Oligosaccharides in Human Milk and Bacterial Colonization

In the first year of life, a stable population of microflora is established in the human gut as a complex ecology that includes the intestinal mucosa and more than 400 interdependent species of bacteria. During this period, a child is vulnerable to enteric and other diseases, but breast-fed infants have a lower rate of morbidity and mortality than do artificially fed infants. The lower incidence of disease in infants while they are breast-feeding has been attributed to many causes: less exposure to contaminated water, better nutritional status, and psychological factors. Although these factors probably contribute, the most direct determinant of reduced disease incidence among the breast-fed seems to be factors in the milk itself. This protection is thought to result from two major kinds of components in human milk: those that provide a prebiotic effect that enhances colonization by Bifidobacterium bifidum, and others that inhibit the colonization and growth of pathogens during this vulnerable period. Three major classes of human milk components are known to inhibit pathogens: secretory antibodies; multifunctional agents, such as fatty acids and lactoferrin; and homologues of host cell surface glycoconjugates. Evidence is mounting that these homologues, consisting of glycoconjugates and oligosaccharides, have a major role. Glycoconjugates are molecules that have conjugated carbohydrate moieties (glycoproteins, glycolipids, glycosaminoglycans, and mucins); milk oligosaccharides have carbohydrate moieties conjugated to lactose. These milk carbohydrate moieties often share structural motifs with the cell surface glycoconjugates that pathogens adhere to when gaining entry to host target cells. Indeed, specific human milk oligosaccharides and glycoconjugates have been shown to block binding of specific pathogens to host cell glycoconjugate receptors. This article presents three examples: 1) A fucosylated oligosaccharide from human milk inhibits the pathogenicity of stable toxin of Escherichia coli in vivo, and the blocking effect has been demonstrated in vitro. 2) Fucosylated structures also inhibit binding by Campylobacter jejuni to host cell receptors and limit its colonization in vivo. 3) A glycoprotein, lactadherin, inhibits rotavirus in vitro and in vivo. High concentrations of lactadherin in human milk are associated with reduced incidence of symptoms of rotavirus infection in nursing infants. These findings, along with other examples of pathogen inhibition by milk glycoconjugates, suggest that this is a general mechanism of protection by human milk. Furthermore, human milk oligosaccharides survive transit through the intestine of the breast-feeding infant, implying that they are available to protect against gastroenteritis over the entire length of the intestinal tract. However, pronounced individual variation in the expression of oligosaccharides in human milk suggests heterogeneity in the ability of individual human milks to inhibit specific pathogens. Oral supplementation of vulnerable infants with glycoconjugates, oligosaccharides, or their homologues may be feasible. The milks of other species also contain homologous oligosaccharides and glycoconjugates, suggesting that this mechanism of protection in early life may be common to many mammals. The presence of a large spectrum of oligosaccharides and glycoconjugates in human milk may represent a major mechanism whereby breast-feeding protects infants from enteric and other pathogens during the period when the microflora of the gut are becoming established. BREAST-FEEDING PROTECTS INFANTS AGAINST DISEASES Evidence that breast-feeding protects infants against diseases during their vulnerable first year of life was first convincingly reported from the Massachusetts General Hospital in 1935 (1). In this study of 20,000 patients, breast-feeding was associated with a lower incidence of morbidity and mortality, especially of enteric disease, otitis media, and respiratory infection. Although subsequent studies yielded mixed results regarding the efficacy of breast-feeding against these diseases, more recent studies using relatively sophisticated experimental design and data analysis have yielded findings that mostly support the earlier report. A noteworthy example is a study performed in Lahore, Pakistan (2). The population was divided into rural poor, urban poor, and urban upper middle class; the efficacy of breast-feeding against enteric disease was determined by the same type of analysis used for testing the efficacy of drugs (Fig. 1). Breast-feeding was effective in preventing disease among all three populations, with the protection most pronounced in the first 6, and especially in the first 3, months of life. This protection was most pronounced among the poor but was also clearly demonstrated in the upper middle class, whose socioeconomic status and level of hygiene is closer to that of many western societies. A prospective study in a Scottish population on the effect of