 Thank you very much for the introduction and thank you for the invitation to come to the meeting today and share some of the data. There we go. Am I doing this correctly? I'm pushing the green button. Oh, there we go. Oh, it caught up. Forget it. There we go. So we were initially charged with trying to come up with a question that was the basis for the presentation. We have this long standing interest in the interactions between intestinal microbes, the immune system, and inflammatory disorders. And in some cases those inflammatory disorders extend beyond the bowel into other parts of the body. And so this is the basic question that I posed was do these immune networks have the potential to address some of our public health issues? It's broken down into a number of different categories. And I'll start with a little bit of basic background. So we became quite interested in the idea, and this is almost a decade ago, of the possibility that there were microbial interactions, some of those occurring very early in life, that were going on to impact adulthood, and that those interactions involved a balance of immune cells. And everyone knows Sarkis mesmenian has this beautiful paradigm for the balance of the pro and anti-inflammatory cells. So I'm using that scale, the balance, a paradigm here. And the basic concept is that the gastrointestinal tract microbes are impacting this balance throughout the body, and that when there's homeostasis or balance between the pro and anti-inflammatory activities, that health is the outcome. And when there's an imbalance, that that's when there's a possibility for disease. And ultimately, what we would strive for is healthful longevity. So not just to live a long time, but to have those years be healthy and meaningful as healthy and meaningful as possible. And we envision that this involved this balance between the immune activities and the microbiome. So that's the background concept. So about six years ago, six or seven years ago, I met Tina Clark-Dur at a Department of Defense Era of Hope Meeting. We were both doing breast cancer research, and she oversees a cohort of women. She and Pam Horne Ross oversee a pretty large cohort of women called the California Teacher Study. And this has many subjects who are followed prospectively in interesting ways. They endeavored to do this in the early 2000s, I think. And we've incorporated a lot of Tina's data as preliminary data to support concepts of things we see in mouse models. And I hope everyone will let me take the liberty to include some of that data and to credit Tina with that. So we had this basic question, do the maternal and infant microbial ecology offer opportunities to impart good health to future generations? And many of you have presented fabulous data looking at infants, looking at pregnant women, and trying to determine whether or not there are changes in those subjects that may translate into adulthood. And we attempted to do that by proxy in a lot of Tina's California Teacher Study women. For the purposes of the discussion today, though, I'm going to present a lot of my own mouse model data. And we were interested in looking at mother-infant relationships in the mice. And for those of you who don't do a lot of work with animal models, we've certainly discussed it a lot. There are a lot of good reasons to use mouse models. And one of them is because you can create genetically uniform animals. They can have different kinds of mutations in a very controlled sort of way. You can control their microbiome, as we've discussed. And you can introduce all sorts of substances in controlled ways that are very difficult to do in human subjects. But there's another advantage, and that is that mice have very short generation time. So we use this opportunity with these short generation times to start to examine some of the things that might happen, be very difficult to capture in a human subject where it might take a very long time to have a generation, or some of that information is by proxy as with Tina's data. And we were going to use these mice to start to examine what would happen if we manipulate the microbiome in the immune system in interesting ways and then follow them for several generations. So we had a lot of difficulty getting funding for this, even though we thought this was the greatest idea ever. We finally got, they took pity on us. Leona Samson and John Essigman agreed to fund a pilot project through NIHS. And it was on perinatal micro-exposures, and we thank them very much for the opportunity to do that. So some of the data should be credited to that grant. So we have some interesting data, and I'm not going to spend a lot of time talking about it right now, but I want you to think about it as I'm giving you some of the background material. And that is that when we looked generations, not just the mom and the kids, even though we have a lot of data suggesting that the children are hugely impacted by things we do to the mother's microbiome, we wanted to go one generation further. And when we did that, we discovered some absolutely shocking things. So animals that were of a wild type background, they're outbreak Swiss mice, they are animals that were not manipulated in any way other than the conventional housing that we do in the animal facilities. And they were eating standard chow, the standard rodent chow. But an enormous number of