Human Milk

Chapter 117

Human Milk: Its Components and Their Immunobiologic Functions Dolly Sharma University of Buffalo School of Medicine, Buffalo, NY, USA

Lars Å. Hanson, Marina Korotkova and Esbjörn Telemo Göteborg University, Göteborg, Sweden

Pearay Ogra University of Buffalo School of Medicine, Buffalo, NY, USA

Chapter Outline Introduction2307 Components2308 Immunoglobulin2308 Lactoferrin2309 α-Lactalbumin2310 Oligosaccharides and Glycoconjugates 2310 Lipids and Milk Fat Globules 2311 Nucleotides2312 Defensins2312 Cytokines2312 Hormones and Growth Factors 2313 Anti-inflammatory Components 2314 Soluble CD14 2314 Leukocytes in Milk 2315 Neutrophils2315 Macrophages2315 Lymphocytes2315

INTRODUCTION The newborn shows many special characteristics; one is that it is born sterile and another is that it has a complete, yet immature, 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 acquire microflora, which is a necessary component of normal life. The common 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 germ-free animals (Hanson et al., 2003). This stimulus helps the immune system to develop the capacity to respond Mucosal Immunology. http://dx.doi.org/10.1016/B978-0-12-415847-4.00117-8 Copyright © 2015 Elsevier Inc. All rights reserved.

Immunobiologic Functions 2316 Breast-Feeding and Infant Mortality 2316 Protection against Gastroenteritis 2317 Protection against Infections of the Respiratory Tract 2318 Protection against Infections of Other Sites 2320 Breastfeeding and Allergy 2320 Active and Long-term Effects of Breast-Feeding on the Infant’s Immune System 2322 Breast-Feeding and Vaccine Responses 2322 Long-term Protection against Infections 2323 Breast-Feeding and Obesity 2323 Long-term Effects of Breast-feeding on Autoimmune and Other Inflammatory States 2324 Breast-feeding and Malignancies 2325 Possible Basis of the Long-term Effects of Breast-Feeding 2325 Conclusion2326 References2327

with specific immunologic tolerance, avoiding the development of allergic and autoimmune disease. Immediately after delivery, perhaps the most significant factor that determines the microbial colonization of the newborn gut is its mode of feeding (Coppa et al., 2006). Waiting for its own immune system to take over its host defense, the infant requires help from its 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 FcRn (Hanson et al., 2003). However, such antibodies, once in contact with microbes in tissues, will activate a proinflammatory cytokine-mediated defense, which causes symptoms, tissue 2307

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derangement, and high energy consumption. This process can be deleterious for the young infant requiring energy for growth and development (Moret and Schmid-Hempel, 2000; Read and Allen, 2000). The higher animals, humans and chimpanzees, have a fully developed additional specific immune defense system; the secretory IgA (S-IgA) antibodies, which in humans make up 70–80% 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 numerous other factors are also supportive of defense, as reviewed subsequently.

COMPONENTS Immunoglobulin The S-IgA antibody system was first noted in and isolated from human milk as an IgA with an additional structure, subsequently named the secretory component (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. S-IgA binds microorganisms found on the infant’s mucosal membranes and is crucial in preventing activation of the complement cascade and phagocyte recruitment, which are essential in the newborn’s proinflammatory defense mechanism (Hanson, 2007). Lactogenic hormones, produced during late pregnancy and lactation, are crucial for the homing to the mammary glands (Roux et al., 1977; WeiszCarrington 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 (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. In a study by Dahlgren et al. (1987), it was found that after initiation of an IgA response in the Peyer’s patches with Escherichia 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. 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.

S-IgA makes up 80–90% of the immunoglobulins in milk. In colostrum, it may reach about 12 g/L and then level off around 1 g/L (Goldblum et al., 1982). An exclusively breast-fed 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). 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). The stability of the S-IgA molecule is due to its superior resistance to gastrointestinal proteolytic enzymes 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. This ratio 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 such as 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. 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. 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 immature immune system. 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 24–74% of bacteria in the colon are coated by IgA and fewer by IgM and IgG (van Der Waaij et al., 1996). It seems that milk S-IgA may enhance the growth of such bacteria in the gut of the breast-fed infant by providing this binding site for these bacteria, which are of low virulence. Milk S-IgA antibodies also bind and neutralize toxins and viruses. In contrast, milk S-IgA antibodies do not, or do only to a limited extent, support systemic immunity (Ogra et al., 1977; Klemola et al., 1986). The maternal IgG reaching the fetus via the FcRn receptors in the placenta results in IgG antibodies in intestinal tissue fluid at a concentration of 50–60% of the intravascular level (Brandtzaeg et al., 1991). Postnatally, only traces of S-IgA and S-IgM are detected in the intestinal tissue fluid, whereas IgG is more prominent. These IgG antibodies may

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participate in mucosal defense in conjunction with the milk antibodies. In their limited amount, they may be important for the defense of the non-breast-fed infant.

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, similar to 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 for by the increased intake of mature milk by the infant. A healthy breast-fed infant obtains 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 weight of 78 kDa 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 breast-fed infants (Goldblum et al., 1989). Human infants, similar to adults, have a receptor in the gut for uptake of LF and large LF fragments (Kawakami and Lonnerdal, 1991). LF and certain of its fragments (lactoferricin (LF-cin)) are bactericidal for a wide range of gram-negative and gram-positive bacteria. LF destabilizes the outer membrane of gram-negative bacteria, binding and releasing lipopolysaccharide (LPS); thereby reducing the inflammatory response of the neonatal immune system (Hanson, 2007). This increases the bacteria’s sensitivity to killing by lysozyme (Ellison, 1994). Human LF interacts similarly with the cell wall lipoteichoic and teichoic acid of gram-positive bacteria, increasing their sensitivity to killing by lysozyme (Leitch and Willcox, 1998). By enzymatic release of LFcin from LF, its microbicidal activity is markedly enhanced (Bellamy et al., 1993). Serine protease activity has been discovered in human LF (Hendrixson et al., 2003). LF appears to cleave the surface proteins of H. influenzae, possibly contributing to the antibacterial capacities of LF. 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 1000-fold (Edde et al., 2001). LF interacted with macrophages, but was also microbicidal in cooperation with lysozyme.

The effect of LF on the invasiveness of Shigella flexneri was studied in 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 actinmediated movements (Gomez et al., 2001). LF inhibits bacterial adhesion by binding to the bacterial surface, the initial step of Salmonella enterica serovar Typhimurium infection (Bessler et al., 2006). LF also exhibits antimycotic effects by interacting with mannoproteins in the cell wall of Candida albicans, damaging the cell wall structure (Nikawa et al., 1994). The synergistic effects of human recombinant lactoferrin in combination with anti-staphylococcal and anti-candidal antimicrobial agents have demonstrated in vitro efficacy against these respective neonatal pathogens (Venkatesh and Rong, 2008). LF suppresses transcription of β-lactamase in Staphylococcal aureus capable of producing this enzyme, contributing to its antibacterial properties, and also has been shown to augment the inhibitory properties of penicillin against Staph. aureus strains (Lacasse et al., 2008). LF has demonstrated antiviral properties, specifically against enterovirus 71 and rotavirus (Yen et al., 2011). This protein has the ability to interact with cell surface glycosaminoglycans, essentially blocking viral entry and inhibiting cell-to-cell spread of herpes simplex virus 1 and 2 (Jenssen et al., 2008). Bovine LF has shown anti-human immuno­ deficiency virus (HIV) activity in vitro by altering the viral entry process (Berkhout et al., 2002). On 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 breast-fed infant, preventing symptoms caused by released proinflammatory cytokines, which cause tissue damage by activating inflammatory cells. 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, such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and granulocyte–macrophage colony-stimulating factor (GM-CSF) (Zucali et al., 1989; Mattsby-Baltzer et al., 1996; Choe and Lee, 1999). Studies have shown that this effect may be due to LF entering monocytic cells and their nuclei, interfering with the transcription factor NF-κB (Haversen et al., 2002). This factor controls the transcriptional activity of proinflammatory cytokine genes. Togawa et al. (2002) also noted the inhibitory effect on the production of the proinflammatory cytokines TNF-α and IL-1β via NF-κB and a simultaneous upregulation of the anti-inflammatory cytokines IL-4 and IL-10. The antimicrobial and anti-inflammatory effects of human LF and certain of its fragments have been tested in experimental models. 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

