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Human milk is amazing. It is a baby’s first meal, and contains all the essential, nourishing ingredients they need. As well as water, milk contains all three primary macronutrients: fat, carbohydrate and protein1. But there’s more. Within this mix, the third largest component is an ingredient that is, bizarrely, indigestible2. It turns out that this ingredient — a class of molecules called human milk oligosaccharides (HMOs) — is not there to feed the baby at all.
When these intriguing molecules were first characterized in the 1930s, their role wasn’t known3. As research uncovers their secrets, scientists are increasingly realizing that HMOs have many important functions, not only in the gut but also throughout the body and brain3.
A newborn’s world
“Babies have a huge challenge — how do they survive the onslaught of microbes?” asks Ardythe Morrow, director of the Human Milk Research Lab at the University of Cincinnati, and a human-milk researcher for more than 30 years.
Speaking at Abbott’s HMO talk in Paris in March 2018 Morrow described how, from the moment of birth, a baby is thrust into a world full of viruses, bacteria and fungi — some of which are friendly, some less so. As well as avoiding the worst pathogens, the baby also needs to populate its own gut with beneficial microbes. And the baby’s immune system needs to know how to tell the good ones from the bad — which is where milk comes in.
Morrow has observed first-hand across the world how breast-fed babies thrive, with improved health and survival. They have fewer infections and faster recovery than babies who are bottle-fed. Breast milk clearly provides protection against infectious disease and also against inflammatory conditions. But how?
Observations from the turn of the last century provided the first clues. “In the orphanages in Chicago in the 1900s — when they fed the babies whatever type of formula they had — there was enormous mortality,” says Morrow. Babies were more likely to fall ill or die if they did not have breast milk. Around the same time, scientists discovered differences in the bacteria in the stools of breast-fed versus bottle-fed babies. In the 1920s, they confirmed that there was something in human milk that fed healthy, protective, beneficial bacteria. And they named it after those bacteria as the ‘bifidus factor’3.
In parallel, researchers were investigating the components of human milk. Chemists initially identified a large fraction of non-lactose carbohydrate, which they dubbed gynolactose, in the 1930s. Its role remained unknown, and it wasn’t until the 1950s that it was identified as the same entity as the bifidus factor3. Chemical and biological breakthroughs subsequently revealed that there were in fact hundreds of these factors, of different lengths, which seemed to be there to feed the baby’s gut bacteria. This realization inspired the field, and research into the newly renamed human milk oligosaccharides took off. By 2000, researchers had discovered more than 100 HMOs, which provide food for Bifidobacterium bifidus and other bacteria species. This prebiotic effect of HMOs became well-established, but it wasn’t the full story4.
Up to this point, research into human milk had been a niche pursuit. But now it had caught the attention of people researching the gut microbiota5. “When this work started to transcend the typical human-milk research,” recalls Morrow, “it became really cool in the human microbiome research world.” And it turns out that being cool helps accelerate the science.
Rachael Buck is director of preclinical research, Gut Health Platform, in Abbott’s nutrition business, and has studied the immune system since the early 1990s. “The most important time for immune development is during infancy,” said Buck, speaking at the HMO talk in Paris.
With her immunology training came the supposition that HMOs, which comprise as much as 10% of the dry weight of breast milk1, were more than just food for bacteria. And so, she dedicated herself, her team and their research partners to studying why they are important, what they do, and how they interact with the infant’s immune system. This was more than just an academic pursuit. “Human milk is a treasure trove of benefits,” says Buck. “My goal was to study all the components of breast milk and determine whether we can add them to formula.”
A key concept to emerge from the flourishing body of research in this field was of the 'immune gap' between breast-fed and formula-fed babies6.
Buck and her research team found significant differences in the levels of circulating signalling molecules called cytokines. Breast-fed babies have fewer inflammatory cytokines than do formula-fed infants. Although it was unclear what this difference meant, it was widely known that inflammation — especially hyperinflammation — underlies a lot of severe health conditions including necrotizing enterocolitis, where the gut starts to die within the (typically premature) baby. And if inflammation persists throughout life, even at a lower level, it can contribute to other diseases including inflammatory bowel disease, asthma and arthritis. So modulating the inflammatory response early in life is probably beneficial.
