Mother lode

Superhero sugars in breast milk make the newborn gut safe for beneficial bacteria

POWER PUNCH  Certain sugar molecules in human breast milk act a lot like superheroes, fostering beneficial microbes and banishing harmful ones.

Todd Churn

A bonanza of potent disease-fighting compounds has been discovered in a surprisingly common source — the breasts of every nursing mother on the planet. Human milk, the only substance that evolved to feed and protect us, seems to contain a trove of medicines just now being unlocked by scientists.

“We go down to the bottom of the ocean to find new compounds and test them out against diseases,” says nutritional scientist Lars Bode of the University of California, San Diego. “But if we just look at the natural compounds in human milk, we’ll be surprised at what we find.”

At the forefront of breast milk’s potential lies a diverse set of sugar molecules called human milk oligosaccharides. Although sculpted by 200 million years of mammalian evolution, the sugars don’t feed infants at all. Instead, they play the role of microbial managers, acting as liaisons between the infant’s newly available intestinal real estate and the throngs of microbes that seek to call it home.

These oligosaccharides serve as sustenance for an elite class of microbes known to promote a healthy gut, while less desirable bacteria lack the machinery needed to digest them. And recent research has shown that the sugars act as discerning bouncers as well: The molecules prevent pathogens from latching onto healthy cells, routing troublemakers into a dirty diaper instead. The oligosaccharides also defuse bombs by calming an infant’s emerging immune system so it doesn’t overreact against friendly bacteria.

Feeding the troops Many oligosaccharides provide ready-to-eat meals for favorable microbes, giving beneficial species a leg up over less appealing ones. Newborns with healthy microbes are apt to thrive. Todd Churn
Oligosaccharides found in human milk might even hold keys to staving off disease in vulnerable children, a notion that keeps Boston College biochemist David Newburg toiling in the lab late at night. In some scenarios, he suggests, the sugars might even supplant the use of antibiotics.

Newburg and others hope to extract knowledge from breast milk components to thwart disease in babies not receiving this superfood. High on the list are premature infants clinging to life in neonatal intensive care units without access to breast milk and toddlers in developing countries who are susceptible to deadly diarrheal diseases after weaning. Even adults undergoing gut-stripping chemotherapy or antibiotic treatments might stand to benefit from the protective sugars.

But extracting or producing human milk oligosaccharides on an affordable, industrial scale won’t be easy. Synthesizing the sugars is an expensive headache because the molecules — about 200 of which have been identified so far — are put together in a dizzying array of configurations. Some scientists are placing their bets on pulling oligosaccharides out of cow’s milk, although the sugars found there are less abundant and qualitatively different from their human counterparts. Others are turning to genetic engineering with hopes of designing microbes that can mass-produce the most useful oligosaccharides.

Along with major players such as the Gates Foundation and the formula and dairy industries, researchers and clinicians are striving to understand and in some ways re-create the health-promoting powers of a tailor-made substance that nature has had millennia to perfect. They have big shoes to fill.

Complex sugar supercrew

The train of milk oligosaccharide discovery started rolling in 1900, when microbiologists noticed that the feces of breast-fed infants contained different amounts of some bacterial species than those of bottle-fed infants. Around the same time, chemists discovered a novel carbohydrate component in human milk, later revealed as the oligosaccharides. By 1954, scientists had dubbed these sugars the “bifidus factor,” as they fueled the growth of Bifidobacterium species abundant in the feces of breast-fed infants.

Fast-forward to 2013, and researchers are still trying to identify the sugars and unravel all their capabilities. The collection of different oligosaccharides in human milk is a ball-and-stick modeler’s dream (or nightmare), but the ingredient list for the oligosaccharides’ recipe is deceptively simple: Only five simple sugars (called monosaccharides) serve as building blocks.

Simply indigestible In addition to nourishing a baby with fats, proteins and ordinary sugars such as lactose, breast milk harbors complex sugars that a newborn largely cannot digest. Some 200 types of oligosaccharides, formed by five simple sugars joined in various structures, help cultivate an infant’s microbial garden and provide many indirect benefits. L. Bode/Glycobiology 2012

When two of these monosaccharides — glucose and galactose — get together, they form lactose, which people can digest and use for energy. From there, more and more monosaccharides jump on, forming a conga line of sugars. But the real complexity comes into play when new branches diverge from the line, producing a dazzling array of chemical structures.

