of such neoplastic disease.375 Therefore, with adherence to reasonable maternal hygiene and in the absence of intense chemical contamination, generally few proven or well-defined contraindications to natural breast-feeding exist. Current recommendations regarding the transmission of infectious virus in human milk and their implications for the breast-fed infant are summarized next.

Human Immunodeficiency Virus Infection. Recently, serious concern has been voiced regarding the potential risk of the transmission of HIV from infected mothers to their suckling neonates through the process of breast-feeding. The possibility of postnatal transmission of this virus from mother to child has been considered in a large number of infants breast-fed in the United States and in other parts of the world. In some of these infants, breast-feeding has been implicated as one of the major risk factors for acquisition of HIV infection. Since 1985, small but significant numbers of infants with HIV infection possibly acquired through the process of breast-feeding have been reported. 376 In virtually all reported cases, maternal seroconversion for HIV antibody probably occurred after delivery of the infant. More than 50% of these mothers acquired the infection through heterosexual transmission, and about 30% through blood transfusion. Few of the mothers were judged to be intravenous drug users, Although acquisition of HIV infection before delivery cannot be ruled out with certainty, the likely route of transmission in these infants has been presumed t o be through breast-feeding. The most convincing observations are based on several maternal-infant pairs in whom maternal seroconversion to HIV antibody occurred 4 months or longer after delivery.377

A number of studies have demonstrated HIV in milk.378-382 The findings include isolation of HIV from milk supernatants collected from symptom-free women and from cellular fractions of maternal milk, recovery of HIV virions in the histiocytes and cell-free extracts of milk by electron microscopy, and detection of viral DNA by polymerase chain reaction (PCR) assay in greater than 70% of samples from HIV-seropositive lactating women. Limited epidemiologic studies carried out to date, however, have failed to demonstrate the magnitude of risk of HIV infection in breast-fed infants. Cohort st~dies''~ in different populations have suggested increased, reduced, or similar transmission rates in breast-feeding and in non-breast-feeding (bottle-feeding) infants of seropositive mothers ( Table 5-11) . Thus, it appears that although precise epidemiologic data are still lacking, a majority of breast-fed infants born to HIV-seropositive mothers remain uninfected despite the presence of HTV DNA in the milk in a high proportion of such mothers. Nevertheless, the risk of acquisition of HIV infection through breast-feeding must not be ignored. On the basis of meta-analysis of available data, it has been estimated that the additional risk of HIV infection through breast-feeding may be as high as 22%. 384 Some studies have suggested that breastfeeding contributes up to a 50% increase in the overall vertical transmission of HIV infection. 385 Despite the potential risk of HIV infection in infants of HIV-infected breast-feeding mothers, consideration of cessation of breast-feeding must be balanced against other beneficial effects as outlined in this chapter. In a 1990 study, breast-fed HIV-infected children progressed to acquired immunodeficiency syndrome (AIDS) at a slower rate than that noted for bottle-fed ~hildren."~ Current estimates indicate the overall risk of acquiring HIV infection from breast-feeding to be about 16%. Of all HIV-infected infants, 47% may be infected by means of breast-feeding. Among those breast-fed for 3 months or longer, the rate of infection was estimated to be approximately 21%, and among those breast-fed for 2 months or less, the rate of infection was approximately 13%.' It is, however, important to realize that a number of other risk factors contribute to the increased transfer of HIV through breast-feeding. Associated maternal factors include younger age, multiple deliveries, high virus load, lower number of CD4+ lymphocytes, and maternal mastitis. Other risk factors associated with the maternal milk include high viral load in the milk, long duration of breast-feeding, especially mixed formula feeding and breast-feeding, low levels of antiviral factor in the milk (low CTL, sIgA, lactoferrin, lysozyme). Evidence of oral candidiasis in the breast-feeding neonate also appears to be a risk factor for development of breastfeeding-associated HIV infection. ' Current recommendations from the American Academy of Pediatrics state that in populations such as that of the United States, in which the risk of death from infectious diseases and malnutrition is low and in which safe and effective alternative sources of feeding are readily available, HIV-infected women should be counseled not to breast-feed their infants nor to donate milk. All pregnant women in the United States should be counseled and encouraged to be tested for HIV infection. Data are not available about the safety of breast-feeding by mothers receiving highly active antiretroviral therapy (HAART). In geographic areas in which infectious diseases and malnutrition are important causes of death early in life, the feeding decision may be more complex. The World Health Organization (WHO) states that if a mother is infected with HIV, replacement of human milk to decrease the risk of HIV transmission may be preferable to breast-feeding, provided that the risk associated with replacement feeding is less than the potential risk of HIV transmission. Implementation of this suggestion has many obstacles. The WHO policy stresses the need for continued support for breast-feeding by mothers who are HIV negative or of unknown HIV serostatus, improved access to HIV counseling and testing, and government efforts to ensure uninterrupted access to nutritionally adequate human milk substitutes.'

