Metabolic Endotoxemia | Kiran Krishnan | Microbiome Labs

My name is Kiran Krishnan. I'm the Chief science officer at Microbiome Labs. I'm a research microbiologist and have been studying the microbiome and probiotics for a better part of the last decade now. And so what we're going to be talking about today is our major clinical focus with regards to the microbiome. And we want to convey to you what we think is the most root cause of, the root cause of most functional conditions. So we are very much targeted, we're very much targeting chronic disease, things like obesity, diabetes and so on. And we want to talk about what we think is the absolute root cause of many of these conditions. Okay, so we've developed a probiotic product and you guys are all very familiar with probiotics, but there's a very different approach to this particular probiotic. These are bacillus endospore based probiotics. What we were looking for when we developed a probiotic product was something called a keystone strain. So as it turns out, there are strains within the microbiota that kind of hold up a lot of the other organisms within the microbiome. They play, they act like a keystone within the microbiota population itself. So we were looking for these strains that would be naturally derived from that our ancestors would actually pick up either from the environment or through maternal transfer, and whether or not these strains could have a significant protective role in the gut. So when we did find these keystone strains, which you'll find in the spore based product we developed called Megaspore, we were looking at what is the most important role of a true probiotic. So assuming we have a real probiotic that can get into the gut, that can colonize, that can create functional change, what should we be looking for in a clinical setting as the most important function of a true probiotic? If you're familiar with probiotic research, a vast majority of product research is focused on gas bloating, frequency of bowel movements. Those are important things. But we wanted to go a little bit deeper to find true functionality. So to us, the most important feature of a true probiotic is one that protects its host and protects its host from something that we do every single day. That's probably the most toxic thing we do. And you guys are all doing it right now. And that's eating food. So eating food, as it turns out, is chronically the most toxic thing we can do because of a condition called metabolic endotoxemia. Metabolic endotoxemia is basically described as a chronic innate immune activation. It becomes low grade inflammation in the body, but it's subclinical and it is chronic and it's associated with high levels of LPS lipopolysaccharide that exists in the circulatory system after a meal. So this is a postprandial phenomenon. So metabolic endotoxemia, it's a condition, some papers say 33%. Most recent studies on this show that maybe around 50% of the healthy population actually suffer from this condition, where after you eat, you get a huge influx of LPS lipopolysaccharides that infiltrate from the digestive system into the circulatory system. And I'll explain all of that in greater detail as well. It is characterized by high serum lps levels within 5 hours after your meal, and then it's meals that are high in fat and high in calories and actually drive this condition a little bit better. What I'll be able to show you in the upcoming slides is the molecular link between diet and the risk for obesity and everything that falls under cardiometabolic syndrome. So we'll talk about the key molecular link and how the microbiome plays a role in all of that. And this all comes back to metabolic endotoxemia. The increase in serum endotoxin then correlates with increase in the number of key inflammatory markers. Things like interleukin 1, alpha and beta, interleukin 6, interferon, gamma, triglycerides, even postprandial insulin is affected by the presence of LPS in the circulatory system after a meal. Chronic metabolic endotoxemia and the inflammation that it causes is associated with numerous conditions, things like, of course, diabetes, cardiovascular disease, obesity, autoimmunity, and so on. I'll give you more information about this in the upcoming slides. So this is lps. This is an endotoxin. LPS is a normal component of gram negative bacteria and it sits within the cell membrane of gram negative bacteria. Bacteria use it as an inflammatory immunogen. They use it for binding, they use it for signaling within the microbiota. So it's a normal part of gram negative bacteria. And as you know, the vast majority of bacteria within the gut fall in the gram negative category. So there's a lot of LPS in the gut and it's normal for this LPS to actually be excreted. As bacteria die off within the microbiota, bacteria are constantly dying off. But as it turns out, the process of eating increases microbial turnover during the digestion aspect of it. So that's why you end up getting a lot of die off during the eating process, especially postprandial. And then that die off releases a lot of lps. So it tends to circulate at fairly low levels in healthy individuals. And the toxicity is mainly derived by this lipid A portion, the bottom part of it, which is a lipid a tail. And the types of fats that the bacteria uses to make its LPS will determine how toxic LPS is to your immune system. Right. So we've all been looking for the link between saturated fats and obesity and diabetes and chronic disease. One of the molecular links is that when bacteria use saturated fats from the diet and make lps, they make a more toxic version of LPS because the lipid A tail is completely different than when they use omega 6 fatty acids. For example, when, when they use omega 6 fatty acids, they actually make a much more non toxic version of LPS than when they use saturated fat. So that's one of the connections. Now, just to show you what's happening here in terms of the breakdown at the end of the day, what is supposed to happen is the microbes that live in the lumen and the upper part of the mucosal layer, they're normal commensal bacteria. That's where they exist. When they die during the digestive process, they will release the LPS into the lumen and in the upper part of the mucosal layer, where we end up with problems. If the mucosal layer is compromised, then you get LPS migrating through the mucosal layer. The moment they start interacting with