Introduction
How do HMO modulate the immune system in infants with CMPA? Emerging evidence and clinical implications.
This symposium was held on 9th June 2023 at EAACI congres 2023 in Hamburg with the participation of Prof. Alexandra Santos (chairperson), Prof. Liam O'Mahony, Prof. Anna Nowak-Wegrzyn and Dr. Ralf Heine.
Video
Hello, everyone good afternoon.
Welcome to this Nestle health, science, Industry, Symposium, um, entitled how do human milk, oligosaccharides modulate, the immune system in infants with cow's milk, allergy emerging evidence and clinical implications? Um, my name is Alexandra Santos and I'm, a professor of Pediatric Allergy at King's, College London and I'm delighted to share this session.
And to have with me, a World level experts in the field talking about a variety of interesting talks around and around this topic.
So we'll start with Professor, um Mahoney, um, who will talk about mechanisms of human milk, oligosaccharides in modulating immune response.
Thank you Alex for the introduction.
And so first of all here are my conflicts of interests and I'm delighted to speak to you about the development of the early life, microbiome so we're, all, uh, born sterile.
So we start to pick up microbes true diverting process.
The first round comes from moms and the local environment.
And then over the months that follow microbes that come from other sources like older, siblings, The, Wider, family, Network, pets, environmental sources.
They all become much more important.
But actually today, there's a number of limitations to us picking up, many of these microbes from the use of antibiotics to smaller family sizes.
And perhaps most important for today's discussion is around when you get these microbes, they have to be supported by something and what they're supported by is diet and human with garlic saccharides as I go into is one of the most important early supporters of some of those early microbes, especially bifidobacteria.
So at the same time that the microbiome is developing, we know the immune system is developing.
And as we heard this morning from Peter, broden's, talk, it's, not that the cells, the early life, immune cells, can't respond, but actually how they speak to each other is different.
So they have to learn to communicate properly with each other and have to learn how to communicate in concert with each other.
So they can be very effective and Regulatory networks that can be established.
And of course, we would say, who's, the conductor of this it's, the microbiome and it's, the microbiome composition as well as its metabolic outputs that are coordinating and driving these the development of these communication.
Pathways between the immune system cells, early in life.
And just to show you how important the microbiome is for that early life or say, how important diet is for the early life microbiome development.
This is a study that is not published yet.
But we looked at the variance in microbiome composition between kids at 6 and 12 months of age.
And basically what I'm showing you over here on the left is that 20 to 25 percent of the variants are the difference between kids can be explained by their diet at both 6 and 12 months of age.
And when you compare that to things like your C-section delivery, which is very very important, it's, far less important in determining the composition of the microbiome early in life.
And when we look at those dietary factors, I know, this is quite small here, but the main standout dietary factors, that's contributing to this microbiome development is breastfeeding and Then, followed by other plant-based foods, like Sesame, legumes, beans and so on.
But breastfeeding is a standout alone as the most important contributor to that early life, microbiome development.
And what is it in the breast milk? That's driving that.
So one of the most important factors are the human milk, alligosaccharides, the hmos, these are basically sugars that are chemically modified in such a way, they're unavailable to the host for energy use, but they're available to certain bacteria that have very specialized enzymatic systems that can degrade and digest and utilize this source of energy.
There's.
Many different hmos have been identified over 150 different types.
And and its composition can be or the comp, the HMO composition of a mom's breast.
But it can be very very different, depending on many different factors.
And this can be important for outcomes, such as food allergy.
So here are examples of two different studies in looking at HMO composition of breast milk and looking for an association with risk of food allergy, or in this case, Cosmic protein allergy in offspring.
And there is an association.
And one of the things that we would suggest is that Association is mediated via the HMO effects on the immune system, either directly or directly via the microbiome and I'll show you some examples of how that might occur in a moment, but it's, not just about allergy.
All it's also infection is an infection risk is very important.
So this is one study, um, where kids were given two hmos and their early life, uh incidents of things like bronchitis or use of antibiotics was significantly reduced in those kids that got hmos early in life and I'll talk a bit more about some of the mechanisms by which we think that may be mediated.
So I want to come back to something I mentioned at the beginning that the microbiome is important, both for effector as well as regulatory responses.
And so I think something that will improve regulatory responses.
Of course, is important for allergy prevention.
But also you need an e-factor response to protect against infection.
And that regulatory response will keep that anti-infective response in check.
So it won't overreact.
So these are all connected responses.
So what are the mechanisms through which hmos may modify allergy risk as well as infection risk? So number one is hmos can be utilized by specific microbes.
So you get expansion of those so we'll go into that in a bit more detail in a moment.
In addition, HMO, utilization by microbes generates different metabolites that can then have an effect on the immune system.
And thirdly, hmos can have Direct effects on both epithelial as well as host immune cells.
So hmos can have direct as well as indirect effects via the microbiome on the host immune system.
So let's start with the direct effect, or the indirect Effects by increasing the levels of microbes in the early gut and I guess my favorite group of microbes are bifidobacteria and select the group bifida bacteria are very good utilizers of hmos.
