Judith Lukaszuk, PhD from NIU educates us on recent studies on the gut microbiome

Dr. Lukaszuk

Hi, My name is Judith Lukaszuk, and I am a registered dietitian and nutritionist, and a licensed dietitian and nutritionist in the state of Illinois. I have my master’s degree in nutrition and my PhD in exercise physiology and today I’m going to talk to you about the gut microbiome. I was asked to give an update for 2015 and 2016, and just 2015 alone has 1,700 different studies published on the gut microbiome. I won’t be going over all 1,700 today. I’ll just be giving you the highlights of those. But 300 of those articles alone are showing that the microbiota actually supports both innate and acquired immune responses. So it plays a big factor in how you react from an immune perspective.

Now just talking about some terminology, when we talk about microbiome, we talk about the genes as well as the bacteria. When we talk about microbiota, it’s just referring to the bacteria itself. Now your microbiota starts to change at the time that you’re born. So if you’re born vaginally, you have more lactobacillus in your large intestines, and if you’re born C-section, you have more staph in your bacteria. It seems to normalize around the age of three, but then our microbiotas continues to change throughout life based on age, genetics, environment, and of course, antibiotics. So I’m just going to give you a brief overview of where we’re going with this lecture today.

So just an overview we’ll start out with is, “What types of bacteria are found in the large intestines?” And then perhaps what treatment options we have, “If you have an over-abundance of bad bacteria, what do we do to help improve that and increase the good bacteria to outweigh the bad?” We also are going to look at the gut microbiome. Now just an overall perspective, we’ll be discussing the effects of the microbiome on weight, and what bariatric surgery does to the microbiota, as well as what the microbiota has a role to play in energy balance.

But just some recent studies that were done up in Canada. In Canada a researcher looked at children, from birth all the way up to the age of three, and he found that even a transient change in the microbiota significantly changed their incidence of asthma at the age of three. So a transient change in microbiota simply means that they were put on a course of antibiotics that changed their microbiota enough to increase their autoimmune disease risk. There’s also researchers worldwide that are looking for a specific microbial marker in the large intestines that will help identify colon cancer tumors before they get out of control. Then lastly, there is a study out by Lee, and what he showed is that…Lee actually was looking at the different effects of the microbiota on your health, and he found that 26% of your HDL change was due to the microbiota changes, so a significant increase on risk of CAD.

From an autoimmune perspective, most things that have been studied in 2015 and ’16 looked at IBD. So we’re talking about Crohn’s disease, we’re talking about ulcerative colitis, but I will bring up rheumatoid arthritis and some other things that were in the research as well. We also will be discussing the gut brain axis. We know that it plays a very significant role in your ability to digest foods. So not only does the vagus nerve increase hydrochloric acid, which helps us digest proteins and break them down, but it also regulates how your enzymes are released from your pancreas so you can break down that food that you’ve consumed.

And it also affects motility going through the gastrointestinal tract. So the vagal nerve is a huge player in your ability to break down and metabolize food. Last, but not least, we will discuss an Alcat Study that was done last spring. I was the primary investigator, and I did this out of the university I work at, which is Northern Illinois University in DeKalb, Illinois. And it was the first study to show that avoiding foods that you’re intolerant to actually reduced total body inflammation, so this is the first time that those study results are actually being presented.

Okay, so just an overview of the microbiota. The things that are bold and asterisks, those are found predominantly in the large intestines. They have anti-inflammatory properties. But we know that Firmicutes and Bacteroidetes, they actually comprise 80% to 90% of the bacteria that’s found in your large intestines. Actinobacterium takes up about 10% of the bacteria that’s found in your large intestines. And then Proteobacteria, it is a phylum. Those are the large categories of bacteria, however it’s less than 10% in your large intestines. It’s said to have more of an inflammatory role than the others, which have more of an anti-inflammatory and butyrate producer role. And you’ll find out the benefits of butyrate as we go on with further slides.

Now under Firmicutes itself, you’ll see this thing called Clostridium leptum. It’s underlined. That’s a genus. And then underneath the Clostridium Leptum, Faecalibacterium prausnitzii, that’s called a species, or bacterium. So there are a tremendous amount of bacterium underneath each category of phylum, and I think that that’s where they’ll see most new research being done. Because there’s a variance in what you’ll see with the phylum in how they move with different disease states. So I think that what they’ll ultimately be doing is looking more on a smaller level at how the bacterium itself is moving. Now just to give you an idea, some of the different phylums and conditions. So the only similarity here is obesity and type-II diabetes. You can see that across the board here there’s an increase in Firmicutes and a decrease in Bacteroidetes.

And then with high-fat diet, guess what? If you eat a high-fat diet, you actually increase your risk of intestinal dysbiosis, and it has very much the bacteria effect of obesity and type-II diabetes, where you’re increasing Firmicutes, and you’re actually increasing Proteobacteria, remember, I said that that that one has inflammatory properties? And you’re decreasing Bacteroidetes. So that’s what the asterisks are accounting for, for the high-fat diet. But otherwise, when you look at some of these autoimmune things, celiac, rheumatoid arthritis…yeah, celiac and rheumatoid arthritis both have decrease in Actinobacterium.

But otherwise, it’s pretty much all over the board. There’s very variable results with phylum differences with diseases. And we know that just changing the diet for one day can significantly change the bacteria that’s in your large intestines. So that might be some of the problem, but we know environment changes it as well. So again, I think that in the future, you’re going to be looking at not phylums, but you’re going to be looking at species and how they change in these different conditions.

So if you have an over-abundance of bad bacteria, what do you do about it? Well, there’s fecal microbiota transplantation. And essentially what that is, is there’s three forms that they can do this. One is they can give you a poop pill. I don’t think they would call it poop pill, because it wouldn’t be a very enticing factor to take it. But that would be one option. The other option is they do a slurry of fecal material, and then they put it down an NG tube into your stomach. Then the other option would be a southern option, would be given via enema. So there is that possibility that you can actually have a fecal sample from a donor, and it might actually benefit you, and that’s what we’ll see in a lot of studies that were done.