Cytomegalovirus Infection. CMV infection is a common perinatal infection. The virus is shed in the milk in about 25% of infected mothers. Although breast-feeding from infected mothers may result in seroconversion in up to 70% of breast-feeding neonates, the infection often is not associated with clinical symptoms of disease. Low-birthweight infants (born at less than 1500 g), however, may exhibit evidence of clinical disease, with thrombocytopenia, neutropenia, or hepatosplenomegaly seen in 50% of breastfeeding-infected babies. The decision to breast-feed a premature baby by an infected mother should be based on weighing the potential benefits of human milk versus the risk of CMV transmission. ' Hepatitis B Virus Infection. Hepatitis B surface antigen (HBsAg) has been detected in milk of HBV-infected mothers. Nevertheless, breast-feeding does not increase the risk of HBV infection among these infants. Infants born to HBV-positive mothers should receive hepatitis B immune globulin (HBIG) and the recommended series of hepatitis B vaccine without any delay in the institution of breastfeeding.'

Hepatitis C V i i s Infection. The RNA of hepatitis C virus (HCV) and antibody to HCV have been detected in the milk from infected mothers. Transmission by means of breastfeeding, however, has not been documented in anti-HCV-positive, anti-HIV-negative mother. According to current guidelines, HCV infection does not contraindicate breast-feeding.'

Rubella. Rubella virus has been recovered from milk after natural as well as vaccine-associated infection. It has not been associated with significant disease in infants, however, although transient seroconversion has been frequently demonstrated. No contraindication to breast-feeding exists in women recently immunized with currently licensed rubella vaccines.

West Nile V i i s Infection. The RNA of west Nile virus has been detected in human milk, and seroconversion in breastfeeding infants also has been observed. Although West Nile virus can be transmitted in milk, its extent of transmission in humans remains to be determined. Most infants and children infected with the virus to date have been asymptomatic or have had minimal disease.' Infection Due to Human T-Lymphotmpic Viruses 1 and 2. Epidemiologic studies strongly suggest the possibility of mother-to-infant transmission of HTLV-1 by breast-feeding. In the United States, currently it is recommended that HTLV-1-infected women should not breast-feed. On the other hand, the status of maternal-infant transmission of HTLV-2 through the process of breast-feeding has not been well established, and until additional information is available, breast-feeding should not be recommended in seropositive women.'

It is apparent that human colostrum and milk are richly endowed with a wide variety of cellular and soluble components that participate in many nutritional, immunologic, and anti-infective processes of specific benefit to the neonate. The function of the products of lactation and maternal breast-feeding best characterized to date is nutritional support, and modulation andlor compensation for the transient mucosal immune deficiency against infectious and dietary macromolecules in the autologous infant.

In general, it is quite safe for the mother to collect her milk for later feeding or to directly breast-feed her own neonate. Nevertheless, increasing concerns regarding contamination of human milk by infectious agents have resulted in the limited use of either milk banks or wet nursing. Because of the transfer of infectious agents from maternal blood to milk (see Table 5 -10), several national advisory committees have recommended that patients who have known transmissible infectious viral or bacterial diseases should not breast-feed. 388 Other clinical situations in which withholding breastfeeding is appropriate because of high metabolite content in the milk include presence of galactosemia (galactose from lactose), phenylketonuria (phenylalanine), and other amino acid disorders in the infant.

As shown in Tables 5-9 and 5-10, many drugs, infectious agents, and environmental agents can be transferred to the infant in maternal milk. Rather than stopping breast-feeding, a nursing mother should avoid use of any drug unless it is absolutely essential. Many organohalides and fat-soluble environmental products, such as DDT and PCBs, may be present in higher concentrations in human milk?2 Although not much is known about their risk to the infant, it is generally agreed that unless the degree of exposure in the mother is extremely high, the benefits of breast-feeding outweigh the possible risks associated with environmental contaminants. Caffeine, alcohol, and nicotine also present potential hazards to the infant (see Table 5 -9). It is advisable to reduce or preferably discontinue the intake of tobacco, caffeine-containing products, and alcoholic beverages during lactation and nursing.

Both international389 and organizations have endorsed breast-feeding as the optimal means of feeding for the healthy term infant. In general, the percentage of mothers initiating breast-feeding in developing countries is 80% or higher and often 90% or more. 392 The health and economic consequences for bottle-fed infants in these countries are severe, however. In the United States, at one point in the early 1970s, the rate of breast-feeding initiation was as low as 25%. This low point was followed by an increase to a high of 61.9% in 1982. After 1982 a slow decline was observed (to 52.2% in 1989), after which a modest increase has been observed since the early 1 9 9 0~~~~

The pattern of breast-feeding initiation is accompanied by concomitant changes in maintenance of breast-feeding to 6 months, from 24% (1984) to 18% (1989) to 21.6% ( 1995).3939394 These changes took place despite goals set by the U.S. Surgeon General for 75% of infants to be breastfeeding in the first week of life and 35% at 6 months.395 These goals were reestablished for the year 2000.3%

Within the United States, a variety of demographic patterns appear to be associated with breast-feeding behavior. Older mothers, mothers with a college education, and higherincome mothers all are more likely to breast-feed. By contrast, black and Hispanic mothers, mothers of lower socioeconomic status who are participants in the Women, Infants, and Children (WIC) program of the U.S. Department of Health and Human Services and mothers who live in the southern regions of the United States are much less likely to breast-feed. The low rate of breast-feeding for mothers enrolled in WIC is of particular concern, as that agency has a specific policy to encourage breast-feeding. Many states, however, now depend on formula manufacturer rebates to fund part of their WIC programs, creating something of a conflict of interest. The disturbing part of the demographic pattern of breast-feeding in the United States is that the infants of lower-socioeconomic-status mothers, who would accrue the greatest health and economic benefits from breast-feeding, are those least likely to be breast-fed.

