Zonulin, intestinal permeability and immune mediated disorders: facts and fantasies

Dr. Blyweiss

Hello, and thank you for taking time out of your busy schedule to join us today. My name is Dr. David Blyweiss, Chief Medical Officer for Cell Science Systems. The purpose of this webinar series is to broaden your understanding of the immunological differences between innate and specific immunity, and how those differences manifest in disorders ranging from gluten and other food sensitivities, to more severe manifestations such as celiac disease.

In this lecture, entitled “Zonulin, Intestinal Permeability & Immune Mediated Disorders: Facts & Fantasies”, Dr. Alessio Fasano, Chief of the Division of Pediatric Gastroenterology and Nutrition for Massachusetts General Hospital and Director of the Center for Celiac Research, will be discussing his research into zonulin, intestinal permeability, and immune-mediated disorders. Be sure to stick around at the end of the lecture, as I’ll be giving you information on some of Cell Science Systems’ innovative laboratory testing.

Dr. Fasano

Nevertheless to say that I feel so inadequate compared to Dave. He got such cool videos. Where is he? Dave? Where did you get those? Anyhow, so…YouTube? That’s not cool.

All right, by the way, how much time do I have? It’s an hour what? We’re not going to go two hours, because otherwise, you guys will kill me. Also because this is such a controversial issue, that I would like to leave us a little bit more time for discussion because I’m pretty sure that there is a lot of stuff that we can discuss about this issue of leaky gut.

As a matter of fact, this is an interesting topic, if you wish, because it’s been a black box and it still is for many, many years, because there are lots of misunderstandings, there are very few facts, a lot of fantasies. what I want to do in the next hour and a half, let’s put it this way, is really to be factual and tell you what we know about this situation of leaky gut. And again, it’s an interesting story if you are interested in how stuff works, and how we stumbled upon it in the entire business of understanding a little bit more the situation.

The reality of the story — and this is the objective — is that, again, if you allow me to be a little bit philosophical here, we are whatever we are because we are really the quintessential of this interplay between what mother nature decides to put together as a project; i.e., genetically speaking, what we are, and the environment that surrounds us. It doesn’t come as a surprise that if you take identical twins who are genetically identical and you split them at birth, one of these kids is raised in the North Pole and the other one is raised in Africa. At the end of the day, they will be very two different individuals. The way that I would like to conceptualize, if you’ll allow me to, my Italian nature has to surface here and there. It’s like to have a wonderful, superb marble block like Michelangelo was given by the De Medici in Florence.

The marble block is just a morph. It doesn’t tell you anything. It depends on who really would work on this marble block, this sculpture, what you’re going to have to have out of there. And if you visit Florence and you have the chance to see not the fake David outside, but the one that’s really in the office, it’s astonishing. And again, Michelangelo got the chance to work on this marble block when he was 19, a kid. And this marble block was sitting there for 50-60 years because there was a lot of discussion how precious as a beautiful marble block, and they didn’t want to screw it up and give this task to people that would not do the job right.

And the year would come. This young kid would come across and say, “I know what is in there. I can pull it out. Give me the chance, and you will see something astonishing.” To get out of the metaphor, the marble block is our genetic makeup. Michelangelo was the environment that carved out of the marble block, this beautiful statue.

The question is how does this interplay between genes and environment translate in what we are, actually, as human beings? Now, if we want to be factual and molecular and mechanistic, the question is how the genes are within our body would interact with the environment that results in our body so that we become whatever we are? Because under normal circumstances, this interplay cannot happen because they are very tightly controlled, regulated barriers that separate the environment outside of our body to our insides, our genes. But if we need to interact, there must be some way, some pathway, some shortcuts that these two different worlds can find themselves.

Now, here comes the biased view of this process. Because as a gastroenterologist, I really do believe that the largest interface where this interaction occurs are the guts. And the reason why, we’ll see in a moment, there are all the anatomical, functional, and objective findings that seems to suggest that that is the case.

Typically, as we know in terms of the physiological path, when you push physiology to the limits, you from health to disease. This interaction with the environment most likely is also at the heart and soul of a problem that deals with inflammation and autoimmunity. And the question is how we can connect all these dots of our genes, the environment. We discuss a little bit about the macrobiome that is a third key element in this interplay, the battlefield that will eventually get to the clinical outcome, so that we can be in a state of health, or lose that state of health and switch to disease.