So this trend line is a trend line from nearly 4 000, infants and it's, just showing the average biflo bacterium levels in the first four years of life.
And as you can see, they Peak between a couple of months of age up to maybe one year of age.
And then they start decreasing and are up to sorry, three months of age, and then they start decreasing the box plots are from one study that we recently did looking at kids born during the pandemic.
So these were born during a lockdown period.
And actually they have more bifidobacteria, uh at six months, but really a lot more at 12 months compared to this trend line.
So why might that be? So actually in these kids, there was a hugely increased race of breastfeeding and a reduced rate of antibiotic usage.
And the numbers are here.
So in the coral cohort, the cohort born during the pandemic Which is higher levels of biflo bacteria.
53 percent of moms were still breastfeeding at six months of age.
And in an Irish context that would normally be 15 so it's way higher and 35 percent were still breastfeeding by 12 months.
And if you look at the antibiotic numbers, only seven percent had an antibiotic at six months and only 17 had at 20 at 12 months of age, and previously that number would be about 80 percent of kids.
So we think those two factors and I think this is a really good example of how maybe a lifestyle change in one way can very positively impact that early life, microbiome, particularly around biffs.
So more breastfeeding, more hmos, more biffs.
And we think that's a good thing.
And why do we think bits are so important? So many people have looked at this and I'm just going to show you some examples from our own work.
So this is a germ-free mouse model, which we gave two different infant derived bifidobacterial strains to the mice and germ-free mice have very low levels of tea regulatory cells in the gut.
But you can see the mice that got this bifidobacterium.
You have a huge expansion of T regulatory cells in the gut.
So we think some of these biffs are really important for t-regs, but as you can see, not all biffs do it.
So this bifidobacterium strain didn't have any impact on T regulatory cells in the gut.
But actually it colonizes to the same number of cells so it's, not that it doesn't survive.
But it doesn't engage in the same way with the immune system.
Also if we take these cells from these mice, we put them into mice that we cause inflammation in this case, we're infecting mice with salmonella and we're causing this huge increase that we can visualize in N of Kappa B activation.
These bifidobacterium-induced T cells, dampen down all these inflammatory activities, and it doesn't really matter how we induce this inflammation it's always dampened down.
And then if we go to humans, we've done a lot of human studies with this bifidobacterial strain, excuse me.
And in this study, it was an eight-week feeding study.
And when we looked at il-10 secretion after eight weeks, everybody that was fed to before bacteria had an increase in their il-10 secretion.
And on average, there was a 10 to 15 percent increase in the numbers of circulating T regulatory cells.
So it's, not just in mice.
It also seems to be in humans that bifidobacterium engage this regulatory immune response, some bifidobacterium, not all.
And how do they do that? So the bifidobacterium cells and particularly some of these early life, bifurobacteria are covered by these extracellular polysaccharides that we think are very very important for their immune regulatory effects.
So in this particular one I'm showing it here, we knocked out the extracellular polysaccharide.
And what we could show when we co-incubated these cells with monocyte derived.
Dendritic cells from humans was that we now got a tnf response, the pro-inflammatory response to the microbe.
But when the EPS is on the cell surface, we don't get any tnf response.
And these numbers here are that's a hundred bacteria, 100 bacterial cells to one dendritic cell.
So an awful lot of bacteria when the EPS is on there, you don't get any tnf response, but when you don't have the EPS on the microbe.
Now you get this pro-inflammatory response.
And we even took that further to look in most models where we isolated the EPS, we put in the nodes of mice.
And when you look at an allergic Airway, uh disease model, you have a lot less acidophils coming into the nose of these mice and that excuse me dependent on tlr2 and il-10.
So just as an example of one of the factors that's associated with the cell wall, epipheral bacterium, that seems really really important for the immune system.
But again, it's, not just about allergy.
So this is a lethal influenza model in mice it's, a H1N1 it's, a mouse adapted influenza.
And a hundred percent of these mice die between day, eight and day nine.
But if we give one shot of this bifidobacterial cell wall into the nose of the mice like reader before, or after infection, doesn't matter about 50 to 60 percent of the mice survive.
And these mice have actually cleared the virus so it's, not that they die a day or two later.
And if you look at the viral titers here, you can see the huge kickoff in viral titers that happen in the placebo mice.
Whereas you don't get any increase in the viral titers in these biblical bacterial treated mice.
And when we look at cytokine levels in the lungs of these mice, what we can see it's, not that there's a dampened immune response, actually, early there's, a much more pronounced immune response in the bibliobacteria treated mice.
But then when you get a bit later, it switches, so the placebo might now have this huge immune response, whereas the bifidobacterial treated mice have a controlled immune response.
So you have this early effective and dealing with the virus and a controlled immune recovery, thereafter.
So that's the biffs.
And what about the second one, which is metabolites, so I know, Ralph is going to talk about the study in much more detail later, I just wanted to mention it here.
This is a study again done with a HMO supplementation in infants.
And what these authors did was they did a metabolomic profiles.
And you can see a whole range of different metabolites changed with HMO supplementation, even things like 12-3 die home, which was shown by Susan Lynch's group.