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helical region, was effective at significantly reducing the infection and its inflammatory symptoms in the urinary tract (Haversen et al., 2000). Breast-fed infants do, in fact, have milk LF, and LF fragments are detectable in the urine (Goldblum et al., 1989). This may be one reason breastfeeding seems to protect against urinary tract infection, as discussed later. The anti-inflammatory capacity of LF and certain of its peptides isolated from human milk was also illustrated in a rat model of dextran sulfate-induced colitis (Haversen et al., 2003). Significant protection against tissue damage, with reduction in IL-1β levels in blood, in TNF-αproducing cells, and in CD4+ cells, resulted. LF fragments from the LF-cin 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. LF also induces enterocyte growth and proliferation, making it an essential regulator of intestinal development of the newborn gut (Buccigrossi et al., 2007). Bovine LF has been shown to exert similar biologic activity compared to human LF and, when added to infant formulas, may act as a growth factor in gut-related disease (Buccigrossi et al., 2007).

α-Lactalbumin α-Lactalbumin is the predominant protein component of breast milk, comprising up to 25% of total protein found in human breast milk. This protein serves many crucial functions from a physiological and immunological standpoint (Lonnerdal and Lien, 2003). The total concentration of α-lactalbumin in breast milk varies geographically, with mean values indicating higher concentrations found in samples of breast milk obtained from the United States compared to other nations. The concentration of α-lactalbumin typically displays an inverse relationship with duration of lactation (Jackson et al., 2004). α-Lactalbumin facilitates the absorption of essential minerals and is crucial for protein synthesis of the developing neonate (Lonnerdal and Lien, 2003). Infant rhesus macaques fed α-lactalbumin supplemented formula experience fewer and milder intestinal E. coli gastrointestinal infections than those fed control formula (Bruck et al., 2003). Multimeric α-lactalbumin 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 converted by unfolding the protein with oleic acid as a required cofactor (Svensson et al., 1999, 2000). The resulting structure, called HAMLET (human α-lactalbumin made lethal to tumor cells), is a Ca2+-elevating and apoptosis-inducing 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 C18:9cis oleic acid (Svanborg et al., 2003). HAMLET kills cancer cells, but not healthy, differentiated cells (Gustafsson et al., 2005). Although cancer cells have inactivated their apoptotic pathways, HAMLET is capable of destroying them, both those of human origin and those 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 bel-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, and 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 breast-feeding on childhood leukemia, further discussed later. The long-term effect might be due to HAMLET reaching sites of proliferation such as 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. S. pneumoniae demonstrate apoptosislike activity similar to tumor cells when induced by HAMLET, causing degradation of DNA and shrinkage of cells (Hakansson et al., 2011; Hallgren et al., 2008).

Oligosaccharides and Glycoconjugates Human breast milk contains prebiotic oligosaccharides, which demonstrate an immunomodulatory role in the postnatal period. Milk fat globules contain a wide spectrum of glycoconjugates and oligosaccharides, many of which are important in host defense because they function as analogs of microbial ligands on mucosal epithelial surfaces, thereby inhibiting binding of pathogens to target ligands of the host cell (Newburg et al., 2005). Oligosaccharides 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). Human milk oligosaccharides (HMOs) make up the third largest solid component of human milk (Newburg et al., 2005).

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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). Some 90 different oligosaccharides have been isolated from milk (Newburg, 1997), but with time-of-flight mass spectrometry, some 900 fucosyloligosaccharides have been discovered (Stahl et al., 1994). Oligosaccharides in human milk seem to have quite substantial effects on the intestinal microflora and the capacity of several microbes to infect the infant. HMOs affect the growth of bifidus bacteria in the newborn gut and, therefore, are influential in the composition of neonatal gut flora (Kunz and Rudloff, 2008). They may add to the observation that breast-fed infants have fewer serogroups of fecal E. coli than non-breast-fed (Rueda, 2007) (Gothefors et al., 1975). These infants also less often carry pathogenic E. coli, Klebsiella species, and other Enterobacteriaceae strains. HMOs demonstrate varied immunomodulatory roles. HMOs containing sialic acid components have the ability to downregulate leukocyte adhesion to the endothelium (Kunz and Rudloff, 2008). Oligosaccharide preparations bearing functional resemblance to HMOs were shown to enhance delayed-type hypersensitivity responses in a dose-dependent manner when tested in a murine influenza vaccination model, suggesting that supplementation with oligosaccharides in infant formulas may aid in the development of the immune system during infancy (Vos et al., 2007). These structures can be regarded as a new class of antimicrobial agents. α1,2-linked fucosylated glycans inhibit binding by Campylobacter, stable toxin of enterotoxigenic E. coli, and major strains of calicivirus (Morrow et al., 2005). Other studies have defined in detail the milk oligosaccharide structures involved in the inhibition of bacterial adherence. An elegant example is the work by Ruiz-Palacios et al. (2003). Investigators demonstrated that the mechanism by which Campylobacter jejuni binds to the H-2 antigen in human intestinal mucosa is an essential step in the infectious process. This binding was inhibited with high avidity by human milk fucosyl-α1,2-linked molecules. Sialylated oligosaccharides from milk prevented cellular adhesion of E. coli (Korhonen et al., 1985). Otitis-causing 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). Studies evaluating the passage of HMOs through the gut of infants have demonstrated that most of them survived

intact (Chaturvedi et al., 2001). Lower concentrations appeared in the urine. The fecal and urinary oligosaccharides of breast-fed infants resembled those in their mothers’ milk. Formula-fed infants had fewer oligosaccharides in feces and urine and these oligosaccharides had a different composition (Rudloff et al., 1996). Human milk contains various glycoconjugates. One is mucin, which is found particularly on milk fat globules. Through their sialic acid content, mucin and milk fat globules also inhibit binding of S-fimbriated E. coli to epithelial cells (Schroten et al., 1992). Mucin also inhibits rotavirus replication in an experimental model, presumably via its carbohydrate moiety (Yolken et al., 1992). Human milk κ-casein can inhibit adhesion of Helicobacter pylori to human gastric mucosa (Stromqvist et al., 1995). The content of fucose-containing carbohydrate moieties seems 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. Gangliosides are acid glycosphingolipids distributed throughout most body fluids of vertebrates, including human breast milk. Gangliosides function as target receptors for certain pathogenic bacteria and therefore prevent binding of pathogens in various body tissues, specifically, the GI tract (Rueda, 2007). The glycoconjugate GM1 is a ganglioside, which functions as a cholera toxin receptor and prevents adherence of Vibrio cholerae to cells (Iwamori et al., 2008; Holmgren et al., 1983) and binds the analogous toxins of E. coli, V. cholerae, and C. 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 Shiga-like toxin from enterohemorrhagic E. coli. Prevention of their adherence to epithelial cells has been shown (Newburg et al., 1992).