these animals are developing lesions. Some of them we had hoped to keep on the study for a longer period of time. I'm going to present a little bit of preliminary data later on showing that five-month-old, these are outbreak Swiss mice, five-month-old animals not manipulated in any way were developing 100% of them bronchial alveolar carcinoma. I mean, I just thought that this was very unusual and very interesting, and I wanted to share a little bit of that with the group. But let's do a little bit of background first. So Fiona Powery's lab has done a lot of work over the years. We got started actually working with these mouse models in around 1999 in earnest. And this is a fabulous display. I'm just going to very quickly show you this. You recognize this would be your gastrointestinal tract epithelium and your microbes, which you can manipulate in various ways. And then there's this whole cascade of events that occurs. Some of those are innate immune cells and others of those are adaptive immune cells. And you can actually dissect them out with different kinds of mouse models, which lets you be very creative to answer scientific questions. So there you go. There's the innate immune system in solitude, and you can actually work with a mouse model. Many of you know this. It's rag deficient mouse that has these cells without mature adaptive immune response. And that's a platform that we've used quite extensively. So what we've done is we've taken this possibility of using a rag deficient mouse that doesn't have any lymphocytes of its own and substituting in these various adaptive immune cells in different sorts of creative ways. And using the Powery paradigm, which became very popular in the late 1990s, we started doing a series of experiments where we're mixing and matching different kinds of cell transfers and these different cell populations. And we found many of the things that Dr. Powery had discovered earlier. But we decided because I have the good fortune of working in one of the most wonderful places in the world, actually. And Bruce Horowitz, who is now at Harvard, was at MIT at the time. And James Fox is the director of the Division of Comparative Medicine, where I work at MIT. And the three of us started working together on these model systems. And in particular, Bruce had gotten some funding that allowed us to develop these systems. And what we did was we mixed and matched a targeted infection with helicobacteropaticus that you'll see this icon show up several times throughout the presentations. And we discovered some very, very interesting things when we started working with the rag deficient mice and helicobacteropaticus and the adaptive transfer paradigm. So I'm going to start moving pretty quickly through this material because I'm presenting it because I want you to have background to the things that we're going to do a little later, but in and of itself is not the end of the presentation. So what we found was that the uninfected animals that were maintained with their, essentially, their tachonic flora, and this in particular works most effectively on a 129 strain background, but you can recapitulate elements of it on other strain backgrounds, we found that the uninfected animals develop virtually no pathology, even if you develop, follow them for very long periods of time, whereas their counterparts that were infected with helicobacteropaticus as a targeted infection, and that was gastric gavage, ended up getting this profound inflammatory bowel disease. And the, not the rag mice, but their wild-type counterparts that were helicobacteropaticus infected didn't develop any pathology. So this led us to put together a paradigm that showed that innate immunity was sufficient for inflammatory bowel disease, and I'll talk about carcinoma in a minute, and that the cells of adaptive immunity were suppressing it. We hypothesized that this was an inflammation-associated paradigm, and that the regulatory T-cells were actually what was very important in that process. And so we set up a series of experiments probing that paradigm, and we found that as the animals went on for longer periods of time, they went on to develop cancer in the intestinal tract. So this would be typical of inflammatory bowel disease-associated carcinoma, and this is in the six to 12-week time frame where you can see these invasive lesions, the development of amusinous carcinoma. And then if you follow it out for longer periods of time, you get these, while the uninfected control has virtually no pathology at all, everybody recognizes that relatively normal colon, you get these musinous carcinomas or these poorly differentiated carcinomas. These grow out into the lumen, these grow out into the abdominal cavity, and they become quite large. They're very impressive actually. I think they're not bigger than the mouse, but they're very large lesions, very significant lesions. And what we found was that helicobacteriopaticus was actually, as a targeted infection, doing a lot to upregulate pro-inflammatory cytokines in these different settings, and these are all cytokines you recognize IL-60, NF-Alpha, and IL-17. And in particular, IL-17 appeared to be a big player, and if you modulated the immune system by looking at the paradigm of IL-10 and IL-17, what you find is this exquisite presentation. All these brown cells, and actually I've got an even higher power