Now what’s the difference between the fecal microbiota transplantation and bacterial consortia transplantation? Bacterial consortia transplantation refers to…and they’ve done the studies in mice. What they did is they took the stool sample of mice, they isolated the bacteria, they put it in a petri dish, they grew it. Once it got stronger, they put it in a pellet. They gavaged it down the mouse. And when Lee did a study between the fecal microbiota transplantation and bacterial consortia transplantation, he found that both of them were comparable in improving the intestinal lining in mice, but that’s in mice. We’d have to confirm that in humans as well. Now there’s a number of ways that you can alter your short chain fatty acids. One is eat a high fiber, low protein, low fat diet.

Now you’ve heard of that show, Wife Swap? Well they did a diet swap. Okay, so O’Keefe did a study a study in 2015 where he had 20 people, from…African-American individuals, eating a Western diet, so cheeseburger, French fries, high-fat diet. And then he had 20 individuals that he got from South Africa that were living in rural environments. So they were eating more high fiber, low protein, low fat diet. And he switched their diet for two weeks, so they did a diet swap. What he found, interestingly, is that the African-Americans eating the Western diet, by switching to the high fiber, low protein, low fat diet, their butyrate production increased twofold. And he also found that they had a lot less inflammation, and they measured that by looking at pulp formation in the colon. So, very, very promising results showing that diet does essentially effect your microbiota.

Something else that affects butyrate production, which we’ll be talking about…short chain fatty acids are what break down…you eat dietary fiber, they break down to short chain fatty acids, and there’s butyrate, acetate, and propionate. Butyrate has been shown to be the most beneficial on increasing colonocytes. It feeds the colonocytes. It keeps the colon nice and healthy. And flaxseed oil is a great precursor of butyrate. Now you’re probably wondering, “Okay, what about flax seeds and flaxseed powder?” Well I did see a study in 2014 that looked at flaxseed powder, and they found that even though it did increase insulin sensitivity, it did so independent of changing the microbiota. So again, we’ll probably have to look at more mechanisms since the action there.

So if you’re trying to actually increase butyrate production, I would stick with the flaxseed oil, and not go with the powder or the seeds until they do further studies on that. Now there are also a number of studies done on oral sodium butyrate, and these were done in mice. And it showed that it increased insulin sensitivity and increased energy expenditure, because it increased mitochondrial activity. And right now, Harstra, H-A-R-S-T-R-A, is doing a study on people.

 

And he’s giving them four weeks of oral sodium butyrate to see if it changes metabolic syndrome, meaning increases insulin sensitivity, and he wants to see what kind of microbiota changes that can occur with such supplementation as well. There have also been a lot of benefits from butyrate enemas. They’ve been done in ulcerative colitis, to decrease inflammation in the colon, and they’ve been very successful. Seems to be very well tolerated, and so that’s something potentially that one could do to increase their good microbiota.

Additional things that are out there. There’s a prebiotic. It’s called Akkermansia muciniphilia, and it now has a patent on it. There was a researcher named Patrice Cani from Catholic University, and he did a study on his post-doctorate work showing that it has great effects on metabolic syndrome. It increases insulin sensitivity. It decreases metabolic syndrome. It helps you control your blood sugar better. And so he now has a patent on this as a potential treatment for metabolic disorders, and he’s doing studies on going with that form of prebiotics. So you’ll see, probably in the future, not only Akkermansia muciniphilia, but other things that are also equivalent precursors to butyrate. The A. muciniphilia makes a tremendous amount of butyrate, but it also makes a mucous lining that keeps the colon nice and healthy. There are other things such as tailored probiotics.

Dr. Nissle, back in World War I, actually discovered a probiotic that has anti-inflammatory properties. Now the FDA gets a little bit sensitive about making claims about prebiotics, probiotics, and health. So it’s not available in the United States, but it is sold in Canada, and it’s under the brand name of Mutaflor. And what he found is that it actually reduced inflammasomes. Inflammasomes are protein molecules that go on and signal other molecules that are inflammatory. And then Johnson & Johnson, here in the United States, markets something called VE202, and it is 17 different strains of Clostridia, and it helps your T cells become T-reg cells, and that kind of normalizes your immune system from getting out of control. So that’s shown a lot of benefit too.

And really what I’ve seen with all the readings I’ve done is they’re not going to be using just one strain of a probiotic. They’re going to be using multiple strains to see if that will benefit more. Just like the VE202, it’s a great butyrate producer, but it uses 17 different strains of Clostridia. So for the most part, FMT at this point has benefited mostly the bolded things here, IBS, IBD, and recurrent C. diff. It was done initially on C. diff patients that were refractory to any other treatment, and the C. difficile just kept coming back.

But you’ve got to be careful, because there was a study done in New England. It’s not published, it was just like an abstract, and it was announced in the news that a woman was treated for refractory C. diff. And instead of them screening stool donors, she simply had her daughter donate a stool sample, and they put the fecal sample from the daughter up into the mom. It did take care of the C. diff. The C. diff totally abated, it went away.

However, the daughter was obese, and the mom continued to gain weight over the next three or four months of 40 pounds. So now they’re saying that if you do get an FMT sample, you better get a lean donor because of the study results. And there’s other studies that I’ve came across that said the same thing. Now the other conditions listed, they’re doing most of the studies now on diabetes, and insulin resistance, and metabolic syndrome, just because they’re such big players in what we’re seeing with blood sugar out of control now and obesity. And so the other conditions, they did use FMT with variable results. Some of them had long-lasting results. Some of them just had transient results. And I don’t think FMT is something that you want to be doing every week. I think that would be a hard sell.

Okay, so let’s look at some of the studies, what they found on the gut microbiome and its effect on wellness. This was a really nice study, now remember, Harstra is the one that I brought up before that is doing a four week study looking at the effects of oral butyrate on metabolic syndrome. Now they have found this diagram to be true in laboratory mice, and it’s hypothetical for how things occur in humans. So you can see the right hand of the graph, it’s obese and type-II diabetics, and the left hand of the graph is in lean individuals. So essentially, you see the lightning bolt on the right side. You’ve got dietary fiber, and there’s microbial degradation, but it’s not working adequately. So the short chain fatty acids that are supposed to be produced, they’re not being produced. There’s enzymes that are not being activated, regulated.