Unlike the linear, orderly synthesis of DNA or protein molecules, oligosaccharides assemble Wild West–style. “For all we know, the linkages could be totally random,” says milk biochemist Daniela Barile of the University of California, Davis. “They’re very unpredictable and there are hundreds of possible structures.”

Although a smorgasbord of oligosaccharides have already been spotted using an ultrasensitive detection technique called mass spectrometry, Bode expects newer techniques to uncover still more. Harder will be figuring out all of the sugars’ roles and which are most important for health.

Breast-fed bacteria

The growing list of jobs the oligosaccharides perform mirrors the burgeoning diversity of their structures. At UC Davis, researchers focus on the sugars’ primary function — feeding and nurturing the beneficial bacteria that live in the infant gut.

To sniff out which oligosaccharides the various species of bacteria consume, microbiologist David Mills’ team extracts Bifidobacterium from infant feces and feeds the bacteria different oligosaccharides.

The bouncers Certain oligosaccharides block pathogens seeking to commandeer cells in the intestines. Some microbes mistakenly latch on to oligosaccharides instead of cells and earn a free ride out of the body. Todd Churn
Healthy, full-term, breast-fed infants tend to have a gut microbiota dominated by various species of the oligosaccharide-consumer Bifidobacterium . But premature infants typically harbor fewer bifidobacteriaand more potentially pathogenic microbes such as Escherichia coli and Clostridium difficile . Of preterm infants, 3 to 7 percent will develop a potentially life-threatening illness called necrotizing enterocolitis, an inflammatory disease that destroys the intestinal lining and exposes the infant bloodstream to hordes of bacteria normally cordoned off in the gut. Ten to 50 percent of babies with NEC ultimately succumb to the disease, with the lowest birth weight infants at most risk.

Breast-feeding halves an infant’s chances of getting NEC. While scientists don’t yet know whether a preemie’s distorted microbiota causes NEC, observational studies have shown that changes in the microbiota precede disease onset.

UC Davis neonatologist Mark Underwood recently teamed up with Mills to conduct a small clinical trial in premature infants in the neonatal ICU that were being fed either their mother’s milk or infant formula (a decision made independently of the trial). The researchers spiked the formula or the breast milk with one of two Bifidobacterium strains: one that consumes oligosaccharides and one that does not.

Fecal analysis revealed that the babies who were fed breast milk plus an oligosaccharide-consuming strain of bacteria, Bifidobacterium longum ssp. infantis, had more beneficial microbes. “When you put in the breast milk and the B. infantis together, then you get B. infantis colonization,” says Underwood. But when B. infantis don’t get nourished by the oligosaccharides in breast milk, Underwood hypothesizes, “they fade away quickly and don’t get established.”

While the study wasn’t large enough to establish protection against NEC, Underwood hopes that the trial will pave the way for larger tests. Especially in infants whose mothers produce little or no breast milk, bolstering formula with a combination of oligosaccharides and probiotics could help prevent NEC in the most vulnerable infants.

Depending on blood type, the stage of lactation and other factors, the milk oligosaccharide profile varies from woman to woman. This could explain why some infants develop NEC in spite of being breast-fed, Bode says. Reporting in Gut in 2012, his team showed that a specific human milk oligosaccharide helps prevent NEC in rats. In the study, only one oligosaccharide, a branched beauty called disialyllacto-N-tetraose, dampened the disease. And research by Mills and Underwood showed that the milk oligosaccharide content from women delivering at preterm differed from those giving birth at full term. Bode envisions one day mixing NEC-busting oligosaccharides into the breast milk of mothers who lack them and boosting premature infants’ chances of getting out of the neonatal ICU alive.

Reprogramming cells By snuggling up to cells lining the gut and modifying which genes are switched on or off, oligosaccharides can induce cells to make proteins that form a nonstick coating for some pathogens. With nothing to cling to, many bad guys get flushed. Todd Churn
Immune tuners

Beyond feeding bacteria in the gut, the milk oligosaccharides might cultivate a healthy microbiota by dialing down the immune system. Bode argues that a toned-down immune response is crucial for successful colonization of the gut by microbes. The sugars were discovered as “food for bugs,” Bode says, “but I believe they’re so much more than that.”

A tiny fraction of the sugars appear to find their way into a newborn’s bloodstream. Bode and others have detected oligosaccharides in the urine of infants, suggesting that the sugars and their influence could extend well beyond the gut. “Milk oligosaccharides may be able to reduce inflammation throughout the body,” he says. In one study, oligosaccharides reduced interactions between inflammatory immune cells and cells that line blood vessels. “The oligosaccharides,” Bode says, “are able to keep the immune system in check.”

