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Effects of Milk Osteopontin on Intestine, Neuro Development and Immunity
Bo Lonnerdal
Wednesday, Sept. 25, 2019 · 1:18 p.m. · 39m 16s
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very much not our yep and take a elevate piece a little bit
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here i really appreciate being invited via an and
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i to share this with my distinguish co chairs audible
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current right oh graph it's been a pleasure to yeah i work with them to get this program together
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you heard about one single pro ten this morning they can button talked about like to for an hour
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talk about austere content with just a i think it's a little
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bit more you on the radar screen for most of you and
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i will try to tell you a bit about or skip on ten and what it possibly can do
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was the part that is a very acidic phosphor related
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like a broken and extensively post translation of the modified
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it's about twenty eight thirty different phosphate groups
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and or several dry cans along the molecule
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more importantly almost departing contains an origin
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imply seen a sport eight what is abbreviated
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for the amino acids or g. d. domain which is actually an integration by the site
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yeah uh that works as a receptor forced upon them and also happen binding domain which is recognised
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by the c. t. forty four receptors so austere
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plankton houses several eh receptors on the cell's surface
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it's emphasise by a riot that issues including
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the mammary gland brain bone immune orleans and intestine
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and as you can recognise it was first localised in the
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bone that's because that's how we got its name of course
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it's present in most body fluids i just know serum town and see a set of them
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it's involved in a lot of biological functions cell proliferation cell survival
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by administration again coming back to bone but a many of them in
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immune function look aside recruitment inflammation new model model battery functions will healing
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and initiation of cell mediated immune responses now
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we'll show you some examples of this later on
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we started the an ally seeing austere point in in in breast milk is collection now
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then i each uh sponsored breast milk tea a study that did sometime ago when we use that
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mail to bank to look out or stay pointing concentrations and as you can see it's very high
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the first couple weeks come last rum bottles in transitional milk
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and then it maintains a stable but relatively high level up to one year of lactation
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and it's actually the fifth or sixth most abundant human way protons it's kind
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of surprising that you see so let them all of it and the literature
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and it's a very high like i said this this came
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out a few years ago from a danish group of researchers
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showing that can last from again and human colours terms very hyenas dupont in internet does lower
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but in particular cows milk is low one most important and
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not only that but infant formula is even lower during the processing
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where you take here skim milk and whey proteins and make it into infant formula
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most you point then is partially lost so it's very low concentration of or skip on them
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they expect this the danish group be
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exposed austere content in to hear gastric juice
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and found out that it seemed like it was re system yeah resisting brodie onassis
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and later on it was found that the male cost departing can survive innovative approach goddesses
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in twenty days all most cops and we have verified that as you would see later on
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a few years ago bovine austere point then became commercially available
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from a or left words in denmark and therefore
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the possibility existed to add it to infant formula
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why would you do that well like i said the amino acid composition all the the ball
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point for most appointed and human yeah his sport
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surely different this about seventy percent to model g.
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but the first for elation yeah regions are very similar
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big like oscillation regions or similar even if the the video monastic rates vary a bit
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but importantly the integrated binding sites and the the apology sporty tacit
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regions or totally concert between human and bovine yeah oh strip on them
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so both of them can bind to the same types of standard receptors
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we did a a preliminary trial on this bill one most dupont and this was done together we sure and donovan
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one of my previous p. h. d. students and we fad infant formula with ball point was to point
