Human Milk: Its Components and Their Immunobiologic Functions

Human Milk: Its Components and Their Immunobiologic Functions

Chapter

104

Lars Åke Hanson, Marina Korotkova Department of Clinical Immunology, Göteborg University, Göteborg, Sweden

Esbjörn Telemo Department of Rheumatology and Inflammation Research, Göteborg University, Göteborg, Sweden

The newborn shows many special characteristics: one is that it comes out sterile and another that it has a more or less complete, but tiny, immune system. To provide the neonate with a microbial flora, mammals are delivered next to the mother’s anus. This initial exposure helps the offspring to pick up a microflora, which is a necessary component of normal life. One major reason is that the normal microflora on various mucosal membranes plays an important role in defense against potential pathogens. The intestinal microflora is also the major stimulus to expand the infant’s immune system as seen in studies of germfree animals (Hanson et al., 2003). This stimulus, especially when containing gram-negative bacteria, also helps the immune system to develop the capacity to respond with specific immunologic tolerance, avoiding the development of allergic and autoimmune diseases. Waiting for its own immune system to take over its host defense, the infant needs help from the mother. In higher animals such as humans and monkeys, this is partially handled by the active transplacental transfer of IgG antibodies via the Brambell receptor, an Fc receptor called Fcn (Hanson et al., 2003). However, such antibodies meeting with microbes in tissues will activate proinflammatory cytokine-mediated defense, which causes symptoms, tissue derangement, and high energy consumption, which can be deleterious for the young infant needing available energy for growth and development (Moret and Schmid-Hempel, 2000; Read and Allen, 2000). The higher animals, humans and chimpanzees, have in addition fully developed another major specific immune defense system, the secretory IgA (S-IgA) antibodies, which in humans make up 70% to 80% of all antibodies produced

and reach the infant via the mother’s milk. Among their advantages for the infant are that the S-IgA antibodies stop or limit exposure to microbes via mucosal membranes, preventing infections. In the young infant, this form of immunity is especially important because it efficiently decreases the risk of damaging and energy-consuming inflammation in tissues. Human milk provides specially adapted, noninflammatory host defense to the infant with S-IgA antibodies as a major component, but also via numerous other factors supportive of defense as reviewed subsequently.

COMPONENTS Immunoglobulins The S-IgA antibody system has been described in detail earlier in this book. It was first noted in and isolated from human milk as an IgA with an additional structure, later named the secretory component (SC) (Hanson, 1961; Hanson and Johansson, 1962). Later it was realized that milk S-IgA antibodies resulted from the enteromammary link as part of the homing of IgA-producing cells from the intestinal lymphoid tissues to various exocrine or mucosal sites, including the milk-producing mammary glands. Lactogenic hormones, produced during late pregnancy and lactation, are crucial for the homing to the mammary glands (Roux et al., 1977; Weisz-Carrington et al., 1978). The enteromammary link explains why the mother’s milk contains S-IgA antibodies against so many of the various bacteria in her gut microflora. These include a wide variety of microbial antigens (e.g., several O and K antigens of Escherichia coli)

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(Carlsson et al., 1976, 1982). The presence of S-IgA antibodies to many different microbes suggests that memory cells reflecting previous exposures have also been directed to the mammary gland. Agents from the respiratory tract have also induced production of milk S-IgA antibodies, again illustrating that the lactating mammary glands are part of the whole integrated mucosal immune system. It has usually been assumed that the homing of the IgA-committed lymphocytes occurs at random, but this does not appear to be true. In studies by Dahlgren et al. (1987), it was found that after initiation of an IgA response in the Peyer’s patches with E. coli O6 carrying type 1 pili, O6 S-IgA antibodies were primarily found in the bile, whereas the S-IgA antibodies to the pili mainly appeared in the milk. S-IgA makes up 80% to 90% of the immunoglobulins in milk. In colostrum it may reach about 12g/l and then level off around 1g/l (Goldblum et al., 1982). An exclusively breastfed infant will receive about 125 mg/kg/day at 1 month of age and about 75 mg/kg/day by 4 months (Butte et al., 1984). The stability of the S-IgA molecule is due to its superior resistance to gastrointestinal proteolytic enzymes as compared with dimers of IgA or other isotypes of immunoglobulins (Lindh, 1975). This stability and developmental delay in the production of intestinal proteases explain the outcome of balance studies involving feeding infants of low birth weight preparations of human milk (Schanler et al., 1986). Another reason for the relative stability of milk S-IgA antibodies may be its ratio of IgA1 to IgA2. It is nearly equal in milk, whereas it is about 85/15 for IgA in serum (Telemo and Hanson, 1996), reflecting the proportion of IgA1- and IgA2-producing plasma cells in the mammary glands (Goldman and Goldblum, 1989). This may bring the advantage that IgA2 is more resistant than IgA1 to IgA proteases produced by bacteria like Haemophilus influenzae and Streptococcus pneumoniae, which are common mucosal pathogens (Plaut, 1978; Putnam et al., 1979). On the other hand, milk often contains S-IgA antibodies against these enzymes, protecting the milk S-IgA1 antibodies (Gilbert et al., 1983). The major protective role of the milk S-IgA antibodies is to bind microbes and macromolecules, to prevent or limit their binding and possible attachment to epithelium with subsequent passage through mucosal membranes (see Chapter 39). Such functions of milk S-IgA antibodies are especially important for modulating the effects of the early microbial colonization of mucosae in the upper respiratory and gastrointestinal (GI) tracts in the neonate with its small and immature immune system. The prevention of adherence or attachment by microbes to mucosal membranes is efficiently handled by the milk antibodies. For instance, human colostrum was found to contain S-IgA antibodies against the intimin and bundle-forming pili on enteropathogenic E. coli in a Brazilian study (Loureiro et al., 1998). Such antibodies were present in the colostrum and milk from mothers of prematures and small-for-date neonates as well (Delneri et al., 1997). Milk S-IgA antibodies also bind and neutralize toxins

and viruses. On the other hand, milk S-IgA antibodies do not, or only to a limited extent, support systemic immunity (Ogra et al., 1977; Klemola et al., 1986). An additional function of S-IgA that has recently been proposed is that its carbohydrate moiety appears to be a receptor for the type 1 fimbrial adhesin on E. coli (Nowrouzian et al., in press). It seems that milk S-IgA may enhance the growth of such bacteria in the gut of the breastfed infant by providing this binding site for these bacteria, which are of low virulence. The maternal IgG reaching the fetus via the Fcn receptors in the placenta results in IgG antibodies in intestinal tissue fluid at a concentration of 50% to 60% of the intravascular level (Brandtzaeg et al., 1991). Postnatally, only traces of S-IgA and S-IgM occur in the intestinal tissue fluid, whereas IgG is more prominent. These IgG antibodies may participate in mucosal defense in conjunction with the milk antibodies. In their limited amount, they may be important for the defense of the non-breastfed infant. The major function of the pIgR is to bring IgA onto mucosal membranes and into exocrine secretions; it also aids IgA dimers in providing host defense within mucosal epithelium during its transport through the cells onto the mucosal surface (see Chapter 7). Free SC is present in human milk and may play a role in host defense on its own. For example, it can reduce the effects of Clostridium difficile toxin A on brush border membranes (Dallas and Rolfe, 1998). It also limits infection by enterotoxigenic E. coli by binding to the bacterial fimbriae (De Oliveira et al., 2001). In a study by Cruz et al. (1991), it was noted that mothers with intestinal infections caused by Giardia lamblia or Shigella spp. had a temporary decrease in the homologous SIgA antibodies in the milk. In addition to IgA, human milk also contains antibodies of the IgM and IgG isotypes (Goldman and Goldblum, 1989), but only traces of IgD (Keller et al., 1984) and no IgE (Underdown et al., 1976). Milk IgG amounts to about 0.1 mg/ml in colostrum, although IgG4 is proportionally a little higher in milk than in serum (Keller et al., 1983). IgM in colostrum is present at about 0.6 mg/ml (Goldman and Goldblum, 1989). Lactoferrin Human milk is low in protein compared with milk from other species; yet, a major protein in colostrum and mature milk, lactoferrin (LF), is, like S-IgA, primarily for defense not nutrition. LF amounts to 5–7 g/l in colostrum, decreasing to 1–3 g/l in mature milk (Goldman et al., 1982; Hennart et al., 1991). This decrease is compensated by the increased intake of mature milk by the infant. A healthy breastfed infant is obtaining about 260 mg/kg/day at 1 month of age and 125 mg/kg/day by 4 months of age (Butte et al., 1984). LF is a single polypeptide chain glycoprotein consisting of two α-helix linked lobes, each binding iron. LF belongs to the transferrin family and is also present in other exocrine secretions in addition to milk, as well as in the secondary granules of neutrophils (Lonnerdal and Iyer, 1995). It has a molecular

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

weight of 78 kD and two glycosylated sites with glycans showing structural microheterogeneity (Spik et al., 1994). LF is only partially degraded in the gut, the iron-saturated form being more resistant than the apo form (Brock et al., 1978). Human LF is relatively more resistant to trypsin and chymotrypsin than many other proteins, and this may account for the presence of significant amounts of LF and large LF fragments in the stool of breastfed infants (Goldblum et al., 1989). Human infants as well as adults have a receptor in the gut for uptake of LF and large LF fragments (Kawakami and Lonnerdal, 1991). A pepsin-derived fragment of LF, lactoferricin (LF-cin) is bactericidal, but it is not known whether LF-cin appears in the infant’s gut (Lonnerdal, 1991). LF and certain of its fragments are bactericidal for a wide range of gram-negative and gram-positive bacteria. It appears that the iron-binding capacity of LF is less important as a defense factor than previously believed. Neisseria can even utilize the iron from LF by expressing species-specific receptors for human LF, permitting internalization of the iron-saturated form of LF (Schryvers and Lee, 1989). LF is bactericidal by destabilizing the outer membrane of gram-negative bacteria, binding and releasing LPS. This increases their sensitivity to killing by lysozyme (Ellison, 1994). Human LF interacts similarly with the cell wall lipoteichoic and teichoic acid of gram-positive bacteria, also increasing their sensitivity to killing by lysozyme (Leitch and Willcox, 1998). By enzymatic release of LF-cin from LF, its microbicidal activity is much enhanced (Bellamy et al., 1993). Recently, serine protease activity was discovered in human LF (Hendrixson et al., 2003). Thus, LF appears to cleave surface proteins of H. influenzae, possibly contributing to the antibacterial capacities of LF. It also has antimycotic effects by interacting with mannoproteins in the cell wall of, for example, Candida albicans, damaging the cell wall structure (Nikawa et al., 1994). Antiviral effects also have been described (Valenti et al., 1998). Thus, on the one hand LF is antimicrobial, which is obviously useful for the infant. On the other hand, LF is also strongly anti-inflammatory, which is a most useful capacity for the breastfed infant, preventing symptoms from released proinflammatory cytokines which by activating inflammatory cells cause tissue damage. Thus, nutrient-provided energy is saved for growth and development of the infant. It is striking that LF has been shown to prevent production of several proinflammatory cytokines like IL-1β, IL-6, TNF-α and GM-CSF (Zucali et al., 1989; Mattsby-Baltzer et al., 1996; Choe and Lee, 1999). A recent study showed that this effect may be due to LF entering monocytic cells and their nuclei, interfering with the transcription factor NFκB (Haversen et al., 2002). This factor controls the transcriptional activity of proinflammatory cytokine genes. Togawa et al. (2002) also noted the inhibitory effect on production of the proinflammatory cytokines TNF−α and IL-1β via NFκB and a simultaneous upregulation of the anti-inflammatory cytokines IL-4 and IL-10.