A couple years ago that it was a negative regulator of T regulatory cell induction in the gut and HMO, um supplementation reduced the levels of this molecule.
But again, I, let Ralph talk about this in more detail later.
But the other class of metabolites that we all know in lower short chain fatty acids.
So these are acetate propionate and butyrate.
They can be made by a whole variety of different microbes, there's multiple different enzymatic, Pathways to make them and I'm, not going to go through all of these today, put I suppose, the main point is that these engage with wholesale receptors like GPR, 109a, 43 41 and also their important hdac Inhibitors.
So they influence epigenetically at the immune system.
And all of these effects actually induce a regulatory response and protect against allergic sensorization.
And other authors have shown that they have really good effects against virus infection if the respiratory tract as well.
And we already had shown a few years ago that actually having a high level of these short chain, fatty acids, early in life is associated with a lot much lower rate of allergen sensorization.
So this is correlating at those kids with the highest levels versus the lowest levels of Bute rate, proprietor acetate at one year of age, and then their allergen sensorization by five or six years of age and that's, the Annie It's, both Aero as well as food allergens.
And even if we look at some disease outcomes, those kids with the highest levels of Bute rate, they have a lot less asthma, allergic rhinitis and food allergy.
Whereas those we propionate or acetate, the outcomes were not as significant as what you see with butter.
It really seems to be the one that's important.
So that's an example of a your metabolite that's secreted by microbes, following HMO supplementation.
And then lastly, what's the Direct effects of hmos on these cells.
So hmos can actually provide decoy receptors for certain pathogens.
And we decoy receptors for the human blood group antigens.
So pathogens bind to the hmos rather than binding to the host receptors on host cells.
Hmos have been shown to have a direct effect on epithelial cells and have an effect on epithelial barrier.
Function hmos can be directly recognized by some innate immune cells through pattern recognition receptor, such as you know through toe like receptor 2 or DC, scientists, C type lectin receptor.
And this results in cytokine secretion and a Cell Activation program that influences many other different cell types, Downstream of this act direct activation.
And finally, very recently, hmos were shown to directly stimulate il-22 secretion from intestinal dendritic cells and il-22 is a really critical barrier, cytokine within the gas.
So to summarize and what I hope I've shown you is that human breast milk contains many immuno margin three and microbiome modulating factors, but hmos are one of those, and we would suggest they're one of the more important ones.
These HMO effects can be direct or indirect so direct effect on cells of the host or indirect, perhaps through microbial expansion or microbial metabolism.
And we think that the development of the immune Regulatory and immune effector.
So both go hand in hand are really impacted by hmos.
So thank you for your attention.
Thank you very much Liam for this very insightful and exciting talk and we'll move on to the second Talk of the session by Professor Anna, Novak, wedge, green, um, talking about clinical implications of 2fl and lnt in the nutritional management of cow's milk, allergy.
Okay, just touch it.
Okay.
Thank you Alex for this introduction.
Thank you for having me today, I will be sharing some of my experiences pediatric allergists.
And here are my disclosures and I do receive honorary for this presentation from Nestle.
So here are my objectives I'll talk about uh, just to refresh your memory, um that Cosmic protein allergies immune-mediate that disease we'll talk about the role of hmos, particularly for children with Cosmo protein allergy and then I reviewed the evidence from clinical studies on adding two hmos to FL and lnnt to the hypoallergenic formulas, both extensively hydrolyzed and amino acid base for children with Cosmic protein allergy.
So, uh, calcium proteinology is an immune mediated disease.
It could be IG mediated or non-ig-mediated.
And it is one of the most common food, allergen allergens affecting maybe two to three percent or even higher in the first year of Life.
The treatment at this point is avoidance and substitution for the eliminated nutrients for every infant.
The first year of life is the critical time for development of the immune system.
And also of the maturation of the gastrointestinal tract.
The intestinal barrier, the digestion and it's heavily influenced by establishment of healthy microbiome.
And we know that children with Cosmic protein allergy have also reactions in the gut.
So local immune reactions, which suggest that there's a defect at the mucosal level, and you know, very simplistically, we think calcium creatine allergy reflects over activation or overexpression of the th2 immune responses over the normal anti-infected th1.
Immune response, increased gut, permeability as well as altered gut, microbiome microbiota so dysbiosis and what's relevant to my presentation is also to remind you that infants and children with calcium protein allergy are at a very high risk to develop infectious complications, uh, predominantly lower as well as upper respiratory tract infections.
And this is, you know, reflective of that imbalance between the th1 th2 also delayed, maturation of the th1 response.
In addition to that imbalance.
So normal normally there's, a maturation of the immune system over the first three to five years of life and children who are born with separate disposition to aw have much delayed, maturation, uh.
What does it mean that those children have? Uh more infections? It means that unfortunately they're being treated with antibiotics.
So we, although we know that most of the infections in infancy are viral, uh, etiology yet children are being treated with antibiotics.
So on average receive up to three courses in the first, uh, three years of life.
So, and we know that, um, you know this reflects this impaired immunity th1 immunity, and the gut environment plays a big role in that.