Other groups discovered further differences between breast- and bottle-fed babies. In 2008, a large trial in Belarus7 found that breast-fed babies had a cognitive advantage over their bottle-fed contemporaries, an effect that was apparent in IQ at age six. Clearly there were ingredients in human milk that were having profound effects across the body — and which were absent from formula.
How were these effects mediated? The gut microbiome is hugely important for modulating the immune system — 70–80% of the body’s immune cells line the intestines8 — so some of the differences were coming from effects of human milk in the baby’s digestive tract. But, in a surprising discovery, Buck’s team found HMOs in breast-fed babies outside of the gastrointestinal tract9. HMOs were able to slip through the gut, into the bloodstream and wider circulation, to have direct effects on immune and other cells. The early signs of a gut–immune–brain axis were emerging.
Although HMOs were a chief suspect in the health benefits of human milk, laboratory research was hampered by the lack of material for testing. “There was unanimous agreement that HMOs have multiple benefits, but nobody could make them in sufficient quantity,” recalls Buck.
Narrowing down the field
With around 200 HMOs to choose from, researchers faced a very big task if they were to untangle their effects and add them to formula. Several lines of evidence helped Buck, Morrow and other researchers converge on one lead candidate. Of all the HMOs, the most abundant is 2′-fucosyllactose (2′-FL), which is also one of the smallest, comprising just three molecules10.
Although being plentiful is a good sign that a molecule is important, researchers need more biological evidence to be sure that something is clinically useful. Epidemiological studies provided clues. It turns out that only 75–80% of women secrete 2′-FL in their milk; when tested, the babies of these secretor mothers had more beneficial bacteria in their guts than did babies of non-secretor mothers. 2′-FL seemed to be an important component of breast milk11.
More compelling evidence came from studies of the effects of HMOs against infectious diseases. Morrow’s team discovered that babies of 2′-FL-secretor mothers seemed to be protected against infectious diarrhoea12. Other teams found that 2′-FL was effective against Campylobacter jejuni13 — one of the most common causes of bacterial diarrhoea and infant mortality — and Pseudomonas aeruginosa14 — a nasty opportunistic bacterium that can infect many parts of the body. Tests showed that 2′-FL could stop these bacteria interacting with human cells from the gut wall and the respiratory tract. Technically known as an ‘anti-adhesive’ molecule, it seemed that 2′-FL was preventing these disease-causing bacteria from binding to the cells, which potentially could stop them from gaining a foothold in the body (see ‘System-wide effects’). Many common viruses, including norovirus, rotavirus and E. coli, are known to use a similar infection mechanism, and thus could also be foiled by 2′-FL3.
But research was slow without a synthetic version of 2′-FL of sufficient quality and in sufficient quantities. Several groups had tried different approaches. “For many years, people tried to chemically synthesize it from scratch by using kind of a ‘brute force’ approach,” says Morrow. Researchers looked at using enzymes and yeast to create 2′-FL, but with no success. One of the main stumbling blocks was that the processes were too time- or labour-intensive; if 2′-FL was to be added to essential nutrition for a newborn, it could not be expensive. The breakthrough came following the development of methods for adapting bacteria to be biological factories over the past decade15. “The molecule they make is identical in structure to the 2′-FL found in breast milk,” says Buck.
Having a ready source of high-quality 2′-FL enabled researchers to clarify just how versatile this molecule is. Animal studies showed that 2′-FL could attenuate the severity of necrotizing enterocolitis16, speed recovery following intestinal surgery17, and reduce symptoms of food allergy18. Buck’s team and collaborators also showed19 that this little molecule had a direct biological effect on gut motility, suggesting it might reduce discomfort caused by colic or irritable bowel syndrome.
“The body of research was so compelling,” says Buck, “we were committed to launching an infant formula with 2′-FL.” Abbott had already conducted the first ever clinical trial of an HMO-enhanced infant formula20, and was now in a position to create a 2′-FL-enhanced version. Studies showed that this product was safe. The big test would be how it stacked up against breast milk.