Although demographic studies indicate who is breastfeeding, they do not explain the behavioral differences among groups of mothers. One of the more complete models designed to explain breast-feeding behavior includes components that address maternal attitudes and family, societal, cultural, and environmental variables.397 Individual studies have shown that the maternal decision-making process is closely related to the social support and influence that come from the family members surrounding the mother. 398 The husband in particular appears to have a strong positive influence, whereas the mother's mother may have a negative influence on the breast-feeding decision. Social support appears to be different among ethnic groups, as are maternal attitudes; such differences may provide one explanation for differences in breast-feeding behavior among ethnic

Clearly, human milk contains a wide variety of soluble and cellular components with a diverse spectrum of biologic functions. The major milk components identified to date exhibit antimicrobial, anti-inflammatory, pro-inflammatory, and/or immunoregulatory functions; cytotoxicity for tumor cells; ability to repair tissue damage; and receptor analogue functions, as well as other metabolic effects. The relative contributions of different milk components to these biologic effects are summarized in Table 5 -12.

The bulk of antimicrobial effects are associated with milk immunoglobulin, especially the sIgA isotype, which makes up to 80% of all immunoglobulins in the human body. Clinical observations have demonstrated that milk antibodies protect against a large number of intestinal pathogens such as Campylobacter, Shigella, E. coli, V cholerae, Giardia, rotavirus, and respiratory pathogens such as respiratory syncytial virus. The milk antibodies also effectively neutralize toxins and a variety of human viruses. The role of small amounts of IgG and IgM in milk has not been fully examined. Recently, it has been suggested that milk IgG may hydrolyze nucleotides and DNA.40' In general, milk IgA antibodies induce antimicrobial protection in the absence of any inflammation, a characteristic of other complement-binding immunoglobulins such as IgG and IgM.

Significant numbers of PMNs, macrophages, and epithelial cells are observed in the milk. Their precise function in the milk remains to be determined. It is possible that their primary task is the defense of the mammary gland itself. The lymphocytes present in the milk transfer immunologic information and may offer significant T cell-mediated immunologic defense to the suckling neonate. Breast-fed infants seem to become tolerant to their mothers' HLA, which may have important implications regarding immune responsiveness and allograft rejection. 402 Lactoferrin, a major milk protein, also may play an important role in antimicrobial defense. It can kill bacteria, fungi, and viruses without causing inflammation. Lactoferrin also has been found to block mechanisms that result in the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-1 p) by inhibiting nuclear transcription factor (NFKP) activation me~hanisms.4~~

Recent observations have suggested that a-lactalbumin may exist in human milk as large complexes binding to oleic acid. Such complexes, referred to as human a-lactalbumin made lethal to tumor cells (HAMLET), induce apoptosis of all malignant cells tested to date. These complexes have remarkably little to no effect on normal cells. 4049405 Additional recent studies have suggested that milk contains large numbers of cytokines, chemokines, and growth factors. Although their precise role in the milk remains to be determined, some of these may act as signals for recruitment of pro-inflammatory and or immunoregulatory cells to the mucosal sites. TGF-P may be important in downregulating immune response and induction of tolerance, thereby decreasing the risk of allergic disease. IL-7 may promote development of a yS T cell population in cryptopatches which are small aggregates of lymphocytes in the intestinal crypts, and in the maintenance of thymus It also has been shown that increased levels of IL-1 b, TNF-a, IL-6, and possibly other cytokines increase the level of leptin, an appetite-regulating hormone that is present in significant quantities in mammary epithelial cells, milk fat globules, and milk.407*406 Leptin has several immunologic effects, including differentiation and proliferation of hematopoietic cells and regulation of monocyte-macrophage function. It also influences T cell response by enhancing IL-2 and IFN-y production (for TH1 cells) and IL-4 and IL-10 production (for TH2 cells). Leptin is absorbed by the breast-feeding infant408.409 and may be associated with prevention of obesity observed in breast-fed infants.

Another important recent observation with milk relates to the presence of antisecretory factor (AF). It has been observed in samples of milk, possibly induced by exposure to enterotoxin-producing bacteria.41° It possesses significant effect on intestinal fluid secretions. The precise function of AF remains to be determined. Preliminary observations, however, suggest that AF may be highly effective in treatment of inflammatory bowel disease. 4113412 Human milk has been found to possess large quantities of soluble CD14 and soluble Toll-like receptors TLR-2 and TLR-4, important elements for innate immunity!133414 CD14 promotes differentiation in expression of B cell function and anti-inflammatory effect of lactoferrin. Intestinal epithelium does not possess CD14, and it is possible that milk CD14 facilitates phagocytosis of organisms that require expression of this ligand. The TLR-2 and TLR-4 bind to a variety of microorganisms and may play an important role in downregulating inflammatory responses in the mucosal sites.