image here, are all expressing IL-17A. And some of these cells look very much like they might be lymphocytes and others that look like they're cells of the innate immune compartment, but there's massive, massive upregulation of IL-17 under certain circumstances in these model systems. So we went on with this hypothesis that what's happening is there's a lot of inflammation. That inflammation, if it's unchecked, is eventually leading to cancer, and I went on to test in a bunch of different settings using either blocking the inflammatory cytokines with the NT-TNF, adding exogenous IL-10 into a RAG deficient mouse, or transplanting the regulatory cells that I told you about earlier, that you could actually restore. You could take an intestine that had these lesions and restore health, and we found, in fact, that that was the case. So that allowed us to put together a paradigm like this, and it involved an interaction between IL-10 and all this stuff is published in 2003-2004 timeframe. IL-10, Tregs, pro-inflammatory cytokines, and that inhibition of the regulatory cells blocking those pro-inflammatory cytokines ends up ultimately blocking the growth of the tumors that were occurring in the intestinal tract. So as a result of a lot of this work, I was able to actually get funding, and I want to make sure that I credit individuals and funding every time I possibly can throughout the presentation. I don't know how much time I'll have at the end, but go ahead and allow me to do this if you don't mind, and I'll credit NIH grant. NCI was actually the funding source for this, and this was interrelated roles of IL-10 and TGF-Bate and colon cancer. That grant has been renewed, and it goes through 2015, and it is the basis for what allowed us to take a lot of these next steps in these model systems. So what did we say here? We were focusing on helicobacteriopaticus infections. We were focusing on the possibility of using adoptive transfers, and we were using rag-deficient mice in all kinds of mix-and-match creative ways. So the next thing we did was we moved on, and we moved on to a model system that allowed us to look at discrete polyps as an intestinal cancer model in comparison with the more overt inflammatory bowel disease cancer model, still speculating that inflammation was important in this paradigm. So for those of you who are familiar with min-mice, APC min-mice, they have a lot of immune dysregulation spontaneously. It's not entirely clear what's chicken and egg, whether it's cancer and inflammation, or inflammation and cancer, but there does seem to be an uncontrolled, massive uncontrolled inflammatory response. There's premature thymic involution, and a number of things that are very interesting in this model system. So just looking at untreated black-six wild-type mice and untreated min-mice side-by-side, you already see enormous differences in this model system, and these are stocked from Jackson Labs. So we've gone on to begin to probe this model system in a lot of interesting ways, and one of the things we wanted to do was probe the microbiome. I'm not going to present a lot of data, but I do want to credit Eric Almslab, and you may have noticed that Eric is also a collaborator on the grant with Tina and David Halfler and Tim Wong and others, and I'll show you a little bit more about that grant in a few minutes. We tried to make it as comprehensive and multi-institutional as you can on not a really large budget. Mark Burnham-Smith did a lot of this work from Eric Almslab. So we set up to use the adoptive cell transfer paradigm in the mice that I just described to you, the min-mice and the wild-type mice, and that's the same basic paradigm that we had used in the 129-RAG deficient mice only. These are immunologically intact C57 black-six min-mice, and what we found was that the regulatory cells, the same subset of cells that were purified using the power-y paradigm of purification, that same subset of t-regulatory cells was actually able to inhibit the development of tumors in the min-mice, and this fit our hypothesis of this pro-inflammatory series of events, that we had the cytokines to support that, and that IL-10, the anti-inflammatory cytokine, and its priming of the regulatory cells was important in that, and so there's two things. One of them is that this paper, it's a cancer research paper that was published in 2005, so anyone who wants to take a look at it, you're welcome to. I also wanted to thank the Shower Lab because David Shower was the senior author on this paper. We went into this together, one of his graduate students, Jane Sohn, actually had a lot of the insights that helped us develop this model further. So this is just summarizing that data, and as you can see, if you look at this panel on the left, we have the sham-treated mice. These are the min-mice that got the regulatory cells, and there were enormous differences in numbers of tumors between these animals. They were highly significant in their differences, and in fact, you could take animals that had established tumors like this, as Jane pointed out, if you caught it really early, you looked at two to three days right after we had transferred the cells, and you would actually see these involuting polyps, and the involuting polyps were packed with apoptotic figures. So the Treg cells induced regression of established adenomas, and here's that data, so we were