So in this right hand side of the equation, looking at HDAC inhibition, that stands for histone deacetylases inhibition. When that is working appropriately, what happens is you use more fat as an energy source. So that’s a good thing. It also increases insulin sensitivity, so it has an epigenetic effect. Now looking at the left side of the equation, when butyrate goes to GPC, that’s G-protein coupling receptor signaling. That affects epigenetics. That affects your metabolic rate. Because we know GPCR acts like an inbox for your e-mail. It detects light, and it tells whether it’s carbohydrate, protein, or fat signaling that’s coming in. And then it will store things in the inbox or put it in junk mail, what have you. But it has a play in PYY, which is a hormone that regulates satiety, how full you feel after you’ve eaten food.

And then one other thing, when we go through the intestinal epithelium here, you see that they have intestinal gluconeogenesis, that’s IGN. So we know that the intestines can make glucose. It actually produces glucose by itself, and that’s said to have a beneficial effect. But they think that that’s dysregulated as well in people that are obese and type-II diabetic. So round, and round, and round we go. So if we follow this thing around to the bottom portion where it says LPS, that’s lipopolysaccharide. We know that LPS…there’s a low grade of inflammation always in obese and type-II diabetics, and with that low-grade inflammation, you’ll see LPS going up. And essentially what that can do is it can actually cause endotoxemia, and that causes more insulin resistance. So round, and round, and round this goes. We have decreased satiety, because the GPCR signaling is not working right, so it plays a role on PYY.

Butyrate also plays a factor with serotonin. Serotonin is the one that makes you feel satisfied, right? So essentially what we have as the outcome here is with increased lipogenesis. People are making more fat. Now they did a study on bariatrics, and they showed that bariatric surgery actually induced chronic changes to the gut microbiome. This is Tremaroli, and they were following these bariatric individuals for nine years just to see how well they kept their weight off, and what their success rate was in keeping their weight off for nine years, post-bariatric-surgery. The person on the left is an obese well subject, and they were matched to the pre-bariatric people’s weights. Then the bariatric surgery, the RYGB, VBG, they were both matched on body weights, and they were matched on body fat. They did a fecal sample on these women nine years after they had bariatric surgery.

And from the graphs on the right, the predominance of red, you can see that they had a preponderance of an increase in Proteobacter in E. coli, but most of that red is Proteobacter. Now remember, I did mention Proteobacter in the beginning of the lecture, and it’s said to have more of an inflammatory effect. So they’re not really sure why this is, but this is what they showed in people that had bariatric surgery. So then what they did is they took the fecal samples from each of these individuals, and they put the fecal samples into the mice.

And they found that both of the groups that had bariatric surgery, the mice had retained more lean body mass, and they had a lower respiratory quotient, like 0.7, which means that they’re using more fat as an energy source. So that is the one way that bariatric surgery, people are said to have a lower body weight long term, is that it’s actually affecting their microbiota. Simply they took the bariatric surgery’s fecal material, put it in a mouse, and found the same thing in those mouse. In the obese well individual, they put the fecal sample in the mouse, and the mouse grew. It got more obese. So again, the microbiota is playing a factor on how much we weigh.

Now this was a really interesting study by Chevalier. And what they did is they exposed mice to cold. I feel like I’m in Chicago again. As a matter of fact, this morning I refused to wear my winter coat, and so I was exposed to cold at least acutely. So I could probably eat anything I want today. I’ll be hypermetabolic. But what they found in these mice is that mice that were exposed to cold temperatures for at least 10 days, what it did is it increased their insulin sensitivity, it increased their thermogenesis so they actually burned more fat. But essentially, it’s a white fat, brown fat. So let’s go back to that. So white fat is stored…when you’re young, you have a predominance of brown fat. It’s found around the subscapular. It’s found around the kidneys. It’s found underneath the arms. And as you get older, that brown fat tends to go away, and we have an increase in white fat.

So what they know is that these mice that were exposed to cold temperatures for at least 10 days, they had a white fat browning effect. So that white fat became more beige, which means that it did increase in sensitivity. It increased its metabolic rate. It increases your ability to use fat as an energy source. And then they looked at mice for 30 days. So the mice that for 10 days, that were exposed, they continued to lose fat. They continued to lose body weight.

The ones that were exposed for 30 days there was a change, in that their intestinal absorptive capacity, both their villi and their microvilli increased in response to them being cold. And because their caloric intake was higher, now they’re able to absorb more, because they have greater absorptive capacity through the villi and microvilli, so they gained weight. And so then when they looked at microbiota changes, they found that it was very consistent with what you’d see with somebody that’s obese, increased Firmicutes, decreased Bacteroidetes.

But more importantly, what they found is mice that were exposed to cold had a decrease in the Akkermansia muciniphila. Boy, that’s a hard word to say. But that’s the prebiotic that I said that’s the great butyrate producer. So what they found is that A. muciniphila was actually reduced in the mice exposed to 30 days of cold exposure. And because of that reduction, it increased the absorptive capacity of the gut to allow for weight gain, fat storage. When they supplemented these mice with the A. muciniphila, they found that their intestinal absorptive capacity did not increase, which means that the mice continued to lose weight. So this might be a major player.

It’s definitely showing the microbiota affects thermogenesis, for sure. And in Miami, I don’t think that you’ll ever have this effect. But certainly in colder climates, you are going to see this effect. So I think what this study is showing is that the major player to prevent the intestinal absorptive capacity from increasing is that you have to supplement with that prebiotic. And if you do that, the gut absorptive capacity will not increase, so then your body weight won’t increase. You’ll continue to lose fat.

Now let’s talk about nitrate-nitrite. You probably see a lot of the sports nutrition supplements being sold in health food stores. A lot of athletes take this because it makes them look more vascular, and they like to look in the mirror, and they like to look at their veins when they’re lifting weights and so forth. But essentially what we know about nitrate is nitrate can actually be reduced in the mouth. And the way that happens is when it’s exposed to the anaerobic bacteria on the posterior of your tongue, it converts, or it’s reduced from nitrate to nitrite. And we find nitrate specifically in green leafy vegetables, but specifically beet juice is a great source of nitrate.

