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The intestinal epithelium of the adult gut supports a complex, dynamic microbial ecosystem and expresses highly fucosylated glycans on its surface. Uncolonized gut contains little fucosylated glycan. The transition toward adult colonization, such as during recovery from germ-free status or from antibiotic treatment, increased expression of fucosylated epitopes in the colonic epithelium. This increase in fucosylation is accompanied by induction of fut2 mRNA expression and [alpha]l,2/3-fucosyltransferase activity. Colonization stimulates ERK and JNK signal transduction pathways, resulting in activation of transcription factors ATF2 and c-Jun, respectively. This increases transcription of fut2 mRNA and expression of [alpha]1,2/3-fucosyltransferase activity, resulting in a highly fucosylated intestinal mucosa characteristic of the adult mammalian gut. Blocking the ERK and JNK signaling cascade inhibits the ability of colonization to induce elevated fut2 mRNA and fucosyltransferase activity in the mature colon. Thus pioneer-mutualist symbiotic bacteria may utilize the ERK and JNK signaling cascade to induce the high degree of fucosylation characteristic of adult mammalian colon, and we speculate that this fucosylation facilitates colonization by adult microbiota. bacterial colonization; fucosylation

Breast-feeding is a highly effective strategy for preventing morbidity and mortality in infancy. The human-milk glycans, which include oligosaccharides in their free and conjugated forms, constitute a major and an innate immunologic mechanism by which human milk protects breast-fed infants against infections. The glycans found in human milk function as soluble receptors that inhibit pathogens from adhering to their target receptors on the mucosal surface of the host gastrointestinal tract. The [alpha]1,2-linked fucosylated glycans, which require the secretor gene for expression in human milk, are the dominant glycan structure found in the milk of secretor mothers, who constitute the majority (~80%) of mothers worldwide. In vitro and in vivo binding studies have demonstrated that [alpha]1,2-linked fucosylated glycans inhibit binding by campylobacter, stable toxin of enterotoxigenic Escherichia coil, and major strains of caliciviruses to their target host cell receptors. Consistent with these findings, recently published epidemiologic data demonstrate that higher relative concentrations of [alpha]1,2-linked fucosylated glycans in human milk are associated with protection of breast-fed infants against diarrhea caused by campylobacter, caliciviruses, and stable toxin of enterotoxigenic E. coil, and moderate-to-severe diarrhea of all causes. These novel data open the potential for translational research to develop the human-milk glycans as a new class of antimicrobial agents that prevent infection by acting as pathogen anti-adhesion agents. KEY WORDS: * oligosaccharide * anti-adhesion agents * innate immunity * receptor analogs * histo-blood groups

Human neonates are born with an immature and naive acquired immune system, and many of the innate components of mucosal immunity are not fully developed. Thus, the innate immune system of human milk is an important complement to the mucosal barrier of the developing gut. The nursing mother provides her infant many protective agents through milk, a growing number of which have been identified as isolates of milk in laboratory models of infection. The number, the potency, and the importance of these protective agents are probably greater than previously thought. For example, many potent protective agents are not found in milk until digestion releases antimicrobial agents such as fatty acids and peptides. An alternate conformer of [alpha]-lactalbumin forms from milk in the stomach and inhibits cancer cells. Many of the protective constituents of human milk inhibit different aspects of a pathogenic process, creating a synergy, where much lower concentrations of each component become protective. Some components have a temporal and a spatial specificity that would cause their protective role to go unrecognized by most laboratory models of infection. Some protective components had remained underappreciated because of technical challenges in their isolation and testing. Recent reports suggest that human milk contains a highly potent mixture of protective agents that constitute an innate immune system, whereby the mother protects her infant from enteric and other diseases. These human-milk components may represent a rich source of novel classes of therapeutic agents against human pathogens. KEY WORDS: * innate immunity * disease * human milk * neonates * protection

The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique[beta](1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.