able to show it didn't just stop the progression of it, it actually suppressed things that were already existing, and it was through an apoptosis-mediated mechanism of the epithelial cells. So we went on to take this paradigm and tweak it even further by introducing the helicobacteriopaticus. So you probably see some emerging themes here. We're using a lot of these model systems in the same way. We're introducing helicobacteriopaticus. We're using min-mice or rag-deficient min-mice on a different strain background. This is actually a C57 black strain background, not the 129 of the earlier models. And we went on to show that the cells of adaptive immunity were able to suppress carcinoma and that the innate immunity was sufficient for carcinoma in the same ways that we had shown in the earlier model systems. So one of the things that we discovered serendipitously, although I have to say we weren't entirely surprised when we saw it, was that the introduction of helicobacteriopaticus into the APC min-mice actually caused mammary tumors to develop. So for those of you who are very familiar with this model, you know that when it's kept under clean health status conditions, you very rarely see mammary tumors develop, but they do have a genetic predilection to mammary tumors, and in fact, when we challenged them with the gastric gavage of helicobacteriopaticus, what we found was this very rapid development of a high number of mammary tumors. And this paper, let's see if we have it here, this paper was published in 2006, and we were able to show that there was this continuum of progression, which actually starts from around the mammary lymph nodes and turns into a carcinoma. And it was sufficient to use the innate immune response alone, so the rag deficient min-mice displayed all these characteristics. And a lot of this work was actually funded by a Department of Defense Award. I think I mentioned to you earlier, it was at the Year of Hope meeting. We had this hypothesis that anti-inflammatory regulatory cells could actually inhibit mammary tumor formation, or breast cancer potentially, by inhibiting microbial activities that were in the intestinal tract. So we were not looking at just what was happening within the intestinal tract. We had moved into an extraintestinal arena, when was this funded? In 2005, and that's where we were. All right. So we got invited to do a review paper, and that review paper is the basis for how I'm going to present the rest of the data. The individuals here deserve a lot of credit. This is Rao Verrata. He was the first author on this paper. He was a postdoc working with me at the time. This is Theophilus Putihitas, also working with me as a postdoc at the time, and of course we were working with Dr. Fox at MIT. And this was published in Cancer Research, and it displayed the ideas beautifully. So there was a review figure that basically said, we had this hypothesis that these gastrointestinal bacteria were doing these amazing things in other parts of the body, and these were our two possibilities as far as we could tell. Systemic elevation of cytokines or activated cells that were happening as a result of the GI infection, or the possibility of translocation of intestinal bacteria or antigens. And frankly, I could talk for hours on these topics, but I'm not going to do that. I'm giving you this information as a matter of background. So the U01 grant, the one I had mentioned earlier with Tina Clark, is Erdman as a PI, Kopi Aum, Kopi Tim Wang at the Columbia University. And we had set up this paradigm where we were going to probe all these different things with the helicobacteriopaticus infection. Tim was going to look at stem cells and stem cell distribution. Erica Aum looking at the microbiome. And Tina Clark, this is her initials right here, looking at this possibility of this perinatal window. And David Halfler is also working with us at Yale. Here's some of Tim's data. And again, I'm going to move through this very quickly. This was looking at the distribution of stem cells that were mobilized as a result of the helicobacter challenge in the bowel because, as everyone knows, Tim Wang's focus is looking at stem cell migration and its contributions to cancer. And so when he did some of these studies, and many of these actually occurred separately, from my own facility and my own models, he was able to recreate the same things that we had seen earlier, which is always very reassuring. And we wanted to use this adoptive cell transfer paradigm to probe this concept. And this is beginning to look pretty familiar to you at this point, with IL-10 inhibiting and Tregs inhibiting. And what we found, and this is all published in the 2006 paper, was that the bottom line was that the regulatory cells did appear to be inhibiting. This is the epithelial interactions over on this side. The regulatory cells were inhibiting, and their potency to inhibit was increased by earlier exposures to microbes. And so that is the sort of hygienic concept that we went on to follow up on. So we had this is right from that review paper, and it's talking about modern hygiene practices and in crediting all these individuals who had done all this work on the hygiene hypothesis. And we were quite sure that the anti-inflammatory cytokine IL-10 and the regulatory cells were the key to what was going on in all of this. So we've gone on to do a