And not only does it decrease your blood pressure, but it seems to have a beneficial effect on protecting stomach against bacteria. What we know is that if you use a lot of antibacterial mouthwashes, and if you spit a lot throughout the day, you don’t get that nitric oxide conversion. And you’re spitting out a lot of nitric oxide, which means that you could have an increased propensity towards having increased blood pressure. So when you are producing the saliva, you’re supposed to swallow it. This picture on the left hand side is showing you the intersalivary recirculation of nitrate in the body. It’s very efficient as long as you don’t use the bacterial mouthwashes or spit throughout the day.

Proton pump inhibitors, okay, they’re this very, very commonly used strong antacid, oftentimes used to heal gut ulcers. But we know that it decreases most of your hydrochloric acid that you would otherwise produce. So from a perspective of gut health, we know that when you look at people on proton pump inhibitors, we’re talking about Nexium, Prevacid, Prilosec, Aciphex. Some of them are over-the-counter, some of them are prescription, but it tends that people stay on those long-term, and that’s never the way they were intended to be used.

They were intended to be used for eight weeks, and then you get off of them. And usually it takes eight weeks to heal a stomach ulcer. So people on PPIs, they have an abundance of oral and upper GI tract commensals, increased Lactobacillus, especially Strep. And a lower abundance of gut commensals, simply because your pH is so alkaline when you’re taking them. So the outcome of this study said that you are altering your microbiota by taking these PPIs. And really, reduce unnecessary prescribing of them. Don’t let people stay on them long-term.

Now this is a very interesting study with a very interesting title, too. And I’ll let you guys look it up to see the title. I couldn’t believe that they actually published a study with that title in it. But anyways, gut microbiome and cancer. What they found is that alkaline agents such as CTX and Cisplatin, they’re significantly attenuated when people take antibiotics or if they use them in germ-free mice. And so they know that the microbiota is involved in reprogramming intratumoral myeloid cells. So they know that gram-positive bacteria, such as Actinobacteria, actually makes chemotherapy work better. And specifically, the microbiota is important for immunomodulators such as anti-interleukin-10R and toll-like-receptor-9.

So their proposal is to use a symbiotic approach. So you’re going to do everything that you normally do. You’re going to diagnose the cancer. You’re going to find out the extent of the tumor, any nodal involvement, any imaging that you need to do. And then the second step here would be metagenomics, diagnosis of dysbiosis, what bacterias are too high, what are too low. And then treat them therapeutically, before the chemotherapy starts, with a prebiotic, potentially a probiotic as well, to increase those good bacteria and work synergistically, and actually augment the benefits of the chemotherapy, so the chemotherapy will be more effective.

So we have a lot of people walking around with autoimmune diseases. And that’s essentially that your immune system never turns off. It’s constantly active. Most of the studies have been done on inflammatory bowel disease. So this was looking at fecal microbiotic transplantation in pediatric Crohn’s disease, they had at least a moderate to active disease. Now PCDAI is a pediatric Crohn’s disease activity score. Their scores were 10 to 29. Under 10 is remission. Over 30 is a very active Crohn’s disease score. And they had their parents donate their fecal samples to these kids. And their follow-ups were at 2, 6, and 12 weeks. So what they looked at for markers, or PCDAI, how they felt, the CRP level, which is inflammation in the body, and then fecal calprotectin which is correlated very highly with E. coli, which is correlated with inflammation in the large intestines.

Essentially, Crohn’s disease, what they’ll see, and it’s pretty consistent, they’ll see a decrease in Bacteroidetes, decrease in Firmicetes, specifically Clostridia genus, which is F. prausnitzii, and an increase in Proteobacter. So looking at the baseline levels here. The PCDAI, remember, that under 10 is remission, over 30 is very active. So these people started with a score of like 19.7, and at two weeks they seemed to have the most benefit on decrease. Okay. And then at six weeks it went up a little bit more.

But at 12 weeks, still 5 out of the 9 people at 12 weeks were pretty much still in remission, under 10. CRP, a little bit different, starts at 2.4, had the most benefit at 2 weeks. And then it kind of creeped up around 6 to 12 weeks. Then at 12 weeks, for most people, was not very much different than what the baseline score was. Calprotectin, again, the most benefit was at 2 weeks, and then a variable response at 6 and 12 weeks.

What they found from this study is that the parents that had the most different microbiota versus their children did the best. So the children were more likely to engraft and accept that new fecal sample if it was much more diverse than what they had in their body already. So that’s something that we need to check into in the future. Because if you’re giving somebody a fecal sample from somebody that’s very similar in bacteria, you’re not going to get much benefit there.

This was a study that looked at FMT for ulcerative colitis. They had a treatment group of 36 people. They had a placebo group of 34 people. And then they did a retention enema. So they essentially gave these people an enema once a week for six weeks. Now remember that these people didn’t necessarily know they were in placebo, but they had to sign a consent form saying that, “We know we possibly might be in a placebo group.” Because if somebody’s putting an enema up you, you don’t know what they’re giving you. So what troopers to participate in the study knowing that you’re going to get an enema once a week for six weeks. And what they did is they tried to define them all like standardized scores for Mayo clinic score, IBD questionnaire scores. And essentially they didn’t find any difference in the scores at all, how the people felt at the end of the study.

However, this was the first study to show that in seven weeks, active ulcerative colitis, because these people had very active ulcerative colitis that really was refractory to any other treatment. Nine out of 38, 24%, people were in remission versus 5% for placebo. And they did find that it definitely improved bacterial diversity. So they said that future studies should actually intervene before the inflammation gets so great, because it’s a very toxic environment for ulcerative Crohn’s disease once it gets underway and gets a good foothold. So they’re saying that they’d probably have a better success rate if you look at people that are newly diagnosed with ulcerative colitis, with FMT. Because that’s still a pretty low percentage, 24%.