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The antimicrobial and anti-inflammatory effects of human LF and certain of its fragments have been tested in experimental models. Thus, an E. coli infection was induced in the urinary tract of mice that were given human LF and synthesized LF fragments to drink. It was found that LF as well as peptides from the antibacterial surface-exposed helical region was effective in significantly reducing the infection and its inflammatory symptoms in the urinary tract (Haversen et al., 2000). Breastfed infants do in fact have milk LF and LF fragments coming out in the urine (Goldblum et al., 1989). This may be one reason why breastfeeding appears to protect against urinary tract infection, as discussed later. In a model of gut-related systemic infection, recombinant human LF protected against E. coli, reducing the number of colony-forming units in the blood a 1000-fold (Edde et al., 2001). The LF interacted with macrophages, but the LF was also microbicidal in cooperation with lysozyme. The effect of LF on the invasiveness of Shigella flexneri was studied with HeLa cells. It was found that the invasiveness was impaired by both disruption of the integrity of the bacterial outer membrane and inhibition of the actin-mediated movements (Gomez et al., 2001). The anti-inflammatory capacity of LF and certain of its peptides isolated from human milk was illustrated in a rat model of dextran sulphate-induced colitis (Haversen et al., 2003). Significant protection against tissue damage, with reduction in IL-1β levels in blood, in TNF-α-producing cells, and in CD4+ cells, resulted. LF fragments from the LFcin region of LF appeared similarly effective in experiments of shorter duration. It seems that LF may be an important milk component not only in amount, but also in protective functions. Lysozyme This glycoprotein enzyme splits peptidoglycans from bacterial cell walls by hydrolysing the linkage between N-acetyl glucosamine and N-acetyl muramic acid (lysozyme is also called muraminidase) (Chipman and Sharon, 1969). As mentioned earlier, lysozyme cooperates with lactoferrin and also with S-IgA antibodies in attacking E. coli (Adinolfi et al., 1966). In contrast to other protective milk proteins like S-IgA and lactoferrin, lysozyme increases in concentration during lactation. In colostrum about 70 ng/ml is found and in mature milk 20 ng/ml by 1 month and 250 ng/ml by 6 months of lactation (Goldman et al., 1982). Exclusively breastfed infants obtain 3–4 mg/kg/day at 1 month and 6 mg/kg/day at 4 months of age (Butte et al., 1984). a-Lactalbumin Multimeric α-lactalbumin, one of the major human milk proteins, has been found to induce apoptosis in all transformed, embryonic, and lymphoid cells, but not in mature epithelial elements (Hakansson et al., 1995). Further elegant work based on this observation showed that these effects were mediated by oligomers of α-lactalbumin, which had been

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converted by unfolding the protein with oleic acid as a required cofactor (Svensson et al., 1999, 2000). The resulting structure, called HAMLET (human a-lactalbumin made lethal to tumor cells), is a Ca2+-elevating and apoptosisinducing state of α-lactalbumin with its folding altered to a molten globule-like state. In its native conformation, α-lactalbumin acts as a specifier in the lactase synthase complex.The change into HAMLET may be supported by the conditions in the stomach of the nursing child: low pH is known to release Ca2+ and activate lipases that release free fatty acids from milk triglycerides in addition to the oleic acid already present in milk. α-Lactalbumin changes its conformation so that its strongly bound Ca2+ is released and in its apo conformation exposes a new fatty acid binding site, highly specific for the C18:1, 9cis oleic acid (Svanborg et al., 2003). HAMLET kills cancer cells, but not healthy, differentiated cells. Although cancer cells have inactivated their apoptotic pathways, HAMLET is capable of destroying them, both those of human origin and from various other species. HAMLET has shown antitumor activity in more than 40 different lymphoma and carcinoma cell lines. The apoptosis induced is independent of p53 and bcl-2. A significant antitumor effect has been shown in vivo using infusions of HAMLET in a xenograft model of human glioblastoma and local treatment of skin papillomas in humans (Svanborg et al., 2003). HAMLET enters cells, translocates to the perinuclear area, enters the nucleus where it accumulates and binds to histones, and disrupts chromatin organization. In the cytoplasm, ribosomes and mitochondria are targeted. It is possible that the activity of HAMLET is one explanation for the effect of breastfeeding on childhood leukemia, further discussed later. The long-term effect might be due to HAMLET reaching sites of proliferation like the intestinal mucosa where purging of premalignant perversions of the rapidly growing enterocytes and lymphocytes may occur (Svanborg et al., 2003). Instead, HAMLET may drive the lymphocyte population away from malignancy and toward maturity. Its possible role in protection against mammary cancer is unknown. Oligosaccharides and glycoconjugates The milk fat globules and skim milk contain a wide spectrum of glycoconjugates and oligosaccharides, many of which are important in host defense because they function as analogues of microbial ligands on mucosal epithelial surfaces. They may be an important part of the explanation of the fact that breastfed infants have a lower incidence of diarrhea, respiratory tract infections, and otitis media (Newburg, 1999). Oligosaccharides make up the third largest solid component of human milk. They consist of five different monosaccharides in various combinations: glucose, fucose, galactose, N-acetylglucosamine, and sialic acid. The oligosaccharides are chains of 3–10 monosaccharides. They are produced in the Golgi apparatus of the epithelial cells lining the alveoli and smaller ductules in the mammary glands (Kunz and Rudloff, 1993; McVeagh and Miller, 1997). The oligosac-

charides are higher in concentration in early lactation, whereas lactose is higher in later lactation. Some 90 different oligosaccharides have been isolated from milk (Newburg, 1997), but with time-of-flight mass spectrometry, some 900 fucosyloligosaccharides have been shown (Stahl et al., 1994). The urine of formula-fed infants has only minute amounts of the oligosaccharides typical of human milk. In contrast, breastfed infants have those oligosaccharides in the urine in amounts of 1% of the daily intake (Rudloff et al., 1996). The oligosaccharides in human milk appear to have quite substantial effects on the intestinal microflora and the capacity of several microbes to infect the infant. First, they may add to the observation that breastfed infants have fewer serogroups of fecal E. coli than non-breastfed (Gothefors et al., 1975). They also less often carry pathogenic E. coli, Klebsiella, and other Enterobacteriaceae strains. Second, the milk oligosaccharides may act as receptor analogues, preventing mucosal adherence of various microbes or microbial toxins. Thus, sialylated oligosaccharides from milk prevented cellular adhesion of E. coli (Korhonen et al., 1985). Otitiscausing strains of S. pneumoniae and H. influenzae could be blocked by human milk and milk oligosaccharides from adhering to pharyngeal epithelial cells (Andersson et al., 1986). Subsequently, many more bacterial and viral strains, including HIV-1, have been studied in this respect (Kunz and Rudloff, 1993; McVeagh and Miller, 1997; Newburg, 1997). More recent studies have defined in detail the milk oligosaccharide structures involved in the inhibition of bacterial adherence. An elegant example is the work by RuizPalacios et al. (2003). They showed how Campylobacter jejuni bind to the H-2 antigen in human intestinal mucosa as an essential step in the infectious process. This binding was inhibited with high avidity by human milk fucosylα1,2linked molecules. These structures can be regarded as a new class of antimicrobial agents. Studies of the passage of human milk oligosaccharides through the gut of infants showed that most of them survived intact (Chaturvedi et al., 2001). Lower concentrations appeared in the urine. The fecal and urinary oligosaccharides of breastfed infants resembled those in their mothers’ milk. Formula-fed infants had fewer oligosaccharides in feces and urine and these oligosaccharides had a different composition. Human milk contains various glycoconjugates. One is mucin, which is found particularly on milk fat globules. Via their sialic acid content, mucin and milk fat globules inhibit binding of S-fimbriated E. coli to epithelial cells (Schroten et al., 1992). The mucin also inhibits rotavirus replication in an experimental model, presumably via its carbohydrate moiety (Yolken et al., 1992). Lactadherin is a mucin-associated 46 kD glycoprotein that binds to rotavirus and inhibits its replication. In a study in infants with rotavirus infections, it was considered likely that the protection against symptoms related to the milk content of lactadherin, but not to butyrophilin, also associated with mucin, or S-IgA antibodies to rotavirus (Newburg et al., 1998). Human milk κ-casein can inhibit adhesion of

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

H. pylori to human gastric mucosa (Stromqvist et al., 1995). The content of fucose-containing carbohydrate moieties appears important. Degradation products of casein such as caseophosphopeptide and caseinoglycomacropeptide inhibit adhesion of actinomyces, as well as streptococci obtained from the oral cavity (Parker et al., 1984; Neeser et al., 1988). Breastfeeding may be protective via such components. The glycoconjugate GM1 is a ganglioside that prevents adherence of Vibrio cholerae to cells (Holmgren et al., 1983) and binds the analogous toxins of E. coli, V. cholerae, and Campylobacter jejuni (Otnaess et al., 1983; Ruiz-Palacios et al., 1983; Laegreid et al., 1986). The milk glycolipid Gb3 is also a cell membrane receptor for Shigella dysenteriae and a Shigalike toxin from enterohemorrhagic E. coli. Prevention of their adherence to epithelial cells has been shown (Newburg et al., 1992). A large glycoprotein neutralizing respiratory syncytial virus has been described in human milk (Laegreid et al., 1986). Another high-molecular-weight component in milk interferes with the infectivity of hepatitis A virus (Zajac et al., 1991). A 90kD protein that is abundant in milk has been shown to correspond to the Mac-2 binding protein that bridges macrophages and various microorganisms (D’Ostilio et al., 1996). It has been shown that higher levels of this protein in milk relate to the lower risk of respiratory tract infection in breastfed infants (Fornarini et al., 1999). The carbohydrate moiety of S-IgA has been found to bind E. coli type 1 fimbrial lectins (Wold et al., 1990). This may be one reason that in vivo 24% to 74% of bacteria in the colon are coated by IgA and fewer by IgM and IgG (van Der Waaij et al., 1996). This binding capacity to E. coli type 1 fimbriae by S-IgA may play a role in the persistence of such bacterial strains in the gut. This is suggested by the fact that IgA-deficient individuals have fewer type 1 fimbriated E. coli in their intestinal microflora than IgA-replete controls (Friman et al., 1996). Lipids and milk fat globules Lipids hydrolyzed by gastric lipase, bile salt–stimulated lipase, and lipoprotein lipase result in fatty acids and monoglycerides that can attack enveloped viruses, Giardia lamblia, Entamoeba histolytica, and certain bacteria (Gillin et al., 1983, 1985; Resta et al., 1985; Isaacs et al., 1986; May, 1988). The lipid-rich fraction from milk binds and neutralizes Shiga toxin. This fraction was enriched in glycosphingolipids but the nature of the neutralizing moiety is not yet known (Herrera-Insua et al., 2001). The free fatty acids released by the major lipase in milk, the bile salt–stimulated lipase, are efficient in killing G. lamblia (Hernell et al., 1986). Milk fat globules have already been mentioned to carry mucin. Furthermore, S-IgA is strongly associated with the milk fat globule membrane (Schroten et al., 1999). This may support the protective capacity of milk S-IgA through the intestinal passage. The surface of the milk fat globule membrane has numerous small vacuoles adsorbed to it (Patton and Huston, 1988). These exosomelike structures probably carry the MHC class II molecules found in

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milk fat globule membrane fractions (Wiman et al., 1979b; Newman et al., 1980). The origin and possible function of these MHC-rich structures are unknown. It is unlikely that they emanate from the mammary epithelial cells, which do not express these molecules. It is possible that these exosomelike structures are produced by the numerous dendritic cells accounting for a large proportion of the cells found in the mammary gland (Telemo et al., unpublished observations). Nucleotides Nucleotides are present in human milk, making up about 2% to 5% of its total nonprotein nitrogen, which is more than in ruminants (Cosgrove, 1998). The nucleotides amount to 53–58 mg/l in human colostrum and about 33 mg/l in mature milk (Kuchan et al., 1998). Nucleotides participate in several biochemical processes and may support the breastfed offspring in various ways. They function as building blocks of nucleic acids. This may be especially important for the very rapid early growth of the infant’s immune system, which expands primarily in response to exposure to colonizing microbes, especially on the mucosae in the gut. Nucleotides are also important in various biosynthetic pathways, by transferring chemical energy, as coenzyme components and as biologic regulators. The maturation of the gut mucosa may be enhanced by nucleotides. Enzymes capable of limited digestion of nucleotides are present in human milk and fetal intestine homogenate could, when incubated with human milk, add to such degradation (Thorell et al., 1996). Some studies have shown the advantage of adding nucleotides to formula to try to achieve some of the effects of nucleotides in human milk. Thus, such addition to formula in prematures resulted in higher serum levels of IgA and IgM (Navarro et al., 1999), greater responses to immunization against H. influenzae type b (Hib) and diphtheria toxoid, higher numbers of natural killer (NK) cells and activity (Carver et al., 1991), and less (Pickering et al., 1998; Ostrom et al., 2002) diarrheal disease (Brunser et al., 1994). Long-term breastfeeding resulted in higher serum antibody responses to oral poliovirus vaccine compared with a formula + nucleotide, or formula group. The response to the Hib vaccine was similar in the breastfed and formula + nucleotide groups (Pickering et al., 1998). Studies of nucleotides and polyamines in human milk suggested that levels of putrescine and spermine in mature milk were lower in atopic than nonatopic mothers. But there was no relation to atopy in the offspring (Duchén and Thorell, 1999). Defensins Defensins form an interesting group of antimicrobial heat stable peptides. Their cytotoxic activity is nonspecific, and they act against gram-positive, as well as gram-negative bacteria, mycobacteria, fungi, and some enveloped viruses (Lehrer et al., 1993). The β-defensin LBD-1 gene was expressed in human mammary tissue and in a mammary gland epithelial cell line, and the defensin was present in