series of experiments where we're doing adoptive transfers, using that same paradigm in the min-mouse model, and then probing what's happening to tumor rates and changes in the microbiome. And so there were significant changes in the min-mice, the white stripe is the wild type animals. There were significant differences in the min-mice who are on the inside, and the min-mice who had received the hygienic T-cells, commensurate with these big increase in intestinal polyps, and as it turns out, mammary tumors as well. So we followed groups of animals. We followed them for extended periods of time. If we used hygienic cells versus the bacteria prime cells, we were actually able to dramatically increase the lifespan of the min-mice in this setting, as opposed to the animals that got the hygienic cells, which actually had a truncated lifespan. This all fits together in the paradigm in the way that I described. So the preliminary data that I wanted to share with you that relates to the hygiene hypothesis is the beginning of the sort of the mediest part of the discussion, and that is that Tina has taken individuals from this California teacher's cohort, taken their history by proxy, and someone had brought up earlier that it was very important you had exposure to animals, as it turns out some of the strongest protective exposures that we see against inflammatory diseases and all these types of cancer in women was actually how close they had lived to a stable or a dairy, and whether or not they had worked or mother had worked or they had worked under those conditions. So it was like a farm association. So the farm association was actually quite protective. We had put together this paradigm. It's not published, at least I don't think it's been accepted yet, but we're looking at all these cancers with the idea that this is merely an extension. These different types of carcinoma that are developing are developing with inflammatory underpinnings, and they're just an extension of all the other inflammatory conditions that we're seeing in a public health context. And so we're going on now with Eric Olm's lab at MIT to begin to look at the profiles of some of these women, unfortunately usually making associations with their early life exposures by proxy, but still looking like there's tremendous potential to begin to elucidate what the roles of early life exposures in these women may be in their later health outcomes and the establishment of their microbiome. And so the last thing that I want to present in this context is sort of dated to give you the backdrop is the interesting discoveries we had with probiotic microbes. And Jim Versalvic is actually here in the audience today, and a lot of this is to his credit that we ended up evolving the thing, learning the things that we found, evolving in the ways that we did. We became quite interested in the possibility that you wouldn't have to expose an individual to pathogens in order to get the additional priming of the regulatory cells. And we could go on about the details of that mechanism, but we thought if we could create that same phenomenon by feeding a beneficial bacteria, that the possibility existed for us to be able to use that in some kind of constructive public health setting, which is sort of the direction that we're heading in. So what we had done was we set up a series of experiments looking at simply exposing mice to yogurt. And this slide is actually the upshot, sort of at the end, the conclusion that we'll come to, but I want to make sure that I describe exactly how it unfolded. So first, we fed a bunch of mice probiotic yogurt with the idea that probiotic yogurt was actually something that human subjects would potentially be eating regularly. As it turned out, every time there was scientific criticism, and this is very legitimate, it was always based on there's calcium in there, there's vitamin D in there, there's protein in there, and on and on and on. So we also, in parallel, started running experiments using a purified organism, and that purified organism is one that Jim had shared with us. So this is the team of people. I won't spend a lot of time talking about them. They are wonderful people. They make every day fun for me, and I guess that's the way science should be, right? So one of the things we found was that the mice, the control mice, whether they were on a C57 black six background, or whether they were outbred Swiss mice, they were wild type mice, were developing this difference in their fur coat, and the difference in the fur coat was measurable in lots of different ways. I'm not going to present all the data. It's actually published in plus one in January. And one of the things you'll notice when you look at all these, the ones that were eating the probiotic have this patch of hair five days after they've been shaved and the controls don't have anything at all. So what we discovered was that there was a massive shift in hair follicle cycling commensurate with feeding these purified organisms to the animals, and it had a lot of different attributes to it. One of them is it was an antigen phase shift. You can see the hair follicle roots here. We actually went on to quantify this, and you can see the difference between a telogen hair up here and an antigen hair, and it ended up that when we plotted all this out that eating those organisms was causing hair to grow like