Now this is looking at FMT for people that were refractory for Crohn’s. All these people were put in the treatment group, and the FMT was placed via gastroscopy. And the most benefit by far was shown one month after the FMT. And they know that it changed the microbiota, because it decreased the CRP, it decreased erythrocyte sedimentation rate. It increased IgM at one month after. And within a week, it increased CD3, CD4, and CD4 to CD8 ratio. So again, very good effects on the microbiota by giving the FMT sample.

They said that people did feel better. Their productivity outside of work improved, because they felt better. And this one had 23 different donors that donated, and they did fresh versus frozen. It’s a lot easier to do frozen, because the person doesn’t actually have to be there. And this study actually said that there was much better clinical improvement, 16 out of 23 people, and much better remission, 13 out of 23, with a fresh FMT sample versus that of a frozen, in regards to clinical improvement and remission. And you can see that in the numbers.

Now across the board with Crohn’s disease, they’re showing a decrease in this bacterium called F. prausnitzii. And they know that this F. prausnitzii is actually predictive of relapse in patients in remission. So we know that when F. prausnitzii is actually abundant, it produces these bioactive peptides named MAM. And MAM helps express and block NF-kappa-beta activation and interleukin-8. And so what they’re saying is that perhaps in the future they’ll be using F. prausnitzii to constitute new drugs for Crohn’s disease, because they know across the board, Crohn’s disease individuals seem to be very deficient in this.

Okay, so we’re wrapping up the studies on IBD. Stay with me here. So there was another study done on the gut microbiome and inflammatory bowel disease. And they wanted to see if there was a geographical location effect. So they recruited 30 people from Germany, 30 people from Lithuania, 30 from India. They did three sigmoid biopsies on each individual. And what they found is that the microbiota is definitely influenced not only by age, but based on geographical location, what environmental pollutants that you’re exposed to, and what diet that you’re eating. And two consistencies that they saw, again across the board, were Clostridium Leptum is there was a decrease in F. prauznitzii in Crohn’s disease patients, and there’s a reduced papilio bacteria in Crohn’s disease and ulcerative colitis in all three origins, meaning people from Germany, Lithuania, and India. So we’re seeing some consistencies with IBD.

Now what kind of diet should you use for IBD? Well, just so you know, Crohn’s disease seems much more responsive to diet treatment than ulcerative colitis does. We know that Inulin is a very strong and potent prebiotic. It increases production of F. prausnitzii. And we know that if you eat a plant-based diet, like either the South African diet or a vegan diet, it does increase your short chain fatty acids, it significantly increases butyrate. We know that glutamine and arginine are very beneficial. They have an anti-inflammatory effect. They’ve shown that in mice, and they’ve shown that in pigs. And the same with threonine and tryptophan is that they’ve shown to have an anti-inflammatory effect in reducing colitis in pigs and mice.

Tryptophan, of course, is a precursor for serotonin, and butyrate does affect production of serotonin, which we’ll be talking about in a couple more slides. Now they do recommend calcium and vitamin D. Not necessarily because of its bone-strengthening effects. That’s part of it. But vitamin D has an anti-inflammatory role, and so that’s why they recommend vitamin D specifically for that. Omega-3s, we know that’s a mild anti-inflammatory. And then tailored probiotics. They’re saying that either they can make them and you can swallow them, or they can make them and you can take them as an enema. Either one of those might be beneficial.

Now this is looking more at the metabolic response to the microbiome. So this is looking at postprandial glucose receptors. This was a study done in Israel. They looked at 800 subjects that weren’t previously diagnosed with type-I diabetes. But they did recruit people that either had an overweight BMI or obese BMI, as you can see by the numbers there. They did a continuous glucose monitor on them for one week. They logged their activities. It was a very precise and specific study. They looked at food intake, exercise, sleep. And they gave them 4 different standardized meals of 50 grams of carbohydrate, because they knew that people would respond differently to different meals, and they did. Some people had a high glycemic index response to some of the meals, and some people didn’t.

And so what they did is based on the results of that, they made up an algorithm that they could use that would predict the PPGR to meals, so postprandial glucose response. And we know that PPGR was positively associated with BMI, systolic blood pressure, and C-reactive protein, which of course is inflammation in the body. So of this 800, they took a sub-sample of 26 people to look at good diet versus bad diet. Good diet means that it didn’t really affect their postprandial glucose response that much.

Their glucose stayed lower. Bad diet means that they know that these meals definitely increased their glucose response. They had them follow those diets for one week. One week was enough time to show that the good diet reduced Bifidobacterium, so it will allow for greater weight loss. It increased Bacteroidetes, which lowers BMI, and lowers resting blood sugar. And it increased Alistipes, which lowers BMI. So again, very, very beneficial when you can control that postprandial glucose response.

 

All right, so this is looking at, “Is there something less invasive that we can do than a fecal microbiota sample to find out what’s going on with the bacteria in the body?” So this was a study done in rheumatoid arthritis individuals. They had 77 in the treatment and 80 in the control. And they found that dysbiosis can be detected not only in the dental plaques on your teeth that they scraped off, but it can also be detected in saliva and the gut. And when they’re referring to the gut, they’re referring to the large intestines fecal sample. And they found that there were certain decreases in things across the board. Haemophilus SPP depleted in rheumatoid arthritis at all sites. Lactobacillus salivarus too high at all sites, and negatively correlated with CRP, anti-CCP, which is used to detect rheumatoid arthritis, and RF, which is also used to detect rheumatoid arthritis, its rheumatoid factor.

They also found that transport and metabolism of iron, sulfur, zinc, and arginine were altered by the microbiome. So this gives us another area that we can actually investigate and look into, because certainly that would not be beneficial, and we want to see how we can change the microbiome so those things are not altered. But the outcomes of this study said that use the oral microbiome for prognosis and treatment. It’s not very invasive. You just get a spit sample, and find out what kind of bacterias people have a preponderance of.