breast-feeding on morbidity and mortality due to enteric diseases (3) showed that even in developed countries breast-feeding during the first 3 months of life can be demonstrated to confer protection against gastrointestinal illness. Curiously, in this study the benefits persisted beyond the period of breast-feeding itself. This phenomenon is explicable if breast-feeding contributes to establishing a healthy ecology of the gut, the ramifications of which extend beyond weaning.FIG. 1.: Efficacy of breast-feeding in preventing enteric disease was high in both rural (village and mud hut) and urban (slum) poor, especially during the first 6 months of lactation, and the protection persisted for the 2-year duration of this study. Note that breast-feeding was also efficacious in preventing enteric disease, albeit to a lesser extent, in the upper middle class, a population whose living conditions are closer to those of developed countries. (Reprinted, with permission, from Kluwer Academic/Plenum Publishers. Hansen et al. Adv Exp Med Biol 1991;310:1–15).COLONIZATION BY MICROFLORA The adult human intestine represents a complex ecosystem of more than 400 species of bacteria, interdependent among themselves and with the human gut per se. The human intestinal microflora typically contains more cells than the entire number of cells in the human body. These intestinal bacteria have many functions, such as digesting food components that are indigestible by human intestinal enzymes, and can be a source of important factors such as vitamins. Moreover, a stable, healthy intestinal microflora is thought to contribute to overall health by excluding foreign, potentially harmful bacteria (4). The formation of a stable, interdependent ecosystem of intestinal microflora during the first year of life requires selective acquisition from among the many bacteria in the environment. Important environmental sources of bacterial exposure include the mother and other individuals with whom the infant has intimate contact (5). The colonization of the gut by new bacteria is influenced by the current resident bacteria. Moreover, colonization by any new strain of bacteria is influenced by intestinal epithelial cells, which in turn are affected by the bacteria that colonize the gut, as is discussed in greater detail in the review in this issue by Walker. Diet greatly influences the selection of intestinal microflora during this first year of life. For example, in the gut of the breast-fed infant, bifidobacteria predominate and enterobacteria are present (6), whereas in the artificially fed infant, bifidobacteria, bacteroides, clostridia, and streptococci are dominant (7). Bacteroides dominate the adult flora (8). Human milk, normally the principal food during this first year of life, contains factors that stimulate the growth of commensal bacteria. Prebiotics (9) enhance colonization by specific bacteria; for example, bifidus factor stimulates growth of Bifidobacterium bifidum (formerly called Lactobacillus bifidum) (10,11). Lactose affects colonization directly, by selecting for bacteria able to use this unique sugar, and indirectly, when fermented to acetate, making the gut acidic and stimulating the growth of some bacteria while inhibiting colonization by many pathogenic organisms. Human milk also contains three types of components that inhibit colonization by harmful bacteria. One is the secretory immunoglobulin A (sIgA), which is produced by the immune system of the mother against enteric pathogens to which she has a history of exposure (12). Another type is the multifunctional agents that serve as nutrients while protecting against a broad spectrum of pathogens. Examples are fatty acids that, when released from the triglycerides of milk in the stomach of the infant (and in expressed human milk that has been stored) destroy envelope viruses (13) and also act against other pathogens such as Giardia(14,15). Lactoferrin, the major protein in human milk, is active against a broad spectrum of pathogenic bacteria, and its digestion products include lactoferricin, a porin that is toxic to many strains of bacteria (16). The third class of milk components that are active against pathogens is molecules containing structural carbohydrates, the glycoconjugates and oligosaccharides. The oligosaccharides are complex carbohydrate structures that are generally attached to lactose (17). The glycoconjugates contain complex carbohydrate structures that are attached to proteins, lipids, and other structures (e.g., glycoproteins, glycolipids, mucins, and glycosaminoglycans). The carbohydrate portion of these molecules is largely indigestible and passes through the intestinal tract of the infant mostly intact. These carbohydrate structures potentially act as decoys to which pathogens bind. It is this fraction of human milk that will be the focus of this review. HUMAN MILK OLIGOSACCHARIDES Oligosaccharides are, collectively, the third largest solid constituent in human milk, after lactose and fat. Human milk oligosaccharides consist of