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the milk (Tunzi et al., 2000). It was found in amounts of 1–10 ng/l and showed antimicrobial activity against E. coli (Jia et al., 2001). Cytokines Numerous cytokines, including several chemokines, have been found in human milk. Because cytokines are the signaling molecules of the immune system, their presence in milk might be of importance for the infant. First to be found was transforming growth factor (TGF) (Noda et al., 1981). An experimental study in TGF-β1 null mice showed that neonates developed progressive inflammation in various organs with overexpression of MHC class II molecules. These mice were rescued if foster-fed by normal mothers. Uptake of intact TGF-β1 from the intestine could be demonstrated (Letterio et al., 1994). Later studies have suggested that TGF-β in milk may promote S-IgA antibody production by acting as an IgA switch factor on B lymphocytes. This could partly explain the decreased risk of developing allergy by prolonged breastfeeding (Saarinen et al., 1999b). The anti-inflammatory effect of TGF-β is also due to its direct suppressive effect on T cells. IL-1β was also identified early in milk (Söder, 1987) and noted to inhibit production of IL-2 by stimulated human T cells (Hooton et al., 1991). TNF-α is present in milk in substantial amounts, around 600 pg /ml (Rudloff et al., 1992). The milk TNF-α has a chemokinetic effect on human milk phagocytes (Mushtaha et al., 1989). It also enhances production of the pIgR, potentially contributing to the uptake of IgA dimers by the lactating mammary epithelium (Nilsen et al., 1999). Soluble TNF-α receptor is present in milk. It is capable of diminishing the biologic activity of the TNF-α in milk (Buescher and McWilliams-Koeppen, 1998). Human milk contains IL-6, which may be involved in the synthesis of IgA (Saito et al., 1991) and IgM (Hirano et al., 1990), and which activates T cells. IL-6 also influences the terminal differentiation of B cells (Kono et al., 1991) and may enhance production of α-antitrypsin by phagocytes, which may help explain why this component is found in the stool of breastfed infants (Perlmutter et al., 1989; Davidson and Lonnerdal, 1990). The high levels of IL-6 reported (Saito et al., 1991) may have been excessive to judge from later studies not using neutralizing antibodies but ELISA for the measurements (Rudloff et al., 1993).This may be true also for measurements of other cytokines in human milk. IL-7 is important for thymic as well as extrathymic T-cell development. It promotes the development of Peyer’s patches and cryptopatches in the intestine and specifically the Tγδ cells (Laky et al., 2000, 2003). Recent studies suggest that thymic size may be influenced by breastfeeding because of the presence of IL-7 in the milk (Collinson et al., 2003). The immunomodulating and anti-inflammatory cytokine IL-10 is also present in human milk (Rudloff et al., 1993). Its activities may be supportive to breastfed infants especially in the gastrointestinal tract. IL-12 and IL-18 are present as well in milk (Bryan et al., 1999; Takahata et al., 2001). Some of

the cytokines in milk originate from the milk cells, as reviewed later, but others like IL-18 are produced by the mammary gland epithelium (Takahata et al., 2001). The macrophage-colony stimulating factor (M-CSF) and the granulocyte-CSF (G-CSF) are both present in human milk, as well as macrophage migration inhibitory factor (MIF) and IFN-γ (Bocci et al., 1993; Eglinton et al., 1994; Gilmore et al., 1994; Hara et al., 1995; Flidel-Rimon and Roth, 1997; Magi et al., 2002). IFN-α appears in milk, but is poorly transferred from the blood even if high doses are given (Kumar et al., 2000). The chemokines IL-8, eotoxin, IL-16, and RANTES (regulated on activation, normal T cell expressed and secreted) are also found in human milk (Bottcher et al., 2000a). Their biologic significance in the breastfed offspring is unknown but is discussed further in the section on breastfeeding and allergy. It is not clear what other effects milk cytokines may have in the offspring. They may be involved in the fact that the spontaneous expression of integrins on CD4+, CD8+, and CD19+ blood lymphocytes at 6 months of age is significantly lower in breastfed than formula-fed infants (Bottcher et al., 2000a). In another study, it was found that at 6 months of age breastfed infants had significantly fewer CD4+ T cells and a higher number of NK cells than age-matched formulafed infants (Hawkes et al., 1999). It is unknown whether milk cytokines are involved in the fact that at 4 months of age the thymus is twice as large in infants who have been exclusively breastfed as compared with formula fed (Hasselbalch et al., 1999). At 10 months of age, those who were still breastfed had a larger thymus than those who had stopped breastfeeding between 8 and 10 months. Actually, there was a significant correlation between the number of breastfeeds per day and the thymus size (Hasselbalch et al., 1999). These striking findings have been discussed as to possible origin and relevance (Prentice and Collinson, 2000). Hormones and growth factors Numerous hormones and growth factors are found in milk (Koldovsky, 1995), but it is not always clear how functional peptide hormones can be in the breastfed infant. Low enzymatic capacity and enhanced uptake in the intestine of young infants may support a transfer of biologic activity (Koldovsky, 1994; Playford et al., 1995).This is more evident in the less mature neonate (Britton and Koldovsky, 1989). Nonprotein hormones are well absorbed in the human gastrointestinal tract as noted for thyroid hormone (Bohles et al., 1993) and steroid hormones. Transfer of function has clearly been shown in animal experiments for epidermal growth factor (EGF) (Berseth et al., 1990) and insulinlike growth factor-1 (IGF-1) (Philipps et al., 1997). Rat milk containing an added GH-releasing factor (GHRF)-like immunoreactivity increased GH production in 2- and 8-dayold suckling rats (Kacsoh et al., 1989). Removal of suckling rats from their mothers resulted in decreased GH levels in plasma, with a return to normal when put back to the mothers (Kuhn et al., 1990). A similar observation was made for

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

luteinizing hormone (Baram et al., 1977). This might have been due to a temporary deficiency in the transfer of milkborne luteinizing hormone-releasing hormone (LHRH). There is also evidence for effects of erythropoietin (EPO), transferred via the milk to the offspring. EPO is produced by epithelial cells in the mammary gland and is taken up by receptors in the intestinal tract (Semba and Juul, 2002). The receptors are also found in the spinal cord and brain and on endothelial cells. It is believed that milk EPO could be important for development of the gut, the central nervous system, and the immune system of the infant. Anoxic or phlebotomized lactating rats or mice induced an increase in hemoglobin (Grant, 1952) or an enhanced erythropoiesis in their offspring (Carmichael et al., 1978). This was presumably due to an increase in EPO in the milk. Prolactin, EGF, and insulinlike growth factor (IGF-1) are all present in milk and are potential immunomodulatory agents that may act locally in the gut (Grosvenor et al., 1993). Prolactin can induce IL-2 by binding to a receptor, which is a member of the IL-2 receptor superfamily (Viselli et al., 1991). In this way, prolactin enhances T-cell activation (Reber, 1993) and thereby stimulation of macrophages and NK cells (Bernton et al., 1988). Trophic effects on cultured human fetal cells have been shown by growth factors in human milk, including IGF-1, EGF, fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) (Hirai et al., 2002; Itoh et al., 2002). Leptin, the obese gene product, is a 16kDa cytokinelike protein (Zhang et al., 1994). It is produced by adipocytes and its blood level reflects the body lipid content (Frederich et al., 1995). Leptin is also produced in the placenta (Senaris et al., 1997), stomach mucosa (Mix et al., 2000), and mammary epithelial cells, and it is present in milk fat globules (Smith-Kirwin et al., 1998). The receptor for leptin (Ob-R) belongs to the cytokine class 1 receptors making up the receptors for IL-6, leukemia inhibiting factor (LIF), granulocyte-colony stimulating factor, and glycoprotein 130 (Tartaglia et al., 1995). The Ob-R exists in several isoforms, of which the Ob-Rb carries the intracellular motifs required for the JAK-STAT transduction pathway.This receptor is expressed at high levels in the hypothalamus, in vascular endothelium, and on T cells (Lee et al., 1996; Lord et al., 1998; Sierra-Honigmann et al., 1998). ObRa, the short leptin receptor isoforms, are found in choroid plexus, vascular endothelium, kidney, liver, lung, and also in the circulation (Lollmann et al., 1997). Leptin regulates food intake within a system for controlling feeding behavior (Friedman and Halaas, 1998). An increase in adiposity increases the level of circulating leptin, which decreases appetite. Starving leads to diminished leptin levels and increased food intake. The characteristics of starving with decreased body temperature, decreased immune and reproductive functions, and stimulation of the hypothalamus-pituitary-adrenal (HPA) stress axis are counteracted by leptin (Ahima et al., 1996; Takahashi and Ide, 2000). Leptin stimulates proliferation and differentiation of hematopoietic cells (Mikhail et al., 1997) and upregulates

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functions of monocytes/macrophages (Santos-Alvarez et al., 1999). Leptin also modifies T-cell responses, increasing production of IL-2 and IFN-γ from TH-1 cells and IL-4 and IL-10 from TH-2 cells (Lord et al., 1998). The increase in IL-1β, TNF-α, and IL-6 induced by infections also induces an elevation of leptin. This may be one explanation why appetite decreases during infections. In a recent study, the anorectic response in breastfed or formula-fed infants to mild immunologic stimuli was analyzed. Breastfeeding was found to protect against the reduced energy intake caused in formula-fed infants by immunization with the quadruple diphtheria, pertussis, tetanus, and hemophilus (DPTH) vaccine (Lopez-Alarcon et al., 2002). Most likely, this anorectic response was due to the significant increase of leptin in the formula-fed infants. It is likely that leptin plays a role in several functions in the neonate, including host defense and proinflammatory capacity. Leptin in milk seems to be taken up by the infant (Savino et al., 2002). As discussed later, it may be one factor to explain why breastfeeding can decrease the risk of developing obesity. Antisecretory factor A component named antisecretory factor (AF), which inhibits the gut fluid secretion induced by cholera toxin, has been described (Lonnroth and Lange, 1984, 1986). As little as 10−11 to 10−12 mol of recombinant AF inhibits the intestinal fluid secretion induced by cholera toxin. It also acts against other toxins such as Clostridium difficile. AF is produced in the pituitary gland, and by lymphocytes and epithelium in the intestinal mucosa, the placenta, and the mammary gland (Lange and Lonnroth, 2001). AF has a molecular weight of 41 000 kD and binds like a lectin to certain polysaccharides. Its mode of action is not fully understood but it potently blocks neural GABA and chloride transport through isolated nerve cells (Lange et al., 1985, 1987). AF protects against diarrhea in suckling piglets (Lonnroth et al., 1988). In humans with diarrhea, AF increased by day 3, when the diarrhea began to subside. Five days later, the AF levels decreased (Torres et al., 1993; Lange and Lonnroth, 2001). We have recently demonstrated AF in human milk from Pakistani and Guatemalan mothers (Hanson et al., 2000). AF is structurally related to a group of food-induced lectins, the production of which can be induced by processed cereals containing various sugars and amino acids. Such diets gave significantly enhanced protection against diarrhea in suckling piglets (Lonnroth et al., 1988).Those piglets with diarrhea caused by enterotoxigenic E. coli had received significantly less AF in the milk than those without diarrhea. In humans, there is still little data, but our initial study showed induction of AF in milk from lactating mothers using a specially prepared food. Although the groups were small, a significant protection against mastitis was observed (Svensson et al., 2004). Anti-inflammatory components Human milk is rich in anti-inflammatory agents of diverse nature. Vitamins A, C, and E as well as the enzymes catalase

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and glutathione peroxidase all act as antioxidants (Garofalo and Goldman, 1999). There are protease inhibitors that block the action of trypsin, chymotrypsin, and elastase, decreasing inflammation. The E prostaglandins are cytoprotective. Platelet-activating factor (PAF) acetylhydrolase degrades PAF, which may act as an ulcerogen, possibly linked to necrotizing enterocolitis. This mechanism may relate to the fact that breastfeeding seems to protect against necrotizing enterocolitis (Furukawa et al., 1993; Muguruma et al., 1997) (see further in the section, “Protection Against Infections of the Respiratory Tract and Other Sites”). Human milk contains several components that inhibit the complement system (Ogundele, 1999). Lysozyme, lactoferrin, α-lactalbumin and other ligand chelators, complement regulatory proteins, and further soluble inhibitors of complement activation may all act synergistically. Lactoferrin inhibits production of proinflammatory cytokines, and milk S-IgA antibodies protect without inducing inflammation as mentioned earlier. In addition, milk shows a general paucity of proinflammatory components including complement factors (Goldman et al., 1986). Soluble CD14 The soluble form of the CD14 molecule, originally detected and described in serum, has been shown to possess a variety of biologic activities. One of its important functions is to mediate LPS reactivity to cells that do not express the CD14 LPS receptor on their surface but do express the Toll-like receptor 4 (Vidal et al., 2001). Thus, it can mediate activation of intestinal epithelial cells that do not express CD14. It recently has been reported that colostrum and milk contain high concentrations of soluble CD14, up to 20 times higher than the serum concentration (Labeta et al., 2000). Apart from aiding LPS signaling, soluble CD14 can directly promote the expansion and differentiation of B lymphocytes (Filipp et al., 2001). It can also synergize with lactoferrin in its anti-inflammatory role (Baveye et al., 2000) (see earlier discussion). Leukocytes in milk Human milk contains live, activated neutrophils, macrophages, and lymphocytes. During the first days of lactation there are as many as 1−3 × 106 leukocytes/ml (Smith and Goldman, 1968; Ogra and Ogra, 1978; Goldblum et al., 1982). Their numbers decrease gradually to less than 1 × 106/ml over the next 2 to 3 months. Thus, many millions of leukocytes reach the infant per day via the milk. Flow cytometry indicates that about 4% are lymphocytes (Bertotto et al., 1990; Wirt et al., 1992; Keeney et al., 1993). Spontaneous production of IL-1β, IL-6, and TNF-α by the milk cells was less than by blood mononuclear cells from the same mother (Hawkes et al., 2002). After stimulation with endotoxin, milk cells, including monocytes/macrophages, neutrophils, and lymphocytes, produced less of these cytokines than did the blood cells. By 4 to 6 months of lactation, epithelial cells rise to about 80% of milk cells. (Ho et al., 1979; Brooker, 1980).

Neutrophils Milk neutrophils show several characteristics suggesting that they have been activated. These include increased surface expression of CD11b and part of the Mac-1 heterodimer, as well as decreased expression of L-selectin (Keeney et al., 1993), which agrees with their diminished adherence, polarity, and motility (Thorpe et al., 1986). Blood neutrophils show similar characteristics on exposure to human milk or a fraction enriched in milk fat globules (Keeney et al., 1993).