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in the infant rhesus monkeys we believe that this is a very very nice model for the human infant
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eh rhesus monkey male composition is very similar to that but human note
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yeah we can use it they can get a
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formula and feed themselves from basically one double one
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and they're weaned at about the same age as human infants about four to six months
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so this was a a limited number of monkeys as you can
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see there were four breastfed once and that we had six each
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eh given formula without us department and with us to point
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them at the same concentration roughly as you have in breast milk
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and in this case which we unfortunately of course not can do without
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studies on turn in advance we got the did you know that a messenger or nay
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so we could do transcript yeah the transcript or makes on the on the interesting samples
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yeah there were no adverse effects a tall growth and clinical parameters was similar
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yeah breastfed monkeys were a little bit leaner which they often are and what
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we showed in the yeah gina race
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was that there were one thousand seventeen jeans
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that were different between the breast fed and the formula fed infants but when bovine
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also pardon was added to the formula this didn't manage to two hundred so they
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they it made them remarkably more similar to breastfed infants and then
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there were of course some differences between the regular formal of a ones
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and those that perfect formula with ball wine it was dupont
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are we concluded there from this one that it's safe from book was we were not too concerned about it
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taking your regular dairy fraction and adding it to work uh cows milk formula
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but uh we believed that the preliminary data from the infant monkey
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yes said that we could potentially have their biological activity
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is by adding this eh almost you from the uh_huh uh_huh
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because we saw these differences in in gene expression profiles the
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pathway analysis i don't show here but the pathways mostly affected
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by the ball one what's the point in was those related to
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immune moderate worry functions and to sell a a proliferation and differentiation
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i i don't have the opportunity to do a clinical study where we
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added a a similar to what we had done in the infant rhesus monkeys
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we added the ball wine was dupont been to uh infant formula in this was done at
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food on university of shanghai in china yeah doctor penny did a fantastic job on this study
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it's a good site to to your do clinical city or is
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that is similar to what what i have done in your mail
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virtually all the inference there in shanghai in that
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area of the city or breast fed from birth
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about eighty percent or breast for that one month of age
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so that means that it's harder to find the formula fed infants than it is to find the breastfed infants
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but they have ten thousand births at that hospital and therefore recruitment great was quite decent
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yeah or the formal or uh was done by a chinese company called biased time
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and or learn what was uh yeah providing the ball when all's dupont
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in this case we were fortunate because we were allowed to use fuel
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concentrations all or skip on them as you can see him very
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many of these clinical trials only one dose level is chosen because these are
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work intensive they're expensive and a lot of times you you can just have one concentration
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most dupont in this for is relatively expensive to four at
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the formula and therefore it would be of interest to see
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if a lower level than what you see on an
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average in breast milk would have similar effects on now
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so this is the breast milk average level this is about half that level but with
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individual variation among women there are women that will have this level in breast milk also
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we had a regular intern formula without well why most upon ten and whether reference group on breastfed infants
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and this was a randomised controlled trials double blind
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and are we did of course on top and the palm a train we mesh rated birthday and every
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month up to six months of age with talk venous blood that one four and six months of age
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and the parents pay out together actually with the clinical staff
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yeah they visit them to make sure what that information was obtained properly
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there was a feeling and died three john and with the recorded formula intake
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and also there was a very detailed morbidity journal that was supervised by definition also