lactose at the reducing end, with a carbohydrate core that often contains a fucose or a sialic acid at the nonreducing end (17). When they were initially discovered and studied, these human milk oligosaccharides were thought to be the accidental consequence of the high levels of glycosyltransferases in mammary epithelial cells (required for formation of the abundant milk glycolipids and glycoproteins) in proximity to the high levels of lactose unique to these cells. However, the idea that these oligosaccharides may have a biologic purpose is supported by two lines of evidence in relatively recent investigations. First, it was discovered that the approximately 100 milk oligosaccharides that had been isolated and characterized represent only a very small portion of the total that remain to be characterized. The potential for many thousands of fucosylated oligosaccharides in human milk was suggested when the total human milk oligosaccharide fraction was analyzed by time-of-flight mass spectrometry: Evidence was found for the existence of oligosaccharides with up to 32 sugars and up to 15 fucoses (18). The huge potential for structural isomers suggests that there may be tens of thousands, or perhaps even hundreds of thousands, of potential human milk oligosaccharide structures. Such a large number of possible structures greatly increases the probability that some are biologically active. A second, independent line of investigation has identified several biologic functions attributable to the milk oligosaccharides. Oligosaccharides have been found in human milk that inhibit the adherence of Streptococcus pneumoniae to target cells in vitro (19). Human milk oligosaccharides also inhibit binding by enteropathogenic E. coli(20), binding and infection by invasive strains of C. jejuni(21), and the toxicity of stable toxin of E. coli(22). The findings from both lines of investigation, taken together, suggest that many more biologically active oligosaccharides remain to be discovered. MAMMALIAN CELL SURFACE GLYCOCONJUGATES ARE PATHOGEN RECEPTORS Mammalian cell membranes contain glycoproteins and glycolipids, and the membrane structure is such that the oligosaccharide moieties of these cell surface glycoconjugates protrude into the extracellular region (Fig. 2). These complex carbohydrate moieties in and can act as receptors for such as can also act as receptors for and pathogens. are the viruses and bacteria adhere to the host target cell to the pathogenic surface of glycoconjugates. Glycoconjugates glycoproteins, and can in These glycoconjugates can also to and can in These same cell surface glycoconjugates can be used by pathogens bacteria, and for binding and gaining entry to host cells as the first in glycosyltransferases that human milk oligosaccharides are to the glycosyltransferases that the oligosaccharide moieties of cell surface receptors. the milk oligosaccharides are to have some structural motifs to those found at the cell These human milk oligosaccharides can act as homologues to the cell surface receptors that are the of specific pathogens. as they to the pathogens and inhibit their ability to to the host cells. pathogen binding to the host is first in specific milk oligosaccharides can protect against Three examples will be discussed in which specific human milk components inhibit specific two of the components are oligosaccharides, and is a of Escherichia coli in E. coli are are at high for by these organisms. E. coli both a which toxin in that it to in the host cell and a stable which to the extracellular of in intestinal cells, a of pathogenic in secretory Human milk reduced mortality in stable toxin of E. coli(22). The was found to in the oligosaccharide fraction of human milk and was active at its When this oligosaccharide fraction was over a the was found in the fucosylated oligosaccharide fraction that to When the active fucosylated fraction was into its on by only of the the by stable toxin in vivo. When this was into its components by the was found in of its This was isolated to at its in human milk into in its this fraction stable toxin in as as the milk from which it was isolated 1). This fraction is present in human milk at a of approximately per high level of at such a suggests high of and for its and fucosylated milk oligosaccharides inhibit binding to in vitro system used to study stable toxin was used to the mechanism by which the fraction may protect against by stable cells, line from intestinal were conditions that the expression of many of intestinal epithelial expression of on the and expression of among these is a protein with extracellular that to stable the and the of to levels of result in of from the by of and This of and to the of the gut is the secretory by stable The to a on the extracellular of the of cells, blocking binding by stable toxin This seems to be the mechanism whereby this oligosaccharide the stable toxin from secretory The mechanism whereby a milk oligosaccharide inhibits the ability of stable toxin to secretory When stable toxin to