intestinal epithelial cells, they'll cause massive recruitment of immune cells like dendritic cells, macrophages that come into that space. And you can actually measure this. I'll show you some biomarkers that you can use to measure this. They'll come into this space and they'll cause a bunch of inflammatory responses. Once that inflammatory response kicks in, the tight junctions in between the epithelial cells start to open up and falter, and then LPS ends up leaking through the tight junctions and entering the basolateral circulation. So now we've got LPS from the lumen into the circulatory system. And this is happening after you eat a meal. Now, if we had a really sturdy mucosal layer, it acts as a physical barrier against LPS migration. If you had a really diverse microbiota and you were producing a lot of secretory iga, the secretory IGA would actually neutralize LPS in the lumen so you wouldn't have the translocation if you had a really diverse microbiome, it would actually produce enzymes that break up LPS in the lumen. So it's not there to translocate into the circulatory system. Also, one of the reasons why a high fat meal increases LPS translocation and is high fat meals induce silomicron production. These are the things called lipid rafts that actually grab dietary lipids and take them through the cell into the circulatory system. Right. Because LPS has this fatty acid tail, these lipid rafts show up and then they end up accidentally grabbing LPS and bringing it into the system. So now what's happening when you have a dysbiotic flora, when you have a dysfunctional mucosa, when you have high fat coming in with the diet, you end up getting a lot more LPS into the circulatory system. And we'll talk about what LPS does once it enters the circulatory system. So this is immune activation by lps. This has been extremely well studied. Basically what starts to happen is LPS enters the circulatory system. It's immediately sought after and bound by something called lbp, which is LPS binding protein. This LPS and LBP complex actually circulates around and then dendritic cells and macrophages. Other parts of the immune, Innate immune system that express CD14 receptors will look for this LPS and LBP complex. When it sees the LPS and LBP complex, they bind together. So the LPS, LBP binds to the CD14 and toll like receptor complex. When that happens, you get a release of NF Kappa B in the cell. The release of NF Kappa B then subsequently releases things like TNF, Alpha Interleukin 1, Alpha and Beta Interleukin 6, MCP and so on. So this is an immune cascade that occurs when LPS is seen in the circulatory system. Now, this particular immune cascade has been extremely well studied with respect to bacteremia or septicemia. So this is the same thing that happens when patients are undergoing septicemia. And as you know very well, that's a very dangerous condition. So when you eat food, you're basically getting a mini septic reaction inside the body. Now this can happen systemically. This can be localized into the brain, this can be localized into your joints, it can be localized into the enteric nervous system. And we'll talk and show all of the data that shows this kind of effect as well. Now, the American Diabetic association has been studying this LPS endotoxemia quite dramatically because they're really looking for the leading cause of type 2 diabetes. We'll show you some data that exemplifies that. Type 2 diabetes is really driven by LPS endotoxemia. It's the primary insult that starts the pathogenesis and the process of type 2 diabetes. So what they were able to show is that in CD14 knockout mice, you don't get any immune activation from LPS. Now, why that's important is because we now know that LPS induces inflammation through a CD14 pathway. The reason that's so important is that when you look at obesity, when you look at cardiometabolic syndrome, when you look at cancers, Alzheimer's, Parkinson's, all of these are driven by CD14, activated immune responses. So CD14 is at the crux of the immune responses that drive these conditions. LPS is the biggest driver of CD14 activation. Now, in your patients, you can look for something called soluble CD14. You can have them come in three to five hours after they've eaten a meal, draw blood, send it out for soluble CD14. If you see elevated levels of soluble CD14, it more than likely means that they have this metabolic endotoxic response to food, and that's probably what's driving their condition. So what are the clinical manifestations of LPS translocating from the lumen after a meal into the circulatory system? So basically, anything under cardiometabolic or metabolic syndrome is driven by LPS. So things like heart disease, of course, that's interleukin 1 alpha and interleukin 1 beta. If you look at lipid problems, so hyperlipidemia, these are also issues. High levels of triglycerides are driven by LPS, endotoxemia, hypertension, of course, type 2 diabetes. I'll show you some of the data by American Diabetic Association. Dementia and Alzheimer's and Parkinson's, or anything neurological. The two biggest neurodegenerative conditions, Parkinson's and Alzheimer's, are all driven at the root, caused by LPS endotoxemia. I'll show you those papers as well. Cancers, we all know cancers are driven by inflammation. CD14 type of inflammation is a major driver of cancer, and it certainly dramatically affects the prognosis of cancer treatments. So now we know that the microbiota plays a significant role in how effective cancer treatments are going to be. This is one of those mechanisms that you can use to determine that polycystic ovarian issues, non alcoholic fatty liver disease. So all of these things that fall under metabolic syndrome are associated or driven by lps, endotoxemia. So here are some of the studies that have looked at this. This is an interesting study because the top part of the study, they looked at high fat feeding in obese and non obese. So overweight and normal weight individuals. Right. So what they were looking at is if we put somebody on a high fat diet for eight weeks, and these aren't great fats, of course, they're not eating avocados and stuff every day. These are also poor fats. So these are very probably oxidized vegetable oils and so on. But will a high fat diet actually increase the endotoxic response of food? And as they showed, both in overweight and normal weight individuals, after eight weeks of high fat feeding, there's