Okay, so the gut brain axis, it’s a two-way street, and it affects just about everything. So the way that I can simply just recall it is you have a fire in the gut, and you don’t extinguish that gut fire. And then it becomes a fire in the brain, and you don’t extinguish that gut fire. And then it becomes a towering inferno, which leads to autoimmune disease. And so the gut affects the brain, the brain affects the gut. And all these other things, behavior, anxiety, autism, pain, they’re all affected.

They’re all affected by your microbiota. And when your microbiota gets out of control, and you end up with dysbiosis, you start to have decrease in some of the precursors that are going to make you feel satisfied from eating, like peptide YY. Serotonin precursors that make you feel satisfied. So you’re not feeling satisfied from that food. And furthermore, when you eat the food, you might actually feel sick because of this intestinal permeability issue, brain inflammation issue, and out of control autoimmune disease.

So De Palma took germ-free mice, which is the standard. They’re taking out the microbiota to see what the effects of the microbiota is. So he looked at germ-free mice with stress, and how he did this is he took their moms away. And so maternal separations cause a tremendous amount of stress. It actually altered the hypothalamus pituitary axis. Then adult mice were colonized with the microbiota of the stress mice, and it led to distinct microbial changes. After colonization, behavior was altered in stress, but not control, mice. This is huge. So stress-induced changes actually caused intestinal dysbiosis. And dysbiotic gut was required to induce anxiety-like behavior. So this is showing you that the gut affects the mind, the mind affects the gut. They are linked.

And this is showing you that serotonin, we make about 80% of serotonin in our gut. And although the tryptophan enzyme is rate limiting, the typical tryptophan that we have in our diet is not limited at that point. So you can actually supplement with tryptophan, and when it over-exceeds that rate limiting capacity to generate the 5-HTP, then you’ll stop making serotonin. So there’s a feedback mechanism on it. But serotonin affects so much. Not only our emotions and suppresses your appetite, but it also affects your gastrointestinal secretions, your motility, your colonic tone, your pancreatic secretions. So it plays a huge role in your ability to digest food, and digest it adequately.

There are a number of bacterial strains that we could actually give people, that have studies on them, to show that they actually produce serotonin. And so that’s something that we can look at in the future as well. If somebody is depressed, perhaps give them a probiotic, and at that point they call them psychobiotics. And so Faecalibacterium, specifically, may actually protect against depression. And Enterbacteriaceae and Alistipes, it could actually indicate that there’s some kind of intestinal permeability issue going on.

They actually did a survey on 100 college students to see what they eat, as far as fermented food, pickles, kimchi, sauerkraut. And they found that the ones that ate more fermented food, and they exercise, because exercise relaxes you, the beta endorphin effect, and so forth, they had reduced anxiety. So they found that when they gave students a mix of Lactobacillus helveticus and Bifidobacterium longum, they showed less anxiety and depression.

 

Okay, so this is the vagal nerve. So this little hickeymajigger that sticks down there is called the uvula. And no one really knows what role it plays. Maybe it moistens your throat. But anyways, when you say, “Ah,” and you open your mouth, and you say, “Ah,” your uvula should go up, and the upper palate around it should arch like an M. Okay. If those don’t arch up, and the uvula doesn’t go up, that means that you have some sort of vagal nerve dysfunction. So there’s a number of exercises that you can do to actually improve the vagal nerve. Now what’s so important about the vagal nerve? Well, one is it increases hydrochloric acid, but it also affects motility of foods going through your digestive tract, and it also helps release digestive enzymes from the pancreas so that you digest your food. So it’s a tremendously huge role.

Now if you don’t have enough hydrochloric acid being produced, which you don’t if your vagus nerve is dysfunctional, you’ll kind of feel like the food’s just sitting in your stomach, because it is. You need hydrochloric acid to allow that food to digest, and to transit into your small intestines. And so one way that you could actually increase your hydrochloric acid naturally, put lemon juice or lime juice in your water, and that increases hydrochloric acid production naturally. So there are a number of exercises that you can do to actually strengthen your vagus nerve, if you go, “Ah” in the mirror and see that it doesn’t increase.

You can gargle, but you have to gargle to the point that your eyes are watering. At the point that your eyes water, you know that you’ve stimulated the vagal nerve. Okay, it usually takes about one minute. I wouldn’t do it with mouthwash, because you might choke on it. I would just do it with water. Singing and chanting, because you’re enervating your vagal nerve when you hum. It enervates the vagal nerve. You can do posterior tongue brushing. My 11-year-old practices this every night. She brushes her tongue, and she comes out, and she goes, “I think I stimulated my vagal nerve,” because she gags.

So the gag reflex is stimulation of your vagal nerve. Tongue stroking, so everyone in here can do this. You put your tongue on the roof of your mouth, right behind your teeth. And you’re going to stroke all the way back as far as you can go, and then come back up, and then you’ll have your tongue on the back of your front teeth again. That’s something that you could do when you’re sitting at your desk. No one will even know what you’re doing. That stimulates the vagal nerve. There’s something else called the dive reflex. The vagal nerve is the tenth cranial nerve. It’s parasympathetic nervous response. So relax and digest. That’s what they say. Relax and digest. The vagus nerve helps you relax so you can digest.

The dive reflex is, you want to accumulate a lot of saliva in your mouth, and then you dive your tongue into it. And at the same time that you’re diving your tongue into that saliva, you put your head in a bowl full of ice water for one minute. That stimulates the parasympathetic nervous response. That stimulates the vagus nerve. And they say that it causes a relaxation effect for up to 60 to 90 minutes after you’ve done it. So dive reflex is something that we can easily do, and very effective. Ear lobe pressure, so just at the base of each ear lobe, you just put some gentle pressure on each ear lobe, and you hold for one or two minutes, and that is stimulating the vagus nerve.

Now there are other ways that we can stimulate the vagus nerve too, coffee enemas. If you’ve read anything by Datis Kharrazian, he has a number of books out on, “Why is my brain not working?” And coffee enemas actually will stimulate the nicotinic cholinergic receptor response, which means that it gives you the urge to defecate. So once you put the coffee enema up, and they do recommend organic coffee, make sure it cools off before you put it up, it gives you the urge to defecate.