Lipids and Milk Fat Globules The antimicrobial lipid components of human milk act synergistically to block the steps required for pathogen replication, hence blocking their ability to establish active infection (Isaacs et al., 2005). 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. The free fatty acids released by the major lipase in milk, the bile saltstimulated lipase, are efficient at killing G. lamblia (Hernell et al., 1986). When injected into skin lesions of mice, oleic acid has been shown to be effective at eliminating methicillin-resistant

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Staph. aureus through cell wall disruption and may provide a novel therapeutic approach against the organism (Chen et al., 2011). 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.

Nucleotides Nucleotides are present in human milk, making up about 2–5% of its total nonprotein nitrogen, which is greater 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 breast-fed 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, particularly on the mucosa 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 premature infants resulted in higher serum levels of IgA and IgM (Navarro et al., 1999); greater responses to immunization against H. influenzae type b, tetanus, and diphtheria toxoid (Schaller et al., 2004); 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 (Gutierrez-Castrellon et al., 2007) (Brunser et al., 1994). Long-term breast-feeding has resulted in higher serum antibody responses to oral poliovirus vaccine compared with a formula plus nucleotide or a formula group (Pickering et al., 1998). Compared with healthy term infant subjects fed a control formula, those fed formula fortified with nucleotides had a reduced risk of developing diarrhea and had significantly higher serum concentrations of IgA over the next 48 weeks (Yau et al., 2003).

Defensins Defensins form an interesting group of antimicrobial heatstable peptides. In humans, defensins are the major microbicidal 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). α-Defensins exhibit anti-HIV-1

activity in vitro (Kuhn et al., 2005). In a cohort of African women 1 month postnatally, women with detectable levels of α-defensins (≥50 pg/mL) had significantly higher levels of mean HIV-1 RNA levels in their breast milk (Bosire et al., 2007). In another case–control study, α-defensin concentration had a significant inverse correlation with intrapartum and postnatal HIV transmission and, therefore, may have had a role in preventing mother-to-infant transmission of HIV to breast-fed infants (Kuhn et al., 2005). The β-defensin LBD-1 gene has also been expressed in human mammary tissue and in a mammary gland epithelial cell line; the defensin was present in 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 all mammalian milks. Because cytokines are the signaling molecules of the immune system, their presence in milk might be of importance for the infant in inhibiting excessive inflammation and controlling epithelial proliferation (Penttila, 2010). It has been suggested that a newborn’s country of birth and mother’s ethnicity have an influential effect on the cytokine profile that child develops throughout its infancy via breast milk (Holmlund et al., 2010; Amoudruz et al., 2009). First to be discovered 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 major histocompatibility complex (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 breast-feeding (Saarinen et al., 1999b). The anti-inflammatory effect of TGF-β is also due to its direct suppressive effect on T cells. When studied in a mouse model, passive transfer of allergen along with TGF-β through breast milk was proven to induce development of regulatory CD4 T lymphocytes in their progeny, further protecting against development of asthma (Verhasselt, 2010). In a multivariate regression analysis of the risk of development of infant wheeze in breast-fed children, a significant inverse relationship was demonstrated between the concentration of TGF-β transmitted through human milk and the percentage of infant wheeze within the first year of life (Oddy et al., 2003). In addition to 12 human studies, animal studies have demonstrated the role of TGF-β found in human breast milk in the prevention of allergic diseases in infancy and early

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childhood (Nakao, 2010; Oddy and Rosales, 2010). A study in which investigators quantified and compared TGF-β concentrations in mature milk of allergic versus nonallergic mothers found that levels of the cytokine were secreted in significantly lower concentrations in allergic (0–2400  pg/mL) compared to nonallergic mothers (0–6250 pg/mL), further demonstrating that certain cytokines found in human milk may play a role in the development of atopy in early childhood (Rigotti et al., 2006). 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). 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 also 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 breast-fed infants (Perlmutter et al., 1989; Davidson and Lonnerdal, 1990). 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 breast-feeding because of the presence of IL-7 in the milk (Collinson et al., 2003; Ngom et al., 2004). In a mouse model in which recombinant IL-7 was labeled with a fluorescent dye and traced from the stomach across the gut into the lymphoid organs, thymocyte subsets and peripheral T cells were found to be significantly higher in the offspring of normal wildtype mothers compared to knockout mice who received milk from IL-7-knockout mothers. This finding provides direct evidence that IL-7, which is maternally derived, can traverse the intestine of the offspring, stimulate production of T lymphocytes in the thymus, and support survival of T cells in secondary lymphoid organs (Aspinall et al., 2011). When breast milk concentrations of IL-7 levels were compared between breast-feeding HIV-infected mothers who transmitted HIV to their infant after the neonatal period and HIV-infected women who did not transmit the virus to their infant, IL-7 concentrations that were undetectable (<30 pg/mL) were significantly associated with less HIV transmission, indicating that IL-7 may be a cytokine that is necessary for effective transmission of the virus from mother to child (Walter et al., 2007). The immunomodulating and anti-inflammatory cytokine IL-10 is also present in human milk (Rudloff et al., 1993; Rigotti et al., 2006). Some of the cytokines in milk originate from the milk cells, as reviewed later, but others, such as IL-18, are

produced by the mammary gland epithelium (Takahata et al., 2001). The macrophage colony-stimulating factor and the granulocyte colony-stimulating factor are both present in human milk, as well as macrophage migration-inhibitory factor and interferon-γ (IFN-γ) (Bocci et al., 1993; Eglinton et al., 1994; Gilmore et al., 1994; Hara et al., 1995; FlidelRimon 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). Current evidence regarding the protective role of breast-feeding in development of allergy and atopic disease is still conflicting. Cytokines TGF-β, IL-10, and IL-12 and soluble CD14 were analyzed from milk samples obtained from allergic versus nonallergic mothers 1 month postpartum. No direct correlation was made between levels of the studied immune factors with development of atopy in infants of these mothers (Snijders et al., 2006). It is not clear what other effects milk cytokines may have in the offspring. 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 compared with formula-fed (Hasselbach et al., 1999). At 10 months of age, those who were still breast-fed had a larger thymus than those who had stopped breast-feeding between 8 and 10 months. Actually, there was a significant correlation between the number of breast-feedings per day and the thymus size (Hasselbalch et al., 1999).

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 are in the breast-fed 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 (thyroxine) (Bohles et al., 1993) and steroid hormones. However, thyroxine concentrations have been found to be equally low (0.17–1.83 μg/L) in breast milk and formula milk and do not seem to affect plasma thyroxine levels compared between breast-fed and formula-fed infants (van Wassenaer et al., 2002). Transfer of function has clearly been shown in animal experiments for epidermal growth factor (EGF) (Berseth et al., 1990) and insulin-like growth factor-1 (IGF-1) (Philipps et al., 1997). Rat milk containing an added growth hormone (GH)releasing factor-like immunoreactivity increased GH production in 2- and 8-day-old suckling rats (Kacsoh et al., 1989). Removal of suckling rats from their mothers resulted in decreased GH levels in plasma, which normalized when the babies were reunited with their mothers (Kuhn et al., 1990).