That brings me to the concept of the intestinal tract, and this superbly sophisticated and unbelievable organ. Now, when I teach — I teach functional systems — in general, there’s this misconception that the intestinal tract is not a big deal. They are much more noble and sophisticated organs. And I am always in argument with my colleagues. Because after all, what is the intestine? It’s a long tube that makes this magic change from…put one thing on the top that’s food, and get something out of the bottom that is poop. That’s it.

And it’s much more than that. And typically, when I talk about how important is this organ, and we discussed so many times during this day and a half, that it’s not impinged on the GI function, but the function of the entire body, I make a quick statement and I survey with my students that if you allow me, I will do the same over here. Because again, my colleague neurologists say that the brain is the most important organ in our body. The cardiologists say that it’s the heart. The pulmonologists say that it’s the lungs, and so on and so forth.

Let me ask you this. How many of you guys have ever had a stroke? How many of you guys have ever had a heart attack? Just one, okay. How many of you guys have ever had an episode of pneumonia? How many of you guys have ever had an episode of diarrhea? Did I make my point?

But the question is how do you define the gastrointestinal tract? Because any kind of definition is quite reductive for what we’re going to discuss today. The best and I believe most comprehensive definition of gut came from one of my students. I teach also in elementary school. Forgive the spelling here. “The intestine is a long tube, with a clean open on the top, and a dirty open on the bottom.”

 

Appreciate the quintessential here. What are you asking for? What is going on? I heard some “Fish”. I ask her, and she said, “This is the noise that my dad makes from the dirty end.” You can imagine his face when I met him.

Anyhow, what is so important about the intestine? And why do we have to spend time to discuss the matter if this really goes way beyond the gastrointestinal diseases and so on and so forth? Well, the father of all physicians already appreciated that the intestine was the crossroads of all human diseases. This is, again, his statement, “All disease begins in the gut.” And this was a long time ago. I told you what I would have used here, that the gut is not like Las Vegas, and I showed you yesterday the reason why. What happens in the gut does not stay in the gut, but today, we’re going to go a little bit more in-depth about that.

The reality of the story is that the intestinal mucosa is truly the battlefield where friends and foes need to be recognized every day, 100 times a day. And this is because it will give you the balance between tolerance; i.e., state of health, or an immune response; i.e., inflammation, therefore, the state of disease.

And imagine, again, let’s go back on this one, gene environment interaction. This is the largest port of entry of environmental factors that can dictate this balance, this yin and yang. Now, if you shrink yourself millions of times and put yourself on this battlefield, you will see quite a challenging environment. Hundreds of craps a day come through. Some are good friends; nutrients, for example. Some are definitely enemies, like pathogens, that can really harm us. And the intestine has to really decide many, many times a day who’s a friend that should stay there, and who is a foe that needs to get out of there?

Just as a comparison, the second-largest interface that is the lung…the lung has a very monotone function. Everything that goes through needs to get out of there. That’s important for their pollens, bacteria, viruses, particles; they don’t need to stay there.

The skin, the third largest interface with the environment. Not one, we have seven layers to make sure nothing comes through. The intestine can’t do that because otherwise, we’ll be out of business in the blink of an eye. Appreciate the sophistication of the decision making, a process that the intestine has to go every single day. And it’s mesmerizing to me that despite this complexity, mistakes are rarely made. But what are made, we pay a dear price.

For example, celiac disease, that we briefly mentioned yesterday, is the ideal paradigm to understand how we make these mistakes. Because a friend, a nutrient, a protein that come from food will become a foe. This is the classical example of this decision making that goes bad, that goes in the wrong direction.

Now, I’m assuming that you’re not going to see anything here, but when I said anatomy’s functional mechanism suggests that the intestine is much more sophisticated and complex that we deeply appreciate, I really mean it. This is a cross section of the gut with the lumen being here, and the part of the body over here. And these are color-coded cells that are part of this mesh work of mechanistic and very sophisticated interacting cells that makes this decision making possible every day. Starting from the inner lumen, this red stuff here is this layer of a single-layer cell, the epithelial cells, that is pretty much what defends us against this uncontrolled passage of crap from the environment. Single-layer cells, single-layer. And again, going back to the comparison that I told you yesterday, it’s like the walls of a middle-aged city. It surrounds the city so that we are defended by this uncontrolled entrance of enemies.