Macrophages Several phenotypic and functional characteristics of milk macrophages suggest that they are activated (Ozkaragoz et al., 1988; Keeney et al., 1993). Human milk macrophages produce cytokines like IL-1β, IL-6, and TNF-β spontaneously, although significantly less than blood mononuclear cells (Hawkes et al., 2002). Even after stimulation with LPS in vitro they produced less of these cytokines than blood leukocytes. Milk macrophages were also found to produce GM-CSF spontaneously (Ichikawa et al., 2003). Whereas peripheral blood monocytes differentiated into dendritic cells (DCs) first after exposure to GM-CSF and IL-4, human milk macrophages did so after exposure to IL-4 only. Such milk DCs were able to stimulate T cells, suggesting that they might induce T cell–mediated responses in situ. Peripheral blood monocytes became capable of producing GM-CSF and turned into CD1-positive DCs on exposure to milk after addition of IL-4 only. Milk macrophages unexpectedly expressed the marker DC-SIGN, which is a DC receptor for human immunodeficiency virus (HIV) (Cameron et al., 1994; Granelli-Piperno et al., 1996). Thus, unstimulated milk macrophages may add to the risk of HIV-1 transfer via the milk from mother to infant (Ichikawa et al., 2003). Milk macrophages are capable of antigen presentation (Oksenberg et al., 1985) and they synthesize prostaglandin E2 and plasminogen activator (Le Deist et al., 1986), lysozyme, and C3, the third component of complement (Cole et al., 1985), as well as platelet-activating factor acetyl hydrolase (Furukawa et al., 1993). Milk macrophages may be one source of these components. Milk macrophages stimulated with concanavalin A showed enhanced phagocytosis of zymosan particles, whereas endotoxin had the reverse effect. Phagocytosis of zymosan but not latex particles increased PGE2 secretion from the cells (Blau et al., 1983). Macrophages from milk were found to have a receptor for S-IgA. Binding to the receptor with increasing concentrations of S-IgA caused a progressive increase in PGE2 production and in oxidative burst (Robinson et al., 1991). Enteropathogenic E. coli opsonized with colostral IgA were killed by colostral monocytes (Honorio-Franca et al., 1997). A function of milk neutrophils and macrophages may be to defend the mammary glands against infections, possibly aided by the opsonic function of fibronectin, IgG antibodies, and C3 present in milk (Nakajima et al., 1977).

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

Lymphocytes The great majority of the milk lymphocytes are T cells (83%), with some B cells (6%) and a small number of NK cells (Bertotto et al., 1990;Wirt et al., 1992).The milk T cells include both CD4 and CD8 cells, with a higher proportion of CD8 cells (Bertotto et al., 1990; Wirt et al., 1992). Almost all the CD4 and CD8 cells carry the CD45RO marker associated with activation and immunologic memory. The T cells mainly have the α/β receptor, but with a relative increase in β/β compared with blood T cells (Bertotto et al., 1990, 1991; Gibson et al., 1991). There appears to be quite a selective compartmentalization of milk T cells, presumably because of directed homing to the mammary glands. Thus, Vδ1 and Vγ2, but not Vδ2, were significantly overrepresented on milk T cells compared with blood (Lindstrand et al., 1997). On the other hand, lymphocytes in milk share carriage of the receptor CCR9 for the epithelial thymus-expressed chemokine (TECK) with essentially all small intestinal CD4 and CD8 cells and similar cells from tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium, synovial fluid, and seminal fluid. The CCR9 receptor is only expressed by a small subset of lymphocytes in the colon and TECK is not found in the colon, stomach, skin, lung, or salivary glands (Kunkel et al., 2000). These findings may reflect an important mechanism for targeting and specialization of the immune system, including that of the mammary gland and milk. Milk T lymphocytes may be another source of milk cytokines, especially IFN-γ (Bertotto et al., 1990; Eglinton et al., 1994).The CD45RO T cells are the major origin of this cytokine. Other cytokines like macrophage-inhibiting factor and monocyte chemotactic factor are produced by milk T cells (Keller et al., 1981). Several studies have suggested that milk cells are taken up by the offspring and that milk lymphocytes are even capable of transferring immunologic information (Beer and Billingham, 1975). In a number of animal models such transfer has been illustrated (Weiler et al., 1983; Schnorr and Pearson, 1984; Sheldrake and Husband, 1985; Jain et al., 1989a; Siafakas et al., 1999). In mice hetero- and homozygous for green fluorescent protein (GFP) transgenic markers, it could be shown that GFP+ maternal cells were transferred to GFP-offspring via both placenta and milk (Zhou et al., 2000). Milk transfer resulted in GFP+ maternal cells that infiltrated the gut wall and mainly ended up in the liver. In an experiment in sheep it was shown that maternal technetium-labeled milk lymphocytes deposited in the duodenum of the lamb were subsequently found in the gut mucosa and in the mesenteric lymph ducts and in the cortical zone of mesenteric lymph nodes. A consequence of such uptake in human infants seems to be the appearance of tolerance to maternal HLA antigens. As a result, transplantation of a kidney from a mother to her offspring gives better results if the offspring has been breastfed than if it has not. If the offspring has not been breastfed, the result is the same as if the kidney came from the father (Campbell et al., 1984;

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Kois et al., 1984; Zhang et al., 1991; Zhang and Miller, 1993). These observations were confirmed by foster-feeding experiments in rats (Deroche et al., 1993a). Breastfed individuals also have a lower frequency of precursors of cytotoxic T cells reacting with maternal HLA than non-breastfed (Zhang et al., 1991). Tolerance to maternal HLA could also be induced by HLA-carrying milk fat globules (see earlier discussion). It has been noted that breastfeeding may result in transfer of tuberculin positivity to the infant, presumably via T-cell transfer (Ogra et al., 1977; Schlesinger and Covelli, 1977). However, this was not confirmed in another study (Keller et al., 1987). As discussed further later, priming to vaccines appears to take place via transfer of milk cells. Recent investigations in B cell–deficient mice showed that B cells could be transferred from phenotypically normal dams by foster feeding (Arvola et al., 2000). Such B cells could settle, produce antibodies, and save the antibody-deficient mice.

IMMUNOBIOLOGIC FUNCTIONS Breastfeeding and infant mortality In addition to its strong anti-infectious capacities, breastfeeding is, with about six suckings per 24 hours, strongly contraceptive. Actually, more conceptions are prevented by breastfeeding than by all family planning programs in the Third World (Rosa, 1975). Decreased fertility means less crowding in the family with less risk of infection and more food available. These effects add to the lower infant mortality provided by breastfeeding based on its anti-infectious effects (Aaby et al., 1984; Hobcraft et al., 1985; Reves, 1985; Hanson et al., 1994b). Hobcraft et al. (1985) actually showed that birth interval independently determined infant and child mortality along with socioeconomic status and maternal age. An interpregnancy space of less than 2 years increased the risk of death before age 5 by 50% on average. About 97% of the approximately 130 million children born each year are delivered in the areas with the most poverty and therefore the highest infant mortality rate (IMR). As an overcompensation for the high IMR, the highest population growth is seen in these countries and the link between birth rate and IMR is quite strong, but also complex (Hanson et al., 1994b). Saving children’s lives seems to be the most efficient way of reducing fertility in the poorest parts of the world because a reduction in the IMR is usually followed by a reduction in fertility (Hanson et al., 1994b). Breastfeeding is one important factor in this connection because it reduces both mortality and fertility. The World Health Organization (WHO) has determined that increasing breastfeeding by 40% would reduce respiratory deaths by 50% and diarrhea deaths by 66% worldwide in children younger than 18 months of age (WHO, 1996). It is obvious that breastfeeding has a significant capacity to reduce the IMR. This is illustrated by our observations that even partial breastfeeding reduced the risk of neonatal septicemia in a Pakistani community with an odds ratio of 18

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(Ashraf et al., 1991). Similar observations were made in another study of early-onset septicemia in a Pakistani population (Bhutta and Yusuf, 1997). This really influences the IMR because the mortality for neonatal septicemia in that setting is about 60% (Khan et al., 1993) and neonatal septicemia is a major cause of death in infancy, second only to diarrhea. These two diseases during the first 6 months of life are the cause of 80% of the mortality of the first 2 years of life (Khan et al., 1993). Previously it was determined that the risk of dying of diarrhea for a non-breastfed child in a poor country is 25 times that of an exclusively breastfed child (Feachem and Koblinsky, 1984). In a study avoiding confounding factors as much as possible, it was observed that compared with exclusively breastfed infants, those supplemented with formula or cow’s milk had a 4.2 times higher risk of dying of diarrhea (Victora et al., 1987). Infants who received only formula or cow’s milk had a 14.2 times higher risk. The risk of dying of respiratory tract infection was 1.6 times higher for breastfed infants also given a supplement, compared with those who were only breastfed (Victora et al., 1987). Feeding with only formula or cow’s milk increased the risk 3.6 times. For completely weaned infants, the risk of dying of other infections as well was increased by 2.5 times. A study from Rwanda demonstrated that breastfeeding significantly reduced case fatality in measles, diarrhea, and acute lower respiratory diseases (Lepage et al., 1981). An extensive, well-controlled analysis by Habicht et al. (1986) found that each additional month of breastfeeding decreased the IMR by 6.2 per 1000, and with breastfeeding plus supplement, 3.8 per 1000. In contrast, artificial feeding increased postnatal mortality by a factor of 1.8 to 2.6. Obviously, breastfeeding can substantially reduce the IMR by preventing deaths from infections causing the largest number of deaths in early life, such as neonatal septicemia, diarrhea, and pneumonia. It should be noted that about 80% of the deaths from diarrhea in a poor area occurred in cases of prolonged diarrhea (Zaman et al., 1993). Thus, it might be that to be so significantly protective against deaths from diarrhea (Feachem and Koblinsky, 1984; Victora et al., 1987), breastfeeding must prevent prolonged diarrhea very efficiently. This notion is supported by the studies of Sazawal et al. (1992) and Victora et al. (1992), who showed that breastfeeding protected against acute as well as persistent diarrhea. A review of the literature studying the relationship between early breastfeeding practices and neonatal mortality showed that breastfeeding helps prevent hypothermia and hypoglycemia in newborn babies. This contributes to a decrease in early neonatal deaths, especially among lowbirth-weight and premature babies (Huffman et al., 2001). Exclusive breastfeeding reduced mortality in the late neonatal period in developing countries where most deaths at that age are due to sepsis, acute respiratory tract infections, meningitis, omphalitis, and diarrhea. A recent study from the Dhaka slums showed that exclusive compared with partial or no breastfeeding in the first few months of life resulted in a

2.23-fold lower risk of infant deaths from all causes (Arifeen et al., 2001). The reduction was 2.40-fold for deaths in acute respiratory infections and 3.94-fold for diarrhea. A WHO collaborative team studied the protection against infant and child mortality caused by infectious diseases (WHO, 2000). This review showed that breastfeeding provides important protection against those infectious diseases that account for more than two-thirds of the 12 million deaths among children younger than 5 years of age. Pooled odds ratios from several large studies showed a significant protection of 5.8 for infants younger than 2 months old, decreasing to 1.4 from 9 to 11 months. Slightly lower protection was seen for boys than girls. During the first half year, protection against diarrhea was significantly better against acute respiratory infection (OR = 6.1 compared with 2.4). During age 6 to 11 months, similar levels of protection were noted (OR = 1.9 and 2.5, respectively). In the second year of life, the pooled OR from the studies ranged between 1.6 and 2.1. An ecologic study of the effect of breastfeeding on infant mortality was performed in Latin America (Betran et al., 2001). It was reported that 55% of infant deaths from diarrhea and acute respiratory infections were preventable by exclusive breastfeeding during the first 3 months and by continued partial breastfeeding throughout infancy. Exclusive breastfeeding could protect against 32% of such deaths in infants 4 to 11 months old. Altogether, 13.9% of infant deaths of all causes could be prevented by breastfeeding, which corresponds to 52,000 children in this region yearly. It seems well founded that non-breastfeeding is the most common basis of immunodeficiency (Hanson, 1998b). The WHO/UNICEF Breastfeeding Friendly Hospital Initiative makes a very positive difference when promoting breastfeeding and decreasing disease and mortality in infancy (Nicoll and Williams, 2002). Breastfeeding has been suggested to protect against sudden death in infancy (SIDS). This may help reduce infant mortality (Klonoff-Cohen et al., 1995). A protective effect has not been seen, however, in studies from England and Scotland (Fleming et al., 1996; Brooke et al., 1997). On the other hand, an independent protective effect of breastfeeding with an odds ratio of 2.9 was noted in a study in New Zealand (Ford et al., 1993). In a Scandinavian population, a weak protective effect of breastfeeding was found (Alm et al., 2002). It has been suggested that the protection may be due to the presence in the milk of S-IgA antibodies neutralizing bacterial toxins possibly involved in the pathogenesis of SIDS (Gordon et al., 1999). Historically, it has been claimed that the reduction of infant mortality during the nineteenth century in Western countries was primarily a result of socioeconomic development. A recent study from Sweden shows, however, that during years in the mid-nineteenth century with poorest harvests and many socioeconomic problems a decrease in infant mortality occurred that appears to have been due to increased breastfeeding (Lithell, 1999).