crazy. And the hair wasn't just growing like crazy. It was really healthy. It was really shiny. And it had these interesting gender correlations that suggested that it was somehow related to reproductive fitness, which is a topic that's discussed in a moment. Paper, we call it the glow of health. So we found other things too. Some of these we set out with more intent to find than others. We were actually interested in understanding the roles of obesity and whether or not obesity was contributing to cancer and whether that might be through a regulatory cell-mediated mechanism. But what we found shocked us, and that was because we were using a number of different diets, some of them mimicking fast food and having low vitamin D and having high levels of fat and being supposed to be mimicking potato chips and french fries and bad things. We found that there was this enormous protective effect even when they're eating the fast food chow when they were also eating the purified lactobacillus roidery. And it sort of suggests, like from an evolutionary perspective, that it may have been protection against some kind of malnutrition or times of hardship. But what this organism was doing was it was simultaneously making the animals lean, which you can see the differences in the subcutaneous fat and the differences in the abdominal fat. But the quality of the fat was completely different. So the animal was in a state of health, superb health actually, and you can see this difference here. So these animals, while they're slender, that were eating the fast food chow and the roidery have fat pathology that really rivals either a very young animal or an animal that was just on control diet, even though they're getting the junk food, which was very interesting to us. It's not the topic of discussion today, but I wanted to make sure everyone understood that. So one of the things that we noticed was that the regulatory cells were increased under that setting, and I showed you that just a moment ago on the slide. And so we wanted to test, using the same paradigm we had used before, but with a bit of a twist here, this is a decade later. And so now we're using GFP-labeled FoxP3 cells as our regulatory cell marker. And there was enough background earlier for everybody to appreciate what those cells are. And they're labeled green here. We inject them into the rag deficient mice, and we were actually able to show that the GFP cells that emerged from donors that had been fed LR were able to decrease the subcutaneous fat levels and make a lot of the fat pathology changes. And so those cells alone were sufficient to do this. And so you probably see where we're going with this. We're going into the same idea that these regulatory cells are inhibiting inflammation and that there are cycles occurring within the body, and those cycles involve food, stuffs, microbes, and different kinds of bacteria, and that you can modulate them profoundly, change the cycling simply by interrupting which microbes you might happen to introduce. And it's back to the same paradigm as before, only this time it's obesity instead of cancer. And it happens to match some work that was done in 2011 that was published in the New England Journal of Medicine, and that was looking at human subjects. And in fact, the humans that had eaten yogurt were actually protected from weight gain more than any other single food item, which is really interesting. And some data from David Hafler's lab was looking at humans eating fast food in restaurants and looking at the IL-17 expression in their T-cells. And actually the whole story fit together beautifully. The animal data was supporting the human data and vice versa. So we have, as I've described, we're using a wild-type littermates, their Outbred Swiss mice. They come directly from the vendor in most cases. We did have a small in-house breeding colony for the studies of looking at the generations that I described. But we specifically were asking the vendor to supply us with wild-type littermates or we were using littermates from in-house. So, and behold, the slender animals had much higher thyroid hormone levels than the controls. And one of the things that was very interesting about this, and this is a group of females that we had followed out, was that they all start out around two months of age. We start them on the feeding of the LR. They're all within normal range, and this will be our normal range of T4 right here. And what we found was that those animals that were getting aroidari even when they reached the age of eight months or older were staying up in the very high normal range. Those animals were very active, and they were slender in comparison to these animals, which became hypothyroid as they aged. So the thing that seems to be most interesting to everyone, everyone about any of the mouse models that we've developed so far is always this. And actually it was a technician in my lab who had noticed this swagger that these mice had and we decided to take a look at what the origins of the swagger might be. And what we found was that whether you worked with yogurt or whether you worked with a purified organism, that they actually had larger testes if they had been eating lactobacillus aroidari or the probiotic yogurt. And again, this fits into our paradigm of some kind of reproductive fitness benefit that might be associated with these