Bode’s team has also produced a slew of studies that demonstrate human milk oligosaccharides’ most provocative power — flushing pathogens out of an infant’s body before they get a chance to wreak havoc. The researchers reported on September 16 in the Journal of Pediatric Gastroenterology and Nutrition that the sugars block the attachment of a nasty strain of E. coli to the cells that line the intestine, thwarting the pathogen’s ability to infect neonatal mice. The microbe is responsible for deadly diarrheal diseases that plague infants and children, especially in developing countries where access to clean food and water is lacking.

Milk oligosaccharides that reach the bloodstream may even usher bladder-infecting E. coli right out of the urinary tract, the research team has also found.

For more than two decades, Newburg’s group has been compiling a hit list of pathogens that fall prey to milk oligosaccharides, starting with the discovery that the sugars could disarm a toxin secreted by some forms of E. coli. “Then we tested the oligosaccharides against various pathogen models — and this is my favorite story — it worked against all of them,” Newburg recalls. “We thought we’d made a mistake.” He and others have since discovered, mostly through cell culture studies, that the sugars may dash the diarrhea pipe dreams of such microbial villains as salmonella, cholera, rotavirus, norovirus, a campylobacter-caused gastroenteritis and multiple strains of E. coli.

Newburg envisions one day giving toddlers oligosaccharide supplements to stave off diseases that tend to creep in following weaning, especially in poor countries where diarrheal diseases abound. But the hunt for potential sources of the sugars has proved challenging.

Sweet defenders Undesirable microbes can latch onto cells that line a newborn’s intestines by binding to sugar-rich receptors that stud the cell (left). Once tethered, the pathogens can colonize and infect the tissues. But oligosaccharides found in breast milk (center) can act as decoys, snagging undesirables and tagging them for removal. Another defense (right) involves oligosaccharides inducing cells to populate their surfaces with unwelcoming receptors by modifying which genes get switched on in the cell nucleus. This thwarts pathogen binding. L. Bode/Glycobiology 2012; S. Egts
A better milk mimic

Milk oligosaccharides possess a seemingly endless spectrum of complexity, and researchers are just starting to understand which structures do what. Synthesizing them in the lab is a tedious and expensive process, Bode says. “If you wanted to synthesize and add oligosaccharides to infant formula at the supermarket,” he says, “the price would be ridiculous.”

But for premature infants, caregivers may be willing to invest in the costly sugars to help prevent diseases such as NEC, he says. Because synthesis is still cost-prohibitive, Bode suggests using donor breast milk in cases when the mother’s milk is unavailable. Frozen, pasteurized donor milk from breast-milk banks is far superior to infant formula for such a use, provided processing leaves the oligosaccharides intact.

Neonatologist Underwood agrees, but says, “the truth is, there’s probably not enough donor human milk to go around.” While Europe has more than 200 milk banks and Brazil has a similar number, the United States has only 13. Though donor milk is becoming more common in neonatal ICUs around the country, Underwood says, general availability, especially for full-term infants, seems a long way off. “There’s a tremendous amount of capacity in the world,” Underwood says, “but the U.S. and Canada are very slow to develop milk banks. They’re expensive, and the regulations are tremendous.”

Formula companies already sell an easy-to-synthesize oligosaccharide called galacto-oligosaccharide as an additive to infant formula, a product scientists once thought similar to the oligosaccharides found in human milk. But now they know better, Bode says. “The oligosaccharides that are currently in formula are structurally very different from those in human milk,” he says. While the synthesized oligosaccharide has been shown to promote the growth of some beneficial bacteria and to offer protection against certain pathogens, the additive falls short when compared with the benefits of the full spectrum of oligosaccharides found in breast milk.

Bode is working to help companies develop better oligosaccharides to add to infant formulas. Although breast-feeding is always the best option, he says, certain circumstances such as low milk supply, illness or the stresses of returning to work may prevent some mothers from breast-feeding. “It’s a little bit romantic to think that formula will disappear from the surface of the planet at some point,” Bode says. “I’d rather help make something better if it’s going to be used anyway.”

Immune tuning Using powers not fully understood, ligosaccharides seem to influence immune cells and modulate their responses to gut bacteria. The result is an immune system that’s quick to dispatch pathogens but more tolerant of beneficial microbes. Todd Churn
While Bode hopes that new synthesis techniques or increased donor milk supply will one day meet the demand for oligosaccharides, at least in the neonatal ICU, Mills and his colleagues at UC Davis are betting on dairy cows.