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A similar observation was made for luteinizing hormone (Baram et al., 1977). This may have been due to a temporary deficiency in the transfer of milk-borne luteinizing hormonereleasing hormone. There is also evidence regarding the 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, in the 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 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 manner, 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, and hepatocyte growth factor (Hirai et al., 2002; Itoh et al., 2002). Leptin, the obese gene product, is a 16-kDa 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). Breast milk produced by women who have experienced preterm labor has been proven to be deficient in leptin, which may essentially contribute to neuroendocrine dysregulation in infants born to these mothers (Mehta and Petrova, 2011). 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. Both infant and maternal serum leptin concentrations have a positive correlation with infant growth parameters and maternal body mass index, respectively (Savino et al., 2010; Eilers et al. et al., 2011). However, breast milk leptin concentrations are significantly lower than levels found in sera of the breast-fed infant and the mother (Savino et al., 2010). 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 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 functions of monocytes/macrophages (Santos-Alvarez et al., 1999). Leptin also modifies T cell responses, increasing production of IL-2 and IFN-γ from Th1 cells and IL-4 and IL-10 from Th2 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. 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.

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 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 and is possibly linked to necrotizing enterocolitis. This mechanism may relate to the fact that breast-feeding seems to protect against necrotizing enterocolitis (Furukawa et al., 1993; Muguruma et al., 1997) (see further in the section, “Protection against Infections of 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. Additionally, human 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. Human breast milk contains significant concentrations of sCD14 (approx. 25–50 μg/mL) (Spencer et al., 2010). 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, by stimulating cytokine production of cells that do not express CD14 (Spencer et al., 2010). Apart from aiding LPS signaling, soluble CD14

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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). In vitro studies have demonstrated that when exposed to purified α-lactalbumin, the degradation of sCD14 is suppressed, allowing for this protein to remain viable in the neonatal gut, further boosting the immunoregulatory response to gram-negative pathogens (Spencer et al., 2010). Although specific polymorphisms of maternal CD14 have demonstrated a positive correlation with development of atopy in children under the age of 2 years, this same relationship has been under debate when this protein is studied in its soluble form (sCD14) in breast milk. Although higher levels of sCD14 have been identified in mothers with an allergic history compared to mothers without this history, they did not appear to influence the development of atopy in exposed infants (Snijders et al., 2006, 2010). Other investigators have demonstrated a protective effect of breastfeeding and higher concentrations of sCD14 in breast milk against the risk of developing atopic dermatitis or asthma in children (Rothenbacher, 2005).

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 2–3 months. Thus, many millions of leukocytes reach the infant per day via the milk.

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 such as IL-1β, IL-6, and TNF-β spontaneously, although in significantly smaller amounts 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 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 stimulated with concanavalin A showed enhanced phagocytosis of zymosan particles, whereas endotoxin had the reverse effect. Phagocytosis of zymosan but not latex particles increased prostaglandin E2 (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).

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 a quite 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 expressed only by a small subset of lymphocytes in the colon, and TECK is not

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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 such as 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 an experiment that studied 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 provides better results if the offspring has been breast-fed than if it has not. If the offspring has not been breast-fed, the result is the same as if the kidney came from the father (Campbell et al., 1984; Kois et al., 1984; Zhang et al., 1991; Zhang and Miller, 1993). These observations were confirmed by fosterfeeding 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 nonbreast-fed (Zhang et al., 1991). Tolerance to maternal HLA could also be induced by HLA-carrying milk fat globules. As discussed further later, priming to vaccines appears to take place via transfer of milk cells. 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 Breast-Feeding and Infant Mortality In addition to its strong anti-infectious and immunomodulatory capacities, breast-feeding is, with about six suckings per 24 h, strongly contraceptive. More conceptions are prevented by breast-feeding 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 breast-feeding based on

its anti-infectious effects (Aaby et al., 1984; Hobcraft et al., 1985; Reves, 1985; Hanson et al., 1994b). Effective breastfeeding has the potential to prevent nearly 720 infant deaths in the United States annually (Chen et al., 2004). 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 infant mortality rate (IMR) is usually followed by a reduction in fertility (Hanson et al., 1994b). Breast-feeding is one important factor in this connection because it reduces both mortality and fertility. The American Academy of Pediatrics states that exclusively breast-feeding of an infant until at least 6 months of age would result in greater than 1 million fewer pediatric deaths annually worldwide. It is obvious that breast-feeding has a significant capacity to reduce the IMR. This is illustrated by our observations that even partial breast-feeding reduced the risk of neonatal septicemia in a Pakistani community with an odds ratio of 18 (Ashraf et al., 1991). Similar observations were made in another study of early-onset septicemia in a Pakistani population (Bhutta and Yusuf, 1997). This 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 in the first 2 years of life (Khan et al., 1993). Previously it was determined that the risk of dying of diarrhea for a non-breast-fed child in a poor country is 25 times that of an exclusively breast-fed child (Feachem and Koblinsky, 1984). In a study avoiding confounding factors as much as possible, it was observed that compared with exclusively breast-fed 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 optimal timing of the initiation of breast-feeding has been a subject widely studied and debated. Edmond et al. conducted a study in rural Ghana, which included >10,000 infants born over a 12-month period, and concluded that newborns for whom breast-feeding was delayed until day 7 of life had a 2.4-fold increase in risk for neonatal mortality. This study suggests that early initiation of breast-feeding, along with exclusively breast-feeding, is optimal, specifically in areas such as sub-Saharan Africa, where infant mortality rates are high and mothers have limited resources and lack access to clean water necessary to prepare formula (Edmond et al., 2006). An Ethiopian case–control study found that children who were never breast-fed suffered a greater risk of death than their breast-fed counterparts [odds ratio (OR) = 13.74, 95% confidence interval (CI) 3.34, 56.42], with pneumonia being the most common cause of death (29.7%), followed by acute diarrheal illness (23%) and malaria (23%) (Girma and Berhane, 2011).

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A study from Rwanda demonstrated that breast-feeding significantly reduced case fatality from 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 breast-feeding decreased the IMR by 6.2 per 1000, and breast-feeding plus supplement, by 3.8 per 1000. In contrast, artificial feeding increased postnatal mortality by a factor of 1.8 to 2.6. Investigators from Israel studied 238 Bedouin infants until 18 months of age and concluded that infants exclusively breast-fed until 3 months of age had significantly lower rates of infection and morbidity. Older infants from 4 through 6 months of age and 10 through 12 months of age also reaped beneficial effects of partial breast-feeding against certain GI infections, otitis media, and asthma (Bilenko et al., 2008). Obviously, breast-feeding can substantially reduce the IMR by preventing deaths from infections, which cause 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). This suggests that to be significantly protective against deaths from diarrhea (Feachem and Koblinsky, 1984; Victora et al., 1987), breast-feeding 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 breast-feeding protected against acute as well as persistent diarrhea. A WHO collaborative team studied the protection against infant and child mortality caused by infectious diseases (WHO, 2000). This review showed that breast-feeding 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 of age, decreasing to 1.4 from 9 to 11 months. Slightly lower protection was seen for boys than for girls. During the first half-year, protection against diarrhea was significantly better than protection against acute respiratory infection (OR = 6.1 compared with 2.4). From 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. Breast-feeding has been suggested to also protect against sudden death in infancy (SIDS). This may help reduce infant mortality (Hauck et al., 2011; 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). In contrast, an independent protective effect of breast-feeding 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 breast-feeding

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 in infant mortality during the nineteenth century in Western countries was primarily a result of socioeconomic development. A Swedish study highlighted, however, that during years in the mid-nineteenth century with the poorest harvests and many socioeconomic problems, a decrease in infant mortality occurred that appears to have been due to increased breast-feeding (Lithell, 1999).