It is beyond the scope of this chapter to explore in any detail the reasons for and possible benefits of the evolution of mammalian life forms. Nevertheless, the passive transfer of the diversity of maternal biologic experiences to the neonate through the process of breast-feeding represents an essential component of the survival mechanism in the mammalian neonate. For millions of years, maternal products of lactation delivered through the process of breast-feeding have been the sole source of nutrition and immunity during the neonatal period and early infancy for all mammals, including the human infant. During the past 150 to 300 years, however, the human societal culture has undergone remarkable changes in rapid succession, which have had a major impact on the basic mechanisms of maternal-neonatal interaction and breast-feeding. Such changes include introduction of sanitation and nonhuman milk and formula feeds for neonatal nutrition, use of antimicrobial agents, introduction of processed foods, and exposure to newer environmental macromolecules and dietary antigens. The introduction of such manmade changes in the neonatal environment has had a profound impact on human homeostatic mechanisms and at the same time allowed new insights into the role of breast-feeding in the developing human neonate.

Comparative analysis of natural (traditional) forms of breast-feeding and artificial feeding modalities of modern times has demonstrated clearly that natural breast-feeding is associated with significant reduction in infant mortality and morbidity, protection against acute infectious diseases (both in the acute phase of the disease and with long-term reexposure), and possible protection against allergic disorders and autoimmune disease, acute and chronic inflammatory disorders, obesity, diabetes mellitus and other metabolic disorders, allograft rejection, and development of a number of malignant conditions in childhood or later in life. Newer evidence suggests a protective role of breast-feeding in modulating many respiratory, intestinal, and urinary tract infections, otitis media, and NEC in the neonate. This information has been recently reviewed by Hanson in an elegant m0nograph.4'~ Despite the overwhelmingly protective role attributed to natural breast-feeding and the evolutionary advantages related to the development of lactation, several infectious agents have acquired, during the course of evolution, the ability to evade immunologic factors in milk and to use milk as the vehicle for maternal-to-infant transmission. The potential for the acquisition of infections such as those due to HIV, HTLV, CMV, and possibly other pathogens highlights potential hazards of breast-feeding in some clinical situations. Thus, it is reasonable to conclude that the development of lactation, the hallmark of mammalian evolution, is designed to enhance the survival of the neonate of the species, and that breast-feeding may have a remarkable spectrum of immediate and long-term protective functions. 

Human milk

Report of the Committee on Infectious Diseases

Experimental analysis of the prenatal development of the mammary gland

Kleinberg DL, Niemann W, Flamm E. Primate mammary developmenk effects of hypophysectomy, prolactin inhibition, and growth hormone administration

Control of mammary growth and lactation by the anterior pituitary: an attempt to correlate classic experiments on animals with recent clinical findings

Gastric suction in healthy newborn infants

Does the newborn baby find the nipple by smell?

Composition of breast milk obtained from mothers of premature infants as compared to breast milk obtained from donors

Comparison of the composition of breast milk from mothers of term and preterm infants

Zinc citrate, human milk and acrodermatitis enteropathica

Milk protein quantity and quality in low-birth-weight infants. 11. Effects on selected essential and nonessential amino acids in plasma and urine

Milk protein quantity and quality in low-birth-weight infants. N. Effects on tyrosine and phenylalanine in plasma and urine

Milk protein quantity and quality in low-birth-weight infants. I. Metabolic responses and effects on growth

Human milk as food

Acetyl-carnitine and free carnitine in body fluids before and after birth

Carnitine and development of newborn adipose tissue

Nucleotides in human milk: sources and metabolism by the newborn infant

Total potentially available nucleotides of human milk by stage of lactation

Textbook of Gastroenterology and Nutrition in Infancy

Dietary nucleotide effects upon immune function in infants

Effect of dietary nucleotide supplementation on diarrhoeal disease in infants

Modulation of the immune system by human milk and infant formula containing nucleotides

Casein content of human milk

Protein and Non-Protein Nitrogen in Human Milk. B o a Raton

Multi-origins of milk serum albumin in the lactating goat

Biosynthesis and composition of milk

Characterization and properties of the human and bovine lactoferrins extracted from the faeces of newborn infants

Vitamin B, absorption in the neonatal piglet. 11. Resistance of the vitamin B,-binding protein in cow's milk to proteolysis in vivo

Tyroxine-binding proteins in human breast milk similar to serum thyroxine-binding globulin

Corticosteroid-binding proteins in human colostrum and milk and rat milk

Biochemical aspects of human milk-Comparison with bovine milk

Human milk lipases and their possible role in fat digestion

Lingual and breast milk lipases

Peptide hormones and hormone-like substances in milk

Breast vs bottle: endocrine responses are different with formula feeding

Hormones and growth factors in human milk

Defense factors in products of lactation

Mucosal immune system

components in the gastrointestinal tract of the newborn

Bacterial and viral coproantibodies in breast-fed infants

Immunoglobulins in feces from infants fed human or bovine milk

Bacterial agglutination studies with secretory IgA prepared from human gastrointestinal secretions and colostrum

Secretory IgA against enterotoxins in breast milk

Isolation and biological properties of three classes of rabbit antibody in Vibrio cholerae