Regional differences in the ontogeny of mouse intestinal [alpha]-2,6-sialyltransferase activities ([alpha]-2,6-ST) and the influence of cortisone acetate (CA) on this expression were determined. High ST activity and [alpha]-2,6-ST mRNA levels were detected in immature small and large intestine, with activity increasing distally from the duodenum. As the mice matured, ST activity (predominantly [alpha]-2,6ST) in the small intestine decreased rapidly to adult levels by the fourth postnatal week. CA precociously accelerated this region-specific ontogenic decline. A similar decline of ST mRNA levels reflected ST activity in the small, but not the large, intestine. Small intestinal sialyl [alpha]-2,6-linked glycoconjugates displayed similar developmental and CA induced-precocious declines when probed using Sambucus nigra agglutinin (SNA) lectin. SNA labeling demonstrated age-dependent diminished sialyl [alpha]2,6 glycoconjugate expression in goblet cells in the small (but not large) intestine, but no such regional specificity was apparent in microvillus membrane. This suggests differential regulation of sialyl [alpha]-2,6 glycoconjugates in absorptive vs. globlet cells. These age-dependent and region-specific differences in sialyl [alpha]-2,6 glycoconjugates may be mediated in part by altered [alpha]-2,6-ST gene expression regulated by trophic factors such as glucocorticoids. sialyl [alpha]-2,6 glycoconjugates; hormonal regulation; ontogeny of the gut; lectin

The oligosaccharide fraction of human milk, the third most abundant solid constituent, consists of hundreds of structures, many of them fucosylated. Oligosaccharides may bear structural homology to cell surface glycoconjugates used as receptors by pathogens, thus protecting nursing infants. The ability of human milk to protect against heat-stable enterotoxin of Escherichia coil in suckling mice has been attributed to neutral fucosylated oligosaccharides of milk. The same phenomenon has been found in human T84 cells, allowing the mechanism of inhibition by the oligosaccharide to be studied in vitro. The oligosaccharide binds to the extracellular domain of guanylyl cyclase, thereby inhibiting the binding of stable toxin. The protective oligosaccharide is a large structure present in too low a concentration to be routinely measured directly; however, its concentration in milk may be inferred by measuring smaller, more plentiful, structurally homologous oligosaccharides. The adhesion by invasive pathogenic strains of Campylobacter to their enterocyte target is also inhibited by human milk fucosyloligosaccharides. Because Campylobacter binds H-2 type oligosaccharide structures, the concentration of protective oligosaccharide may also be inferred from the total oligosaccharide profile. The relationship between oligosaccharide profile heterogeneity in human milk and the incidence of specific gastrointestinal bacterial disease in infants consuming these milks could indicate the significance of these oligosaccharides to infant health. The efficacy of synthetic analogs of active oligosaccharides will confirm their clinical relevance and define minimum structural features essential for activity. KEY WORDS: human milk; oligosaccharides; Campylobacter jejuni; Escherichia coli; enteric disease

The binding of the HIV envelope glycoprotein, gp120, to its host cell receptor, CD4, is inhibited in a solid phase assay by a glycosaminoglycan of human milk; this binding is the essential first step in HIV infectivity. The human milk glycosaminoglycans were identified in this study. Pooled, fractionated human milk contained dermatan sulfate, heparin, heparan sulfate, and chondroitin sulfate. The ability of this glycosaminoglycan fraction to inhibit binding was unaffected by digestion with lytic enzymes specific for heparin, heparan sulfate and dermatan sulfate, but was lost when the milk fraction was treated with lytic enzymes specific for chondroitin sulfate. Furthermore, a purified milk fraction with high specific inhibitory activity contained chondroitin sulfate but not other glycosaminoglycans. This indicates that the ability of human milk to inhibit gp120 binding to CD4 may be attributed to chondroitin sulfate or to a chondroitin sulfate-like moiety rather than to other components of human milk. We speculate that this human milk glycosaminoglycan could limit the rate of postnatal vertical transmission of HIV in breast-fed infants of HIV-infected mothers. INDEXING KEY WORDS: humans; milk; HIV; glycosaminoglycans; chondroitin sulfate

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea. INDEXING KEY WORDS: * bacterial enterotoxin * guanylate cyclase * human milk carbohydrates * cyclic GMP * T84 cells