Protection against Gastroenteritis The most common cause of infant mortality worldwide is infant diarrhea. The beneficial effects of breast milk in reducing morbidity and mortality from diarrhearelated illnesses are varied (Morrow and Rangel, 2004). Oligosaccharides found in human milk protect the neonate against enteric pathogens by functioning as prebiotics and inhibit binding of these organisms to the host cell receptors (Newburg et al., 2005). One of the essential breast milk proteins, sCD14, through its role as a component of the LPS receptor complex, has been shown to retain biologic activity in the neonatal gastrointestinal tract, hence stimulating the immune responses of the newborn and aiding in the prevention of gastrointestinal gram-negative infections early on in life (Blais et al., 2006). The first available data providing 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 breast-feeding protects against diarrhea and remains the major strategic prevention of morbidity and mortality within the first few years of life (Feachem and Koblinsky, 1984; Jason et al., 1984; Victora et al., 1987; Glass and Stoll, 1989; Howie et al., 1990; Lucas et al., 1992; Morrow and Rangel, 2004). 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). To date, 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).

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C. jejuni is one of the most common causes of bacterial diarrhea worldwide (Ruiz-Palacios et al., 2003). By inhibiting binding of Campylobacter to intestinal mucosa, HMOs have been shown to have a significant role in preventing infection by this organism (Ruiz-Palacios et al., 2003). Breast-feeding also determined the severity of Shigella diarrhea during the first 3 years of life of Bangladeshi infants, demonstrating an inverse correlation (Clemens et al., 1986). Among 1476 Pakistani children living in villages and periurban slums, 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 (Jalil et al., 1993). This protection lasted for the 2 years of follow-up. The reduction in the rate of diarrhea by breast-feeding during the first weeks of life was as much as 70–80%, although partial breast-feeding was predominant. In the city slum, the significant protection of partial breast-feeding lasted for 9 months, and in the upper middle class for 6 months. The latter group breast-fed the least (Ashraf, 1993). Although the exclusively breast-fed group was small, the size of the cohort permitted a comparison with breast-fed children who were given only extra water. It is a widespread tradition to give extra water to breast-fed 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). A large meta-analysis that included data from 18 studies conducted from 1980 through 2009 reported that infants between 0 and 5 months of age who were not breast-fed had a relative risk of experiencing diarrhea-related mortality nearly 10 times that of infants of the same age who were exclusively breast-fed (Lamberti et al., 2011). 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 breast-fed group compared with a formula-fed group (Dewey et al., 1995). Another study from the United States showed similar protection by exclusive breast-feeding and formula feeding (Scariati et al., 1997). In Scotland, significant protection by breast-feeding was seen, but only if breast-feeding 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 breast-feeding provided greater benefit in children who showed severe wasting and stunting, protracted illness, and diarrhea as the

only disease (Sachdev et al., 1991). A study from GuineaBissau found that the incidence of diarrhea was higher at both 1 and 2 years of age in weaned compared with partially breast-fed children (Mølbak et al., 1994). The duration of disease was also shorter among the breast-fed. Even if the breast-fed children had poorer nutrition, their morbidity and mortality were lower than those of the non-breast-fed children. A reduction in number and volume of diarrheal stools has also been reported as a consequence of breast-feeding (Khin-Maung-U et al., 1985). A very large and well-controlled study in Belarus showed protection against gastroenteritis by breast-feeding during the first year, but not against respiratory infections (Kramer et al., 2001). In a Mexican study, it was found that breast-feeding provided good protection against infection with G. lamblia but did not prevent chronic carriage of the parasite (Morrow et al., 1992). The risk of non-breast-fed children contracting G. lamblia infection was five times that of those fully breast-fed, whereas non-breast-fed children had a 1.8 times greater risk of contracting infection compared to partially breast-fed children. It is remarkable that breast-feeding does not appear to protect efficiently against diarrhea caused by rotavirus (Duffy et al., 1986; Clemens et al., 1993). However, exclusive breast-feeding significantly reduces severe rotavirus diarrhea during the first year of life. During the second year, however, the risk of severe infection is increased, and so it appears that infections were just postponed by breastfeeding (Clemens et al., 1993). In a 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 breast-feeding tended to result in asymptomatic infections. In a large study of nosocomial rotavirus infections from Italy, significant protection was obtained by breast-feeding (Gianino et al., 2002). None of the breast-fed infants who became infected developed symptoms of diarrhea. It was highlighted in a German study that breast-feeding in infancy does not protect against He. pylori infection 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 He. pylori to human gastric mucosa. The effect seems to be mediated via the fucose-containing moieties of the κ-casein (Stromqvist et al., 1995).

Protection against Infections of the Respiratory Tract Breast-feeding may provide a route for transmission of maternal respiratory pathogens to the newborn (i.e., adenovirus, influenza virus, respiratory syncytial virus (RSV),

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Mycoplasma, and Haemophilus), either through direct contact with infected secretions or through lesions on the breast (i.e., herpes simplex virus 1 and 2, varicella zoster virus). However, the benefits of breast-feeding and providing immunity to a neonate may far outweigh the risks of acquiring infection, making breast-feeding still the optimal choice for infant nutrition except in few contraindicated circumstances: maternal infection with HIV and human T cell lymphotropic virus 1 (Lanariet al., 2012). Numerous studies have suggested that breast-feeding 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 breast-feeding 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 breast-feeding beyond 4–6 months (Duncan et al., 1993). A decrease of 20% in the number of attacks of acute or prolonged otitis was obtained by breast-feeding, as seen in another study (Dewey et al., 1995). The number of episodes of prolonged disease was reduced by 80% in the breast-fed 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, breast-feeding 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 inversely 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 numerous studies claiming protection by breast-feeding against upper and lower respiratory tract disease, but again discordant reports have appeared. It was claimed that the disagreements were due to the presence of confounding factors and a rather weak protective effect of breast-feeding in developed countries (Kovar et al., 1984; Bauchner et al., 1986; Anderson et al., 1988; Wright et al., 1989; Woodward et al., 1990). Possibly, breast-feeding

results in less severe illness (Frank et al., 1982). A study that analyzed data from 2277 children between 6 and 24 months of age concluded that the duration of breast-feeding did positively correlate with prevention of pneumonia. Children who were exclusively breast-fed for 4 months experienced a significantly greater risk of developing pneumonia than children who were fully breast-fed for 6 months, 6.5% versus 1.6% (Chantry et al., 2006). A multicenter trial that studied greater than 200,000 6- to 7-year-old children concluded that breast-feeding in infancy was significantly associated with reduced risk of rhinoconjunctivitis (OR = 0.74, 95% CI 0.59–0.94) and severe eczema (OR = 0.79, 95% CI 0.66–0.95) in study subjects (Bjorksten et al., 2011). In poor countries, protection against pneumonia by breast-feeding is striking, as already mentioned, saving many lives, for example in Brazil (Victora et al., 1987). Cesar et al. (1999) showed that non-breast-fed infants were 17 times more likely than those breast-fed to be hospitalized for pneumonia. They found that this relative risk was 61 for children younger than 3 months of age and decreased to 10 at older ages. In Western countries, several studies have given evidence for significant protection of breast milk against respiratory infections (Bryan et al., 2007; 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). A large study in the United States showed that breast-feeding 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 breast-feeding was stopped before 2 months of age or partial breast-feeding was stopped before 6 months of age. Wheezing lower respiratory tract illness more often caused visits to a doctor or hospital if the child was predominantly breast-fed for less than 6 months (Oddy et al., 2003). Breast-fed infants who are hospitalized with severe bronchiolitis, most commonly caused by RSV, have lower concentrations of IL-8 in their nasopharyngeal secretions compared with formula-fed infants and, therefore, less severe mucosal inflammation. This finding lends further support to the protective role of breast milk during an episode of bronchiolitis in infancy (Dixon, 2010). Schijf et al. used a mouse model to study the immunomodulating properties of human breast milk in protecting against RSV infection in infants. In addition to passive transfer of maternal antibody, investigators demonstrated that specific prebiotic oligosaccharides similar to those present in human milk increased Th1 responses in the lungs of RSV-infected mice and accelerated viral clearance (Schijf et al., 2012). A mother’s breast milk immunomodulatory profile is altered when her infant

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is experiencing a severe case of bronchiolitis. Breast milk of mothers with hospitalized infants with bronchiolitis have significantly greater numbers of viable cells that stimulate cytokine profiles in response to RSV live virus compared to nonspecific mitogens (Bryan et al., 2007). Type 1 interferon responses studied from the nasal secretions of infants experiencing a respiratory infection are greater when breast-fed infants are infected with the influenza virus in comparison to infection with RSV or human metapneumovirus, supporting the role of the innate immune response in protecting against respiratory viruses in infancy (Melendi et al., 2010).