Prevention of bacterial infections of mucosal surfaces of immune secretory IgA

Oral poliovirus vaccination in newborn African infants: the inhibitory effect of breast-feeding

The role of immunoglobulins in the mechanism of mucosal immunity to virus infection

The uniqueness of human milk: host resistance to infection

Pathogenic strains of coli ( 0 1 11) among prematures and the cause of human milk in controlling the outbreak of diarrhea

Secretory antibody directed against rotavirus in human milk-measurement by means of an ELISA

Neonatal meningitis due to Escherichia coli K1

Breast-feeding protects against infection in Indian infants

Prospective studies on the effect of breast-feeding on incidence of infection and allergy

Adverse effects of milk formula ingestion on the gastrointestinal tract: an update

Food and Immunology: Proceedings of a Symposium Co-sponsored by the Swedish Medical Research Council

Development of IgE in newborn human infants

Breast-feeding protects against respiratory syncytial virus infections

Human antibody dependent cell-mediated cytotoxicity against target cells infected with respiratory syncytial virus

Survival of oral human immune serum globulin in the gastrointestinal tract of low birth weight infants

IgE and IgD in human colostrum and plasma

Local production of IgG4 in human colostrum

IgD in human colostrum

The cells of human colostrum. 1. In vitro studies of morphology and functions

Immunologic aspects of human colostrum and milk. 11. Characteristics of lymphocyte reactivity and distribution of E-rosette forming cells at different times after the onset of lactation

Activated neutrophils and neutrophil activators in human milk: increased expression of CD116 and decreased expression of L-selectin

Activated-memory T lymphocytes in human milk

Protection against experimental necrotizing enterocolitis by maternal milk. I. Role of milk leucocytes

lmmunoregulation by breast milk cells

The breast milk macrophage: potential vehicle for immunoglobulin transport

Intracellular immunoglobulins in human milk macrophages: ultrastructural localization and factors affecting the kinetics of immunoglobulin release

Enhanced immunoglobulin A release from human colostral cells during phagocytosis

Functional capacity of colostral leukocytes from women delivering prematurely

Oxidative metabolic response and microbicidal activity of human milk macrophages: effect of lipopolysaccharide and muramyl dipeptide

Phagocytosis and killing of bacteria and yeast by human milk after opsonization in aqueous phase of milk

Human colostral antibody dependent cellular cytotoxicity against herpes simplex virus infected cells mediated by colostral cells

Enhanced cytokine production by milk macrophages following infection with respiratory syncytial virus

Inhibition of growth of Candida albicans by iron-unsaturated lactoferrin: relation to host defense mechanisms in chronic mucocutaneous candidiasis

The cells of human colostrum. 11. Synthesis of IgA and B-1C

Cellular immunity in human milk

Protective factors in milk and development of the immune system

Comparison of the cellular cytotoxic activities of colostral lymphocytes and maternal peripheral blood lymphocytes

In vitro studies on the T-lymphocyte population of human milk

IgA specific helper factor in human colostrum Clin

Human breast milk T lymphocytes display the phenotype and functional characteristics of memory T cells

Phenotype and activation of milk-derived and peripheral blood lymphocytes from normal and coeliac subjects

CD40 ligand expression on the surface of colostral T cells

Ontogeny of expression of UCHLl antigen on TcR-I' (CD4/8) and TcR+ T cells

Lymphocytes bearing the T cell receptor yS in human breast milk

Phenotype of T cells, their soluble receptor levels, and cytokme profile of human breast milk

Intestinal intraepithelial lymphocytes and lymphoepithelial interactions in the human gastrointestinal mucosa

Significance of the cellular component of the maternal immunologic endowment in milk

In vivo distribution of human milk leucocytes after ingestion by newborn baboons

Intestinal absorption of maternal leukocytes by newborn lambs

Demonstration that milk cells invade the neonatal mouse

The immunologic significance of the mammary gland

Evidence for transmission of lymphocyte response to tuberculin by breast-feeding

Decreased response of human milk leukocytes to chemoattractant peptides

Epithelial cell types and their evolution in the rat mammary gland determined by immunological markers

Developmental regulation of cytokeratins in cells of the rat mammary gland studies with monoclonal antibodies

Developmental aspects of complement components in the newborn

Complement system in human colostrum

New areas arising from studies of secretory immunity

A hitherto unrecognized biochemical difference between human milk and cow's milk

Protective effects of human milk in experimental staphylococcus infection

Mucosal immunity

Epidermal growth factor is a major growth-promoting agent in human milk

Detection of a milk factor that facilitates folate uptake by intestinal cells

Antimicrobial activity of human milk against pediatric pathogens

Antimicrobial effects of human milk: inhibitory activity on enteric pathogens

Effects of bile and bile salts on growth and membrane lipid uptake by Giardia lamblia

Role of nonantibody proteins in milk in the protection of the newborn

Gastroenterology and Nutrition in Infancy

Human Lactation: Milk Components and Methodologies

Antiparasitic factors in human milk

Nonimmunoglobulin fraction of human milk inhibits bacterial adhesion (hemagglutination) and enterotoxin binding of Escherichia coli and Vibrio cholerae

Receptor-like glycocompounds in human milk that inhibit classical and El Tor Vibrio cholerae cell adhererence (hemagglutination)