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

Protection against gastroenteritis The first available data giving good evidence for protection against death from diarrheal disease by breastfeeding stem from Swedish communities in the nineteenth century (Brändström, 1984). Several studies have shown that breastfeeding protects against diarrhea (Feachem and Koblinsky, 1984; Jason et al., 1984; Victora et al., 1987; Glass and Stoll, 1989; Howie et al., 1990; Lucas et al., 1992). However, there are many confounding factors in such investigations that cause considerable methodologic problems (Jason et al., 1984; Kovar et al., 1984; Jalil et al., 1990; Victora, 1990). By now, a number of studies have provided results that appear reliable. Most investigations have looked at the role of whole human milk. A few have provided direct evidence that the protection against gastroenteritis caused by V. cholerae, enterotoxigenic E. coli, Campylobacter, Shigella, and G. lamblia is related to the level of S-IgA antibodies in the mother’s milk to these pathogens or their toxins (Glass et al., 1983; Cruz et al., 1988; Ruiz-Palacios et al., 1990; Hayani et al., 1992; Walterspiel et al., 1994; Long et al., 1999). Studies were extended to try to define to which Campylobacter and Shigella antigens the protective milk S-IgA antibodies were directed (Hayani et al., 1992; Torres and Cruz, 1993). Protection by breastfeeding against Campylobacter has been confirmed in one study, but questioned in another (Glass et al., 1985; Megraud et al., 1990). Breastfeeding determined the severity of Shigella diarrhea during the first 3 years of life of Bangladeshi infants (Clemens et al., 1986). Actually, Mata et al. (1969) had already shown in a study from Guatemala that Shigella infection was transient and asymptomatic in one fully breastfed infant and asymptomatic but persistent in two infants who also received food supplements. A fourth infant with inadequate nursing had severe diarrhea. Among 1476 Pakistani children from four different population groups selected to represent the urbanization process and followed prospectively for 2 years, significant protection against diarrhea was seen in the poorest populations. They lived in a village and in a periurban slum (Jalil et al., 1993). This protection lasted for the 2 years of follow-up. The reduction of the rate of diarrhea by breastfeeding during the first weeks of life was as much as 70% to 80%, although partial breastfeeding was predominant. In the city slum, the significant protection of partial breastfeeding lasted for 9 months, and in the upper middle class for 6 months. The latter group breastfed the least (Ashraf, 1993). Although the exclusively breastfed group was small, the size of the cohort permitted a comparison with breastfed children who were given only extra water. It is a widespread tradition to give extra water to breastfed babies during the hot season, although it has been clearly shown that it is unnecessary (Ashraf et al., 1993a). Instead, this extra water diminishes sucking and so the infants have less mother’s milk during the hot season, which is when protection is most needed against diarrhea. The addition of extra water significantly increased the prevalence of diarrhea and even impaired short-term growth (Ashraf, 1993).

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A significant reduction in the incidence of diarrhea has been noted in several other studies, some in developed countries. In an investigation from the United States, a reduction in diarrheal disease of about 50% was seen in a breastfed compared with a formula-fed group (Dewey et al., 1995). Another study from the United States showed similar protection by exclusive breastfeeding and formula feeding (Scariati et al., 1997). In Scotland, significant protection by breastfeeding was seen, but only if breastfeeding extended beyond 13 weeks (Howie et al., 1990). There was also a reduction in hospital admissions in the breastfed group. Similar observations were made in Canada (Chandra, 1979). A modest protective effect was noted in Holland (van den Bogaard et al., 1991). In poor countries it was demonstrated that breastfeeding gave greater benefit in children who showed severe wasting and stunting, protracted illness, and had diarrhea as the only disease (Sachdev et al., 1991). A study from Guinea-Bissau found that the incidence of diarrhea was higher at both 1 and 2 years of age in weaned compared with partially breastfed children (Mølbak et al., 1994). The duration of disease was also shorter among the breastfed. Even if the breastfed children had poorer nutrition, their morbidity and mortality were lower than for the non-breastfed children. A reduction in number and volume of diarrheal stools has also been reported as a consequence of breastfeeding (Khin-Maung-U et al., 1985). A very large and well-controlled study in Belarus showed protection against gastroenteritis by breastfeeding during the first year, but not against respiratory infections (Kramer et al., 2001). In a study from Mexico it was found that breastfeeding gave good protection against infection with G. lamblia but did not prevent chronic carriage of the parasite (Morrow et al., 1992). The risk of non-breastfed children contracting G. lamblia infection was five times that for those fully breastfed. Non-breastfed children had a 1.8 times greater risk of contracting infection than partially breastfed children. It is remarkable that breastfeeding does not appear to protect efficiently against diarrhea caused by rotavirus (Duffy et al., 1986; Clemens et al., 1993). However, exclusive breastfeeding significantly reduced severe rotavirus diarrhea during the first year of life. During the second year, however, the risk of severe infection was increased, and so it appeared that the infections were just postponed by breastfeeding (Clemens et al., 1993). In a recent study from Nicaragua, rotavirus infections were noted to appear already at 2 months of age (Espinoza et al., 1997), but the time of the first occurrence of rotavirus in the stool significantly correlated with the levels of colostral S-IgA antibodies to rotavirus. A long duration of breastfeeding tended to result in asymptomatic infections. In a large study of nosocomial rotavirus infections from Italy, significant protection was obtained by breastfeeding (Gianino et al., 2002). None of the breastfed infants who became infected developed symptoms of diarrhea. Recently it was shown in Germany that breastfeeding in infancy does not protect against H. pylori infection

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in preschool children (Rothenbacher et al., 2002). The infection is known to be acquired in early life. Protection in early life may be mediated by the κ-casein in the milk, which prevents attachment of H. pylori to human gastric mucosa (Stromqvist et al., 1995). The effect seems to be mediated via the fucose-containing moieties of the κ-casein. Protection against infections of the respiratory tract and other sites Numerous studies have suggested that breastfeeding protects against otitis media (Chandra, 1979; Timmermans and Gerson, 1980; Saarinen, 1982; Teele et al., 1989; Owen et al., 1993), but others have not confirmed this finding (Howie et al., 1990) or found only a limited effect (Sipilä et al., 1988). A long duration of breastfeeding was required for protection in other studies (Kero and Piekkala, 1987; Porro et al., 1993). However, a carefully designed investigation has shown a reduction of about 50% in acute as well as recurrent otitis media by exclusive breastfeeding beyond 4 to 6 months (Duncan et al., 1993). A decrease of 20% in the number of attacks of acute or prolonged otitis was obtained by breastfeeding, as seen in another study (Dewey et al., 1995). The number of episodes of prolonged disease was reduced by 80% in the breastfed compared with the formula-fed group. Additional studies support these observations (Clemens et al., 1986; Aniansson et al., 1994; Dewey et al., 1995; Duffy et al., 1997; Scariati et al., 1997; Cushing et al., 1998; Long et al., 1999). Effusions in the ears of children with cleft palate were efficiently prevented by artificially feeding human milk, mostly from the mother (Paradise et al., 1994). The protective capacity of the mother’s milk was also seen in a second study of such cases (Aniansson et al., 2002). In a prospectively followed group, breastfeeding appeared to prevent nasopharyngeal colonization with H. influenzae (Harabuchi et al., 1994). Colonization rate was inversely related to milk S-IgA antibodies against P6, a highly conserved outer membrane protein of nontypable H. influenzae. Colonization was correlated with acute otitis media. In a study by Rosen et al. (1996), nasopharyngeal colonization by pneumococci was not reduced by milk antibodies against the cell wall C-polysaccharide and phosphorylcholine. Antibodies against the bacterial cell wall even correlated with increased colonization. The frequency of otitis media did not vary with milk antibody content. There have also been a number of studies claiming protection by breastfeeding against upper and lower respiratory tract disease, but again disagreeing reports have appeared. It was claimed that the disagreements were due to the presence of confounding factors and a rather weak protective effect of breastfeeding in developed countries (Kovar et al., 1984; Bauchner et al., 1986; Anderson et al., 1988; Wright et al., 1989; Woodward et al., 1990). Possibly, breastfeeding results in less severe illness (Frank et al., 1982). In poor countries, protection against pneumonia by breastfeeding is striking, as already mentioned, saving many lives, for example in Brazil (Victora et al., 1987). Cesar et al. (1999) showed that non-

breastfed infants were 17 times more likely than those breastfed to be hospitalized for pneumonia. They found that this relative risk was 61 for children younger than 3 months old and decreased to 10 at older ages. Solids given were related to a relative risk of 13.4 for all infants and 17.5 for those younger than 3 months of age. In Western countries, several studies have given evidence for significant protection against respiratory infections (Downham et al., 1976; Chandra, 1979; Watkins et al., 1979; Pullan et al., 1980; Howie et al., 1990; Woodward et al., 1990; van den Bogaard et al., 1991; Cushing et al., 1998). The effect on acute lower respiratory tract infections was clear in an Italian study (Pisacane et al., 1994a). In an extensive investigation in the United States, some protection by breastfeeding against pneumonia, as well as otitis media, was observed (Ford and Labbok, 1993). A large study in the United States showed that breastfeeding protected against wheezing respiratory tract infections in the first 4 months of life (Wright et al., 1989). Nafstad et al. (1996) suggested that protection against lower respiratory tract infections was strongest in children exposed to tobacco smoke. In a large Australian cohort, it was found that the risk for four or more upper respiratory infections was greater if predominant breastfeeding was stopped before 2 months of age or partial breastfeeding was stopped before 6 months of age. Wheezing lower respiratory tract illness was more often causing visits to a doctor or hospital if the child was predominantly breastfed for less than 6 months (Oddy et al., 2003). There is an interesting study showing protection against necrotizing enterocolitis by breastfeeding (Lucas and Cole, 1990). The authors suggested based on their findings that the use of exclusive formula feeding in British neonatal units could account for some 500 extra cases of necrotizing enterocolitis per year. Some 100 of these infants would die. The protection against necrotizing enterocolitis by breastfeeding was also noted in another report (Contreras-Lemus et al., 1992). A rat model of necrotizing enterocolitis was used to show that prevention by rat milk but not a milk substitute was paralleled by an increase of IL-10 localized primarily in the cytoplasm of villus epithelial cells (Dvorak et al., 2003). In another study, it was shown that fetal human enterocytes responded to TNF-α and IL-1β with exaggerated IL-8 production (Claud et al., 2003). TGF-β and erythropoietin, which are both present in milk, decreased this induced IL-8 secretion. These studies give evidence of mechanisms that very likely are involved in the protection against necrotizing enterocolitis by breastfeeding. Significant protection against neonatal septicemia by breastfeeding was noted in a study from Pakistan as mentioned earlier (Ashraf et al., 1991), and a similar effect was also obtained in an investigation from Sweden (Winberg and Wessner, 1971). The same protection was obtained by feeding expressed human milk to low-birth-weight infants (Narayanan et al., 1982). Feeding very low-birth-weight children human milk significantly reduced infection and sepsis/meningitis compared with formula feeding (Hylander et al., 1998). In another study from the United States,

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

increasing breastfeeding rates resulted in a decrease in pneumonia and gastroenteritis of 32.2% and 14.6%, respectively. The rates of croup and bronchiolitis increased, in contrast, among those fed formula from birth (Wright et al., 1998). Most likely there was an ongoing viral epidemic, which did not cause disease in those exclusively breastfed. There is evidence that breastfeeding may prevent urinary tract infections (Mårild et al., 1990; Pisacane et al., 1992). This protection might be due both to the effects of human milk factors like S-IgA antibodies binding to the intestinal bacterial flora from which the infections mostly originate (Wold and Hanson, 1994) and the presence in urine of bactericidal lactoferrin derived from the milk (Goldblum et al., 1989). The latter finding agrees with the protection obtained in an experimental model of urinary tract infection with E. coli in mice given human lactoferrin to drink (Haversen et al., 2000). Also, certain fragments of lactoferrin given perorally came out in the urine and were protective. This was discussed earlier in the section on lactoferrin. A few single retrospective studies suggest that breastfeeding may have been less common in children with acute appendicitis or hypertrophic pyloric stenosis, as well as in children who had been tonsillectomized (Pisacane et al., 1995, 1996a, 1996b). It has been suggested that breastfeeding may prevent botulism (Arnon et al., 1982). One investigation has indicated that breastfeeding may delay early colonization with H. pylori (Thomas et al., 1993). This effect might be due to the presence of S-IgA antibodies to H. pylori in milk, as well as the fact that human milk κ-casein specifically prevents adhesion of H. pylori to gastric mucosa (Stromqvist et al., 1995). It is well known that human immunodeficiency virus (HIV-1) infection can be transmitted via breastfeeding. This is discussed later. Breastfeeding and allergy Many studies have been devoted to whether breastfeeding diminishes the risk of developing allergic diseases. Several authors claim to have shown prevention of allergy (Chandra, 1979; Saarinen et al., 1979; Zeiger et al., 1989). Other investigations have shown a decrease in wheezing in infancy and childhood by breastfeeding (Sims et al., 1981; Wright et al., 1989; Burr et al., 1993; Porro et al., 1993).Wheezing is often, but not exclusively, connected with asthma. In schoolchildren with asthma, 80% to 85% of the attacks are reported to be initiated by viral infections (Johnston et al., 1995). It appears that the preventive effect on wheezing by breastfeeding is primarily due to the prevention of these viral infections in early life (Wright et al., 2001). Atopic allergy mostly starts as food allergy in early life and is often caused by cow’s milk proteins, and also soy proteins. Whether breastfeeding prevents such allergy has been much debated (Kramer, 1988; Hanson, 1998a). Breastfeeding influences lymphocyte populations in infants, inducing higher numbers of NK cells and lower numbers of CD4+ T cells compared with formula-fed infants (Hawkes et al., 1999). In infants, the serum IgG antibody