With easy access to a barn full of cows, these scientists have a ready supply of bovine milk at their disposal. The researchers also have a partnership with a cheese producer that sends them regular shipments of various dairy by-products such as whey — a source of oligosaccharides.

Mills and colleagues are hoping to convince the dairy industry to siphon off “waste products” for the production of oligosaccharides. The sugars are at least 20 times more abundant in human milk than in bovine milk, and many of the oligosaccharide structures in human milk are absent or exist in only trace amounts in bovine milk. “On the other hand,” says Mills, processors go through 13 million gallons of cow’s milk per day, “so you could have truckloads of it.”

That helps motivate Barile to keep digging through dairy products in search of more sugars. In 2009, she reported finding 15 distinct oligosaccharides in whey permeate, a substance Barile calls “a by-product of a by-product.” The discovery was initially exciting, Barile says. But the bovine oligosaccharides lack fucose, a monosaccharide that in human milk adorns up to 70 percent of oligosaccharides, tending to lend them pathogen-blocking powers.

Convinced that the fucosylated sugars must exist in bovine milk, even if in trace amounts, Barile’s team refined its separation techniques. As reported in the June Glycobiology, the team eventually identified 50 different oligosaccharides in bovine milk, including structures with fucose. A disease-busting sugar called 2′-fucosyllactose, or 2’FL, that represents more than 20 percent of the oligosaccharides in human milk, comprised just 0.3 percent of the bovine oligosaccharides. The concentration may be low, Barile says, but at least “now we know it’s there.”

Barile, Mills and colleagues just completed a study in which 12 healthy people were given a mixture of purified bovine milk oligosaccharides at escalating doses. Everyone tolerated the treatment well, and the team is in the process of screening the participants’ fecal samples for leftover oligosaccharides as well as changes in gut microbiota.

The cow oligosaccharides “aren’t yet ready for kids in Malawi,” Barile says, but the study is the first step in a Gates Foundation–funded effort to treat diarrheal disease in developing countries.

Not every known human milk oligosaccharide is represented in bovine milk, Barile acknowledges, but the diversity of structures there is far superior to anything that the biotech industry can currently produce, she says. “Maybe in 20 years, technology will catch up, and we’ll have a better understanding of which structures are important.” Until then, she thinks that relying on the natural diversity of structures in bovine milk is a safer bet than cherry-picking some human ones for industrial production.

Newburg’s approach couldn’t be more different. Rather than milking cows (or humans), he manipulates microbes. By adding just the right mix of genes to bacteria or yeast, he hopes to produce oligosaccharides in huge batches through fermentation. In 2002, Newburg cofounded a small company, Glycosyn, with the goal of designing microbes that produce the sugars.

“That’s the way to make it most cheaply,” Newburg says. “Fermentation technology is something you find in every country, no matter how poor it is.” After designing strains of microbes that pump out the oligosaccharides, Newburg envisions inexpensively making oligosaccharides in the countries that need them most.

Breast milk vs. cow’s milk On average, oligosaccharides make up a much greater percentage of the nutrient portion of human breast milk than of cow’s milk, making it difficult to use cows as a source of the protective sugars. L. Bode/Glycobiology 2012
But before that can happen, Newburg’s team must get the microbes on board. “It’s actually quite difficult,” he says. “You try something, and then you fiddle with it and try it again.”

Newburg has set his sights on none other than 2’FL. The molecule holds promise against cholera, campylobacter gastroenteritis and certain E. coli infections. “So it’s an obvious first target,” he says. He expects his team to produce a 2’FL-producing microbe fit for distribution within the next few years, and microbes that make other oligosaccharides are also in the works.

Newburg imagines milk oligosaccharides supplanting the use of antibiotics in some cases. Unlike antibiotics, which breed resistant strains of bacteria, “human milk oligosaccharides have been ‘used’ for millions of years without resistance,” he says. “It could be a game changer.”

Whether derived from a chemistry lab, donor’s milk, dairy products or microbes, scientists hope to deliver the benefits of milk oligosaccharides to children living on life’s vulnerable edges: those born too early and those freshly weaned. Even adults who have had their gut microbiota ravaged by antibiotics or their immune systems pummeled by chemotherapy may, like infants, benefit from the nurturing effects of the oligosaccharides. But researchers will likely never fully re-create nature’s potent elixir or unlock all of its mysteries. Breast, so the saying goes, may always be best.

Former Science News intern Jessica Shugart is a freelance science writer living in California. 

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