So maybe I'll not go into detail over the slide because suddenly I'm beautifully explain how do hmos support the development of the immune system in infants.
And this is true for all infants, but also for children with calcium protein allergy.
So they promote healthy bifidobacteria, which are predominant bacteria, present bacteria communities present in the stool of in the gut of healthy breastfed.
Infants, they assist our gut barrier function.
So are they improving internal permeability so leaky gut.
They provide this pathogen exclusion.
So maybe that's the one there is, you know, one of the components of the anti, um protection protection, anti-infect from infections.
And then as Liam pointed out, they also have an ability to directly stimulate the immune system either through activation of the dendritic cells that then produce il-10 and activate, the T regulator cells or production of other types of cytokines like il-22.
So on to the clinical evidence from the clinical trial.
So the first study was a study which was done to prove that adding HMO to hypoallergenic infant formula does not change hypoallergenicity.
So always whenever you know, there's a new component, added to the hypoallergenic formula.
There's, a concern whether there is a cross-contamination during a process, you know during production.
So it was a study that was done in the U.S multicenter clinical trial, which actually included a lot of children with IGA mediated food allergy.
Those are infants and children are up to four years of age.
And the test formula was Altera.
And this formula has similar concentrations of 2fl and lnnt that are found in human milk.
So those are two sort of very important hmos that are present in I think account were about 30 percent of all of the hmos they're very high in breast milk work in the in the early few early life.
So those are selected for inclusion and uh, the study, uh, confirmed, a hypoallergenicity of this extensively hydrolyzed wave-based formula, which was actually manufactured with a non-parasite, the porcine-derived enzyme, blend so I will not go over this in detail.
But essentially there was a blind double-blind Placebo control challenge, which means once one session of the challenge was feeding with the formula with HMO.
And this second session was feeding on the on a separate day was feeding with the formula without HMO.
And the reactions were evaluating during the challenge two there's, American, Academy of Pediatrics had a formulated or defined allergenicity of our defined criteria for hypoallergenicity of infant formula that it should be tolerated by 90 of children with calcium protein allergy under uh under controlled conditions, such as during oral food challenge.
So we have shown that the formula both test and control formula were accelerated by over 98 of the participants in the trial, which were children exclusively with the IG mediated food allergy.
So that was just one time.
So it didn't really give us.
Uh, you know, we determined a formula is safe.
So now on to the studies that included longer feeding with the formula over a longer period of time.
And actually this study was designed to look at the growth of in infants with calcium protein allergy, not specifically looking at the infections in those in infants.
So the new formula formula with HMO actually has a reduced protein level.
So there is a big concern whether lowering protein in the formula is going to translate into a sub-optimal growth in those infants.
So this was a non-inferiority trial design, which compared a feeling with the formula with and without HMO over several months.
But among the secondary objectives, there was assessment of tolerability safety infected infected morbidity and medication use over the study period.
So it was a large clinical multi-centered clinical trial that enrolled and randomized 194, infants one to one and other some dropouts from the study about 15-20 percent.
Um, the result was positive in terms of the non-inferiority.
So there are similar weight gain and growth longitudinal growth in infants, uh, fed with the test formula with lower protein content.
There was no difference of both formulas were very effective in helping the symptoms because symptoms resolution across all of the time points was not different.
And what was very relevant to my presentation there was reduced, uh, frequency of Upper and Lower tract infections as well as ear infections at 12 months in the pre-protocol analysis.
So there's also some reduction in the lower respiratory tract infections and gastrointestinal infection, although it wasn't statistically significant.
So the primary endpoint of the study was growth and maybe I hope you can see the the lighter green is the control formula.
The uh, dark green is the test formula.
And you can see that they saw track very closely for weight and length.
But uh, this, uh, the outcome.
Secondary outcome of the infection reduction was also very interesting.
So, um, you can see the results of the pre-protocol as well as full analysis set so intention of those infants who are enrolled, um.
And by either type of analysis, there is a reduction in the frequency of the lower upper respiratory tract infections.
Ear infections, also, gastrointestinal infections, acute diarrhea.
And looking at those infections, I should have mentioned that before studying this formula in infants with cow's milk, protein, allergy there's.
A very interesting interesting study that was done in healthy infants, which showed that there was a significant reduction in the uh, lower respiratory tract infections.
And also antibiotic use in healthy infants who were fed with this formula since uh, two weeks of age up to 12 months in contrast in the children with calcium protein allergy.
Those are infants who already develop symptoms before they were entered in the study.
So they were enrolled at an older age and the duration of tree of feeding with formula was shorter 8.8 months.
So there was a relative risk reduction of 34 percent.
But it was statistically non-significant.
However, when looking at the frequency of the um of the infections and reduction in lower and upper respiratory tract infections per month, there was a statistically significant reduction in the upper respiratory tract infections, which I think is, uh interest, very interesting, especially that.
These are the infections that are driving the unnecessary use of antibiotics.
And you know, um making dysbiosis worse, um, the, um.
Now, uh, you know, the conclusions from the study were that the new formula extensively way, uh based uh, supplemented with 2fl and lnt with reduced protein content, supported normal growth and then resolve the symptoms of cow's milk allergy within one month.