a dramatically increased, there's a dramatically increased endotoxic response to the same types of foods that they tested eight weeks prior to that. So it seems that high fat feeding, because it increases chylomicron and lipid absorption, it actually can increase the amount of endotoxins that are getting into the blood from the lumen of the digestive tract. Then they looked at obese and normal weight individuals and what they found was that obese individuals had a more toxic response to food than normal weight individuals did. So the same food, the same meal, an obese individual had a significantly higher toxic response to the food than a normal weight individual did. Then they were looking at type 2 diabetics, right? Because we know that having type 2 diabetes means that your risk for comorbidities are significantly higher. Same thing with obesity. If you have obesity, your risk for things like cancer and dementia and, you know, and diabetes and hypertension are all much higher. Same thing with type 2 diabetes. They were looking for a molecular link between type 2 diabetes and the increased risk of these conditions. What they were able to show here, and this is again American Diabetic association, if you look at the green line, those are individuals with type 2 diabetes and that's the endotoxic response to a same control meal. So the green line are the type 2 diabetics, the dark blue line are the normal individuals, the red line are the obese individuals, and the light blue line are people who are glucose intolerant. So they're pre diabetics. So the diabetics had a significantly increased endotoxic response to the same meal. So when you have diabetes and if you're obese on top of it. Then every time you eat food, even though you're eating the same food as someone else that's of normal weight, your endotoxic response to that food becomes significantly higher, which means all of your cytokines, your pro inflammatory cytokines, increase at a much more dramatic rate than if you're of normal body weight. Now this is a great graph that actually shows you the average increase over normal weight individuals when it comes to glucose intolerant, obese or type 2 diabetics. So when you look at this top line, these are obese individuals. The black bar indicates the endotoxic response to food of a normal weight individual. And then the white bar on top of that graph number A shows the increased endotoxic response if you're obese. And then of course in slide number B, that's a glucose intolerant person. So there is a measurable dramatic increase. And then chart number c are type 2 diabetics. Their amount of increase over normal weight individuals to the same meal is really, really significant. So it seems that there's a direct association of being obese and being type 2 diabetic and the amount of endotoxicity that normal meals present to that individual. High fat feeding does a couple of different things to the microbiome. One of the things it does is it definitely perturbs the microbiota. So you see from this chart that high fat feeding over a period of eight weeks dramatically lowers bifidobacteria levels, for example. And we know that high levels of bifidobacteria are associated with leaner body mass and lower risk for cardiometabolic syndrome. So the high fat feeding does a couple of things. One is that increasing chylomicron expression, which means you're absorbing more lipids and more lps, but it also seems to perturb the microbiome over time. So then the question is, if you have type 2 diabetes and you have obesity, you clearly have a higher endotoxic response to food. But what came first? Do these individuals have higher endotoxic response to food because they're diabetic and they're obese, or are they diabetic and obese because they started off with a higher metabolic endotoxic response to food? So which one actually came first? And the recent studies show that it's actually the metabolic endotoxemia that comes first? So these are individuals that have genetically or sorry, from a microbiota standpoint. So not genetically, but from a microbiota standpoint actually tend to suffer from more potent endotoxic response to food. And that's increased their risk for becoming obese and diabetic. And then once they're obese and diabetic, it actually increases their endotoxic response even further. So they were pre programmed by dysbiotic flora and a non functioning microbiome to actually respond in a more endotoxic way to every single meal that they ate. So every single meal that they ate set them on a path for cardiometabolic syndrome. And now that they have cardiometabolic syndrome, it increases the risk even further. Certainly increases the risk of all the comorbidities. So our question is, can we stop that driver which is at the root cause that LPS translocation across the mucosa and across the intestinal epithelium. And that's where our work lies. So our focus is that if there are microbes that can actually change the microbiome, can it convert a non protective microbiome to a protective microbiome? And if we can stop the LPS from migrating through, we can certainly reduce a significant amount of risk for most of these chronic conditions. So let me share with you some of the other chronic conditions. This is really interesting. So this is a co incubation study where they took LPS and actually incubated it with Oedipocytes. As it turns out, when LPS come in contact with adipocytes, it actually physically swells the adipocytes, so it makes the fat cells bigger. So imagine that every time you eat, you get a translocation of LPS into your circulatory system. That LPS goes around and starts mingling around with your visceral fat and it actually swells the adipocytes and makes them bigger. So there's a significant contact point here. And here's an interesting study from Diabetes Care, Journal of diabetes care in 2012. They were looking at what is the primary insult that starts off the process of diabetes. And they determined that the translocation of endotoxins from the lumen of the gut is the primary insult that sets off diabetes. So the next few slides is going to be the death by PowerPoint side part of the whole presentation. Right. Which means that I'm just going to show you a whole bunch of studies. I'm just going to glance over them so that it doesn't put everyone asleep. But everyone can have a copy of these slides. If you just come by a booth and give us your email, we'll email it to you