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levels against β-lactoglobulin show an inverse relationship to the age at weaning (Hanson et al., 1977). A number of characteristics of the mother’s milk have been linked to the development of cow’s milk allergy in the offspring. Low IgA in the milk relates to such risk (Savilahti et al., 1991; Jarvinen et al., 2000). Low milk IgA and high blood eosinophil count in breastfed infants also correlated with a higher risk of atopic eczema in an 18-month followup (Calbi and Giacchetti, 1998). A link between cow’s milk allergy in the offspring was associated with a lower proportion of macrophages and a higher one of neutrophils in the mother’s milk (Jarvinen and Suomalainen, 2002). Eosinophils in the milk also correlated with the risk of allergy in the infants. There were links to the total number of B cells and their expression of CD23, which is a low-affinity IgE receptor. In an analysis of chemokines in human milk, IL-8 and RANTES were higher in the milk of allergic than nonallergic mother’s milk (Bottcher et al., 2000a). These chemoattractants may influence the transfer and activation of cells in the milk. It has been claimed that cow’s milk proteins (CMP) appear in the milk of cow’s-milk–drinking mothers (Jakobsson et al., 1985; Høst et al., 1990; Husby et al., 1991). However, the specificity of some of the analyses has been questioned (Restani et al., 2000). In favor of the presence of immunologically active CMP in human breast milk is a study showing that 16 of 17 infants with cow’s milk allergy responded to challenge with bovine proteins fed to the breastfeeding mothers (Jarvinen et al., 1999). Similar results have also been reported previously (Jakobsson and Lindberg, 1978). Whether temporary exposure in early life to bovine proteins, in otherwise breastfed infants, brings a risk of developing cow’s milk allergy has been discussed. Most evidence suggests an increased risk (Stintzing and Zetterstrom, 1979; Saarinen et al., 1999a), although this was not seen in a recent large study (De Jong et al., 2002). The timing of exposure and the amount of cow’s milk given were considered crucial for the development of cow’s milk allergy in breastfed infants (Saarinen et al., 2000). Lucas et al. (1990) showed that in preterm babies, early exposure to cow’s milk was a risk factor for allergy if there was a family history of atopy. Human milk contains IL-6, IL-10, and TGF-β, which are all involved in the production of S-IgA by B lymphocytes. Their concentrations correlated with each other and with the concentration of S-IgA in the milk (Bottcher et al., 2000b). The IL-4 level was significantly higher in the milk samples from allergic compared with nonallergic mothers. The major cytokine in the milk was TGF-β (Cummins and Thompson, 1997; Saarinen et al., 2000). Saarinen et al. (1999b) showed that colostral TGF-β1 may have an effect on the immune response to CMP in cow’s milk allergic infants. With a lower content of TGF-β1 there was a more vigorous IgE antibody response, and proliferation of lymphocytes on exposure to CMP was positively correlated with the milk TGF-β1 level. These results are in line with the role of IL-4 as a Th2 promoter and as a switch factor for IgE production, whereas TGF-β is a

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downregulator of both Th1 and Th2 T cells and is a major switch factor for IgA. Wright et al. (1999) showed a relationship between serum IgE in childhood and maternal IgE levels. With low IgE in the mother, breastfeeding was associated with lower total IgE in the child by the age of 6 years. With a high maternal IgE and breastfeeding for 4 months or longer, the child had higher IgE levels compared with those never breastfed or breastfed for less than 4 months. Similar results have also been shown in experimental animals (Jarrett and Hall, 1979, 1983; Bednar-Tantscher et al., 2001). Many factors influence the development of allergies, including heredity, age, dose of allergen on first and later exposures, prevalence of infections, character of the intestinal microflora, and capacity to develop immunologic tolerance to the allergens (Hanson et al., 1997). Therefore, it is very difficult to define the role of breastfeeding as a single factor. Several studies have investigated whether breastfeeding can prevent the occurrence of allergic diseases. In a metastudy of the literature between 1966 and 1999, the conclusion was protection against asthma (Gdalevich et al., 2001). Another metastudy concluded that breastfeeding protected against atopic dermatitis in children with a family history of atopy (Gdalevich et al., 2001). This effect was negligible if there were no first-degree relatives with atopic disease. A review with strict criteria of all papers from 1966 through 2001 on breastfeeding and allergy left only 18 papers after scrutiny. Eight clearly showed protection, three were suggestive, six were indecisive, and one showed possibly a promoting effect (Van Odijk et al., 2003). Lately, studies supporting a low-grade protection (as summarized in Hanson, 2004) have been published.. Oddy et al. (1999) showed in a prospective 6-year followup in Australia a significant reduction in the risk of childhood asthma if breastfeeding was continued for at least 4 months after birth. Multiple attacks of wheezing in the first year of life increased the risk of asthma at 6 years of age in atopic as well as nonatopic children. Breastfeeding decreased the risk of both of these forms of allergy. Maternal asthma did not modify the outcome (Oddy et al., 2002a, 2002b). In a prospective study by Tariq et al. (1998) in the United Kingdom, formula feeding before the age of 3 months predisposed to asthma at the age of 4 years. A family history of atopy was the strongest risk factor. At 2 years in a still ongoing prospective investigation in Sweden, exclusive breastfeeding for 4 months or more decreased the risk of developing asthma, atopic dermatitis, and allergic rhinitis (Kull et al., 2002). Another large follow-up study in Germany showed that the prevalence of atopic eczema during the first 7 years of life increased for each year of age and for each additional month of breastfeeding (Bergmann et al., 2002). Further additive factors were a history of parental atopic eczema and other atopic signs and symptoms in the child, especially specific sensitization and asthma. The development of atopic dermatitis in 15,436 Danish children up to 18 months of age in relation to breastfeeding was subjected

to optimal statistical analyses (Stabell Benn, 2003). Exclusive breastfeeding of children with no allergic parents appeared to increase somewhat the risk of atopic dermatitis, but with both parents allergic there was evidence for postponement or even prevention of the dermatitis. A large German study on breastfeeding and obesity (von Kries et al., 1999) was also used to analyze the relation between asthma and breastfeeding. Mothers with asthma who breastfed induced a significantly higher risk of asthma in their offspring than breastfeeding mothers without asthma (Oberle et al., 2001). Among the children of mothers who had hay fever or eczema, breastfeeding did not bring an increased risk of childhood asthma. In the limited subgroup of mothers living on a farm, the breastfed infants had even less asthma than the non-breastfed (Oberle et al., 2000). Wright et al. (2001) showed protection by breastfeeding against wheezing, which is usually induced by infections, as mentioned earlier. In contrast, breastfeeding by atopic mothers increased the risk of recurrent wheezing and asthma beginning at the age of 6 years in their offspring. This subgroup was 6% of those studied. In a large study from Sweden, among children with a family history of atopy, but without positive skin tests, the risk of asthma at the age of 7 to 8 years was higher in those breastfed for less than 3 months. This increased risk by short-term breastfeeding was not seen among the sensitized asthmatics (Ronmark et al., 1999). An extensive long-term follow-up study from New Zealand demonstrated that breastfeeding increased the risk of specific sensitization against common allergens at the age of 13 to 21 years (Sears et al., 2002). The risk of recurrent asthma between the ages of 9 to 26 years was also increased by breastfeeding, but this risk was not related to parental history of hay fever or asthma. However, the definition of breastfeeding in this study was unclear. A large Japanese study using questionnaires at the age of 12 to 15 years about modes of feeding in early years concluded that there was an increased risk of atopic eczema related to breastfeeding by nonatopic, but not atopic mothers (Miyake et al., 2003). However, with such a long recall of the feeding history the results may be unreliable (Van Odijk et al., 2003). The previous reviews give a rather confusing picture as to whether breastfeeding decreases or increases the risk of allergic diseases in the offspring. Among the reasons may be that certain parameters like mode of feeding are inadequately defined (Sears et al., 2002), or that too long a recall of feeding history is used (Miyake et al., 2003). There could be other factors caused by geographic differences. Thus, it seems that the fat intake of the mother may play a role. For instance, the ratio of n-6:n-3 fatty acids is lower in milk of Australian mothers (Fidler and Koletzko, 2000), who seem to protect against allergy by breastfeeding (Oddy et al., 2002a, 2002b), than in the milk of German mothers, who may rather increase the risk (Oberle et al., 2001; Bergmann et al., 2002). Milk of mothers with children contracting atopic dermatitis showed increased proportions of α-linolenic acid and decreased proportions of its long

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

chain derivatives compared with controls (Businco et al., 1993). In another study, atopic mothers were found to have higher ratios of n-6:n-3 fatty acids in their milk than nonatopic mothers (Yu et al., 1998). Low α-linolenic acid and n-3 long chain polyunsaturated fatty acid levels in the milk correlated with development of atopy in breastfed offspring according to a Swedish study (Duchén et al., 2000). Haby et al. (2001) showed in Australia protection against asthma by breastfeeding, but an increased risk for asthma if the child had had a high intake of polyunsaturated fats. In our recent studies, we fed diets of different fatty acid content to rat dams during late pregnancy and lactation and tested the immune response of the offspring to ovalbumin fed to the dams during lactation. It appeared that a ratio of n-6:n-3 fatty acids of less than 1 in the maternal diet correlated with a significantly better capacity of the offspring to become tolerant to ovalbumin than when the rat dams had had fatty acids with a ratio of 10. The offspring of the rat dams given the low ratio diet responded with less IgM, IgE, and IgG antibodies and less delayed hypersensitivity to the food protein (Korotkova et al., 2004a, 2004b). Another investigation recently suggested that feeding dams ovalbumin before pregnancy could add to tolerance development in the pups via the milk as suggested by foster feeding (BednarTantscher et al., 2001). Prophylaxis against allergic disease has been tried in atrisk families with the mother adhering to a strict diet during late pregnancy and avoiding common food allergens (FälthMagnusson and Kjellman, 1992). No effect was seen in the 5-year follow-up. If the diet was instead applied during the first 3 months of lactation, a diminished risk of atopic dermatitis was noted in the offspring (Hattevig et al., 1989). Reexamining these children at the age of 4 years confirmed that a prophylactic effect remained; eczema was not just postponed (Sigurs et al., 1992). Another large study of prophylaxis concerned diet during the third trimester of pregnancy and through lactation (Zeiger et al., 1989). A significant reduction in atopic dermatitis, urticaria, and/or gastrointestinal disease was seen during the first 12 months of life. The children were followed for 24 months. At that age, the prevalence of allergic rhinitis, asthma, and positive skin tests with inhalant allergens was unaffected. Active and long-term effects of breastfeeding on the infant’s immune system

Breastfeeding and vaccine responses It was noted that vaccine antibody responses after immunization with parenteral and peroral vaccines were better in breastfed compared with formula-fed infants (Hahn-Zoric et al., 1990). Levels of S-IgA antibodies in saliva, IgM in stool, and IgG antibodies in serum were significantly higher; the latter were still elevated 1 to 2 years later.The responses were obtained against tetanus and diphtheria toxoids and live poliovirus vaccine. These findings agree with the higher serum antibody response obtained among breastfed compared with formula-fed children against the H. influenzae type b (Hib) capsular polysaccharide in a Hib–protein con-

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jugate vaccine (Pabst and Spady, 1990). It should be noted that these effects on vaccine responses were also seen for months after the termination of breastfeeding. Enhancement of vaccine responses in serum at 6 months of age to oral poliovirus vaccine by breastfeeding was confirmed (Pickering et al., 1998). Infants had been exclusively breastfed for 2 months and after that given formula or mixed feeding. One control group had been given an iron-enriched formula and another a nucleotide-enriched formula. At 7 months, those on the nucleotide-enriched formula had higher antibody responses to the Hib vaccine antigen and diphtheria toxoid than those in the two other groups. The T-cell response to bacillus Calmette-Guérin (BCG) vaccine was higher in breastfed than in formula-fed infants, although those registered as breastfed might have been so for only 2 weeks (Pabst et al., 1989). The enhancing effect was only seen if the vaccine was given before 1 month of age. Other studies have not shown improvements in vaccine responses by breastfeeding. This is especially true for live vaccines against poliovirus, influenza, and rotavirus. Antibodies against these endemic viruses are common, and impaired responses may be due to neutralization by maternal serum and milk antibodies (Pichichero, 1990; Ceyhan et al., 1993; Karron et al., 1995). Such an effect of breastfeeding was noted early by Sabin for the live poliovirus vaccine. It is still a major problem because the oral vaccine is often given on the day of birth or soon thereafter when breastfeeding has started. Awareness of this problem is often deficient (Rennels, 1996). There are several studies with Hib and tetanus toxoid vaccines in which breastfed infants did not respond better than non-breastfed (Stephens et al., 1984;Watemberg et al., 1991; Decker et al., 1992; Scheifele et al., 1992). These discrepancies may be related to many factors including differences in the definitions used of breastfeeding, its duration and extent. Furthermore, there may be variations in the immune response to these vaccines by the mothers and possibly even the grandmothers. This concept is suggested by our studies of priming and tolerogenic effects of idiotypic and anti-idiotypic antibodies given to newborn rats. Priming effects in the neonates were transferred from one generation to the two subsequent ones (Lundin et al., 1999). A possible enhancing effect of breastfeeding on the immune response of the infant could correlate with the finding that breastfed infants produce more IFN-γ in response to infections with respiratory syncytial virus than non-breastfed infants (Chiba et al., 1987). That salivary S-IgA levels were initially low but increased more during the first 6 months of life in breastfed compared with non-breastfed infants could have a similar basis (Avanzini et al., 1992). Enhancement was also suggested to occur for S-IgA antibodies to E. coli in urine as a function of secretory component and lactoferrin from breastfeeding (Goldblum et al., 1989).