Immediately underneath, you have these green cells. These are immunological cells called dendritic cells. These are pretty much the soldiers that are able not only to defend us, but to have an understanding of what is going on in here. They already have periscopes that they bring out and see into the lumen, and say, “Who is here?” And based on what they scoop, they prepare the rest of the army for peace or war.

Now also here, extremely abundant are blue, are autoimmune cells, they are the B cells. The ones that they have the best capability of weaponry; i.e., producing antibodies including Ig antibodies that can be secreted within the lumen with special tricks. That’s the reason why they’re called secreted IgA.

And finally, the T cells; the ones that when they go wacko, they really go big time. The ones that have the most powerful weaponry, these are the ones that, for example, destroy the intestine’s celiac disease, of the immune process. But look how they are all arranged. And this really speaks volumes. Where is the function of not only surveillance, but interaction with the environment? Because these soldiers are not there just to fight. They are there also to understand and eventually write a peace treaty with the enemy if it’s necessary. And again, I don’t have the time, nor do I have the possibility to go in deep details.

But the beauty of all this done, it’s mind-blowing because this single epithelial cell layer not only is a wall, a morph. They have antennas, they have sensors, that technically we call pattern cognition receptors that you heard about; the toll-like receptor and so on and so forth that will understand who’s here. Not only that, but if this pattern cognition receptor, they feel danger because there is an enemy that they know that can give trouble to us, will already communicate underneath with all soldiers that say, “Guess what, guys? In the lumen, not now, there is somebody that can eventually give us trouble. Get ready, get prepared.”

What kind of language do they use? Secreting specific chemokines, they are chemicals that attract these soldiers on the battlefield. And this is done regularly every day, every day. And keep in mind that this cross talk eventually leads to the decision that we need to keep these folks out, and eventually going after them, or we need to bring them in. Because we need them here for whatever reason.

And that’s where the essence of antigen trafficking that is so important in this interaction between genes and environment, because without these trafficking of antigens, we would not be wherever we are. The genes, epigenetics, and the state of health and so on and so forth will be totally impossible. Do you appreciate, guys, the beauty of nature in doing all this? This is absolutely mind-blowing to me.

Now, I told you that when you push physiology to the extreme, you spill into pathology. And we discussed yesterday about celiac disease as a paradigm of autoimmune diseases. We also mentioned that the recipe for autoimmunity, until the recent past, called for two ingredients: genes, many, because these are multi-factorial diseases. Not a single disease like cystic fibrosis, thalassemia, and the environment that triggers are known most of the time for celiac disease, we know that’s gluten. There is one that triggers that entire business.

The third element that makes this the Holy Trinity that was not clear until the recent past was the breach of these single-layer cells; in other words, a leaky gut. They allowed these enemies to come through and eventually instigate the autoimmune process in people that are genetically skewed to develop autoimmunity.

And this brings me to the leaky gut, facts and fantasies. Now, again, there are very few facts. There are many fantasies. And that’s the reason why this term, “leaky gut”, particularly among traditional medicine folks, it really gives the bumps. In a sense, they say, “Gee, this is so creepy and not cool because this has been totally vilified.” And there is no evidence that this is true, it’s real, that it can do anything, exist, and so on and so forth.

Again, as usual, guys, there is no black and white in life. There are no people that are right and people that are wrong. There is always the compromise, grey, because that’s pretty much what is coming out for what we understand about this leaky gut situation.

What are the facts? Fact is, again, that this intestine is a very long tube. In an adult, it’s roughly 15-20 feet long. The facts are that this tube is covered by single layer cells, as I told you. In the past, these single layer cells were conceptualized as a floor in which each cell is the tile, and the space in between cells is completely sealed with the grout, cement. All this interface with the environment, this exchanged information, was supposed to happen through the cell, and not in between the cell because that space in between the cell was completely and totally sealed.

Now, if you take the intestine of an adult and stretch this intestine, because it is a tube of 20 feet long. But again, there are valleys and hills, they are called faults. And then if you zoom in, they are these finger-like protrusions that I showed you yesterday, the villi, in each cell. The core of the intestine is not smooth, but it has little bumps on the top, they are called microvilli.

If you take the entire surface and you stretch it out, we’re talking about a double tennis court — 3000 square feet of interface with the environment. Imagine how much stuff is going on, on a daily basis. Imagine how much stuff is going on. Not only that…and again, I don’t want to go too much in details, but it is important to appreciate the dynamic nature of these single layer cells that cover the intestine.