So when you reduce and discourage that urge to defecate, you’re actually firing your frontopontine brain, your frontal brain, and it enervates the vagal nerve. It actually makes the vagal nerve much stronger by actually retention. So they say with coffee enemas, start at five minutes of retention, and try to work up to 10 or 15 minutes of retention. Because that’s establishing the gut brain connection again, so the vagal nerve starts to work appropriately, as it should. You’ve got to strengthen it if it’s dysfunctional.

Now these are very exciting results from a study that we did for Alcat, Cell Science Corporation, last spring at Northern Illinois University. And it’s the first study that I know of that actually looked to see that foods that we are eating actually could have an effect on our total body inflammation. So just to give credit to my other researchers that helped me with this, Masih Shokrani is a medical lab research professor at NIU, and then we had some assistance from a graduate student, Jodi Hoppensteadt. And Josephine Umoren is also a nutrition professor at NIU. So it was a pre/posttest double blind study. I had no idea what the subjects were on.

Only Cell Science Corporation knew who was in the placebo, who was in the treatment. They sent us lists for the subjects to avoid, and we gave them guidance on what they needed to avoid for the study time. The treatment group got the correct list of foods to avoid, meaning what was in the red column, and their orange column, and their yellow column, as well as their blue. And the placebo group got a false list of foods to avoid. And they knew this up front, but it was unblinded at the very end of the study. That’s the only way you can do a double blind study is if they don’t know who’s in what group, and I don’t know who’s in what group. So they followed a four week elimination diet based on the Alcat results. They had their blood samples, body composition, and medical symptoms completed day one and day 30.

Just to give you an idea of who would qualify for this study, we did something called the disease symptom inventory, and they had to have a three on at least two of these. Now the DSI is about 20 questions, but when I shrunk it down to 20, you couldn’t read anything. So I figured it was good just to abbreviate it to give you an idea of some of the things that we were looking for. We weren’t looking for people that were totally healthy to come into the study. We wanted people with some inflammation present, some kind of GERD, or eczema, anxiety, something to tell us that they were having food intolerance issues. We had no trouble finding people.

So this is the exciting part. You can see that in the blue, the treatment group, from day 1 to day 2…day 2 is day 30. It’s two time points. But this is looking at SAA, which is systemic inflammation in the body. The treatment group decreased from day 1 to day 30 in their total body inflammation. This is huge. And the placebo group, the one that were following the incorrect list of foods, their inflammation actually increased, and this was at the 0.001 level. So very, very significant results to show that there was decreased inflammation when you stay away from foods that you have intolerances to.

Now what we would expect with a study is when you’re restricting any kind of foods, even it’s 5 or 10 foods or more in some cases, your BMI is going to go down. Once you become more aware of what you’re eating, typically your caloric intake drops and your BMI is going to drop. But you can see that the treatment group had a much more significant BMI drop than that of the placebo group.

Medical system questionnaire. This was after the disease symptom inventory form was given. And the MSQ looks at head, ears, eyes, nose, throat, overall well-being, energy level, joint pain, “How are you feeling?” And so what we saw with that is that people in the treatment group felt significantly better from time one to time two. Placebo group had a benefit in feeling better, but not nearly as much as the ones that were in the treatment group. Now just to pull out one person in particular…and there were probably about 10 of these people that I saw in the study.

The one gentleman came in with lung nodules, and they didn’t know what from, what have you, and he ended up in the treatment group in our study. And his MSQ, his medical system questionnaire score at the beginning of the study was like 160. He was not feeling good. It was very, very, very high. And at the end of the study, it was 11, which means that he felt so much better. So he felt so much better, he was so compliant, he laminated his card of foods. He carried it around with him everywhere.

Any time he went out to eat, he used it. Any time he went to the grocery store, he used it. And when he went back to the doctor, he said, “What are you doing? Your nodules are going away.” And so he said, “Well, I’m following this elimination diet from Alcat Testing.” And he said, “Whatever you’re doing, keep doing it.” He said, “Your nodules are going away.” So this guy was just so, so happy about his results, and said that he was going to follow it forever. So it was very, very cute.

So other things that we looked at, and this is really just showing a main effect for time. It was lower at time two than it was time one. This is myeloperoxidase. You see it a lot high in rheumatoid arthritis. It decreased in both groups. Interleukin-6, now that’s the inflammatory cytokine that’s supposed to drive the boat. Where it goes, everything else follows. And even though it did increase in the treatment group, it increased a lot more in the placebo group here. And then mean fat percent, of course, when they’re losing weight, they’re probably going to lose fat percent, or at least you would hope. And they did, in fact, lose fat percent. The treatment group had a more significant decline in fat percent than the placebo group.

So, very exciting. It showed elimination of inflammatory foods has a positive impact on SAA, BMI, and medical system questionnaire scores. And so Alcat results may actually positively influence inflammatory markers, body composition, and just how you feel. And I know that the people that I did interview, we interviewed everyone at the end of the study, said that they were going to continue eliminating those foods because they felt so much better when they stayed off of them.

Now how do we pull this all together? Because it’s a lot of information. All right. What we know is that the gut microbiota, it does help regulate glucose and lipid metabolism. A lot of the newest studies are being done on metabolic syndrome and type-II diabetes. This has huge implications, because almost 60% of our population is either overweight or obese right now. Type-II diabetes is rampant not only in adults, but also in children now. And coronary artery disease continues to be the number one killer of men and post-menopausal women. So if we can get a hold on how to change and alter the microbiota to reduce our risks of obesity, diabetes, and CAD, that’s going to be a huge player in the market. We also know that the microbiota significantly influence your immune system. Not only your innate immune system, the immune system that you’re born with, but also your acquired immune system.

And then how do we optimize the microbiota then? Well I think that you’re going to see a lot more research done on prebiotics, especially the Akkermansia muciniphila and the tailored probiotics. And then, in one of the studies, they found that some people did significantly better than others. In one of the studies on IBD, they had two donors. And the subject that got donor B’s stool via enema did so much better than the ones that got donor A. So they’re beginning to identify super fecal donors.