Long-term protection against infections A study by Howie et al. (1990) in Scotland showed that breastfeeding for more than 13 weeks resulted in better

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protection against gastroenteritis throughout the first year of life compared with those breastfed less, or not at all. A reanalysis when these children were 7 years of age revealed a continued decreased risk of respiratory tract infections related to breastfeeding (Wilson et al., 1998). Silfverdal et al. (1999) studied the protective effect of breastfeeding against invasive infections and meningitis caused by H. influenzae. A long-term protective effect was found lasting for 10 years after the termination of breastfeeding. The study of Saarinen (1982) on otitis media showed protection lasting for 3 years in infants breastfed previously. The preventive effect on wheezing was seen for 6 to 7 years (Porro et al., 1993) and may have been largely a result of the prevention of infections which would have caused wheezing. This might agree with the finding that long-term prevention of wheezing was seen only among nonatopic children (Wright et al., 1989; Burr et al., 1993). Reports suggesting that breastfeeding may prevent acute appendicitis and frequent infections would if confirmed also mean that breastfeeding provides long-term protection (Pisacane et al., 1995, 1996b). In an investigation by van den Bogaard et al. (1991), children breastfed through 6 months were at the age of 3 years still better protected than non-breastfed children against gastroenteritis, respiratory tract infections, skin infections, and urogenital disease. A recent critical review of the literature supported the presence of enhanced long-term protection against infections of the gastrointestinal and respiratory tract mucosae from breastfeeding (Chien and Howie, 2001).

Breastfeeding and obesity Among the long-term effects of breastfeeding are protection against obesity in children (Von Kries et al., 1999; Gillman et al., 2001; Hediger et al., 2001; Toschke et al., 2002) and against increased blood pressure in teenagers (Singhal et al., 2001), as well as enhancing effects on bone mineralization (Jones et al., 2000). The reduction in obesity seems well documented in large studies. Comparing breast- versus formulafeeding gave adjusted odds ratios ranging from 0.75 to 0.84. With longer duration of breastfeeding a lower incidence of subsequent obesity was found (von Kries et al., 1999; Gillman et al., 2001). One may ask how human milk can have such effects. It has been shown that breastfeeding affects insulin secretion (Lucas et al., 1980, 1981), which may influence long-term energy metabolism. More recent data add to this by demonstrating that some of the cytokines and hormones present in milk may be involved. Thus, IL-1β can inhibit insulin production, whereas TNF-α may block insulin receptors (Hauner et al., 1995; Hotamisligil et al., 1996; Mauricio and Mandrup-Poulsen, 1998). IL-6 may stimulate release of insulin (Shimizu et al., 1995), glucagon, and cortisol as well as stimulate glucose and fatty acid oxidation (van Zaanen et al., 1996; Keller et al., 2001). Furthermore, leptin, which has a cytokine structure, is present in human milk. Leptin, the appetite-regulating hormone, is found in higher concentrations in breastfed than

formula-fed infants. Some of this may come from the mother’s milk (Savino et al., 2002). Furthermore, it has been shown in a rat model that the leptin level in the milk was modified by the ratio of linoleic to α-linolenic acid in the maternal diet (Korotkova et al., 2002). Thus, it may be that the modifying effect on the risk of obesity in the breastfed child may be influenced by the mother’s diet; that is, if milk leptin really is an important factor as suggested by some studies, but not others (Locke, 2002; Singhal et al., 2002; Uysal et al., 2002). Leptin is produced by fat cells and is found in milk fat globules and mammary epithelial cells (Smith-Kirwin et al., 1998). It has many links to the immune system; that is, its production is induced by LPS, IL-1β, and TNF-α and it skews the immune system toward TH1 reactivity (Lord, 2002). During starvation, when leptin levels are low, the thymus, lymph glands, and spleen are diminished in size and cell-mediated immunity is impaired. In starved mice, the sensitivity to septic shock is increased, with LPS and TNF-α playing a role. This state is prevented by giving leptin (Takahashi et al., 1999). Thus, leptin in milk may be supportive of several immune functions in the breastfed infant.

Long-term effects of breastfeeding on autoimmune and other inflammatory states It has been suggested that breastfeeding diminishes the risk of developing type 1 diabetes (Borch-Johnsen et al., 1984; Fort et al., 1986; Mayer et al., 1988; Ford and Labbok, 1993). Mayer et al. (1988) indicated that affected individuals were less likely to have been breastfed than controls after adjustment for maternal age, birth year, socioeconomic conditions, sex, and race with an odds ratio of 0.70. The risk of insulin-dependent diabetes was even less for those breastfed for more than 12 months (odds ratio 0.54). Studies from Finland indicated that the risk of developing type 1 diabetes was significantly diminished by being breastfed exclusively for 2 to 3 months. The risk was less when the infants were 4 months of age or older when supplementary feeding was introduced (Virtanen et al., 1991, 1992). This observation may have many explanations. The authors favor the possibility that breastfeeding limits exposure to a cow’s milk albumin peptide, which may trigger type 1 diabetes (Dosch et al., 1993). Recently it was reported that short-term breastfeeding and early introduction of cow’s milk-based formula increased the risk of type 1 diabetes in genetically susceptible young children because of β-cell autoimmunity (Kimpimäki et al., 2001). A very large ongoing international multicenter study may answer the question of whether avoidance of cow’s milk in infancy prevents diabetes type 1 (Virtanen et al., 1998). Support for protection was also reported recently (Eurodiab, 2002), but introduction of cow’s milk formula or solid foods before 3 months of age did not increase the risk. This complex area was recently reviewed (Darrins, 2001). One study of non-insulin-dependent diabetes in Pima Indians suggested that exclusive breastfeeding for 2 months

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

reduced the risk of disease, which is so common in that ethnic group (odds ratio 0.41, 0.18–0.93). Obesity was also reduced (Pettitt et al., 1997). A few studies have suggested that breastfeeding may protect against multiple sclerosis (Pisacane et al., 1994b) and rheumatoid arthritis (Brun et al., 1995). The prevalence and duration of breastfeeding did not differ between children who developed autoimmune thyroid disease and controls (Fort et al., 1990), but those who developed the autoimmune disease had significantly more often been fed soy-based milk formulas. Several authors investigated the possibility that breastfeeding prevents inflammatory bowel disease. Rather different results have been obtained, with three out of eight studies of Crohn’s disease showing protection by breastfeeding (Bergstrand and Hellers, 1983; Koletzko et al., 1989; Corrao et al., 1998). Similarly, three out of eight studies of ulcerative colitis suggested protection compared with controls (Acheson and Truelove, 1961; Whorwell et al., 1979; Corrao et al., 1998). The significant protection by breastfeeding against these two diseases ranged from 1.5 (Corrao et al., 1998) to 3.60 (Koletzko et al., 1989). In a recent experimental study of colitis in IL-10-deficient newborn mice, it was found that foster feeding by normal mothers reduced the incidence of colitis (Madsen et al., 2002). Diminished TNFα and IFN-γ secretions from colonic mucosa as well as reduced numbers of adhering bacteria were noted. Children with celiac disease were found to have been breastfed for shorter periods than controls (Auricchio et al., 1983; Greco et al., 1988). This finding has been confirmed by other studies (Fälth-Magnusson et al., 1996; Ivarsson et al., 2002), but not by all (Anderson and Brueton, 1985). Some studies showed that the risk of celiac disease was less when gluten was introduced during breastfeeding (FälthMagnusson et al., 1996; Ivarsson et al., 2002). A recent investigation organized in a different manner did not confirm the role of breastfeeding. Among 164 siblings of 97 probands with celiac disease, 85 were found to carry the genes DQAI*0501–DQBI*02 conferring susceptibility. Not based on presence of symptoms, but on interviews, food recording, and a small intestinal biopsy, eight cases of celiac disease were detected (Ascher et al., 1997). The findings suggested that breastfeeding may not protect against asymptomatic, but against symptomatic celiac disease, but other interpretations are also possible.

Breastfeeding and malignancies As recently reviewed, several case-control studies have suggested an increased risk of childhood cancer related to artificial feeding (Davis, 2001). Lymphomas, leukemias, Hodgkin’s disease, and non-Hodgkin lymphoma are among the cancers that may be increased. However, there are also studies not confirming these observations. Because the appearance of cancers may be influenced by so many other factors than early feeding, this is a difficult field of study. One important determinant was the duration of breastfeeding, although this parameter and the exclusivity of breastfeeding were mostly not

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indicated. Significant odds ratios in the positive studies for all cancers ranged from 1.75 (1.08–2.83) to 7.0 (3.6–13.5) (Davis et al., 1988; Smulevich et al., 1999). For Hodgkin’s disease the effect ranged in three studies from 1.47 (1.06–2.0) to 2.5 (p = 0.02) (Schwartzbaum et al., 1991; Smulevich et al., 1999). There was only one positive study showing protection against non-Hodgkin lymphoma, with an odds ratio of 6.8 (1.0–59.0) (Smulevich et al., 1999). For acute leukemia the significant odds ratios were 1.27 (1.09–1.43) and 9.2 (3.1–28.1) (Shu et al., 1999; Smulevich et al., 1999). Breast cancer is rare in traditional societies, where breastfeeding is the norm. Mothers who have breastfed more than three children have a 50% reduced risk of breast cancer compared with never-lactating controls. The same reduction is attained by mothers who have breastfed their first child for more than 13 months (Zheng et al., 2001). In Western countries, the risk of breast cancer is reduced by more than 4% for each year of breastfeeding, in addition to the 7% reduction induced by each pregnancy (Collaborative Group on Hormonal Factors in Breast Cancer, 2002). The suggested reduced risk of malignancies in breastfed children may be related to the recently discovered antitumor effect of milk α-lactalbumin (Svanborg et al., 2003), described in more detail earlier.

Possible basis of the long-term effects of breastfeeding Human milk contains numerous cytokines, growth factors, hormones, and other components that might affect the breastfed offspring. Thus, it may be that the long-term enhancing effects on the IgG2 antibody response to H. influenzae type b by breastfeeding are due to the presence in milk of IFN-γ (Silfverdal et al., 2002). This cytokine enhances the switching to IgG2 antibody production. In children 18 months or older, there was a significant relation between the duration of exclusive breastfeeding and the level of IgG2 antibodies to the H. influenzae type b polysaccharide (Silfverdal et al., 2003). The origin of specific effects on the immune system of the breastfed offspring may be sought primarily among the specific immune factors in human milk. Thus, human milk contains anti-idiotypic antibodies (Hahn-Zoric et al., 1993) that might, for example, influence vaccine responses (HahnZoric et al., 1990). This finding is supported by studies showing that anti-idiotypic antibodies to a bacterial polysaccharide given to neonatal mice via the mother’s milk are as effective as if given directly to the neonate in stimulating the early vaccine response to a bacterial polysaccharide (Stein and Söderström, 1984). Similar effects were obtained with an anti-idiotypic antibody against a viral antigen (Okamoto et al., 1989). Human milk contains numerous cells, including T and B lymphocytes. Several studies have made it likely that such cells can be taken up by the gut of the offspring. Thus, radioactively labeled human colostral cells given to premature baboons resulted in radioactivity in intestinal lamina propria, spleen, and liver (Jain et al., 1989a, 1989b). Recent experiments used labeled human milk cells infused into