Shrink, again, yourself a million times and put yourself in there. Now, these valleys in between these villi, they are called crypts. In there, there are the stem cells. This is one of the few places in which stem cells are active from birth to death. No matter how old are you, your guts are five to seven days old. That’s the life of an enterocyte, i.e., epithelial cells that cover the intestine.

Why do we waste all of this energy? Why do we waste all of this effort to change the cells all the time? Guess what? That’s a really harsh environment. And you can’t afford a mistake, because if you are bombarded by chemicals, radiation, heat, toxins, so that you have genetic modifications…in other words, that these cells can go through genetic mutations and they don’t work the way they are supposed to, we will be in deep doo doo and nature can’t afford that.

Not only that, the other thing that is mind-blowing to me is these cells. They go through a process, a maturation that reminds me very much of the story of the caterpillar and the butterfly, a very sad story. When they’re immature, they’re down there. They are really ugly. They are really unsophisticated. They are very rudimental. They are not able to do too much. In other words, they look like a worm. Then a biological clock will tell this rudimental cell there is time to mature. And when that information is passed to the cell, the cell from the valley, i.e., the crypts, start to migrate along the villus.

And during this process of migration, during this trip from the bottom to the top start to change and mature in one of the most sophisticated, complex cells that we have in our body to become mature enterocytes with all this machinery to digest and absorb the foodstuff, these antennas to see enemies, the capability to have the immune function, the capability to talk with the neighboring cells, the capability to interact with the nervous system and endocrine system; i.e., a wonderful butterfly. Guess what, though? When they reach that level of sophistication, their life is a few hours, after which they fall off in the intestine like butterflies.

And all this is because we really want to make sure that our intestines are covered by the most efficient, sophisticated, active cells with no chance that they are mutated in their function. And again, when I say “function,” I don’t mean just the digested foodstuffs, but all the sophistication I was telling you before.

Until the ’80s, this concept that these cells were linked to each other by a grout was a dogma. Until a Japanese group in the late ’80s said, “Wait a second, this space in between cells is not sealed. There is no cement. There is not a wall.” Rather, they are doors most of the time closed that technically are called tight junctions. With the door concept is also in place with the concept that sometimes, this door that can be opened and creating a shortcut from stuff in the environment to come through in our body.

Now, until then, the major edict for people that were interested in immunology, but secretly autoimmune disease, was how instigator autoimmunity that, in general, proteins can get into our body? Because one thing is for sure, guys, and this is something that never changed in the past 60 years; proteins, they don’t go through cells. It’s like to pretend that two dozens of elephants will go through a little needle. It does not happen. Because proteins, they love water, so they’re hydrophilic. The cell wall, it’s very hydrophobic, and there is no way that proteins can come through. They are huge.

There was always this edict, “How did that happen?” How did this interaction with the environment happen? How did this trafficking happen? The answer was, “We don’t know, but we know that happened,” and that sufficed, and that’s it.

Now, with this finding that we have doors, there was a complete shift of paradigm to say, “Well, if they’re doors, they may open once in a while. Maybe this is the path to which these enemies; i.e., instigators of inflammation, can come through.” But a question was, “If they’re doors, where is the key? How are these doors modulated? Who decides if and when they’re open?” And for almost another two decades, we have no freaking clue about this, until we stumbled upon this mechanism that led to the discovery of zonulin.

Now, I was privileged to get an award, the Pauling Award, not long ago. My typical style, I decided…rather to say, “Gee, the discovery of zonulin, it turns to be a big deal. It was an act of smart thinking, that I was so cool, that I thought about it and I went after it, and I discovered it.”

That is bull poop because the vast majority of discoveries are made by serendipity, that by the way, it’s my favorite English word. Let’s be frank, the English vocabulary is totally unsophisticated. It’s made by what, 600,000-700,000 words? That’s all. The Italian vocabulary is made by almost 2 million words. That’s the reason why we Italians, when we talk, we go on and on, we use our hands, and so on. You’ve got to use the entire vocabulary.

 

But there is one word that I can give right, how can I really express in Italian, the quintessential content of serendipity? The unforeseen that is there. Something that is lined up and is meant to be. There is no way that I can translate this. And this is a story of serendipity that I think that I want to share with you since I have two hours to fill, so I can do that.