And it suggested that there are some immune enhancing properties of the formula supplemented with HMO and introducing the respiratory in infections.
So, um, Professor Hein is going to to Really show you the the you know impact of those, um hmos on the microbiome and the metabolon, uh in this in the study they have just discussed.
So those are.
This was an extensively hydrolyzed way based uh formula now onto a Amino acid-base formula, um and uh, just briefly.
This was a study from Australia, which was an observational single arm study again in infants with the mixed up picture of both IG and non-ige, but um.
So the the outcome was growth of infants with moderate to severe Cosmic allergy who are fed amino acid formula with hmos.
And this is this same concentration of the hmos.
And that since there, it was a single arm study the growth parameters were compared to the who growth standards.
The population of the study was 32 infants who were enrolled about four months of of age, um and uh, they there's pretty good retention in the study, um.
And they were follow up up to 12 months of age.
So I think, what is interesting in the study is that the manifestations of both ige, apparently and non-ige, although we don't have the results of the IG testing by looking at the types of the symptoms.
Um, there are those arcticaria angioedema.
But the vast majority of the symptoms were either gastrointestinal so rectal, bleeding, irritability vomiting or eczema, atopic dermatitis.
So very common manifestation of cow's, milk, protein, allergy so growth parameters were.
So so the Blue Line represents the study infants, the red dotted line, I mean, interrupted line represents the standards, and you can see that they subtract nicely over the study period.
And you know, what was also very interesting was this very prompt symptom resolution.
So you can see at Baseline a lot of crying, fasting spitting up vomiting feeding problems, skin symptoms or respiratory symptoms.
And then over the first month after initiation of the treatment with amino acid formula with HML, there is significant reduction in all of those symptoms.
So, um, just as a reminder formula without atrials also reduce the symptoms in the infants offered with excessively hydrolyzed wave-based formula.
So the summary of clinical results of the Platypus study, um is that infants with mother to severe calcium protein allergy, both IG and non-ig mediated fed with amino acid formula with two HMO achieve normal growth or some catch-up growth during the four months of study period, the all are entroponetic parameters progress along the wa geograph reference, significant clinical symptoms decreased significantly from enrollment to the one month follow-up visit.
So pretty rapidly I was there was a significant Improvement and I should mention that.
The formula was very well tolerated.
There is no Adverse Events and they're, just, uh, two infants that reported some abdominal discomfort.
But that continued in the study.
So clearly, the formulas are having some beneficial effect, uh, beneficial effects in infant with calcium protein, allergy and Professor hyena is going to take over and tell you why and how this is happening.
So in summary, although I didn't talk about lactose I should mention that it is also a very important component in the human milk.
And in addition to HMO is important for development of the healthy immune system in breastfed infants.
And the formulas that until recently hypergenic formulas that contain lactose and did not contain any hmos.
And you know, development of allergic diseases is complex, and there are multiple risk factors.
But microbial dysbiosis is one of the important risks, Cosmic protein allergies immune system, mediated disease and the 2fl and lnt play around in protection against Allergic Disease through several actions.
Both a way based and amino acid-based, formulas, supplemented with 2fl and lnnt, whereas tolerated, well, resolved symptoms and maintain a normal growth supported normal growth, despite lower protein concentration, and it does have a positive influence over the microbiome and Metabolism of the gut in those infants.
Thank you foreign presentation.
Thank you.
And the last talk by Professor Ralph Heine, um entitled evolving our understanding of the impact of 2fl and lnt on the gut, microbiome and metabolome in infants with cow's milk allergy.
Thank you again for the kind introduction.
My tax is to now provide some mechanistic, um background to the observations that we found regarding the infection reduction and what's actually happening with the microbiome in infants receiving HMO supplemented formula.
Um I think breast milk is the the gold standard of infant nutrition.
And it provides a good guide for any future development in the in the formula space.
So um, and and we know that the metabolic and microbiome effects of breast milk are largely driven by human milk, oligosaccharides and there's, hundreds of them different kinds of human organ saccharides in breast milk.
These are very very important in establishing, the early microbiome towards an infant type microbiome.
And this is a point I want to drive home and I will explain this in Greater detail, um, until recently infant formulas did not contain any human milk, oligosaccharides and it's only since, um, they can be made by a bacterial biofermentation that we have now the benefit of breast milk, identical components that can go into into formula.
And and this is also now followed by a rapid rapidly expanding knowledge about what what these actually do in in Vivo.
And in vitro I want to just spend a little bit of time to talk about infant type bifida bacteria, because this is a distinction that's quite important.
The infant type epifidobacteria can see at the bottom here, uh, basically, four species that are, um, very good at assimilating and utilizing, um hmos.
So this, the main one, the first one bifidobacterium long gum, subspecies, infantis or bifido infantis is the all-round master at utilizing hmos.
But the other three that you can see there be brother, B, bifidum and be a long long are also able to utilize, um, bifidos and are typical in the early colonization of the infant microbiome.
You can see that these bifidobacteria have specialized Transporters that allow to assimilate hmos and then have a specialized set of enzymes to break down these hmos into into smaller ingredient sugars.