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human fetal small intestine transplanted into nude mice. The great majority of the cells 20 to 72 hours later were found intramurally in the intestinal transplant (Siafakas et al., 1997). A few cells were also found intact in the spleen and lungs. In a sheep model, labeled milk lymphocytes were deposited in the small intestine of lambs by a catheter or by laparotomy (Tuboly et al., 1995). It was found that colostral lymphocytes from mothers given to lambs appeared in the lymph and reached the mesenteric lymph nodes by 8 hours after their uptake. Blood lymphocytes were noted in the mucosa but not in the mesenteric lymph glands. Antibody and blastogenic responses to tetanus toxoid vaccine were determined in lambs given or not given cells from mothers who had been vaccinated. The lambs given cells from immunized mothers had quicker and higher vaccine responses. Similar specific effects of colostral cells have previously been demonstrated in the pig (Tuboly et al., 1988). Earlier studies suggested that tuberculin sensitivity can be transferred from a tuberculin-positive mother to her offspring via breastfeeding (Ogra et al., 1977; Schlesinger and Covelli, 1977). Human milk contains twice as many γ/δ T cells as peripheral blood, and the milk from a tuberculinpositive mother had a higher specific proliferative response among the milk γ/δ T cells than did cells from tuberculinnegative women (Bertotto et al., 1993). However, another study did not find evidence for the transfer of tuberculin sensitivity via maternal milk but rather via the placenta (Keller et al., 1987). A model of B cell–deficient mice was used to show that foster feeding by normal mothers could transfer immunoglobulin-producing B cells and save the offspring (Arvola et al., 2000). A possible effect of the uptake of leukocytes from the mother’s milk is that the offspring becomes tolerant to the mother’s HLA. Thus, a renal transplant to an adult recipient survives and functions better when it comes from a mother who breastfed the recipient than when it comes from a mother who did not, or if it comes from the father (Campbell et al., 1984). A similar study was performed comparing the outcome of renal transplants when adult donor–recipients were siblings with one shared haplotype. It was found that breastfeeding of donor and recipient was strongly related to good function of the renal transplant: after 5 years, 79% of the transplants functioned among the breastfed recipients but only 15% among the non-breastfed recipients. In addition, the breastfed recipients required much less immunosuppression (Kois et al., 1984). In experiments on rats, a low response to paternal cells could be induced with foster feeding by a mother with the same transplantation antigens as the father (Deroche et al., 1993b). In humans, it was noted that breastfeeding decreased precursor frequencies of cytotoxic T cells directed against maternal HLA alloantigens (Zhang et al., 1991). Thus, breastfeeding appears to specifically downregulate the immune response of children against maternal alloantigens. As mentioned previously, it is interesting to note that human milk fat globule membrane fractions contain MHC class II

molecules (HLA-DR) (Wiman et al., 1979a; Newman et al., 1980). Pabst et al. (1997) demonstrated that breastfed infants at 6 months of age had significantly less spontaneous expression of integrins on CD4+, CD8+, and CD19+ lymphocytes than formula-fed infants. Before vaccination with measles/mumps/rubella vaccines, the breastfed infants had significantly lower blast transformation and lower IFN-γ production without these antigens and also with tetanus toxoid or candida antigen. Two weeks after vaccination only the breastfed infants showed increased production of IFN-γ and increases in CD56+ and CD8+ cells. The authors suggested that breastfeeding enhances Th1-type immune responses. Another illustration of a likely effect of breastfeeding on the infant’s immune system is that the thymus at 4 months of age is strikingly larger in exclusively breastfed infants compared with those partially breastfed ( p = .007) and those formula fed ( p = .001) (Hasselbalch et al., 1996, 1999). This could be significant given the fact that the T cells responsible for downregulating autoreactivity to certain self-antigens, Treg, leave the thymus in neonatal life at a relatively late developmental stage. In rodents this happens after 3 days of age; thus, removing the thymus before this time point dramatically impairs the development of self-tolerance to peripheral self-antigens (Suri-Payer et al., 1996, 1998; Stephens et al., 2001). Breastfeeding might thus protect against the development of autoimmune diseases (see earlier discussion). The nucleotides in human milk that might enhance vaccine responses (Pickering et al., 1995, 1998) can enhance natural killer cell cytotoxicity (Carver et al., 1991) but are considered not to transfer any specificity. Infectious agents in human milk

Bacteria Human milk is not sterile when received by the infant. Cultures show in more than 70% the presence of diphtheroid rods, α-hemolytic streptococci and coagulase-negative staphylococci. Gram-negative bacteria are found in 10% to 15% of milks from healthy women (Law et al., 1989; Sharp, 1989). Pathogens like S. aureus, group B streptococci, Campylobacter, salmonellae, Mycobacterium tuberculosis, and Borrelia burgdorferi may appear in the milk. Swedish breastfed children (16%) have recently been shown to pick up superantigen- and toxin-producing S. aureus by 3 days after delivery. By 2 to 6 months of age, up to 73% carried these bacteria (Lindberg et al., 2000). Further studies suggested that the bacteria originated from the mother’s breast, nose, and skin, and also from the father’s skin. None of the infants had any symptoms; presumably, the bacteria and their toxins were rendered harmless by the protective factors in the milk. The presence of S. aureus at such high prevalence in the gut of breastfed infants may depend on a deficient colonization with other bacteria from the mother’s intestinal microflora, which should have successfully competed with this potential pathogen (Lindberg et al., 2004).

Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

Human immunodeficiency virus type 1 (HIV-1) Part of the tragedy of the HIV/AIDS epidemic is that infected mothers may transfer the virus to their offspring. This may occur in utero, during delivery, or via breastfeeding. The risk of postnatal transmission after 2.5 months of age is 3.2 per 100 child/years of breastfeeding, but earlier postnatal transmission may be more frequent (Nduati et al., 2000; van De Perre, 2000). Breastfeeding may contribute 12% to 14% additional risk, giving a 35% total proportion of all HIV-1infected children in an area because of breastfeeding (Weinberg, 2000). In a WHO document, it is estimated that transfer of HIV1 from mothers without antiretroviral treatment to infants ranges from 15% to 30% in the absence of breastfeeding, to 25% to 30% if there is breastfeeding through 6 months, and to 30% to 45% if there is breastfeeding through 18 to 24 months (WHO, 2001). Countries that already have infant mortality rates 10 to 20 times higher than developed countries can expect a doubling in infant and childhood mortality because of HIV-1 (Goldenberg et al., 2002). About 200,000 to 350,000 infants worldwide may be infected yearly with HIV-1 via breastfeeding. UNICEF estimates that, on the other hand, 1.5 million non-HIV-1-related deaths per year could be prevented in the world through breastfeeding. According to the WHO, infants given formula or other replacement feeds and not mother’s milk show a sixfold increased risk of dying in the first 2 months of life (Coutsoudis et al., 2002). However, a study from Kenya suggests a similar mortality rate and incidence of diarrhea and pneumonia during the first 2 years of life for breastfed and formula-fed infants, but those formula fed had significantly fewer HIV-1 infections (Mbori-Ngacha et al., 2001). The use of breast milk substitutes prevented 44% of HIV-1 infections (Nduati et al., 2000). In an interesting study from South Africa, it was observed that exclusive breastfeeding brought a significantly lower risk of HIV-1 transmission than mixed feeding. The risk was similar to that of no breastfeeding (Coutsoudis et al., 1999). It will be important to try to confirm this finding, which might mean that promotion of exclusive breastfeeding in areas with a high risk of transfer of HIV from mother to infant is a better solution than promoting formula feeding in such poor areas. At this time, exclusive breastfeeding is uncommon in many traditional societies (Ashraf et al., 1993b; Bland et al., 2002). Maternal factors that increase the risk of HIV-1 transfer during breastfeeding are high maternal viral load, low CD4 count, high maternal erythrocyte sedimentation rate, and presence of lesions in the breasts during pregnancy (Fawzi et al., 2002). Human milk cells infected with HIV-1 can be found at all stages of lactation. Around 0.1% to 1% of macrophages and T cells in colostrums and early milk were productively infected in HIV-1-seropositive mothers (Southern, 1998). More than 97% of the infected milk cells were macrophages. When milk cells were mixed with saliva they were disrupted, or bound to and endocytosed by salivary epithelial cells (Southern and Southern, 2002). HIV-1 is also found in the fluid phase of milk.

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The port of entry of the infection in the infant is not known but may be lymphoepithelial tissue of the tonsils, enterocytes, and M cells, as well as mucosa-associated lymphoid tissue in the gut (van De Perre, 2000). Proximal small intestinal enterocytes have been shown to effectively transfer HIV-1 virus particles after they had bound to the CCR5R expressed on the microvilli of the enterocyte. Interestingly, the transfer of virus particles was greatly enhanced if infected macrophages were present at the luminal side (Bomsel and David, 2002). It has been reported that clinical or subclinical mastitis increases the risk of transfer of HIV-1 to the infant (Semba et al., 1999; Willumsen et al., 2003). In women with mastitis, HIV-1 was detected in the milk in 75%, and in 35% if without mastitis. Recently, Willumsen et al. (2003) showed that the viral load in the milk of HIVinfected South African women during the first 14 weeks of lactation was associated with subclinical mastitis and severe maternal immunosuppression. However, there were multiple contributors to the viral load because multivariate models had limited predictive value.These observations indicate that partial breastfeeding may lead to subclinical mastitis, which brings a greater risk of HIV transfer. The study of Coutsoudis (1999) suggests that exclusive breastfeeding with a lesser risk of mastitis does not transfer HIV at the rate of partial breastfeeding (Willumsen et al., 2002). It has been suggested that S-IgA and S-IgM antibodies against HIV may be protective because levels of these antibodies were inversely correlated with transmission (van De Perre et al., 1993). A more recent study investigated S-IgA and IgG antibodies in the milk of HIV-1-transmitting and nontransmitting mothers against HIV-1 gp160 and two HIV-1 peptides derived from gp41 and gp123 (V3 loop) (Becquart et al., 2000). No differences were found between the two groups of mother’s milk. Determination of the 90K (Mac-2BP) protein in the serum of HIV-1 infected mothers and their newborns showed that high levels were associated with lack of HIV-1 transfer (Pelliccia et al., 2000). The 90K protein is known to activate NK cells, which may result in viricidal activity. HIV-2 is a much less virulent virus than HIV-1 and has not been reported to be transmitted via milk (Michie and Gilmour, 2001).

Human T-cell leukemia virus type 1 (HTLV-1) In southern Japan, HTLV-1 causes adult T-cell leukemia/ lymphoma. In one region, almost 60% of new cases each year are caused by viral transfer via breastfeeding (Tsuji et al., 1990). More than 85% of mothers of child carriers are also carriers; 21% of children of carrier mothers get infected, but only 1% of noncarrier mothers’ milk contains HTLV-1, which can also infect marmosets perorally (Tsuji et al., 1990). Although it has not been shown that infants infected via breastfeeding develop T-cell malignancies in adulthood, breastfeeding is discouraged in southern Japan where the virus appears. This may disrupt the epidemic. Antibodies against HTLV have been detected in 10.6% of a high-risk population in Delhi (Varma and Mehta, 1999).

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The virus has also been shown to be transmitted from mother to child in 9.7% of an African population (UretaVidal et al., 1999). In Brazil, it is suspected that within families with adult T-cell leukemia/lymphoma, mothers providing milk to milk banks are a source of contamination with HTLV-1 (Pombo-De-Oliveira et al., 2001). Another related retrovirus, HTLV-2, shows a similar pattern of transmission via human milk (Lal et al., 1993).

Parasites

Cytomegalovirus (CMV)

In summary, human milk contains numerous components like antibodies, cytokines, hormones, enzymes, and major proteins like lactoferrin and α-lactalbumin with multiple activities (microbicidal, tumoricidal, anti-inflammatory, etc.). Thus, breastfeeding brings nutritional, developmental, and anti-infectious advantages to the infant. The significant protection conferred by breastfeeding against numerous infections like acute and prolonged diarrhea, neonatal septicemia, respiratory tract infections, acute and recurrent otitis media, urinary tract infections, and so on is seen in developed as well as in developing countries. In poor countries, breastfeeding is a major public health issue because it can strikingly reduce infant mortality as well as fertility of the breastfeeding mother. In this way, it improves the situation for both mothers and offspring and for society. Interestingly, breastfeeding appears to actively direct the immune system of the offspring. This effect may occur via the uptake of lymphocytes, both T and B cells, and possibly also via anti-idiotypic antibodies and cytokines from the milk. As a consequence, vaccine responses and host defense against infections like diarrhea, respiratory tract infections, and otitis media may be enhanced for years to come. In addition, transplant reactivity against maternal tissues is decreased, presumably because of the tolerance to maternal HLA, which follows the exposure to maternal leukocytes. It might be that the immune system of the offspring also becomes better controlled so that the risk of certain autoimmune and inflammatory diseases, including allergies, may be decreased. Finally, the presence in milk of metabolically active hormones, cytokines, and so on may explain why breastfeeding likely prevents obesity.

CMV is the most common cause of congenital and perinatal infection in humans (Numazaki, 1997). Perinatal transmission occurs during delivery or via the milk. The primary infection in the mother induces an immune response and the CMV becomes latent. Reactivation during pregnancy makes the milk infectious in about 25% of the mothers (Numazaki, 1997). The virus is more often present in mature milk than colostrums, and the fluid portion contains more CMV than the milk cells. CMV-infected milk leads to infection in 69% of the infants, and breastfeeding for more than a month increases the risk of infection (Dworsky et al., 1983). The infection causes no symptoms and it is unclear whether it has consequences in later life. It appears that the immune response of the mother, including milk antibodies and lymphocytes directed against CMV, cannot prevent the infection, but it appears to prevent symptoms. Under this protection, the child is able to develop a protective immune response.

Rubella Rubella virus, a toga virus, is transferred via the milk. Still, neonatal rubella is rarely seen in infants breastfed by mothers with a postpartum infection (Buimovici-Klein et al., 1977; Klein et al., 1980). Vaccination with live attenuated rubella vaccine in seronegative women after parturition resulted in the rubella vaccine virus being in the milk of 70% of these women (Losonsky et al., 1982a). None of the 50% of infants who were infected via the milk showed any symptoms (Losonsky et al., 1982b, 1982a). Such exposure does not affect the immune response of children to later vaccination against rubella (Krough et al., 1989).

Hepatitis viruses There is no evidence that hepatitis B virus is transferred via the milk, causing infection in the offspring (Beasley et al., 1975; Woo et al., 1979). Just as for herpes simplex virus, one may be concerned that an infected mother may transfer virus via cracked or bleeding nipples (Henrot, 2002). Similarly, studies on hepatitis C virus (HCV) have not given evidence for viral transfer via the milk. Of 76 HCV infected mothers, none of their breast milk samples contained HCV RNA, although almost 60% of the mothers had HCV viremia (Polywka et al., 1999). However, Lin et al. (1995) claimed to have found the virus in the milk of 15 HCV-infected mothers who had antibodies in the milk and who did not transfer the virus to their breastfed infants.

There is little or no evidence that parasites cause disease if they reach the infant via the milk (May, 1988). Trypanosomes and schistosomes and some protozoa have occasionally been isolated from milk.

CONCLUSION

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Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

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Chapter 104 Human Milk: Its Components and Their Immunobiologic Functions

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