that way you can take a look at these papers on your own time as well. This one is really interesting because it showed that LPS induced low grade inflammation causes a hypothalamic insulin resistance. So it starts the process of insulin dysfunction in the brain before it even affects the pancreas. And this is irrespective of body weight because most of you are probably seeing this where you're getting people coming into your clinic at younger and younger ages of varying body weights with type 2 diabetes. And the question is, what is driving this epidemic of type 2 diabetes? Well, as they've shown in this paper from 2017, so very recent paper that LPS from the gut has a capability of getting into the hypothalamic region and actually affecting insulin response starting in the brain, even before the pancreas starts getting affected the islet cells. This is a great paper because it gives us a clear metabolic link between cardiovascular disease and metabolic endotoxemia. So it helps answer that question we've all been wondering is why is it that being overweight increases your risk for heart disease, increases your risk for cancer and Alzheimer's and Parkinson's and so on. This paper is a great review paper that shows you the molecular link and it's all tied to metabolic endotoxemia. Here's a few other studies, one of which that's really interesting is it increases the proliferation of CD14, inflammatory immunogens and so on. This one also shows that lipopolysaccharides, it's LPS from the gut lumen, actually increase the leakiness of the tight junctions. So it increases the permeability both in vitro and in vivo. And it does it through an inflammatory process using enterocyte membrane. So they use toll like receptor 4 and CD14 activation to show that that type of inflammation can increase the tight junction permeability in both experimental models, in vivo and in vitro. Oops, sorry, I missed one. Endotoxemia levels, actually, metabolic endotoxemia actually increase fatty liver disease, non alcoholic fatty liver disease as well. We're right now planning a study on that because what we're seeing is that, you know, the liver has to take the brunt of the work of getting rid of LPS once it's in the basolateral circulation. And one of the ways that the liver deals with that is of course depositing fat on itself. So I'll show you. In our study that we published, we saw a significant decrease in triglycerides in just 30 days by taking the probiotic because we were reducing the amount of LPS that was translocating. Here's some studies on autoimmune disease and showing how LPS induction of inflammation in the body can actually start off the process of autoimmune disease. These are several examples on here. Again, you can have these presentations, so you can take a look at these papers on your own. But basically what we're seeing is that LPS endotoxemia affects immunological responses systemically, locally, and of course, affects the endocrine system as well, because we know it affects insulin. We'll also show you data on ghrelin and a couple other interesting things. This one was very exciting, actually. Dr. Campbell brought this paper to my attention. This was in September of last year. They finally figured out what is driving Alzheimer's disease. Right. Over the last three years, we've known that there's an immune process that actually starts the process of beta plaqueing in the brain. So what is driving that destruction of brain tissue is an immune process. It's an innate immune response. But they didn't know what was actually causing that innate immune response. This paper was published in September of 2016 showing that microbiome derived LPS from the gut is translocating past the intestinal barrier, getting into the circulation, entering the brain, and triggering that immune response that's eating away at the brain tissue. So LPS endotoxemia is driving Alzheimer's disease. Here's a whole bunch of conditions. This is basically a collation of a whole bunch of studies, the references to which we can give you. But I just want to show you how pervasive this issue is. Leptin resistance. We know that leptin resistance is at the crux of metabolic disease. Leptin resistance occurs when LPS enters the enteric nervous system and disrupts the communication between the gut and. And the brain. And we saw a really interesting result with this in our study. But that's one of the ways that it can disrupt your metabolic function. The other one is chronic constipation. We all know that the enteric nervous system controls the peristaltic activity and the movement of the bowel. So now, if you have LPS getting in and interfering with neuronal function in the enteric nervous system, you're going to stop the bowels from moving. So you end up with stasis in the bowel. And that, of course, leads to constipation, but even more so, it can lead to sibo, small intestinal bacterial overgrowth. And we know SIBO to Be a precursor to a whole bunch of other issues like Crohn's colitis, ibd, IBS, and so on. Another interesting one is the mood and appetite disorders. So we know that LPS can get in and interfere with things like dopamine receptor binding, with the recycling of serotonin in the brain. And so all of these conditions, which affect mood and which affect your response to stress, can be affected by LPS getting into the brain. The other area is depression by the same mechanism, interfering with dopamine and serotonin function. We have. What is this? This is cognitive decline. So we know that LPS can actually get into areas like the hippocampus and the amygdala and cause inflammation in the hippocampus and amygdala, which actually affects memory recall, gives you that brain fog. A lot of times we've experienced that kind of brain fog after a really big meal that's a super endotoxic meal, high fat, high calories. But a lot of that is driven by LPS actually getting into deep areas of the brain, loss of memory function. Same kind of mechanism, depression I mentioned before, anorexia. This is a really interesting mechanism. If you have a chance to read about metabolic endotoxemia and anorexia nervosa, it's a really interesting mechanism that explains why some people get anorexia end up with anorexia nervosa and some people, and most other people don't. So one thing is the LPS endotoxemia after a meal that drives kind of a depressive feeling after eating food, because food is supposed to trigger dopamine release. Right? That's