If you like, which are then fermented in the bifida bacteria with the outcome of mainly lactate and acetate as the main metabolites and it's.
Now, very important to understand that acetate and lactate can also be then CrossFit to other bacteria, which co-establish in the gut, early on and I, just want to maybe draw attention to the conversion of lactate to propionate by valenella, which is an early colonizer and also very important, the conversion of acetate to butyrate.
And you've already heard in the talk by Professor Omani that butyrate has very strong tolerance, inducing and immune modulating properties, directly on cells.
But um, also on barrier function.
You can see in the right text box all the different effects that butyrite has on the colonic epithelium, but also intolerance development.
And this is the set of enzymes that we don't possess.
So these are glycosyl hydrolyzers that are bacterial.
And you can see that we would struggle to do any of these, um digestive steps in our gut.
But with the help of the bifida bacteria, we can suddenly utilize, um, hmos via bifidobacteria.
And you can see that breakdown products also can be utilized by other bifida bacteria.
And once one sort of infant type, bifidobacterium has established, you can see that based on the microbiome sorry on the HMO breakdown, product, other bifidobacteria follow.
And they also freeze out other unwanted bacteria by competing around nutrients.
And and many bacteria can't utilize these breakdown products that you're seeing above there's, also new discovery on aromatic lactic acids.
And these have previously been unknown to be of importance.
But these are important immune modulators for tolerance development and their modulator T, Cell, responses and monocyte responses.
And they are coming from aromatic aromatic amino acids.
And then an enzyme that was previously unknown until a couple years ago, aromatic lactate hydrogenase can actually make these different types of aromatic lactic acids, which have immune module modulatory function.
Now coming back to our studies.
You've heard very nice summaries of these studies.
So the first one was the cinnamon study of an extensively hydrolyzed formula with two hmrs.
And you can see here that it was predominantly a growth study, but that we also saw a trend in reduced respiratory infections.
And this is, um mirroring.
The findings from a healthy infant study, also which showed a significant reduction in lower respiratory tract infections.
These two collection schedule is shown here.
So they had a baseline sample collected the infants before the intervention and then follow up one month later.
Another two months later, so at three months follow-up, and then all infants had a final specimen taken at 12 months of age.
And you can see the numbers here of the infants initially randomized, 97 in each group, we had follow-up later to a smaller group of infants where we also looked at metabolone, um, looking at what we've what bacteria were actually enhanced or or enriched in in the um microbiome between the two groups we could see that, um bifida bacteria and infant type bifida bacteria here in yellow were preferent preferentially enhanced.
You can see, for example, other bifida bacteria, which we also know are beneficial.
But, for example, here at the lower end, um before the bacterium adolescents is not enhanced because it's not utilizing hmos.
So the HMO effect really selects out the right bacteria before the bacteria for the infantile period.
We then also looked at how the microbiome evolves over time.
And this is done by a so-called cluster analysis where you look at what stages.
The microbiome goes through and I won't have time to go through this in detail.
But you can see here on the right panel, the early colonizers, and you can see that we we have sort of, um, 10, 10 or 12, Main colonizers and the gray area above all the other bacteria.
And as the infant ages, diversification happens, we Then followed these Community types over time, and you can see fecal Community cluster.
One, two, three, four and five.
And infants usually go through these towards, um, a more stable, fecal microbiome pattern.
And we could see when you can compare the control versus test for each column, um that that there were significant differences and um, basically, what we did is we split the cohort into two infants, because we had enrollment ages from 0.5 months to six months and that for uh to look at microbiome, is a huge gradient.
So to get a clearer picture, we looked at those enrolled early under three months where a complementary diet is, not confusing, maybe the findings.
And then the later enrolled group where complementary Diet also May modify more importantly, and you can see that we have significant differences and that's.
The infants who did not receive hmos, ended up with a more adult-like pattern around 12 months.
And if you look at the yellow bars here in the early enrolled or in the late enrolled infants, you can see that about 50 percent of infants in the non-hmo supplemented group had reached an adult like patent Wares, only 25 percent in the uh in in the HMO supplemented group.
And you could argue, why is this important? But if you if you stay for longer in a bifido dominated microbiome pattern, you may also prolong the window for early immune modulation.
So this may actually be additional time.
They have to develop a normal immune responses.
We also looked at metabolites and acetate as I showed earlier is one of the main metabolites.
But the the signature was maybe not as impressive as we were hoping.
And and the reason for that is multiple, but we can also see that, for example, the control infants already start at a very high Baseline, which indicates that, um, some of them would have had, um breast, milk exposure previously so the it's not a black and white picture.
But what we could see is that in the control infants, the early acetate signature goes down, whereas in the HMO group acetate goes up.
And that is almost significant in in this setting.
And we we have to admit that fermentation of dietary fiber at the later visits probably obscures the atrium over effect and that's the technological limitation that we have in in because infants obviously commenced a diet and dietary.
Fiber is also fermented.
So we see a competition of, um fermentation products.
And this is even more complicated and I won't have time to go through it entirely.