Buck’s team compared breast-fed babies against babies fed a control formula and those fed experimental formulae containing different amounts of 2′-FL. Growth rates were similar for all babies, but there were more subtle differences. When the researchers looked at immune markers in the babies’ blood, they found that those fed the 2′-FL-containing formula had certain cytokine levels that were nearly identical to those of breast-fed infants — and both were significantly lower than those of the control group21. Rates of respiratory tract infections were lower in the 2′-FL group than in the control group22, and rates of eczema23 were also lower in the 2′-FL and the breast-fed groups than in the control group. The immune gap was starting to narrow (see ‘Important outcomes’).
In 2016, Abbott had the US launch of its infant formulae containing 2’-FL HMO. It was the first company in the world to launch an HMO-containing formula, and the Chicago Innovation Awards recognized it as a top innovation in 2017. Since then, Abbott has continued to introduce the formula with 2’-FL HMO to new markets around the world. “Now that the technology is there to do it,” says Morrow, “including HMOs will become standard for how infant formula should be.”
A pioneering formula
Nutrition companies have been refining formula milk for decades, adding some of the micronutrients found in breast milk, including nucleotides, lutein, vitamin E and fatty acids (notably docosahexaenoic acid, DHA, and arachidonic acid, ARA). Adding 2′-FL is a significant advance, but is not the end of the HMO or formula milk story. “We know that mother’s milk is a unique living fluid that adapts to baby’s needs,” says Hakim Bouzamondo, global head of nutrition research and development at Abbott. “This unique aspect of mother’s milk has not yet been fully replicated by a formula. But we will continue to pioneer research and move formula in the right direction.”
There are other groups who might benefit from 2′-FL and HMO-related products. Babies born prematurely are at high risk of certain inflammatory diseases. “Necrotizing enterocolitis is runaway inflammation,” explains Morrow. “There is very good evidence that dysbiosis of the microbiota is involved, and may be a driver of that hyperinflammatory response.”
Very premature babies who spend their first few weeks in the neonatal intensive care unit are not guaranteed to have access to human milk, says Ethan Mezoff, Assistant Professor of Clinical Pediatrics at The Ohio State University College of Medicine. This high-risk group, who are often born before their organs are mature, could benefit from formula milk that contained prebiotics to encourage the growth of good bacteria and deter the bad ones, and which could also modulate their immature immune response24. “Pre-term infants have an incomplete gut barrier,” says Mezoff. “Coupled with dysbiosis, one can see how the situation cycles out of control.”
Animal studies have already suggested that 2′-FL might be able to attenuate the inflammation and lessen the severity of necrotizing enterocolitis and have opened the possibility of a future role for HMOs in the prevention and treatment of this disorder in infants25.
Other tantalizing clues from preclinical research point to areas where HMOs in general, and 2′-FL in particular, might prove supportive. Notably, these signals centre around the gut–brain–immune system axis (see ‘Gut–brain–immune axis’). Buck’s team has found evidence that supplementation with 2′-FL increases molecules associated with synaptic function signal strength26. The synapses are the junction points between neurons; stronger synapses suggest improved brain functioning. And indeed, rats given a 2′-FL supplement immediately after birth showed better learning and memory at one year old than did their HMO-free litter mates27.
It is unclear how these neurological effects are mediated, but it is unlikely that 2′-FL goes to the brain directly. The body has an ‘information superhighway’ connecting the gut and the brain. Known as the vagus nerve, it sends signals between the two organs. And these are not just signals about whether the individual is full or hungry. The gut is an organ rich with its own neurons and immune cells. It is capable of sending and receiving sophisticated messages. And quite possibly, the microbiota in the gut help mediate all this information.
There is a lot of work to do here to see if these enticing preclinical results translate into real-world benefits. Further research will ask how 2′-FL and other HMOs communicate with the brain and the immune system — particularly, what they are doing in the circulation away from the GI tract. What’s more, answers to these queries could help provide better nutrition for people of all ages, especially the elderly and those with eating challenges.
With its 2′-FL-enhanced formulae paving the way, Abbott has begun to narrow the immune gap between breast-fed and bottle-fed babies. Continuing on this path will help ensure that babies who, for whatever reason, do not get breast milk, still get the best possible start in life.
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