The real deal how this started was that when I, long ago, I went to Maryland. I was very much interested in diarrhea diseases. Remember, I’m a pediatric gastroenterologist, and the ones that I really was working on very hard, because it was claiming many millions of lives, was cholera. A very bad disease, even now, claims millions of lives, particularly in kids in third-world countries.

And again, the long story short is that I put on a task to develop a vaccine for cholera. At that time, we knew that there was only one major weapon that cholera was making that is called cholera toxin; it’s an extremely powerful toxin. And therefore, I engineered this vaccine by taking the genes of cholera toxin out, leaving everything behind with the idea that this vaccine would not induce disease, but would induce an immune response that would protect these kids.

I did everything under the sun. At the level of the bacteria, the level of animal studies, everything worked great, and therefore, we were ready to go prime-time for clinical trials. Now, when we go talk about clinical trials — and we’re talking about early ’90s, and we’re talking about vaccines — we needed volunteers. And guess where volunteers came from? Where? No. Even in the ’90s, they weren’t making too much money at that time.

Man

Medical students.

Dr. Fasano

Medical students. Not prisoners. Because Belmont banned use of prisoners. Medical students. For $250, that’s not a negligible amount of money, the students were recruited. And they were given three options. Option number one: placebo. You drink water, so you’re going to be fine, and you get $250 for nothing. Option number two, you’ve got this vaccine that we know is safe, and therefore, you got, again $250 for nothing. Third option, you got the real deal. You got cholera, but we’ll take care of you.

 

Many kids signed up, because two out of three said, “What a big deal.” Actually, it turned out to be not two out of three, but only one out of three because the vaccine sucks. In other words, sure, the vaccine didn’t give the 40 leaders of diarrhea that the kids who got the real cholera went through. But still, three or four leaders, diarrhea that meant that it’s unacceptable. And when you feel like that, entire generations of workers and investigators are burned on these situations, and the most logical thing to do is just to give up and say, “Forget it.”

And that’s exactly what I did for two or three days. I got drunk, I was very depressed. Literally, two years of work in the toilet, that was it. But then we went to the drawing board and tried to figure out…long story short, we ended up to discover a second toxin outside the cholera toxin that was making these people sick by opening these gates. Never described before. And this toxin turns out to be the reason why these folks had the residual diarrhea. And then when we started the mechanism of this toxin, we realized it was way too complicated that mother nature did all this machinery just to be the target of a toxin that, by the way, makes us sick.

Reasoning that probably cholera, being a smart fellow, was living in something that we make, we went after the counterpart of this toxin, and that’s how we discovered zomulin. At that time, we had no clue what kind of Pandora box we opened. As a matter of fact, I believe this is one of the most fascinating stories that I’ve been involved in terms of what we’ve learned from there.

First of all, we learn that…again, it’s useless to go through all the details. But what we learn is that, again, these black lines here turn out to be pretty much what we were looking for. This is the serum of people that had a high level of zonulin. We, in the meantime, developed a test to measure zonulin in the blood, in the lab, and so on and so forth. But we still didn’t know what kind of molecule we were dealing with.

And finally, one of my students was able to come up with a sequence of this protein, and it turns to be that zonulin is the precursor of this molecule here that is called Haptoglobin 2. Haptoglobin is a very ancient protein that most of you guys probably don’t even know. But all doctors, when we didn’t have the citrate, the CRP, used Haptoglobin as a bio-marker inflammation. It comes in two flavors; Haptoglobin 1, the ones that we know very well. That is, with us for living, a scavenger hemoglobin. In other words, when the hemolysis, the intravascular hemolysis, this guy will capture and eliminate hemoglobin so to avoid oxidative stress of the tissue that can be destroyed that way. It goes vis a vis with inflammation. And comes as a pre-protein single peptide, then is clipped here in two sub-units; beta and alpha.

Much later, as you’ll see in a moment, came this second variant of Haptoglobin that’s called Haptoglobin 2; same story. It comes in a single protein that is cleaved into the cell. And again, there are two sub-units. The beta sub-unit is identical to the Haptoglobin 1. The alpha sub-unit is two copies of the alpha 1 because there’s a duplication that I will tell you in a moment about.

And therefore, people, they can have three possible arrangements. You can be homozygous for Haptoglobin 2, you have two copies of this guy here, there is this one here. You can be homozygous for Haptoglobin 1, so you have two copies of this guy here. Or you can be heterozygous, so you can have a copy of this, and a copy of this, and that’s what you see here.