Fucosylated oligosaccharides of human milk protect suckling mice from heatstable enterotoxin of Escherichia coli

Inhibition of bacterial adhesion and toxin binding by glycoconjugate and oligosaccharide receptor analogues in human milk

Structural analysis of five new monosialylated oligosaccharides from human milk

Purification of human milk gangliosides by silica gel chromatography and analysis of trifluoroacetate derivatives by gas chromatography

Human and bovine milk: comparison of ganglioside composition and enterotoxin-inhibitory activity

Trace amounts of ganglioside GMI in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli

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

Inhibition of attachment of Streptococcuspneumoniae and Haemophilus influenzae by human milk and receptor oligosaccharides

A human milk factor inhibits binding of human immunodeficiency virus to the CD4 receptor

Non-immunoglobulin fraction in human milk protects rabbit against enterotoxin-induced intestinal fluid secretion

The effect of human milk on the adherence of enterohemorrhagic E. coli to rabbit intestinal cells

Protection of suckling mice from the heat-stable enterotoxin of Escherichia coli by human milk

Protection against cholera in breast-fed children by antibodies in breast milk

Undialyzable growth factors for Lactobacillus bifidus var. pennsylvanicus: protective effect of sialic acid bound to glycoproteins and oligosaccharides against bacterial degradation

Isolation of a glycopeptide fraction with Lactobacillus bifidus subspecies pennsylvanicus growthpromoting activity from whole human milk casein

Isolation and characterization of several glycoproteins from human colostrum whey

Bijidobacterium bijidus 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

Enzymes in human milk: their role in nutrient digestion, gastrointestinal function, and nutrient delivery to the newborn infant

Subcommittee on Nutrition During Lactation, et al. Nutrition During Lactation: Summary, Conclusions and Recommendations

Membrane-disruptive effect of human m i k inactivation of enveloped viruses

The inactivation of the virus of epidemic influenza by soaps

Inactivation of enveloped viruses and killing of cells by fatty acids and monoglycerides

Effect of antiviral lipids, heat, and freezing on the activity of viruses in human milk. 1 Infect Dis IIIUIIUII 602893-2899,1992. 308~1389-1392

Isolation and propagation of a human enteric coronavirus

Human milk kills parasitic protozoa

Cholate-dependent killing of Giardia Zamblia by human milk

Structure of human ladoferrin at 3.1-resolution

Iron in human milk

A bactericidal effect for human milk lactoferrin

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. Immunology 35663-671

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

Human milk banking. 11. Relative stability of immunologic factors in stored colostrum

Human mdk banking I. Effects of container upon immunologic factors in mature milk

Immunologic factors in human milk during the first year of lactation

The effect of trypsin and chymotrypsin on the in vitro antimicrobial and iron-binding properties of lactoferrin in human milk and bovine colostrum

The effect of digestive enzymes on the binding and bacteriostatic properties of lactoferrin and vitamin B, binder in human milk

Ctudes comparatives de la structure de la tranferrine de la lactotransferrine humaines. Finger-printing des hydrolytes proteasiques des deux glycoproteides

Daily ingestion of immunologic components in human milk during the first four months of life

Lactoferrin and secretory IgA in the feces of exclusively breast-fed infants

The persistence of human milk proteins in the breast-fed infant

Enhanced fecal excretion of selected immune factors in very low birth weight infants fed fortified human milk

Molecular forms of lactoferrin in stool and urine from infants fed human milk

Human milk feeding enhances the urinary excretion of immunologic factors in birth weight infants

Breast-feeding increases concentrations of IgA in infants' urine

Origin of intact lactoferrin and its DNA-binding fragments found in the urine of human milk-fed preterm infants: evaluation of stable isotopic enrichment

Lysozyme content of human milk

Human tear and human milk lysozymes

Immunologic components in human milk during weaning

Immunologic components in human milk during the second year of lactation

Quantitative determination of immunoglobulins, lysozyme, and certain electrolytes during a 24-hour period, and in milk from the individual mammary gland

Plasma fibronectin concentrations in breast-fed and formula fed neonates

Breast-feeding and health in the 1980s: a global epidemiologic review

The protective effect of human milk against diarrhea

Anti-inflammatory properties of human milk

Expression of functional immunomodulatory and anti-inflammatory factors in human milk

Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts

Transforming growth factor (TGF)-a in human milk

Transforming growth factor-beta (TGF-P) in human milk

Human lactation. Mammary derived growth factors in human milk

Polyamines in human and cow's milk

Polyamine concentration in rat milk and food, human milk, and infant formulas

Hormones in milk: their presence and possible physiological significance

Milk insulin, GH and TSH: relationship to changes in milk lactose, glucose and protein during lactogenesis in women

Development of the neonatal rat small intestinal barrier to nonspecific macromolecular absorption. 11. Role of dietary corticosterone

Uptake of macromolecules in the neonate

Macromolecular absorption in preterm and term infants

Changes in the organs of pigs in response to feeding for the first 24 h after birth. 11. The digestive tract

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 P-carotene in the newborn infant

Interleukin-10 in human milk

Interleukin-lueficient mice develop chronic enterocolitis

Soluble receptors and cytokine antagonists in human milk

Anti-inflammatory effects of human milk on chemically induced colitis in rats

The presence of plateletactivating factor acetylhydrolase activity in milk

Endotoxin and hypoxia-induced intestinal necrosis in rats: the role of platelet activating factor