one of the ways that we end up becoming addicted to food. But if you have the opposite when you eat and it actually reduces dopamine, reduces serotonin, you end up having a negative association with food. So there's a really interesting mechanism to talk about there. Anxiety. Of course, if you mess around with dopamine and serotonin, it can drive anxiety, chronic pain. This is really interesting. So it looks like LPS can get into the central nervous system and interfere and actually stimulate nociceptors to send out pain signals on a regular basis. So you might be dealing with patients that are coming in that have chronic pain without any real known origin of the pain. There's no injury that they can attribute it to. Well, it's probably just LPS getting in the system and stimulating those pain signals. Parkinson's. So there was a most recent study published, showed that Parkinson's is also Driven by gut derived LPS that can actually cause microglia activation in the brain. It can also affect the central nervous system, so it disrupts motor function. Another very interesting thing is hypogonadism for men. You know, we know that once you hit a certain age over 38, 40, testosterone levels start to drop. There's something called the gelding principle, or gelding theory, I think it might be called, which shows that the type of immune activation that LPS causes in the body actually shuts down, down or slows down testosterone production. So when you have CD14 systemic inflammation because of LPS, you actually start to slow down and shut down testosterone production. So basically the point of this whole slide is to show you how pervasive LPS is. It gets into the circulatory system because of a non protective microbiome and a non protective gut intestinal epithelium. And once it gets into, into the system, it can manifest in so many different ways. Right? So I always say that. I mean, basically, if you look at this chart, this covers more than 80% of the diseases you're seeing or dealing with with your patients. And I always say that if LPS endotoxemia is not the major driver of the condition that you're dealing with, then it's at least a significant factor in preventing the healing from that condition, especially when it comes to things like cardiometabolic syndrome, Alzheimer's, diabetes, cardiovascular disease, obesity. The data is so clear that LPS endotoxemia is a major driver of all of those conditions. So if you don't stop the endotoxins from translocating through entering the circulatory system every time people eat food, you're basically taking two steps forward, one step back with a lot of the treatments. And I think that's driving a lot of the issues and success rate with treatments in general. So what are we doing about it? Right? So we looked at it and said, okay, metabolic endotoxemia is a factor of a dysbiotic flora. When you have a dysbiotic flora, you have a dysfunctional mucosa. When you have a dysfunctional mucosa, you have a dysfunctional intestinal epithelium, the M cells, the panet cells, the goblet cells. None of those things are functioning the way they're supposed to because all of them are controlled by the microbiome and controlled by metabolites and signals that are being produced by the microbiome. So if we fix, if we have a solution to fix, the microbiome itself can we fix the intestinal epithelial layer, can we fix the mucosa and at the end of the day stop LPS from migrating through every time people ate food. That's our focus. So we looked at these keystone strains that I talked about that we've discovered and kind of put together into a very specific formula. And, and as it turns out, in the animal world, they've been doing these studies for quite some time. Because in the animal world, there's a huge economic impact to having leaky gut or metabolic endotoxemia. In chickens, if they have high levels of metabolic endotoxemia, their number of days it takes them to reach catch weight actually reduces because they can't absorb all of the nutrients that they're trying to absorb. They actually end up growing in a different way than the way the farmers want them to grow. Grow. In fact, the mortality rates go up quite significantly. So if you are Tyson Food and you're growing billions of chickens a year and your mortality rates go from 1.5% to 3%, you will really notice it on your bottom line. Right. So these companies have been working with microbiologists and veterinarians and using these types of spore based bacteria to study the impact on gut epithelium, leaky gut hyperpermeability within the tight junctions using these spores. And what they found is that the bacillus subtilis, for example, can induce the expression of all 40 tight junction proteins, all the claudin and occlatin proteins. Right. So they get into the gut, they interact with the intestinal epithelium cells and somehow they stimulate the production of all the tight junction proteins. We're not sure what that mechanism is, but it's seen very clearly in animal studies. So we're presuming that it will do the same in humans as well. In the second study here, we show that there's actually significant histological changes. When you look at a cross section of intestinal lining and intestinal lumen, you see actually increase in the microvilli height. So these strains have the ability of actually regenerating intestinal tissue. So in cases where you have enteropathy, where the microvilli start to shrink over time, like your celiac patients, or your gluten intolerant patients, or your colitis and Crohn's patients, basically what you're doing is you're losing surface area of the intestines. These strains seem to have the capability of regenerating that surface area because they're increasing the height of the Microvilli. They're also improving crypt death and other things as well. The bacillus also seems to protect against intestinal toxins. So mycotoxins that come in with agricultural products like Z, dea, Don, these are all really, really prevalent mycotoxins that are on agricultural products that are really, really significant intestinal epithelium toxigenic products. And it seems like these probiotics seem to be able to alleviate the toxic response of those mycotoxins within the gut itself. So we wanted to study this and we published this paper In August of 2017 in the world Journal of Gastrointestinal Pathophysiology. We wanted to put it in a GI journal so that GI docs at least start seeing it as