It turns out to be that zomulin is this guy here that you can barely see, that is the precursor of Haptoglobin 2 or pre-Haptoglobin 2; totally unexpected because we didn’t know. This protein does different stuff for living. It turns to be its premature pre-Haptoglobin form, losing that tight junction, and when it’s mature, that’s something else.

How it’s released, that’s something else that’s also very cool. I showed you yesterday those two peptides from gliadin that can release zonulin. How do they do that? They do by interacting with this receptor, one of these particular cognition receptors that I was telling you in enterocytes that is called CXCR-3 receptor. This is a receptor that was described a long time ago in general presence of the immune cells like T lymphocytes and B and NK cells. But now, we know they are produced by also enterocytes. And the thing that is very interesting, at least to me, that this receptor has been implicated as a target to impeding T-cell-mediated destruction in autoimmune diseases like MS and Type 1 diabetes. This was described a long time ago.

And again, you see here that when you take epithelial cell, that you see here in red decoration with the cytoskeleton and blue, the nucleus. You expose these cells to either gliadin or bacteria, they are the two strongest stimuli to release zomulin. You see zomulin is produced and packed in these blobs here that are green, and released right away. But if you use casein, for example, that doesn’t happen. Specifically, gliadin, and specifically, those two peptides to interact with this receptor and release zonulin.

And once it’s released, zonulin communicates with the enterocytes to say, “Look, you have a gate with your neighbors’ cells. I want that gate open.” And thus, it’s through a very complex pathway that involves the receptor of epidermal growth factor. And again, it’s not worthwhile to go in detail. Suffice to say, that the steps are, they are instigators including gliadin and bacteria. They release zonulin through the CXCR-3 receptor. Once zonulin is released, we interact with this receptor, and we communicate with the cell, “Please open that gate.” And all this happens in a very short period of time, 20 minutes or so. I showed you yesterday, this picture again, just to reiterate the concept that, again, you can go from closed gate to an open gate in a very short period of time due to this release of zonulin that is CXCR-3 mediated.

Mainly because it costs me three months and $500 to do these slides, I want to show it. This is as much as we understand about how zonulin communicates the cells to open these gates. Whenever they release zonulin, really activated intracellular cascade of events, they are very complex. Suffice to say that this tight junction, this gate, are made by a complex number of proteins. The ones that interact with each other and interact with similar proteins in neighbor cells, this is space in between cells that, again, in the past was thought to be totally sealed. Once zonulin comes in, you have rearranged these proteins that will be released from this edge of the cell. It will leak into the cytoplasm of the cell. So you have a situation from gate close to gate open in a very short period of time. In general, it takes 20-30 minutes.

And again, this is a freeze fracture. So if you see the lateral side of the cell, these are the macro-villi, the surface of the enterocyte of the face of the lumen. This is the cytoplasm. This mesh work here represents the tight junction. The more complex, the closer are. And if you have zonulin released, you go from closed to open in a very short period of time. Now, when the stimulus — gliadin, bacteria, whatever — is removed, everything goes back to the baseline in a very short period of time.

Genetics, this is another cool part to the story. And again, we had no clue what we stumbled upon. Evolutionally speaking, Haptoglobin is a very ancient protein. It came into the picture roughly 450 million years ago soon after the fish split from the rest of the animal kingdom. Fish, they don’t make Haptoglobin.

But the one that we are interested, zonulin, it came much, much later; 2 million years ago, 500,000 years after that we split from our cousins, the chimpanzees. Meaning that zonulin is made only by human beings. No other animals make zonulin, at least as formal as pre-Haptoglobin 2. They make zonulin-like proteins, but not exactly the ones that are seen in humans.

By the way, again, this is something that I do quite often. Any idea how many autoimmune diseases afflict humankind? A little bit more than 60. Roughly 75-80. How many autoimmune diseases are these guys experiencing? Zero. Chimpanzees, they don’t have autoimmune diseases. It’s quite interesting that zonulin tends to be one of the 400 genes that are different between the two.

The gene of zonulin sits on this very interesting chromosome that is Chromosome 16; a tiny chromosome that accounts for roughly 5% of the human genome. But on which they are genes that have been located, they are very important for many, many diseases, particularly, autoimmune disease, cancer, and disease in the nervous system. Among autoimmune diseases in the genes, for example, lupus and Type 1, rheumatoid arthritis sits on Chromosome 16. Cancer, acute nonlymphocytic leukemia, breast cancer, lymphoma and myeloid leukemia, and prostate cancer sit over there. And this is the one…I don’t know, I’m not an expert, but my dear colleague, he knows much better than I do about diseases of the nervous system; Lou Gehrig’s disease, leukodystrophy, MS, and autism genes are sitting over there.