Protection against Infections of Other Sites As advancements in modern technology have contributed to the viability of preterm neonates, necrotizing enterocolitis has become one of the more common causes of mortality in this vulnerable population. Studies have suggested that breast milk consumption in premature neonates may have a preventive role in the development of necrotizing enterocolitis (NEC) (Berman and Moss, 2011). An analysis of 1272 preterm low-birth-weight (LBW) infants (401–1000 g) was conducted to assess the protective effects of breast milk on this group at risk for developing NEC. Investigators established a dose-related inverse correlation of breast milk with NEC development. After 14 days, the risk of NEC in the study subjects decreased by a factor of 0.83 for each 10% increase in proportion of total intake as breast milk (Meinzen-Derr et al., 2009). Another study concluded that preterm LBW neonates whose enteral feeds consisted of >50% breast milk experienced a six-fold reduction in NEC development at 14 days of life, compared to their counterparts who received <50% breast milk as their feeding regimen (Sisk et al., 2007). Significant protection against neonatal septicemia by breast-feeding 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). There is evidence that breast-feeding 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 such as S-IgA antibodies binding to the intestinal bacterial flora from which the infections mostly originate and to 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.

A few retrospective studies suggest that breast-feeding 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 breast-feeding may prevent botulism (Arnon et al., 1982).

Breastfeeding and Allergy Many studies have been devoted to whether breast-feeding 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 breast-feeding (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 school children with asthma, 80–85% of the attacks are reported to be initiated by viral infections (Johnston et al., 1995). It seems that the preventive effect on wheezing by breast-feeding is primarily due to the prevention of these viral infections in early life (Wright et al., 1998). Atopic allergy mostly starts as a food allergy in early life and is often caused by cow’s milk proteins and also soy proteins. Whether breast-feeding prevents such allergy has been much debated (Kramer, 1988; Hanson, 1998). The American Academy of Pediatrics current recommendation for lactating mothers whose infants are at high risk of developing atopic dermatitis is to consider eliminating cow’s milk, eggs, peanuts, and tree nuts. Breast-feeding 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). A number of characteristics of mother’s milk have been linked to the development of cow’s milk allergy in the offspring. In a cohort study that included 4676 children who were breast-fed, Savilahti et al. identified a correlation that linked lower concentrations of IgA casein antibodies and sCD14 in the colostrum of mothers whose infants demonstrated symptoms of atopy by age 4 years (Savilahti et al., 2005). 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 positively 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 compared to nonallergic mothers (Bottcher et al., 2000a). These chemoattractants may influence the transfer and activation of cells in the milk. One study found no difference in concentrations of IL-10 in the breast milk of allergic versus nonallergic mothers (Rigotti et al., 2006).

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Whether temporary exposure in early life to bovine proteins, in otherwise breast-fed 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 another 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 breast-fed 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. A Japanese study that included 92 exclusively breast-fed infants, all of whom had a diagnosis of atopic dermatitis, demonstrated that when allergenic foods (tree nuts, cheese, soy, yogurt) were eliminated from their mothers’ diet, 73% of these infants showed significant improvement of their atopic skin lesions (Uenishi et al., 2011). 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 correlate with one another and with the concentration of S-IgA in the milk (Bottcher et al., 2000b). The IL-4 level was significantly higher in 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 cow milk protein (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 downregulator of both Th1 and Th2 T cells and is a major switch factor for IgA. Wright et al. (1999) demonstrated a relationship between serum IgE in childhood and maternal IgE levels. With low IgE in the mother, breast-feeding was associated with lower total IgE in the child by the age of 6 years. With a high maternal IgE and breast-feeding for 4 months or longer, the child had higher IgE levels compared with those never breast-fed or breast-fed 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 breast-feeding as a single factor. The KOALA study, which compared the breast milk of 315 lactating mothers, with and without an allergic history, 1 month postpartum, concluded that there was no significant correlation between the levels of specific cytokines

(TGF-β, sCD14, IL-10, and IL-12) in the breast milk of the respective groups and the development of atopy in their offspring (Snijders et al., 2006). A Finnish study evaluated 4-year-old children with differing atopic heredity who were exclusively breast-fed for 3 months. The investigators concluded that in children who had a positive atopic hereditary history, breast-feeding had a significant protective effect against atopy (OR = 0.41, 95% CI 0.18–0.95), compared to children without atopic heredity, who experienced a higher incidence of atopy (OR = 2.57, CI 1.16–5.70) (Siltanen et al., 2003). Several studies have investigated whether breast-feeding 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 breast-feeding protected against atopic dermatitis in children with a family history of atopy. This effect was negligible if there were no first-degree relatives with atopic disease (Gdalevich et al., 2001). Oddy et al. (1999) showed, in a prospective 6-year follow-up in Australia, a significant reduction in the risk of childhood asthma if breast-feeding 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. Breast-­ feeding 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. Brew et al. studied the effects of breast-feeding on development of asthma in later childhood. Data from two large cohorts of children, from Australia and Sweden, were compared. The investigators concluded that breast-feeding did significantly reduce the risk of asthma in both groups by 8 years of age in children with a positive family history of asthma; however, the effect was stronger in the Swedish cohort (Brew et al., 2012). 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 breast-feeding (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. 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 7–8 years was higher in those breast-fed for less than 3 months. This increased risk by short-term breast-feeding was not seen among the sensitized asthmatics (Ronmark et al., 1999). A large Japanese study using questionnaires at the age of 12–15 years about modes of feeding in early years concluded that there was an increased risk of atopic eczema related to breast-feeding

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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). Other factors caused by geographic differences may also be involved. 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 the milk of Australian mothers (Fidler and Koletzko, 2000), who seem to protect against allergy by breast-feeding (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 who contract atopic dermatitis showed increased proportions of α-linolenic acid and decreased proportions of its long-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 breast-fed offspring according to a Swedish study (Duchén et al., 2000). Haby et al. (2001) showed, in Australia, protection against asthma by breast-feeding, but an increased risk for asthma if the child had had a high intake of polyunsaturated fats. 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. A Cochrane database that included 523 participants did not establish a protective effect of maternal dietary antigen avoidance during lactation on the development of atopy in breast-fed infants during the first 18 months of life (Kramer and Kakuma, 2012).