In this study specifically, the fecal donor had more Firmicutes, lachnospiraceae, and ruminococcus in their stool. So there are going to be more scrutinizing of people that donate their stool samples. So what we know is that we don’t want anybody with intestinal dysbiosis donating their stool, because you can actually acquire intestinal dysbiosis just by getting a stool sample. We showed that with rats and anxiety, right? You preferably want a lean person. You don’t want them to have any mental health issues, and you don’t want them to have any anaphylactic allergies.

Now this is really interesting. PubMed, Garzorz published something last month. And he showed a male that got a bone marrow transplant, a 46-year-old male got a bone marrow transplant from his sister. She had an extreme anaphylactic allergy to kiwi. And this man developed it after he got the BMT from her. And they specifically linked it to her giving the bone marrow transplant to him, and he got oral allergy syndrome twice when he consumed kiwi. So what that tells me is there’s a lot that we don’t know about FMT.

When we were donating blood back in the ’70s, and they didn’t know to screen for AIDS, and they didn’t know to screen for this and that, I don’t think that we can just jump out and say, “Well, okay. Let’s just do FMT on everybody.” I think that we have to be very careful and very precise on choosing the donors for FMT, specifically for IBD. You want to look for a fecal donor that is high-Firmicutes, and specifically probably F. prausnitzii, because that’s always low. So you can maybe screen out people that are actually getting to be better donators for these individuals. The individuals are going to do better, because the more microbial diversity in the stool sample, it seems like the better that donor does if it’s much different than what they have. But those are some of the guidelines that I would go through after doing a review of 2015 and ’16 literature on this. So I can open it up to questions at this point.

Samy Puccio, Vice President

Is there a relationship between zonulin proteins and microbiota?

Dr. Lukaszuk

That’s an excellent question, because we know that zonulin is affected with intestinal permeability, fire in the gut. And when I did this extensive literature review for this presentation, I didn’t see anything. I did see things about zonulin, but I didn’t see anything in relation to zonulin and the microbiota. So I still think that that’s an untouched area but should require further research to find out.

Jim Hoover, Sales Director

Okay, the question here is, “Did you look at the microbiota post & pre Alcat? And if so, what was the difference?”

Dr. Lukaszuk

Hindsight is always 20-20, and we didn’t even have it on the radar screen at the time that we did the study. But certainly, if we do more studies, if there’s a capability of doing it, that would be fantastic.

Amy Pieczarka, RD

Thank you, Judy. You had mentioned that you measured the myeloperoxidase and interleukin-6…well, I actually had a question about the microbiota as well. You did not measure that. The placebo diet, you said you were not aware of that during the study. Were you aware of that afterwards? Do you know what the …and did people all, were they all on a different placebo or all the same one?

Dr. Lukaszuk

They were all on the same types of foods that we removed, yes. We removed the same kinds of foods for all of them that were pretty obscure foods, let’s say. I don’t want to get into big details, because if we do more studies, people will be able to figure out what treatment group they’re in. But they were unblinded at the end of the study, and we gave them the correct list of foods to avoid then.

Amy Pieczarka, RD

Okay, great, thank you.

Samy Puccio, Vice President

Was there a correlation between the oral measurement of the Firmicutes and Bacteroidetes, and the stool?

Dr. Lukaszuk

Well, they found that in rheumatoid arthritis, but they’ve not looked at it in IBD yet. So that was the first study to do that in rheumatoid arthritis, and that would be a really nice non-invasive parameter to do so. That’s something that we could do for a future study very easily. It’s just a saliva sample. If we find that there’s a correlation across the board for all autoimmunes and other things as well.

Roger Deutsch, CEO and Founder

Dr. Lukaszuk, SAA is something that’s new in the literature. Could you tell us a little bit more about it, and what you think the clinical significance of that profound change?

Dr. Lukaszuk

Well, SAA seems a lot more precise than CRP in inflammation. And so it’s a robust measurement, and we’re very excited with the results that we’ve found with this study, because it is a clear indication that there is a reduction in total body inflammation. Because there was an interaction effect, meaning that the treatment group went down significantly, and the placebo group went up for SAA.

Roger Deutsch, CEO and Founder

Any correlation in the placebo group with the increase in SAA levels and symptomatology?

Dr. Lukaszuk

I don’t know if we looked at that parameter for SAA in the placebo group. You mean in MSQ score?

Roger Deutsch, CEO and Founder

Yes.

Dr. Lukaszuk

The MSQ score did go down. We didn’t correlate it with SAA. But the MSQ score did go down for the placebo group as well. It just didn’t go down nearly as much as the treatment group went down.

Roger Deutsch, CEO and Founder

Thank you.

Moderator

I have a question with regards to I guess the influences of stress. In the study with the mice, the trigger was maternal separation. For humans in modern life, what are the triggers? And are there any clinical insight strategies to address those?

Dr. Lukaszuk

Dysbiosis. It causes anxiety. Usually when you see people with mental health issues now, they can attribute it to microbiota changes. So as I said, it’s a two-way street. The gut affects the brain. The brain affects the gut. And so if you don’t address the gut, sooner or later, your brain will end up foggy, and you’ll end up having anxiety. It could be anxiety because you’re eating a food that you’re intolerant to, because now you’ve got a leaky gut. And your stomach hurts because you ate the food, so then you start hyperventilating, because you can’t take a deep breath, because your stomach hurts. But depression, people don’t want to get out as much, because they don’t feel good, and they’re restricted in what they can eat. So it’s a real issue.

Amy Pieczarka, RD

Judy, for the study that was done on the bariatric surgery and the microbiome being improved afterwards, is that because of the ratio of the Firmicutes to the Bacteroidetes? Did you measure that?

Dr. Lukaszuk

They didn’t find that. They found that the biggest difference was there’s an increase in Proteobacter, which is kind of puzzling I guess you could say. Because it’s said to have more inflammatory effects. And so it might be simply that because Proteobacter is a phylum, so it might be again that it’s a bacterium. There’s a smaller bacteria under there, that there’s a differentiation, and we’re not seeing that separation yet. Yeah.

Moderator

Thank you very much for taking the time to present with us today, and to answer our questions.