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yeah we got tolerance they those two characteristics it's open
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we analysed uh he mythology and with the idea yeah
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flows i talk battery yeah i have to put on university
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we got a red blood cells that we sent to sharon at illinois so we
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have done transcript e. transcript on makes on those that has not been published yet
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with the the zero ferret then at u. c. d. plus many nice it's would
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it be you and and we did an extensive battery o. site the kinds tune
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also that you see and this one is a busy one the bottom line
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here is i just want to show you that we had very low dropout rates
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the always showed some studies that have been done by other investigators
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where you have dropout rates of about forty fifty percent you start wondering
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how about that the data would be we had less than ten percent dropout average after
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the six month the observation period which i think was speaks really well for the both the
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the uh clinical investigator and her staff it who don't university
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parents had twenty four seven axes to yeah to
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the physicians and nurses by telephone and home visits
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we didn't see any significant differences in weight gain nor do we see any difference in height and
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we also didn't have any significant difference in adverse affects in fact
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the they were very low there were no serious adverse affects on
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the the number on adverse effects was a little i'm not significantly different
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no difference in the appetite and formula volume consumed nor in sleeping twenty
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yeah i'm skipping a lot of the other clinical outcomes but
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i would like to uh to emphasise something here and and
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yeah or something that a lot of clinicians and mutation is
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uh like on north where all is that yeah infants fed formula
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have significantly higher t. n. f. alpha this case was not significant but erica is
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allowed who is it the same is due to somebody about the one to go
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they have shown that formula fed infants
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have higher circulating levels of to unify elephant
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and to me that's signifies that it's something we're not doing quite right with these i think it's uh
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a. e. signal that something may be going on down
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in the gut which is a little bit out of whack
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and if we could do something to normal lice these levels
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i think you it is most likely going to be beneficial
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here you can see the t. n. f. l. for this is confirming what you've seen in turkey and
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you can see here that yeah even if the baseline studies with taking it one month
00:13:29
when the the mothers stopped breastfeeding started with
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formula they started to they started with the
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uh with the respective formulas that they were assigned to buy the randomised it control design
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i hear you can see at four months and this was
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exclusive formula feeding an exclusive breastfeeding up to four months after that
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and this was the intervention after that they actually got some weaning foods
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but you can see that with repeated measures analysis yeah those and funds
00:14:01
that received both point was to point them at the higher dose here
00:14:06
they were significantly lower than those fed regular formula and
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there were not significantly different from the bare breast fed ones
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by six months it was different but like i said at this time they had stopped
00:14:19
the having the formula and they were getting weaning foods and there were no significant differences whatsoever
00:14:26
but it seems like it bit maybe a dose response we didn't
00:14:30
see significant difference here but we saw it for the higher doles
00:14:35
we did it yeah uh several inflammatory site the kinds of the picture is
00:14:40
yeah similar i will not show those those we also looked at anti inflammatory site the
00:14:45
kinds and the same thing happened there the profile of side the kinds became more similar
00:14:51
two breastfed infants than those that were fed formula without us dupont
00:15:00
well how does this translate into morbidity well we registered days with
00:15:06
a fever and here you can see similar to what to lead
00:15:10
yeah it showed force we've done it's a higher yeah and sedans
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all the fever and those fed regular formula than impressed quite infants
00:15:19
and then adding a ball line almost dupont and at either these two levels
00:15:25
may then not significant from breastfed infants so a reduction a. n. n. base with fever
00:15:31
so it may be that the immune competence was ordered to make
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them more similar to breastfed infants and more perhaps capable of fighting infections
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we had the subgroups of different types affections
00:15:47
we we did not have a tight as media they're registered in this study
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and we didn't see any individual group differences uh within each category
00:16:00
we also uh yeah they had a their extensive like i said we did flow site on the tree
00:16:06
eh tina west to salsa from universal in no in sweden was in
00:16:10
charge of that and we looked at a lot of different uh immune classes
00:16:16
and what we found to be make this short was that