ACTIVE AND LONG-TERM EFFECTS OF BREAST-FEEDING ON THE INFANT’S IMMUNE SYSTEM Breast-Feeding and Vaccine Responses It has been noted that vaccine antibody responses after immunization with parenteral and peroral vaccines were better in breast-fed 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 last were still elevated 1–2 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 breast-fed compared with formula-fed children against the H. influenzae type b (Hib) capsular polysaccharide in a Hib–protein conjugate vaccine (Pabst and Spady, 1990). Additionally, infants fed formulas that are supplemented with ribonucleotides evoked better antibody responses when vaccinated against Hib, diphtheria toxoid, and polio (Gutierrez-Castrellon, 2007). It should be noted that these effects on vaccine responses were also seen for months after the termination of breast-feeding. Enhancement of vaccine responses in serum at 6 months of age to oral poliovirus vaccine by breast-feeding has been noted (Pickering et al., 1998). Infants had been exclusively breast-fed 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 infants receiving 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 breast-fed than in formula-fed infants, although those registered as breast-fed might have been so for only 2 weeks (Pabst et al., 1989). The enhancing effect was seen only if the vaccine was given before 1 month of age. The majority of children in Africa receive their BCG vaccine against tuberculosis at birth. Infants in this region of the world who are born to HIV-positive mothers continue to be at high risk for acquisition of HIV through breast milk transmission. Investigators in the United Kingdom studied a recombinant vaccine, to be administered at or soon after birth, which included protection against a BCG strain in combination with an African HIV-1 derived immunogen. Protection against Mycobacterium tuberculosis and boosting of natural immunity against natural exposure to HIV-1 in breast milk were achieved, suggesting a means of decreasing mother-to-child transmission of HIV-1 through breast-feeding (Im, 2007). Other studies have not shown improvements in vaccine responses by breast-feeding. 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 breast-feeding was noted early by Sabin for the live poliovirus vaccine. It is still a significant problem because the oral vaccine is often given on the day of birth or soon thereafter, when breast-feeding has started. Awareness of this problem is often deficient (Rennels, 1996).

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There are several studies with Hib and tetanus toxoid vaccines in which breast-fed infants did not respond better than non-breast-fed (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 definition of breast-feeding used, its duration, and its 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).

Long-term Protection against Infections A study by Howie et al. (1990) in Scotland showed that breast-feeding for more than 13 weeks resulted in better protection against gastroenteritis throughout the first year of life compared with shorter term breast-feeding or none at all. A reanalysis when these children were 7 years of age revealed a continued decreased risk of respiratory tract infections related to breast-feeding (Wilson et al., 1998). Silfverdal et al. (1999) studied the protective effect of breast-feeding against invasive infections and meningitis caused by H. influenzae. A long-term protective effect was found, lasting for 10 years after the termination of breast-feeding. The study of Saarinen (1982) on otitis media showed protection lasting for 3 years in infants breast-fed previously. The preventive effect on wheezing was seen for 6–7 years (Porro et al., 1993) and may have been largely a result of the prevention of infections that 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 suggest that breast-feeding provides long-term protection (Pisacane et al., 1995, 1996b). In an investigation by van den Bogaard et al. (1991), children breast-fed through 6 months were at the age of 3 years still better protected than non-breast-fed children against gastroenteritis, respiratory tract infections, skin infections, and urogenital disease. A critical review of the literature has supported the presence of enhanced long-term protection against infections of the gastrointestinal and respiratory tract mucosae from breast-feeding (Chien and Howie, 2001). Investigators in Hong Kong studied the effects of exclusive versus partial or no breast-feeding for the first 3 months of life and risk of hospitalization. A cohort of 8327 children was prospectively followed for 8 years. Exclusive breastfeeding for at least 3 months was significantly associated with reduced risk of hospitalization during the first 6 months of life for respiratory infections, GI infections, and other infections. Partial breast-feeding in the first 3 months also

reduced risk of hospitalization from similar infections, but to a lesser degree. Beyond 6 months of age, this inverse correlation between breast-feeding and hospitalization for infections did not exist (Tarrant et al., 2010). A more recent Tanzanian study established that 666 children born to HIV-infected women who were exclusively breast-fed experienced significantly lower rates of respiratory and diarrheal illnesses in the first 6 months of life; however, this same effect was not evident at 6–24 months of life (Mwiru et al., 2011).

Breast-Feeding and Obesity Childhood obesity is an increasingly significant epidemic in the United States and remains under the influence of a multitude of genetic and environmental factors (i.e., parental obesity, socioeconomic status, smoking, birth weight) (Butte, 2009). Among the long-term effects of breast-feeding 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; Schack-Nielsen, 2007), as well as enhancing effects on bone mineralization (Jones et al., 2000) and increase in neurodevelopmental scores into adulthood (Agostoni et al., 2013). The reduction in obesity seems well documented in large studies. Compared with formula-fed children, breast-fed children have a reduced risk of obesity by 20% by school age, after adjustment for confounding variables (Agostino, 2013; Koletzko, 2009). The Australian NOURISH randomized controlled trial compared infant feeding practices of 612 infants and concluded that formula feeding had a positive correlation with rapid weight gain during the first few months of life (OR = 1.72; 95% CI 1.01–2.94) compared to breast-feeding. Higher protein intake and feeding on schedule (associated with overfeeding) have been postulated mechanisms by which formula feeding contributed to greater weight gain in infants (Koletzko, 2009; Mihrshahi, 2011). With longer duration of breast-feeding a lower incidence of subsequent obesity has been associated (von Kries et al., 1999; Gillman et al., 2001; O’Tierney, 2009). In a Centers for Disease Control study that examined infant feeding practices, infants who initiate their own bottle emptying early on in infancy were nearly 70% more likely to experience excessive weight gain later on in infancy compared to those who rarely emptied their bottles (Li et al., 2008). Infants who are bottle-fed early on in infancy are more likely to empty their bottle later on in infancy, compared with exclusively breast-fed infants, indicating that self-regulation may play a significant role in determining the relationship between breast-feeding and a decreased risk of childhood obesity in later infancy and childhood (Li et al., 2010). It has been shown that breast-feeding lowers plasma insulin levels and decreases fat storage, thereby preventing early adipocyte development (Oddy, 2012). Scientific

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data support this concept by demonstrating that some of the cytokines and hormones present in milk may be involved. 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 stimulating glucose and fatty acid oxidation (van Zaanen et al., 1996; Keller et al., 2001). Human breast milk contains specific hormones and growth factors known as adipokines (leptin and adiponectin), ghrelin, resistin, and obestatin, which are responsible for food intake and energy regulation and are not routinely found in commercial formula milk products (Savino et al., 2009). Leptin is produced by fat cells and is found in milk fat globules and mammary epithelial cells (SmithKirwin 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 breast-fed infant. Leptin regulates food intake and energy metabolism and is found in higher concentrations in breast-fed than in formula-fed infants, whereas the hormone ghrelin is found in lower concentrations (Savino et al., 2004). 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). It is possible that the modifying effect on the risk of obesity in the breast-fed child may be influenced by the mother’s diet, that is, if milk leptin really is an important factor as suggested by some studies (Locke, 2002; Singhal et al., 2002; Uysal et al., 2002). Adiponectin, another hormone secreted by adipocytes, has been found in higher levels in the maternal breast milk fed to overweight 2-year-old children who had been breast-fed for a minimum of 6 months; however, the authors suggested that further research is warranted to establish an association of circulating adipokines with excessive weight gain in breast=fed infants (Weyermann et al., 2007).