well. And then of course this is a great open source journal so it helps us by being able to send the papers out to everybody. But this is basically what we looked at. We worked with University of North Texas, they did the study for us. And we started off by screening 100 healthy college students. The age range is between 19 and 20, all the way to 31. The average age was around 23 or 24. What we were doing is we were bringing in non obese healthy college students and we were giving them a high fat, high caloric meal. On the first study what we were doing is we were looking at, we gave them a McDonald's breakfast. So imagine how easy that was to recruit. We're saying we'll give you 20 bucks and feed you McDonald's if you come in. So we had a huge line outside the lab and we actually had to fight people off and said no, we got enough data. So what we're seeing is when they have an endotoxic response to the meal. We're pulling LPS measurements in the serum at time zero. So they're coming in fasted, that's the time zero. And then once we give them the meal, we're looking at three and five hours postprandial and we're looking for LPS directly in the serum at that point. And what we were looking for is this characteristic increase of LPS within the first three to five hours. We look for a minimum of six fold increase from baseline of LPS in the system once they ate a meal. Now in the pilot study what we did is we took these subjects who had this response or about 16 of them, and then we sent them back with the probiotic for 30 days. We said, just take the probiotic, it's once a day, you take it, come back in. In 30 days, let's redo the same test again to see what it does to your endotoxic response. So this is what the data looked like on the pilot study. So the endotoxic response of the food, the Same meal from 30 days ago was completely blunted. So that gave us the impetus to do a larger version of the study and then look at a whole bunch of other markers. You know, I talked about when LPS enters the circulatory system, you end up getting all of these other inflammatory markers increasing as well. So we looked at 13 different inflammatory markers and some hormones, all of that stuff, including lps, in the expanded study. So in the expanded study, which is what we published, what we saw was that. I'll come back to that. What we saw is an average reduction of 45% in LPS translocating from the lumen into the circulatory system after taking the probiotic for just 30 days. Now, in the placebo group, what was really interesting is not only did we not see a reduction in, we actually saw a 32% increase in the amount of endotoxemia in 30 days. So it seems like the response gets worse over time. Now we're doing a 90 day version of this study to see if that response ebbs and flows over a period or does it constantly get worse. Now, what we saw was about a 60% difference between where the placebo group ended up and where the treatment group ended up in just 30 days. Now, remember, these are college students. They're not doing anything good for themselves in that 30 days. There's no dietary changes. They're not under your care, so they're not doing other therapies as well. This was just kind of like a shotgun thing where we're saying, okay, let's see what happens. We were not that confident that we would see actual changes because they're doing so much to affect their gut in such a negative way, and yet we saw a statistically significant reduction in LPS endotoxemia. Now, let me go back to this graph. The other thing that we saw that was really interesting, if you look at the top left, is ghrelin. So ghrelin levels. As you know, ghrelin controls your hunger, right? And as long as your body's producing ghrelin, you're going to want to eat food. And so if you look at the blue lines, that's the placebo group. When they come in faster, the ghrelin levels are high, as you would expect it. But then we give them a 2000-calorie meal. And ghrelin levels barely drop after the meal. And in fact, within five hours starts to go up again. So that communication between the gut and the brain, where the gut is telling you you have enough food, stop eating, is totally disrupted. After 30 days of the probiotic. We see the way ghrelin is functioning. It's functioning in the classical sense, meaning ghrelin is high at fasting. Then when we give them the meal, ghrelin levels dramatically drop within three and five hours after the meal. So your body is telling you you don't need to eat anymore, and that is restoration of the communication between the gut and the brain. We saw similar things with leptin as well. The cohort did not reach statistical significance for leptin, but we're doing a larger study right now on obesity specifically to look at ghrelin and leptin function. Let me show you a couple of the other things as well. So we also looked at, of course, postprandial or triglycerides in general. If you look at the blue bars, that's the megaspore treated group. We saw a statistically significant drop in triglycerides within 30 days. And we weren't necessarily expecting this. This wasn't a high triglyceride group. The average triglyceride levels was around 140, which would be relatively normal. But in the placebo group, we did not see any change in triglycerides at all. So now we're doing a larger version of just the triglyceride study with people that have really elevated triglyceride levels. We also saw another interesting response in something called MCP1. So MCP1 is a very interesting chemokine that indicates recruitment of immune cells in the intestinal epithelium to during a meal. So if you have elevated levels of MCP1, you know that the microbiome, the intestinal epithelium, is reacting in a real inflammatory way towards the meal that's being consumed. That inflammation in turn will cause opening of the tight junctions. All of this inflammation systemically and localized into places like the brain, and of course, LPS translocating as well. So in the placebo group, we did not see any changes in MCP1. In fact, in the second set of data, we started to see an increase in MCP1, so again, that correlated with their increase of LPS as well. And in the megaspore treated group, we saw a significant decrease in MCP1, which correlated to the decrease in LPS. And then here's one of my favorite charts. It's called a heat map. This is a great way of looking at overall patterns. But if you look at the middle line, all of the