But then what we looked at is the 16 key metabolites that were altered between the HMO supplemented and non-home supplemented formula.
And and also, 17, uh, keg orthologues, which are the enzyme Pathways that these bacteria have.
And you can see all the different enzymes on the right side.
And at the bottom, you see, um, the metabolites.
And what we saw is two main Pathways apart from the acetate that a short-term fatty acid pathway.
We had an effect in the bile assets, and we could see that conjugated, bile acids were decreased.
And we also could see that in the amino acid Pathways that there was decreased breakdown of Branch chain, amino acids and aromatic amino acids.
So so we saw a down regulation of oxidative protein breakdown.
What these mean in a clinical setting, I think needs to be explored and I I, I I can just offer this as a description of what's happening and I.
Think it needs further interpretation.
What this means at the clinical level, the second study, the Platypus study in Australia and we're lucky to have some of the investigators here in the audience was again, a primary a growth study.
But we also looked at microbiome outcomes.
This was a single arm study of about 30 infants with moderate to severe cowsmagology.
And it was a mix of a few infants with ige mediated features.
And the majority of non-ig mediated features.
Again.
We did a detailed shotgun metagenomics analysis.
So all the speciation of the bacteria plus also a quick look at, um short chain, fatty acid profiles in, but not as detailed as for the previous study.
And the visit schedule again, quite similar a baseline sample, a one month follow-up, four months, and then the 12-month sample and I'll show you here.
Now, the firstly, to illustrate that, infants, even at Baseline already had different patterns.
And if you break down the Baseline sample, but by age, you can see that there's already a transition to to different patterns.
But what you can see is, if you look at the top level, the actina bacteria in light green.
And at the bottom panel, the bacteria bifidobacteria CA in in light green as well, you can see that the space that they occupy at Baseline is not very big so there's, a depletion at Baseline of bifidobacteria.
And if you then look at visit one, you can see that the light green space in both graphs has expanded, which means that there is a b photogenic effect.
So an enhancement of infant type bifidobacteria in response to HMO freezing.
When we look more in detail around the HMO, utilizing bifida bacteria, they're, two groups, there's, the infant type epiphidobacteria.
So the four that are introduced on the earlier slide.
There are also two others, bifida bacteria, pseudocortendulatum and catenolatum that can both utilize, um, HMO and dietary fiber.
So they're more transitionary bifida bacteria, which appear towards the end of the first year.
And you can see that we had early amplification from Baseline to visit one of the probably early infant type bacteria.
And then later on a further strengthening of the signal of ambifidobacterial enhancement, which also includes the strands in under five and six.
So we do see, um an enhancement and I also want to draw your attention that some infants at v0 did not have any before the bacteria that could utilize, um, HMO and could also not amplify with the bacteria as we went along.
And this is an important factor to also bear in mind that some infants have a very deranged microbiome and may not be able to utilize HMO anymore.
Um, finally, we did a taxon set enrichment analysis.
And that compares just the which bacteria are the most enhanced or the most suppressed and I won't have time to go through this in detail.
But we saw that bifidobacteria like nuclear stridium and bacteroides were generally enhanced.
And you can see at what levels that was a significant, whereas um, some abundances, for example, E coli and klebsiella, which are proteobacteria were reduced.
So so we see a picture evolving of increase in early stage before the bacteria or HMO is simulating bifida bacteria and the reduction in proteobacteria, which is really the opposite of what describes the dysbiotic stage.
So we see a partial correction of microbial dysbiosis.
And just finally, very briefly, we could not distinguish fiber effect from HMO effect because it was a single arm study, but we can see that, um, the infants had a significant increase in acetate from visit zero to visit one, which could be attributed to an HMO effect.
And we also see that butyrite and propionate rise later on hopefully most likely due to a mixed fiber and HMO effect and cross-feeding, for example of acetate into butyrate by butyrate producers, which we also saw emerging and I won't have time to go through that in detail.
So in summary and in conclusion, and we we saw in infants with Cosmic protein allergy that's, breast milk, identical HMO promoted, an enrichment in infant type, bifida bacteria.
And the stress is really on infant type.
So these are very specific HMO, utilizing bifida bacteria and have reduced abundance of proteobacteria, suggesting a parcel reversal of the dysbiosis that we see commonly in Cosmic allergic infants.
You also could show that hmo's supplementation slowed the premature shift towards an adult type microbiome.
And we saw some metabolic signatures, changing mainly regarding fecal acetate, but also bile acids and amino acid.
Breakdown, but clearly we also need further well-designed studies to look at these immune modulating effects and amitabulomic effects in in Greater detail.
So there's a lot of work ahead of us.
Thank you very much.
Thank you very much.
So I would like to invite the three speakers to join me here on the stage please for the question and answer session.
And this is now open for discussion.
So please use the microphones in the room, um to ask to ask your questions and I think I will get started while people think about, um questions.
So, um, at what stage of the infant's immune system are development, rhmos most important and particularly non-breastfed infants.
What is the key window I? Think the earlier the better right? Like we saw the the the best effect when uh, you know, they're breastfed or like, you know, you you introduce formula with HML, um since birth, essentially so, and we know that the the first year of life is the most critical I think hmos are important.