Long-term Effects of Breast-feeding on Autoimmune and Other Inflammatory States Perhaps the most significant short-term immunological benefit of breast-feeding, worldwide, is protection of the infant against a variety of infectious diseases (Schack-Nielsen, 2007). Longer term, it has been suggested that breast-feeding diminishes the risk of developing inflammatory bowel

diseases, childhood cancers, and type 1 diabetes, further supporting the effects of breast-feeding on the infant’s immune system (Schack-Nielsen and Michaelsen, 2007; 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 breast-fed 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 breast-fed 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 breast-fed exclusively for 2–3 months. The risk was even 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 breast-feeding limits exposure to cow’s milk albumin peptide, which may trigger type 1 diabetes (Dosch et al., 1993). Short-term breast-feeding and early introduction of cow’s milk-based formula have been shown to increase the risk of type 1 diabetes in genetically susceptible young children because of β-cell autoimmunity (Kimpimäki et al., 2001). Crume et al. conducted a retrospective cohort study that established that adequate breast-feeding (>6 months duration) was associated with a significant reduction in childhood obesity rates in children who were exposed to maternal diabetes in utero (Crume et al., 2011). One study of non-insulin-dependent diabetes in Pima Indians suggested that exclusive breastfeeding for 2 months reduced the risk of disease, which is so common in that ethnic group (OR = 0.41, CI 0.18–0.93). Obesity was also reduced (Pettitt et al., 1997). A few studies have suggested that breast-feeding may protect against multiple sclerosis (Pisacane et al., 1994) and rheumatoid arthritis (Brun et al., 1995). The prevalence and duration of breast-feeding 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 soybased milk formulas. Several authors have investigated the suggestion that breast-feeding prevents inflammatory bowel disease. Rather varied results have been obtained, with three of eight studies of Crohn disease showing protection by breast-feeding (Bergstrand and Hellers, 1983; Koletzko et al., 1989; Corrao et al., 1998). Similarly, three 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 breast-feeding against these two diseases ranged from 1.5 (Corrao et al., 1998) to 3.60 (Koletzko et al., 1989). In an experimental study of colitis in IL-10-deficient newborn mice, it was found that foster-feeding by normal mothers reduced the incidence of

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colitis (Madsen et al., 2002). Diminished TNF-α and IFN-γ secretions from colonic mucosa as well as reduced numbers of adhering bacteria were noted. Individuals who are raised in developing countries with high infectious exposure rates tend to be at low risk of developing certain autoimmune illnesses, such as Crohn disease, and other allergic states. When these same individuals emigrate to industrialized nations, they preserve this low risk; however, the offspring of such immigrants typically experience lower infectious exposures in their developed country of birth than their mothers and have shown to be at higher risk for immune-mediated disease. Breast milk studies have found significantly higher levels of cytokines in milk of immigrant mothers from developing nations compared with those levels in milk of mothers from industrialized countries. This highlights the fact that a mother’s country of origin probably influences the immunomodulatory characteristics of her offspring (Amoudruz et al., 2009). Children with celiac disease were found to have been breast-fed for shorter periods than controls (Auricchio et al., 1983; Greco et al., 1988). This finding has been confirmed by other studies (Ludvigsson and Fasano, 2012; Fälth-Magnusson et al., 1996; Ivarsson et al., 2002), but not by all (Anderson and Brueton, 1985). Some studies have demonstrated that the risk of celiac disease was less when gluten was introduced during breast-feeding (FälthMagnusson et al., 1996; Ivarsson et al., 2002). Another investigation organized in a different manner did not confirm the role of breast-feeding. Among 164 siblings of 97 probands with celiac disease, 85 were found to carry the genes DQAI*0501–DQBI*02 conferring susceptibility. Based not 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 breast-feeding may not protect against asymptomatic, only symptomatic, celiac disease.

Breast-feeding and Malignancies Several case-controlled studies have suggested an increased risk of childhood cancer related to artificial feeding (Davis, 2001). Lymphomas, leukemias, Hodgkin disease, and nonHodgkin lymphoma are among the cancers that may be increased. However, there are also studies that contradict 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. 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 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, in which breast-feeding is the norm. Mothers who have breast-fed 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 breast-fed 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 breast-feeding, 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 breast-fed children may be related to the antitumor effect of milk α-lactalbumin (HAMLET) (Svanborg et al., 2003), described in more detail earlier.

Possible Basis of the Long-term Effects of Breast-Feeding 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 breast-feeding 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 breast-feeding and the level of IgG2 antibodies to the H. influenzae type b polysaccharide (Silfverdal et al., submitted for publication). The origin of specific effects on the immune system of the breast-fed 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 suggested 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 the intestinal lamina propria, spleen, and liver (Jain et al., 1989a, 1989b). Recent experiments used labeled human milk cells infused into human fetal small intestine transplanted into nude mice. The great majority of the cells 20–72 h

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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 h 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 breast-feeding (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 milk 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 breast-fed the recipient than when it comes from a mother who did not, or when 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 breast-feeding of donor and recipient was strongly related to good function of the renal transplant: after 5 years, 79% of the transplants functioned among the breast-fed recipients but only 15% among the non-breast-fed recipients. In addition, the breast-fed recipients required much less immunosuppression (Kois et al., 1984). In experiments in 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 breast-feeding 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 breast-fed 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 for measles/mumps/rubella, the breast-fed infants had significantly lower blast transformation and lower IFNγ production without these antigens, and the same was true for tetanus toxoid or Candida antigen. Two weeks after vaccination only the breast-fed infants showed increased production of IFN-γ and increases in CD56+ and CD8+ cells. The authors suggested that breast-feeding enhances Th1type immune responses. Another illustration of a likely effect of breast-feeding on the infant’s immune system is that the thymus at 4 months of age is strikingly larger in exclusively breast-fed infants compared with those partially breast-fed (P = 0.007) and those formula-fed (P = 0.001) (Hasselbalch et al., 1996, 1999). This could be significant given the fact that the T cells responsible for downregulating autoreactivity to certain self-antigens, Tregs, 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). Breast-feeding might thus protect against the development of autoimmune diseases (see earlier discussion). In rural Gambia, investigators conducted a cohort study of thymic function, via measurement of T cell receptor-rearrangement excision circles (TRECs), in 138 infants who were breast-fed during the first 8 weeks of life, and they found that season of birth was a strong determinant of thymic development. Infants who were born in the hungry season had significantly lower TREC counts compared to those born during the harvest season (0.97 and 2.12 TRECs/100 T cells, respectively, P = 0.006). Breast milk analysis 1 week postpartum also demonstrated significantly lower IL-7 concentrations from mothers of infants born during the hungry season compared with milk from mothers who gave birth during the harvest season (Ngom et al., 2004).

CONCLUSION In summary, human milk contains numerous components, such as antibodies, cytokines, hormones, enzymes, and major proteins such as lactoferrin and α-lactalbumin, with multiple activities (microbicidal, tumoricidal, antiinflammatory, etc.). Breast-feeding provides nutritional, developmental, and anti-infectious advantages to the infant. Significant protection conferred by breast-feeding against varied infections such as acute and prolonged diarrhea,

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neonatal septicemia, respiratory tract infections, acute and recurrent otitis media, and urinary tract infections is observed in developed and developing countries. In poor countries, breast-feeding is a major public health issue as it can strikingly reduce infant mortality as well as fertility of the breast-feeding mother. In this manner, breast-feeding provides significant benefits for both mothers and offspring, in addition to society. Interestingly, breast-feeding 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 such as diarrhea, respiratory tract infections, and otitis media may be enhanced for years to come. The presence of metabolically active hormones, growth factors, and cytokines in milk may explain why breast-feeding probably prevents obesity. It may be suggested that the immune system of the offspring is controlled with more precision over time so that the risk of certain autoimmune and inflammatory diseases, including allergies and malignancies, may be decreased.

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