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the the group that got the high level almost a pontoon
00:16:23
i don't increase proportionately cells as compared to standard formula and
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those that were fed have that level of most upon them
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yeah uh we also had the effects on the model
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sites yeah there were other subgroups that we're all of
00:16:40
that also but we just concluded and this was part
00:16:43
h. two years ago yeah that austere pomp an infant formula
00:16:48
at the higher dose level made the inference yeah similar when it came to
00:16:54
the distribution of different subclasses of immune cells and therefore may affect the immune function
00:17:04
so what to conclude from this era clinical trial and this today is the only
00:17:10
one that has been done in in france more studies certainly need to be done
00:17:16
edition of austere point and resulted in no
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significant differences in adverse effects formula intake robustly
00:17:24
as compared to those fed yeah regular formula immune cell
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distribution inside the kind responses were more similar between infants fed
00:17:32
the higher level last open to the press that level and the breastfed infants
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base with fever was fire and infants fed regular formula
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but not in those that were given baldwin most appointment
00:17:45
and therefore we concluded that feeling infants formula with added baldwin hosted point
00:17:51
and resulted in how comes more similar to those of breast it once
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with that we moved on and we actually analyst yeah almost you pointed in the
00:18:03
class more of these infants and here you can see that already at one month
00:18:10
there were apparent differences when it comes to human all skip
00:18:15
on thin and the four different groups these other breastfed infants
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these are the infants fed baldwin most important of the
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two levels and those were the infants fed regular formula
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this became highly significant that four months of age where they had been exclusively
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fed breast milk or regular formula or formula with one or stop from them
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and this persisted eh up to two months after the the intervention
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and this made a steel hypothesise and you
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will see from the mouse that it's most likely
00:18:49
there's most likely or to print up regulation all androgynous sentences all
00:18:55
the uh human or skip on the cost by bovine pasta point then
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and here you can see the analysis of bowl wine almost upon turn
00:19:06
in the ceremonies for the past month these infants and you can see that
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at each sampling location there was much higher levels all ball one was dupont than yeah
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right in the in the last month when the formula contained but when all's dupont and
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note the scale these levels of baldwin most appointed or
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considerably lower than those of human almost appointment but it
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shows that's certainly there was sampling of the ball when
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all's dupont and we know that the one most upon them
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yeah and thursday and joe site eh we're done vocal microscopy
00:19:44
so some of the ball and roasted pumpkin goes into the systemic circulation
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yeah
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then we wanted to explore in the laboratory
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what the biological functions of male cost accountant or
00:20:02
and we used to knock out mouse which i will explain in a in a few seconds
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the reason why we did that was that if you go when they do cut mad on all steep mountain
00:20:13
you will get a couple of thousand responses it's in bone metropolis it's in cancer it's in
00:20:19
just about anything but if you go in and search for male cost upon ten i think varies by now
00:20:26
five publications so we really don't know what the austere point
00:20:32
and present it to a new born does to the new board
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we hypothesise thronged the literature that cost your point and i not only have
00:20:45
effects on the immune system but also on the brain and the development of the cat
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and what we used here is like i said
00:20:54
a knock out mouse model and it's important to recognise
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what we really did we had why fight mouse dance
00:21:04
where while tight tops and then we hard knock out dance
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that produces milk which is totally ball way the
00:21:13
austere point and and she fed normal wild type mice
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so they they have the capacity to and dot
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generously sent the size of steep mountain in all
00:21:27
tissues just like normal but they don't want to get that source of milk or stripped from them
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and then we did the whole battery all passed away whatever
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show your first is it's important when you have a knockout model
00:21:42
that we didn't have an effect on the l. t. v. open but they are locked in a normally
00:21:47
willow that the organ distribution and the pups like radio label line
00:21:52
and then we had these two groups that i've just mentioned those that were all still pointed a a
00:21:58
negative that they're going to get austere pomp and then the note and those that lost the point though
00:22:06
we looked at the intestine morphology willow gave them an l. p. s. challenge
00:22:12
yeah i would hope that site the kind response to the
00:22:15
challenge we locked out normal immune function by outside the kinds
00:22:21
i will update the brains and we also did a battery of cognitive tests to evaluate their brain function
00:22:30
and the first part of this was uh on the brain
00:22:33
what the those they'll cost chip on the on the brain
00:22:38
yeah it is abundantly expressed in the brain and invert brains it's been shown without off the pomp
00:22:44
and expressing cells here from embryonic they sixteen and
00:22:49
increased remarkably during the first week oppose me from like
00:22:53
and most important hostage don't the standard a proliferation
00:22:56
of neural progenitor cells isolated from about rap pretty