Well, the HR most maybe yeah, the first I mean, like physiologically, right during uh, breastfeeding.
So, uh, first, six months of life for sure, but the earlier I think the higher importance.
Yeah, okay.
Can you? Okay, great.
So why why whether we want to know it or not breastfeeding does not protect against food damage.
And so I struggled to understand why we're always trying to mimic the infant guts, it looks like that of a respect baby for the prevention of transient effect on breastfeeding.
But for the other end of the outcomes, we don't see anything, particularly nothing.
Thank you very much Karina.
So, um and I think that the infection benefit may be the one that is evolution.
Foreign uterine is probably one of the key players here.
But um is it going to be 100 protective? Probably not, but, um, I think we're, not having anything is probably not, uh, not ideal.
Because then then you end up with more prolonged dysbiosis and I.
Think it also highlights for Cosmic allergy.
If you aiming for outgrowth that your intervention has to come early, so you have to have an early diagnosis.
And so so these are all things that we have to work out.
But, um, obviously, we don't have all the answers just yet.
Thank you very much.
We have another question there, please from Naples Italy just to to have the more More Concept to the discussion at the Karina opener, every got some data regarding the influence or the quality of the diet, or the lactating matter on the presence and the abundance and the level of this beautiful compounds because this could be the solution because if you are continuing to analyze mother drinking or eating junk Foods, maybe these are this Factor could decrease the Abundant of this protective compound.
This could limit the potential of breastfeeding in protecting against the occurrence of a Fidelity.
So maybe it's better to investigate in a detail, the presence of this compound.
But in correlation, with the dietary habits of the mother, the age of the mother, the smoking habits of the mother, the Sporter activity and many other factors could influence the presence of the absence of this kind of stuff.
But the question is, did you look at innate and acquired immunity compounds in this full sample that we collected? Because this could be the solo.
The explanation why these children experience the less infectious diseases question or comment, I mean, this is certainly something that we are looking at so the aromatic lactic acids, which have emerged as a new candidate immune modulatory candidate.
They were not discovered when we designed our studies.
So they were not on the radar, but I think that these and butyride and short chain fatty assets and other maybe a yet unknown metabolites could could be markers and I think to your question or your comment on breast milk, composition and HMO composition and maternal nutrition.
Obviously, that is an effect.
But, um, for example, malnutrition Ultras breast milk composition.
And we also know that there's strong genetic differences between mothers and that 20 of mothers, for example, as non-secretors don't make much 2fl, which could, for example, also autoimmune responses.
So so I think there is a breast milk is not equal breast milk and different stages of breast milk.
Also may confer different composition in hmos.
So so I think it's a very complex picture.
And to mimic all of this in formulas is a big ask here, but I think we have to start somewhere and and we're seeing very encouraging results.
Now in the infection space, already and I think the the next Frontier will be what happens with regard to tolerance development, either immune modulation reducing allergic symptoms or or fast outgrowth I think that's of great importance.
Thank you.
I think we have got a question there, can you either come to the microphone here in the front or a shout out? Please either? Hello, Amina, Warner from allergy UK, um mine's, much more practical question, really, um, a lot of mums when they make up, um feeds and things put, um, these shots of boiling water.
In does that destroy, you know, the boiling water if that just came on to feed with hmos, would that destroy the HMO? Well, we, um.
Firstly, boiled water is not recommended if I can say the so-so, 70 degrees, let boil the water, but let it cool to at least 70 degrees and then make up the formula.
But I don't think there's a significant effect on HMO composition.
Yeah, because many mums use machines and uh, it comes to just boiling water comes first.
And then they top it up with that.
Yeah, okay.
Thank you.
Thank you so much for your question, um.
So maybe I can ask a question to Liam, um, what what are the immune mechanisms of this effect in the prevention of infection or reduction of infection and reduction reduction of medication use in infants? Having I don't know, the formula or breastfeeding things? So I think and I guess to comment on where the discussion was before this as well, I think, you know, the epigenetic landscape of the immune cells is what's critical, early in life, and they have to get from this stage to this stage, and they start that in utero.
So that already depends on Mom's diet and Mom's exposures and then they're born at somewhere along that line.
And then they need to get to the end within a few months.
And you know, those factors that promote, you know opening up at the th1 type of immune response.
And the closing of the th2 I think that timeline is really what's critical.
And that th1 response is what's needed to deal with infection.
And closing of the th2 is what protects against allergy So.
Many Factors will do that microbes are one bifidos are one, you know, short chain fatty acids, help, um, you know, breastfeeding helps, but actually they're I think you need many of them to really get there as quickly as you can and I think that's.
Maybe one of the reasons why breastfeeding from an allergy point of view is on its own, not significant.
But when it comes to infection, it really is so maybe that's telling us it's directly more of that th1 type of immunity, rather than blocking of the th2.
Maybe let's see right? I think I think we are running out of time.
I had a couple more questions to Anya, but I think we are running out of time and so I'd like to, um to close the session.
And thank you very much to these excellent speakers.
And thank you all efforts.
Thank you.