The role of platelet activating factor and tumor necrosis factor-alpha in neonatal necrotizing enterocolitis

Serum PAF acetylhydrolase increases during neonatal maturation

Absorption of plateletactivating factor acetylhydrolase by rat intestine

Presence of IgE suppressor factors in human colostrum

Identification of the complementary regulatory protein CD59 in human colostrum and milk

Breast-feeding as prophylaxis against atopic disease: prospective follow-up study until 17 years old

Role of infant feeding practices in development of Crohn's disease in childhood

Infant feeding practices and ulcerative colitis in childhood

Reduced risk of IDDM among breast-fed children

Infant feeding in childhood cancer

Effect of breast-feeding on antibody response to conjugate vaccines

Antibody responses to parented and oral vaccines are impaired by conventional and low protein formula as compared to breast-feeding

In-vivo immune responses of breast-and bottle-fed infants to tetanus toxoid antigen and to normal gut flora

Effect of breast-feeding on immune response to BCG vaccination

Maternal donor-related transplants: influence of breast-feeding on reactivity to the allograft

Breast-feeding and maternal-donor renal allografts

Influence of breast-feeding on subsequent reactivity to a related renal allograft

Influence of breast-feeding on the cytotoxic T cell allorepertoire in man

Effect of breast-feeding on responses of systemic interferon and virus-specific lymphocyte transformation in infants with respiratory syncytial virus infection

Development of secretory immunity in breast-fed and bottle fed infants

Prolactin and growth hormone in the regulation of the immune system

Structure and biological action of lactoferrin

Anti-idiotypic antibodies to polio virus in commercial immunoglobulin preparations, human serum, and milk

Cytokines, chemokines, and colonystimulating factors in human m i k the 1997 update

Differentiation of cord blood lymphocytes into IgAproducing cells in response to breast milk stimulatory factor

Human milk stimulates B cell function

A colostral protein that induces the growth and differentiation of resting B lymphocytes

Soder 0. Isolation of interleukin-1 from human milk

Human colostrum contains an activity that inhibits the production of IL-2

Interleukin-1 beta in human colostrum

Tumor necrosis factor-a in human milk

Cytokine production in mononuclear cells of human milk studied at the single-cell level

Normal breast epithelial cells produce interleukins-6 and 8 together with tumor-necrosis factor: defective IL-6 expression in mammary carcinoma

Detection of IL-6 in human milk and its involvement in IgA production

Interleukin-6 in human milk

Presence of interferon-a and interleukin-6 in colostrum of normal women

Properties of the novel proinflammatory supergene "intercrine" cytokine family

Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines

Cytokine, chemokine and secretory IgA levels in human milk in relation to atopic disease and IgA production in infants

Isolation of colony stimulating factor from milk

Human milk contains granulocyte-colony stimulating factor (G-CSF)

Identification of macrophage colonystimulating factor in human milk and mammary epithelial cells

Granulocyte-macrophage colony stimulating factor in human milk

Milk protein concentrations in the mammary secretions of non-lactating women

Escherichia coli-0 antibody content in milk from healthy Swedish mothers from a very low socioeconomic group of a developing country

Antibody production by the mammary gland in mothers after oral colonization of their infants with a nonpathogenic strain E. coli 083

Antimicrobial properties and microbial contaminants of breast milk-an update

The breast-fed infant: a model for performance. Report of the 91st Ross Conference on Pediatric Research

Lactic acid bacteria in food and health: a review with special references to enteropathogenic Escherichia coli as well as certain enteric diseases and their treatment with antibiotics and lactobacilli

Noncultivable viruses and neonatal diarrhea: fifteen-month survey in a newborn special care nursery

Pattern of shedding of two noncultivable viruses in stools of newborn babies

Asymptomatic endemic rotavirus infections in the newborn

Rotavirus infections of neonates

Diarrhea and rotavirus infection associated with differing regimens for postnatal care of newborn babies

Modulation of rotavirus enteritis during breast-feeding

The effects of infant feeding on rotavirus-induced gastroenteritis: a prospective study

Necrotizing enterocolitis

Changes in the incidence of necrotizimg enterocolitis associated with variation of the gastrointestinal microflora in neonates

Clustering of necrotizing enterocolitis: interruption by infection-control measures

Necrotizing enterocolitis

Necrotizing enterocolitis in neonates fed human milk

Necrotizing enterocolitis and human milk

Prophylaxis of necrotizmg enterocolitis by oral IgA-IgG review of a clinical study in low birth weight infants and discussion of the pathogenic role of infection

Donta ST, Myers MG. CIostridium dificile toxin in asymptomatic neonates

Outbreak of necrotizing enterocolitis caused by Clostridium butyricum

Enteritis necroticans due to Clostridium welchii type F

Necrotizing enterocolitis of the newborn-is it gas gangrene of the bowel?

Effect of breast-feeding on morbidity in rotavirus gastroenteritis

The influence of breast-feeding on the incidence of infectious illness during the first year of life

Breast-feeding reduces incidence of hospital admissions for infection in infants

Silverio 1. Breast vs. bottle feeding

Does breast-feeding really save lives, or are apparent benefits due to biases?