00:23:02
again there that them to have the look that he you born high level
00:23:07
and it's a well known regulator on my donation in which of
00:23:11
course is important for a brain function on commission and cognitive development
00:23:18
here you can see like i said we show that milk healed
00:23:21
was a i don't recall and the wind pipe a knockout dance
00:23:26
yeah uh we can fund we hailed
00:23:30
sequentially uh some pops that were fed
00:23:34
by wind pipe down so they got lost a point in the mail
00:23:38
and here is western blocking use an antibody against models all steep mountain
00:23:43
and it's a little bit reversed here i have the models way here
00:23:48
where the allstate pointed with a couple of different molecular sizes and here
00:23:52
you can see this comic if you mains stepson doesn't do a whole lot
00:23:57
and then you see some fragmentation to larger almost appointment
00:24:02
he uh fragments in the and a duty in and the to june
00:24:07
and the aliens of the first second and third porter the small intestine
00:24:12
so he does really survivor throughout the small intestine in relatively
00:24:18
large fragments and these fragments do contain the receptor binding sites
00:24:25
we didn't see any significant effect on an on a weight gain
00:24:30
or body weight or brain weights and the are those that what got no cost appointed and and not
00:24:38
we did histology we didn't see any
00:24:41
apparent differences in morphology between the animals
00:24:46
we looked at the an expression all of austin point and then the brain nothing significant
00:24:53
but when we did western blotting all last year point and in the brain here you can see that
00:25:00
we hide highly significant difference is it's a little bit
00:25:04
bases slide but you had days one to four here
00:25:08
six eight ten here and then you have those that got hosted pomp and in the note
00:25:14
those that didn't get here matched and you can see that there's
00:25:18
a dramatic difference particular between base six eight and perhaps also ten
00:25:24
between those that have been fed and of course the point and not
00:25:33
and then we went on like i said and the the here testing and for
00:25:37
those of you that are not familiar with this this is the possible latency fast
00:25:42
yeah very popular to use yeah uh it tests more memory function than anything else
00:25:49
and then you have a device here where the um the mouse
00:25:54
part will be put into the white compartment and the national tendency
00:25:59
for the most part there is to go into the dark compartment they want to high
00:26:04
yeah unfortunately once they get in that they get a a
00:26:07
small electrical shot which they don't like and they go out
00:26:13
and the nashville tennessee than for the normal mouse pop is that if you do
00:26:17
that one or two times you learned you don't go in there to get shocked again
00:26:22
yeah uh but the of those parts that they haven't
00:26:25
yep yeah and mostly pompano now heidi significantly for a performance
00:26:33
they just couldn't remember as well that they got an electric shock that so they try to yeah that's right yeah
00:26:39
and this is showing that one power call cognitive function
00:26:45
was in had if you do not get them cost account
00:26:50
within another one which is a little bit more related to both
00:26:54
memory learning and then physical capacity it sort of wrote the road test
00:27:00
and it's a rotating apparatus where the mouse is put on top of this rotating wheels
00:27:07
i began if they learn they are quite capable of kind of balancing that by rotating very
00:27:13
slowly and successively when faster and then the
00:27:17
mice yeah or learn how to cope with that
00:27:20
but if you don't get no cost you point then you don't know on as well
00:27:26
again showing doubt cognitive development and memory functions
00:27:31
seem to be impaired if you don't get posted pointed in the mail
00:27:38
by animation of course is very important in your form the milan
00:27:41
shape around are too loud nerve yeah and impart system all quickly
00:27:48
and it's very complex process and i'm not an expert on this at all but it's
00:27:53
really important for the development the proper function
00:27:56
all particularly yeah well the the dandruff sites
00:28:01
and most your point than is normal secreted by astra sites and microbial cells
00:28:06
and may stimulate proliferation of these pictures of cells to promote why the nation
00:28:13
and this is just the graphic pictures showing some of these proteins that or involved in this
00:28:18
and you will see the or abbreviated here but i'm spell that out in the next slide
00:28:24
with the a gene expression studies in in the in the brain
00:28:29
and here you can see that several of the proteins that are involved here
00:28:35
yeah cyclic you click type the force with the one by s. that biased race
00:28:41
my and associated like a propane might in the bigger than just like like a broken
00:28:45
and in particular what many use to to check this might in basic pro team
00:28:51
and you can see that these are normalised to one point all here represents the if you have
00:28:58
a normal in both the us to point the plus and most to point the minus yeah well mine's
00:29:04
but here you can see that in the brain all the austere point the negative ones that didn't get past the point that
00:29:10
that the gene expression of bushy all of these eh is
00:29:15
significantly reduced round they yes six eight ten as compared to normal
00:29:24
so a very specific molecular events taking place in the
00:29:28
in a uh in the machinery involved and my the nation
00:29:33
which may explain why we still these functional deficits yeah in the malls stops
00:29:41
so i'll know cost your part then increases most important expression and the brain
00:29:46
that's well said it's most likely some type of what the current regulation there
00:29:50
and the up regulated most important expression
00:29:53
may increase my animation and promote brain development
00:29:57
in retrospect of course it would have been very nice to have done bailey
00:30:01
test in the study within china but at that time there was no suggestion even
00:30:06
that a male cost a point and may have an effect on the the new one brain development
00:30:13
and a the next started definitely should here in my opinion have that included
00:30:21
yeah we're not that opinion develop that and most importantly
00:30:26
stumbled and the initiation all cell mediated immune responses and
00:30:30
from all t. v. h. one side the kind yeah um yeah picture and inhibits th to cite the kinds
00:30:38
it plays an important role in both acute and chronic inflammation and it nature both
00:30:43
pro inflammatory and ah lifelong button actions depending on the nature of the biological scenario
00:30:49
this was for my review article from an adult experimental animals
00:30:57
so what we did the men make an infection we gave a had a a l. p. s.
00:31:02
you know we should have heard about orders i won't go into that and it's no one that l. p.
00:31:08
s. to up regulate all be an expression microphone just
00:31:11
to deal or for that we or the ball to yesterday
00:31:16
not about those the point about the or for
00:31:19
and here you can see the results in and they
00:31:24
help yes infected yeah animals and you can see here that interfere and gamma
00:31:32
four hours after the l. p. s.