the extracellular of the are in and of from the The of along with its and secretory preventing stable toxin from binding to its extracellular is and secretory the presence of stable Campylobacter a common pathogen of also In in proximity to in contact with the or their are at high for Campylobacter early in life. Campylobacter infection is often in cells in vitro. Human milk inhibits the ability of C. jejuni to cells. This in the oligosaccharide fraction of human milk and in the fucosylated oligosaccharides that to a the concentrations found in human milk, these oligosaccharides inhibit the of pathogenic invasive strains of C. jejuni to cells, but the of A was used to the inhibition of C. jejuni binding in vitro into inhibition of infection in vivo. of were or high in the or the presence of the active human milk oligosaccharide that, at both the presence of the oligosaccharide fraction in several less colonization than in the milk fucosylated oligosaccharides only binding in vitro but infection in Human milk oligosaccharides inhibit the ability of Campylobacter to colonize the of both bacteria per and bacteria per the ability of Campylobacter to colonize the gut is by the presence of human milk oligosaccharides at the found in human findings provide two examples of human milk oligosaccharides that inhibit human pathogens. The of these human milk glycoconjugates to the expression of disease in a breast-feeding human population has been for Campylobacter or stable E. However, this issue was in a third example of a human milk glycoconjugate that inhibits a specific lactadherin inhibition of and is the most common of especially in the developed infection by bacteria and other is less The ability of rotavirus to cells in vitro is by human milk. of this in the protein fraction and has been isolated to a The inhibition is general for all major strains of human rotavirus and is of sialic acid from this its suggesting that a sialic acid attached to a specific carbohydrate of this is for the The lactadherin to blocking its ability to to its host target cell This ability to inhibit rotavirus infection and its has also been demonstrated in in a The the lactadherin levels in milk and presence of in their infants was determined in a population of nursing of the infants were to of milk were for lactadherin and the infants were for symptoms of the infants were with those with were milk with lower levels of lactadherin than those were (Fig. was levels in milk and among These data suggest that the expression of lactadherin in the milk among nursing and that the level of lactadherin expressed is to the presence of in breast-fed concentrations in milk are associated with less disease in breast-fed infants. In breast-feeding infants with those infants whose milk was high in lactadherin were less to symptoms of rotavirus than were those infants whose milk levels of This that lactadherin, which to rotavirus and inhibits its ability to cells in vitro and to in also inhibits the of in seems that large of lactadherin to most of the receptors for host cell surface glycoconjugates, blocking the first in However, when only a of lactadherin is as in levels in their milk, or when lactadherin is as in infants are artificially the has receptors that are able to to intestinal cells and GLYCOCONJUGATES It is noteworthy that in the study of lactadherin and rotavirus the lactadherin and disease was whereas the and disease was This that protection by glycoconjugates of human milk may be at as important as protection by Although in human milk were discovered early and are as agents they to represent only mechanism whereby human milk protects infants. The glycoconjugates and oligosaccharides confer protection at the concentrations found in human milk, probably the large number of possible structures of these milk and potential ability to protect host cells from a of pathogens. It is to that human milk for the protection of infants. The immune of infants are microflora is and is and other of against pathogens are human milk may be important to the of infants from disease and may have protection MILK OLIGOSACCHARIDES The ability of individuals to glycoconjugates and oligosaccharides in human milk as in other and such as have been to oligosaccharide expression in human milk, but oligosaccharide expression is to variation among individual The two and are shown in The in is by a than the in to all human milk oligosaccharides, these contain lactose at the reducing can be in the shown in These also have lactose at the reducing end, but the fucose at the nonreducing end is attached by or the is by type of whereas the are by at the expression of is known to be glycoconjugates in human milk and are as This variation of fucosylated oligosaccharides in of human milk has to be of the two in human milk. In these fucose is attached to lactose by or of these is by a The structures of three of human milk. These structures several common to contain lactose at the reducing end and contain fucose at the nonreducing end of the in or The is