Lars Bode

Human milk oligosaccharides: prebiotics and beyond

Human milk oligosaccharides (HMO) are complex glycans that are present at very high concentrations in human milk but not in infant formula. The significant energy expended by mothers to make these complex glycans suggests they must be important. How do maternal HMOs benefit the breast-fed infant? How are HMOs synthesized in the human mammary gland? How can we provide formula-fed infants with HMOs or HMO-like glycans? This article reviews current knowledge and open questions on the biosynthesis and functions of HMOs.

The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats

Background Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. Objective To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. Methods Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. Results Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98±1.11 to 0.44±0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. Conclusion HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mothers milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.

Structure-Function Relationships of Human Milk Oligosaccharides

Human milk contains more than a hundred structurally distinct oligosaccharides. In this review, we provide examples of how the structural characteristics of these human milk oligosaccharides (HMO) determine functionality. Specific α1-2-fucosylated HMO have been shown to serve as antiadhesive antimicrobials to protect the breast-fed infant against infections with Campylobacter jejuni, one of the most common causes of bacterial diarrhea. In contrast, α1-2-fucosylation may abolish the beneficial effects of HMO against Entamoeba histolytica, a protozoan parasite that causes colitis, acute dysentery, or chronic diarrhea. In a different context, HMO need to be both fucosylated and sialylated to reduce selectin-mediated leukocyte rolling, adhesion, and activation, which may protect breast-fed infants from excessive immune responses. In addition, our most recent data show that a single HMO that carries not 1 but 2 sialic acids protects neonatal rats from necrotizing enterocolitis, one of the most common and often fatal intestinal disorders in preterm infants. Oligosaccharides currently added to infant formula are structurally different from the oligosaccharides naturally occurring in human milk. Thus, it appears unlikely that they can mimic some of the structure-specific effects of HMO. Recent advances in glycan synthesis and isolation have increased the availability of certain HMO tri- and tetrasaccharides for in vitro and in vivo preclinical studies. In the end, intervention studies are needed to confirm that the structure-specific effects observed at the laboratory bench translate into benefits for the human infant. Ultimately, breastfeeding remains the number one choice to nourish and nurture our infants.

Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-gamma, TNF-alpha, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate- or syndecan-1-deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-gamma, TNF-alpha, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

The functional biology of human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a group of complex sugars that are highly abundant in human milk, but currently not present in infant formula. More than a hundred different HMOs have been identified so far. The amount and composition of HMOs are highly variable between women, and each structurally defined HMO might have a distinct functionality. HMOs are not digested by the infant and serve as metabolic substrates for select microbes, contributing to shape the infant gut microbiome. HMOs act as soluble decoy receptors that block the attachment of viral, bacterial or protozoan parasite pathogens to epithelial cell surface sugars, which may help prevent infectious diseases in the gut and also the respiratory and urinary tracts. HMOs are also antimicrobials that act as bacteriostatic or bacteriocidal agents. In addition, HMOs alter host epithelial and immune cell responses with potential benefits for the neonate. The article reviews current knowledge as well as future challenges and opportunities related to the functional biology of HMOs.

Human milk oligosaccharides reduce Entamoeba histolytica attachment and cytotoxicity in vitro

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infants intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.

Heparan Sulfate Plays a Central Role in a Dynamic in Vitro Model of Protein-losing Enteropathy

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a symptom in ostensibly unrelated diseases. Emerging commonalities indicate that genetic insufficiencies predispose for PLE and environmental insults, e.g. viral infections and inflammation, trigger PLE onset. The specific loss of heparan sulfate (HS) from the basolateral surface of intestinal epithelial cells only during episodes of PLE suggests a possible mechanistic link. In the first tissue culture model of PLE using a monolayer of intestinal epithelial HT29 cells, we proved that HS loss directly causes protein leakage and amplifies the effects of the proinflammatory cytokine tumor necrosis factor α (TNFα). Here, we extend our in vitro model to assess the individual and combined effects of HS loss, interferon γ (IFNγ), TNFα, and increased pressure, and find that HS plays a central role in the patho-mechanisms underlying PLE. Increased pressure, mimicking venous hypertension seen in post-Fontan PLE patients, substantially increased protein leakage, but HS loss, IFNγ, or TNFα alone had only minor effects. However, IFNγ up-regulated TNFR1 expression and amplified TNFα-induced protein leakage. IFNγ and TNFα compromised the integrity of the HT29 monolayer and made it more susceptible to increased pressure. HS loss itself compromises the integrity of the monolayer, amplifying the effects of pressure, but also amplifies the effects of both cytokines. In the absence of HS a combination of increased pressure, IFNγ, and TNFα caused maximum protein leakage. Soluble heparin fully compensated for HS loss, providing a reasonable explanation for patient favorable response to heparin therapy.

Human Milk Oligosaccharides Promote the Growth of Staphylococci

ABSTRACT Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infants gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n = 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r = 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland.

Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi-/- embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi-/- embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi-/- embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi-/- fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi+/+ controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi-/- fibroblasts with 2-[3H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi-/- embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi-/- embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi+/+ littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.

Associations between human milk oligosaccharides and infant body composition in the first 6 mo of life

BACKGROUND Evidence linking breastfeeding to reduced risk of developing childhood obesity is inconclusive, yet previous studies have not considered variation in specific components of breast milk that may affect early development. OBJECTIVE We examined whether differences in the composition of human milk oligosaccharides (HMOs) correlate with infant growth and body composition at 1 and 6 mo of age. DESIGN Twenty-five mother-infant dyads were recruited from the University Hospital at the University of Oklahoma Health Sciences Center. Infants were breastfed for 6 mo. Breast-milk and infant measures were obtained at 1 and 6 mo of infant age. HMO composition was analyzed by high-pressure liquid chromatography, and infant growth (length and weight) and body composition (percentage fat, total fat, lean mass) were measured by dual-energy X-ray absorptiometry. Relations between HMOs and infant growth and body composition were examined by using multiple linear regression. A priori covariates included maternal prepregnancy body mass index, pregnancy weight gain, and infant age and sex. RESULTS Higher HMO diversity and evenness at 1 mo were associated with lower total and percentage fat mass at 1 mo. At 1 mo, each 1-μg/mL increase in lacto-N-fucopentaose (LNFP) I was associated with a 0.40-kg lower infant weight (P = 0.03). At 6 mo, each 1-μg/mL increase in LNFPI was associated with a 1.11-kg lower weight (P = 0.03) and a 0.85-g lower lean mass (P = 0.01). At 6 mo, each 1-μg/mL increase in LNFPI was associated with a 0.79-g lower fat mass (P = 0.02), whereas disialyl-lacto-N-tetraose and LNFPII were associated with a 1.92-g (P = 0.02) and 0.42-g (P = 0.02) greater fat mass, respectively. At 6 mo, each 1-μg/mL increase in fucosyl-disialyl-lacto-N-hexaose and lacto-N-neotetraose was associated with 0.04% higher (P = 0.03) and 0.03% lower (P < 0.01) body fat, respectively. CONCLUSION These findings support the hypothesis that differences in HMO composition in mothers milk are associated with infant growth and body composition. This trial was registered at clinicaltrials.gov as NCT02535637.