biomarkers above the middle line reach statistical significance. All of the biomarkers below the middle line did not reach statistical significance, but trended in certain ways. So I like to talk about the ones on the top. Of course, endotoxin is LPS triglyceride, you know, ghrelin. I already showed you MCP1. But a very important one is interleukin 1 beta and interleukin 12, interleukin 1 beta. If you were at the American Heart association meeting last year, you'll know that that's a major target for drug companies right now because it's one of the few cytokines that are found present in every single area where there's plaque formation. It's a self perpetuating inflammatory cytokine. When one endothelial cell expresses it, it actually causes the next endothelial cell to express the same. And it also increases the expression of over 100 different inflammatory cytokines. So it's a major driver of plaque formation in the vascular endothelium. So what companies are looking at is an Interleukin 1 antagonist right now as a treatment for heart disease and of course, everything associated with plaque formation in the vascular system. But we can do it with a probiotic. We saw a statistically significant drop in Interleukin 1 Alpha and Beta with using the probiotic for 30 days without doing anything else. So we're reducing that disease, causing inflammation. So here's a quick conclusion. Metabolic endotoxemia is the main driver of morbidity and mortality. The 2015 paper in the Frontiers of Immunology showed that this mechanism is probably the number one cause of death among non communicable diseases worldwide. And that's because it's a major driver of most of these conditions, like autoimmune disease, diabetes, hypogonadism, et cetera. Metabolic endotoxomy is a feature of a dysbiotic gut. So if we can fix that dysbiosis, reestablish the protective barrier of the mucosa and also the intestinal epithelium cells, we can significantly reduce the driving force and the risk factors for most of the chronic illnesses you're dealing with. The hallmarks are high levels of LPS and LBP, especially postprandial, of course, high levels of interleukin 6, interleukin 1 beta and alpha, interferon gamma, and of course, as I mentioned before, soluble CD14 as well. So these are things you can look for postprandially in your patients. When they come in, it'll be ideal if you can get them getting a fasted serum sample to test for all of these and then have them go out and say, go to McDonald's, eat a meal and come back in and get a second panel drawn up postprandially. Then you'll see the significant difference between all of these megaspore reduces, postprandial endotoxemia and all of the biomarkers associated with it. Because, you know, if we showed that we reduced LPS translocation, but none of the characteristic cytokines actually reduce as well, then we'd kind of be at a loss of what's actually going on. So the fact that the subsequent, the downstream cytokines also reduce shows us with great certainty that we're actually dramatically affecting the overall pathology of these disease conditions. So now I want to talk a little bit about what we're doing in the near future here. So that's megasporbiotic, right? So that is a product that we developed over five years ago, and we were taking a significantly different approach to the idea of probiotics. You know, most probiotics were designed with the idea of let's reseed the gut with good bacteria. So let's take 200 billion of lacto and bifido and throw it in every single day with the hope that it's getting in and colonizing and shifting dysbiosis. However, the problem is the vast majority of products don't survive through the gastric system. So most of what's happening is you're getting a lot of dead bacteria moving through the system right now, in some cases, some of those dead bacteria actually have a metabolic response. So if you look at Lactobacillus rhamnosus gg, there's some, a lot of studies that show that rhamnosus GG has some immunomodulatory effects, both systemic and localized in the gut. However, they've also done heat kill studies where they take GG as a strain, they heat kill it, and they administer the dead version of the strain and it works even better than the live version. Right. So the vast majority of these strains are really not probiotics, because the definition of a probiotic is a live microorganism, when administered in adequate amounts, confers a health benefit to the host. When there's dead bacteria that can kind of give you the same modulatory effects. Then they are actually something called metabolic response modifiers. So there are some strains out there that have a favorable metabolic response. The problem is they're not making any functional changes within the microbiome. So our job was, let's figure out what our ancestors did for probiotics. Right? Because clearly our ancestors didn't have refrigerated bottles and special capsules and 200 billion CFUs of lactobifero strains. Where did our ancestors get their bacteria from? Well, they got it from the environment. So we started studying environmental bacteria to figure out which one of the bacteria that exist abundantly in the environment actually has the capability of surviving naturally through the gastric system once it gets into the intestine can actually make a functional change. So we're going to be publishing a paper this year or submitting it for publication. Hopefully it gets published within the same year where we show that this probiotic megaspore, five strains at 4 billion CFUs, which seems really inadequate when you know about all the rest of the probiotics within the marketplace. But five strains with 4 billion CFUs actually dramatically increases alpha diversity within the microbiome. So it actually increases the microbiome diversity by over 30 trillion organisms. It also increases things like Akkermansia, mucinophilia, faecalum, bacteria, Prosnitzi, bifidobacteria. So these are the gut policing strains. They seem to get in. They use quorum sensing to read the microbial environment. They sit next to bad and overgrown bacteria. They produce up to 25 different antibiotics in the gut, including things like bacitracin, to actually kill off bad bacteria. And that's what they're used for in Europe, Latin America and southeast Asia since 1958 as a prescription drug for the treatment of dysentery and other gut infections. So these strains have been on the market in the prescription