by whose is independent from that the or of oligosaccharides among milk from was by that oligosaccharides of sugars or as This for more than by of human milk oligosaccharides variation was found among expression of glycoconjugates in their In the the are but in some the evidence of any the This suggests expression of oligosaccharides among individual on their of The expression of oligosaccharides over the of was the milk from a The overall of oligosaccharides over the of whose milk oligosaccharide a of to such milk for the first 6 months of those whose milk had produced milk that was in these oligosaccharides during the first 6 months of However, 6 months and year of lactation, the milks from both classes of to contain both types of oligosaccharides, suggesting some in the mechanism of oligosaccharide in These are with the that human milk seems to its protection against disease during the first 6 months of These data also suggest a ability to protect against specific pathogens by breast-feeding whose expression of This phenomenon is a of current MILK OLIGOSACCHARIDES The oligosaccharides of human milk represent a major of by the One a mechanism for their digestion that this be the other if the of these oligosaccharides is protection from disease, they be to remain as they through the to protect even of the the of the milk oligosaccharides after by breast-fed the oligosaccharide of the and of the breast-fed infants were with those of the milk from their The of oligosaccharides in the and of the breast-fed infants that in their milk, suggesting that their is human milk. The and of breast-fed infants were also to the and of infants. Oligosaccharides in the and of artificially fed infants have a from milk and from the and of breast-fed infants. The oligosaccharides in of artificially fed infants probably from of intestinal epithelial cells and from bacteria. oligosaccharides in these infants may from of cells, from the of intestinal oligosaccharides by their into the The of oligosaccharides in the and of breast-fed infants are than those in infants 2) that they may any oligosaccharide The of milk oligosaccharides in the of breast-fed infants as than in the milk they are the other milk components are and as they through the gut, whereas the oligosaccharides remain and in concentrations in milk, and of breast-fed and of and of oligosaccharides, on for the of milk and for and suggests that the of oligosaccharides by breast-fed infants is in the whereas a small is and in the It is to that the small of oligosaccharides in the some protection against tract The of oligosaccharides at the end of the that they are present the intestine in concentrations to protect against pathogens. These results also the that the of the oligosaccharides in milk is than HUMAN MILK GLYCOCONJUGATES INFANTS The three examples of oligosaccharides and glycoconjugates in human milk that have been discussed are of many classes of glycoconjugates found in human milk, oligosaccharides, glycoproteins, mucins, glycosaminoglycans, and glycolipids These all have in common a complex carbohydrate as of their structure and the ability to inhibit binding of pathogen to its host cell surface The of glycoconjugates in human milk that protect nursing infants from specific pathogens is evidence that the glycoconjugates in the may represent a major mechanism of human milk in protecting the infant from Human milk of human milk in includes three of in to nutrients and These three include the produced by the mother against of pathogens to which she has a history of exposure and into the milk. A line of is the multifunctional agents, which may serve as nutrients but also have that can inhibit a broad of pathogens. Glycoconjugates the third line of functions of human milk of are probably all important components of a For example, the glycoconjugates inhibit specific whereas the multifunctional agents to have a these of human milk be as a line of during the period that the exposure to a pathogen and the of secretory against it in milk. the other glycoconjugates be the line of against enteric pathogens. The data human milk lactadherin levels and to symptoms of rotavirus support this serve a protection from disease if the of the mother the of a to a specific milk that inhibits a specific It is also possible that the of these three of protection for pathogens. This of agents in human milk that inhibit common diseases in infants may a in in the study of human milk. than of human milk as a food with some perhaps it is more to of it as a of biologically active agents that also nutritional milk may protect the infant from harmful pathogens during the first year of life, the immune system is and a stable microflora has been while that stimulates the growth of commensal bacteria. These provide for health of breast-feeding in both and developed countries. Furthermore, the of the milk oligosaccharides in and during intestinal transit suggest that supplementation of the oligosaccharides or their homologues may be feasible. by from the of and Human of