00:31:34
was significantly lower and those that had not
00:31:38
been fed austere than in the milk and this was exam jaded at eight hours
00:31:45
and you can see the differently press that them out the twenty they thirty and they for me
00:31:51
and it was particularly pronounced or here in the uh
00:31:57
yeah in that they twenty in that they thought the animals have four hours
00:32:02
on out the uh the eight hours yeah in the date one before he howls
00:32:11
yeah and i thought i thought was the opposite and they're your harvey significantly
00:32:16
hire a t. n. f. out of a response and those that
00:32:21
had been fed and housed upon when they are they're deficient uh no
00:32:28
so a down regulation of t. n. f. alpha one last point that was in the mail
00:32:35
suggesting that pro inflammatory now slightly kinds
00:32:40
maybe down regulated by the mel cost report
00:32:46
so therefore now what's the point in may contribute to resist and the l. p. s. administration
00:32:51
and therefore bacterial infection by all three responses more
00:32:55
work needs to be done about them without a speak
00:33:00
so i'll have to conclude i would like to say that and they'll cost
00:33:05
accountant place important roles in brain in a logical and got development you're out alive
00:33:12
breast milk allstate pomp and by the provides then efficient by activities to the breast better
00:33:18
i think that is what we learn from the clinical trial yeah it's i just
00:33:23
that if you would add something like baldwin austere point that last summer binding sites
00:33:30
the brass knuckles dupont that you may be able to provide some of
00:33:35
these by activities and i think we need to explore much more what
00:33:40
the austin point and and the breast milk is do we
00:33:43
need to be breastfed infant but that is more difficult to assess
00:33:49
okay with that i would like to know i thank my collaborator
00:33:52
stopped gang and the pristine probably the post up with me from
00:33:57
mention and with that i and my presentation
00:34:06
was
00:34:09
yes oh well that was the one who presentation really
00:34:16
outstanding thank you because we don't know much about host appointed told my question relates to do
00:34:24
the effects on the brain itself does the also pointed you you but i'm out
00:34:31
get into the central those still officially got ended interacts with the got enter it new system
00:34:37
yeah we're we're done radio labour man yeah we can detect it in the brain
00:34:41
yes you how does a good that's a good question we see it in serum definitely
00:34:47
yeah if it is then and others have reported that it passes the got the uh the uh got brain barrier
00:34:53
oh yeah alright if there are other routes to reach the brain i don't
00:34:57
know what was what we what we got into the closest till you produce lot
00:35:01
but do you read the do of course appointed induce when you look up to recently got
00:35:06
which may be a a stimulating do entered no system what
00:35:11
i went different pastors i wouldn't include exclude that possibility for jerry
00:35:15
looked really knew the proper isn't isn't digital got notice you're gonna
00:35:19
need to us and we will do that probably haven't done it
00:35:23
thank you okay
00:35:28
yes vanessa i ah there ah i have to patent trial ah that
00:35:37
was passed on to her and actually actually pass it on to write actually
00:35:44
hi ah teasing out the effects are fat ass ha intervention as
00:35:51
being directly related to that but i have to content right at all or are directly related to actually have a passionate actually
00:35:59
so i think it has more to do with the production of
00:36:09
after five things that no ah well actually a nice that were
00:36:20
am i correct when they got and that was the content
00:36:24
they got the increase and this is all a brain masterful yes
00:36:30
we saw higher levels of circulating human host before then when they got one last fall
00:36:37
i i my question is for half an hour ago i asked
00:36:45
ah okay faxes thing have argued stimulation and access
00:36:51
production uh i i i i have i have ah ha ha
00:37:00
in the human human yeah
00:37:03
that's that's possible that's was it's what the t. c.
00:37:06
thought and human we would have limited possibilities of chasing
00:37:14
yes the off that off
00:37:17
you know know how widely distributed off two continents across miles i mean i'm
00:37:27
i don't know no i don't know yeah only
00:37:32
i only know that it's found certainly house no yeah in mouse mail it it's a
00:37:39
but to what we seem to lack the fair and mouse is a highlight different species and it's also
00:37:44
highest fountain one of the relative proportion oh what's
00:37:49
the point in the mouse notice similar to human milk
00:37:52
we have also two weeks ago we have found yeah ah still pointing goats no trouble they haven't looked
00:37:58
within the other species and the mock trial yeah we did but we we have not done no no no
00:38:07
but it's highly likely the so for all the milk proteins that or increases my email it
00:38:14
to you cross you act with the human eye people of this and i would guess that the
00:38:19
the resource to point and if it's there which i think it is would cross track with the
00:38:23
human once we we could check that yes i do do you know whether it's found in other secretion
00:38:31
others have reported in in other types of fluids
00:38:35
yes we have not looked okay thank you for
00:38:39
did did you uh look for any measurable amount off a
00:38:44
boat minus one module marinade into the regular formula that's the controller
00:38:51
i didn't understand the the regular formula that we should control yeah was or any
00:38:55
measurable us coupon yes uh it was the concentration may in that one was about ten
00:39:02
maybe not related as compared to the sixty five one hundred thirty you think
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