space for over 60 years. When we discovered that about nine years ago, we said, we're compelled to bring these strains to the US and then study the heck out of them. Right. So let me show you what we're doing right now. So the first study we're doing is a pre diabetic study. We're taking people with glucose intolerance and seeing if we can revert their intolerance back to normal glucose function. We're also doing an elevated triglyceride study. So now, even though we saw triglycerides drop, we wanted to see what does it do when you have triglycerides of over 300, over 400. So we're looking at people with elevated triglycerides and looking at 90 days to see what we can do. So that's the second study. We're also doing a rheumatoid arthritis study, looking specifically at rheumatoid factor reduction, because we know there's a major autoimmune component to endotoxemia and the types of inflammation that endotoxemia drives. We're also looking at pre autoimmune disease, something we call autoimmune syndrome. You know, when autoimmune disease gets to a point where we can actually diagnose it and label it, then it's really gone too far. Right. Years before that, there's a pattern in your immune system that indicates that you're more TH17, interleukin 17 dominant and that your body's moving towards an autoimmune response that can be measured immunologically. And what we want to show is that, can we rescue people who are on that road and bring them back to normal immune homeostasis? We're also looking at hepatic encephalopathy. We completed one paper. These are liver failure patients who are on xifaxan, who are on lactulose. And what we're able to show is these strains, even in the presence of lactulose and xifaxin, can bring down blood ammonia levels by over 40%. So this is being studied by a huge teaching hospital, acne. We're doing a really cool acne study with the University of California, Davis, where we're showing that changes in the gut microbiome and the LPS endotoxemia and the butyrate production by these strains actually changes the lipidome in the skin. It changes the sebum concentration, sebum profile, and it also changes the microbiome in the skin itself. That has significant impact on, of course, acne, but also on things like psoriasis and eczema. We're doing a study on weight loss that I mentioned before that's at University of North Texas. So full DEXA scans, metabolic pathways and so on. We're also doing a study on gum disease. This is something we're just now starting. It's a multicenter study. There's a really direct relationship between LPS, endotoxemia and inflammation in the gums and the starting of biofilm development in the periodontal tissue in the gums, which drives gum disease. So we're doing a very interesting study there. We're also doing a low testosterone study, which we're still in the development of. We're also doing a C. Diff study with Cleveland Clinic because we're showing that in experimental models, we're showing that these spores can actually competitively exclude Clostridium difficile. And also the spores can help undo the damage that vancomycin does to the epithelium during the treatment of C. Diff. Because there's a great model at Cleveland Clinic where they've shown that the types of damage that vancomycin does to the intestinal epithelium actually makes it easier for C. Diff to colonize down the road. So that's why the reversion rates, the recurrence of the disease is like 40%. Right. It's huge. So the treatment is causing the disease to come back more frequently. We're looking at two steps. Can the spores actually fix what the treatment is doing? And then on top of that, competitively exclude the bacteria themselves. So we have a complete C. Diff treatment system. So we're doing studies with Cleveland Clinic, Thomas Jefferson University, we're doing a really cool diabetes study with metformin there. So people who are coming in with an A1C of around 5.96 who qualify to start metformin are being put on metformin alone or metformin and the probiotic being followed for the next six months to see if the metformin with probiotic dramatically improves their glucose response over people who are on metformin alone. Because we know if you looked at the most recent studies of how metformin works, metformin actually works by increasing butyrate production in the gut. Right. That's what it does. And so when we have a probiotic that increases butyrate production by 40%, we have a significant chance of rescuing newly diagnosed diabetics back into normal glucose function. We're also doing studies at Mercy Hospital groups doing the hepatic encephalopathy study. UC Davis, I mentioned, is doing the acne study. Prodigest is an amazing gut simulated system that has a full GI tract simulated where you can continuously add substrates to it and see what happens to a human microbiome over time. Next month, I'm going in to meet with them because I want to do a human microbiome glyphosate study. Right. We know that glyphosate perturbs the microbiome in a very specific way. It increases clostridia growth, it increases klebsiella salmonella. But no one has ever done it on a human microbiome, and no one's ever done it on a human child microbiome. So we're starting a study where we're looking at how does glyphosate perturb the microbiome of a two and a half or three year old, and then can we rescue that perturbation after a period of time? So that's the idea there. Royal Holloway, London University. That's where the spores come from originally. So we're always doing a bunch of studies with them as well. They're actually looking at using the spores as a vaccine delivery vehicle. Right. So they've published about nine papers right now showing that you could take an antigen, stick it on the spore, deliver it orally, the spores present it to the Peyer's patches, and you get a much more amplified, robust, adaptive immune response than injecting that antigen into your arm or doing it intranasally. So the spores are acting as like a natural vehicle for, for vaccines. So thank you very much. I do want to mention that we're doing a drawing. I think someone at the company told me that if you come to the booth and there's a ticket or something like that, we're giving out an iPad. So come on by, give me your email, we'll send you the PowerPoint. I'll be around here and after lunch at the breaks for questions and all that. But thank you so much for your attention. I really appreciate.