Studies of breast-feeding and infections. How good is the evidence? JAMA 256887-892

The influence of breast and artificial feeding on infantile eczema

Does breast-feeding help protect against atopic disease? Biology, methodology, and a golden jubilee of controversy

Relationship of infant feeding to recurrent wheezing at age 6 years

Secretory IgA antibodies against cow's milk proteins in human milk and their possible effect in mixed feeding

Effect of rotavirus infection and malnutrition on uptake of dietary antigen in the intestine

The secretory immune system of lactating human mammary glands compared with other exocrine organs

Development of the capacity to produce specific antibody to an ingested food antigen in the premature infant

Prevention of atopic disease in "at risk newborns" by prolonged breast-feeding

The relative role of transplacental and milk immune transfer in protection against lethal neonatal herpes simplex virus infection in mice

Neutralizing activity in human milk fractions against respiratory syncytial virus

Prolonged breast-feeding as prophylaxis for recurrent otitis media

The neonate's immunity gap, breast-feeding and cot death

Breast-feeding and incidence of non-insulin-dependent diabetes mellitus in Pima Indians

Do breast-feeding and delayed introduction of solid foods protect against subsequent obesity?

Feeding in infancy and later ability and attainment: a longitudinal study

Breast-feeding and cognitive development

Breast-feeding and later cognitive and academic outcomes

A case-control study of lactation and cancer of breast

Human Milk and Infant Formula

Breast-Feeding: A Guide for the Medical Profession

Production of unconjugated hyperbilirubinemia in full-term new-born infants following administration of pregnane-3,20-diol

Evaluation and treatment of jaundice in the term newborn: a kinder, gentler approach

Management of jaundice in the well baby

Vitamin K deficiency and breast-feeding

Multiple nutritional deficiencies in infants from a strict vegetarian commune

Serum 25-hydroxyvitamin D in infantile rickets

A syndrome of methylmalonic aciduria, homocystinuria, megaloblastic anemia and neurologic abnormalities in a vitamin B,-deficient breast-fed infant of a strict vegetarian

Risks of alternative nutrition in infancy: a case report of severe iodine and carnitine deficiency

A background paper on infant formulas

Significance of cow's milk protein antibodies as risk factor for childhood IDDM: interactions with dietary cow's milk intake and HLA-DQBl genotype

Neonatal herpes simplex infection possibly acquired via maternal breast milk

Hormonal and biochemical changes of pituitary and breast during pregnancy

Postnatal transmission of AIDS-associated retrovirus from mother to infant

Postnatal transmission of the human immunodeficiency virus type 1 from mother to infant: a prospective cohort study in Kigali, Rwanda

Isolation of AIDS virus from cell-free breast milk of three healthy virus carriers. Letter to the editor

Isolation of HTLV-III/LAV from cervical secretions of women at risk of AIDS. Letter to the editor

Virologic and electron microscopic evidence for postnatal HIV transmission via breast milk

The presence of HIV-1 genome in human colostrum from asymptomatic seropositive mothers

Detection of HIV-1 by PCR in breast milk

Breast-feeding and maternal-infant transmission of human immunodeficiency virus type 1

Prevention of mother-to-child transmission of HIV-1 infection

Caesarean section and risk of vertical transmission of HIV-1 infection

Does breast-feeding delay progression to AIDS in HIV-infected children?

UN amends policy on breast-feeding

American College of Obstetricians and Gynecologists Committee statement. Breast-feeding

Guidelines on infant nutrition. I. Recommendations for the composition of an adapted formula

Nutrition Committee of the Canadian Pediatric Society and the Committee on Nutrition of the

Ambulatory Pediatric Association. The World Health Organization code of marketing of breastmilk substitutes

Breast-Feeding in the Developing World Current Patterns and Implications for Future Trends

The resurgence of breast-feeding in the United States

Healthy People 2000: National Health Promotion and Disease Prevention Objectives

Shame and other anxieties associated with breast-feeding: a systems theory and psychodynamic approach

Social support, social influence, ethnicity and the breast-feeding decision

Attitudes toward breast-feeding

Catalytic nucleotide-hydrolyzing antibodies in milk and serum of clinically healthy human mothers

Regulation of parental alloreactivity by reciprocal F, hybrids. The role of lactation

Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance

Conversion of alphalactalburnin to a protein inducing apoptosis

HAMLET kills tumor cells by an apoptosis-like mechanism+ellular, molecular, and therapeutic aspects

Breastfeeding attenuates reductions in energy intake induced by a mild immunologic stimulus represented by DPTH immunization: possible roles of interleukin-Ibeta, tumor necrosis factor-alpha and leptin

Leptin levels in breast-fed and formula-fed infants

Role of leptin in immunology

Food induced stimulation of the antisecretory factor can improve symptoms in human inflammatory bowel disease: a study of a concept

Evidence of protection against diarrhoea in suckling piglets by a hormone-like protein in the sow's milk

Effect of antisecretory factor in ulcerative colitis on histological and laborative outcome: a short period clinical trial

Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14

Soluble CD14 in human breast milk and its role in innate immune responses

Protects Babies