organisms. Makes sense because they're all about the perinatal window. And we went on to show that serum testosterone was also elevated. And so I know what everyone's going to ask. They're going to say what was different about the testes. What was different about the testes was the light excels were bigger and the seminiferous, the light excels that produce the testosterone and the seminiferous tubules were a lot thicker and there was a lot more sperm production. So those were the things that increased the size and made sense with the serum testosterone. So we were looking at reproductive fitness as part of all of this and we were looking at it because we were interested in this generational effect that I'm going to talk about in just a moment. Mother mice were taking much, much better care of their babies if they had been eating the lactobacillus reuteri. And so the effect was profound and it doesn't it's not even captured here with these numbers. I don't know if you can see this, this is about two-thirds survival and almost a hundred percent survival. It was at the first litter in particular that black six moms were not eating any of their babies. They were taking fabulous care of their babies while they're eating the lactobacillus reuteri. So like this is really interesting. But it might have something to do with plasma oxytocin levels. Okay, so we hypothesized that this might have something to do with plasma oxytocin and we call this project My Bacteria Made Me Do It. So we went on to look at everything surrounding fitness in the perinatal window. We looked at wound healing capability. We looked at maternal rearing success rates. We looked at the ability of these animals to get pregnant. And I'll present this data very, very quickly. What we found is that skin wounds extrapolate that to all the fight injuries of the males to the perinatal recovery of the mom that that process is accelerated by eating lactobacillus reuteri or yogurt. I'm just using the purified organism to be more scientifically robust. The wound closure rates are doubled and E. coli K12 does not create the same effect. And when you transplant the FoxP3 GFP cells, it recreates the effect completely, the wound healing benefit. And that's that data right there into black six mice. You can see the wound healing benefit from the mice that are reading LR. Same scenario. We went back to look at the oxytocin. We looked at oxytocin deficient animals and they had a deficiency in their ability to repair wounds after being exposed to the aurel reuteri. And so we put together a hypothesis where oxytocin in the perinatal window was improving almost every element of the fitness. And one of those manifestations was wound healing. So I told you earlier about this funding, looking at this perinatal window. We went generationally and this takes us back to where we were in the very beginning. And that was the possibility that there are perturbations in the microbes and in the immune system that are transcending generations. And with our current hypothesis, knowing that many of these phenotypes are transplantable by immune cells alone, we were hypothesizing that the first generation of progeny has a disrupted immune system and that impacts everything about the pregnancy in the second generation. And nonetheless, the rates of lymphoma were very high in these animals. And this isn't all the data. This is just a group that we had taken off at five months very recently. They were prone to liver cancer. They were prone to skin cancer. And just about every animal in the study had lung cancer without any extraordinary exposures at all. So there were a lot of data, but conceptually it's a very interesting concept. So here we go. Could this offer some kind of opportunity for health in future generations if we understood it? Could we restructure immune networks and would the best way to do that be through microbes? We have data showing that lactic acid bacteria in the people in the audience who are much more of an expert in this area and probably have a lot more data to support this. Is there some kind of interventions using these well characterized strains to benefit for an immediate public health remedy for some types of conditions? And maybe some of those studies are underway. Is there the possibility of setting up more prospective longitudinal studies? A lot of people have talked about this. I'm not the first one to bring this up. And we were also interested in the possibility of probing epigenetics and genetic impact realizing that if hormones like oxytocin and thyroid hormone and activities of the hypothalamus, many of which are vagus dependent, we know this because of vagotomies, perhaps we need to be quite broad in the way that we're thinking about the epigenetic changes and what the impact on the immune system might be from some of these immune hormonal interactions. And so I thank most of these people along the way, but if I didn't get an opportunity to thank everybody, there they are. I'm sorry I ran over. I'm glad to answer any questions, I guess, if there's time. Thank you, Susan, for that very nice talk. We're well over time, so we won't be able to have any questions now, but please feel free to come in at the breaks. We're going to have a short break because we're over time, so we take 12 minutes to just do the basics, get a drink, a bio break, and come back here at 10.45. Thank you.