When zonulin was discovered and cloned, many investigators worldwide went through the same process and asked, “Which diseases is this gene related to?” And they came up with the same three categories; autoimmune disease, including ankylosing spondylitis, celiac disease, Crohn’s, rheumatoid arthritis, lupus, and Type 1. Cancer; breast cancer, lung adenocarcinoma, so on and so forth. The disease of the nervous system, including MS and schizophrenia.

Among the cancers, what really captured my attention is the glioma. And again, the reason why it did is because again, you heard about this issue that there is a gut-brain axis all the time. And controlling the gut permeability has similar mechanisms that control the blood-brain barrier. And these are a couple of papers that just were published on the role of zonulin in control of the gut-brain barrier in a situation of glioma.

This group from Germany published this in 2009, so already four years ago. And what they showed pretty much is that zonulin, that is the stuff in green that is very close to the perivascular part of the blood-brain barrier of the glioma and therefore in the blood-brain barrier, seems to be up-regulated in gliomas. And the more severe the glioma, the more zonulin is produced, the more breach of the blood-brain barrier occurs. They use the zonulin pressure as a biomarker severity of the glioma.

Just published two months ago is this paper talking about the transmigration on neural stem cells across the blood-brain barrier induced by glioma cells. You heard yesterday that the brain has the capability to have stem cells. They’re not completely gone. They, under specific circumstances, can be revitalized to do stuff.

Now, these folks were able to show through cell lines and animal models that zonulin is present in these specific glioma cells, and when it’s released, pretty much what they do, they pretty much increase the permeability of the blood-brain barrier that is measured by this parameter. It’s called transepithelial electrical resistance. The lower it is, the more open are the tight junctions, the leakier is the blood-brain barrier.

And they also were able, by injecting these cells into the systemic circulation of mice, to see that these cells will eventually get to the brain by crossing the blood-brain barrier and reach the intracerebral gliomas. And you see here, the staining in blue…I don’t how much you can see, but you see, this is a cross-session of that cell. You see the circle, and this is the sagittal section along the axis section of a vessel of a capillary of the blood-brain barrier. It looks like, really, that that’s the scheme that they came with.

If this is the blood flow, and this is the brain, and these are the tight junctions that control the blood-brain barrier, what they conceptualized that the astrocytes and the gliomas will eventually, on this other side of the glioma cells, will release zonulin, will make this leak, and allow these cells and other stuff to come through that is pretty much the heart and soul of the program that you have in brain tumors.

 

Again, just think about this. Half an hour ago, maybe 20 minutes ago, I told you how this story started. If when I fail this issue of the color vaccines, somebody would tell me that this would be coming across, I would say, “You must be out of your mind? What are you talking about?” It was totally unpredictable.

Now, again, going back to the genotype of zonulin on this Haptoglobin, I told you that it comes in three different possible arrangements of genotypes. Homozygous for Haptoglobin 1, meaning that you do not produce zonulin. Heterozygous, meaning that you have one copy of the zonulin gene, or homozygous, they have two copies of the zonulin gene.

Now, this is a comparison of several autoimmune diseases. You’ll the disease Chron’s, schizophrenia, and chronic kidney diseases. Look at just for a second, the black column here. We’re talking about the distribution of these three different haplotypes…genome arrangements, genotypes, in the normal population. This is done in different continents, so there is a little bit of difference among the different groups.

But the bottom line is that roughly between 10% and 20% of the human population worldwide do not produce zonulin. Roughly half of the population, 46%, they produce one copy, and the remaining 30%-40%, they produce two copies.

Look at the red column. These are the people that are affected by specific autoimmune diseases like celiac, Crohn’s, schizophrenia, chronic kidney disease. You have a dramatic reduction of the representation of the people that do not produce zonulin. That goes down two, three times. And it’s counterbalanced, but increased of the ones that have two copies, meaning that people with autoimmunity are skewed compared to the general population in terms of zonulin gene presence and number copies. And now, it looks like, by several studies, that the more copies of zonulin that you have, the more severe is the disease.

If you’re homozygous with zonulin and you come down with an autoimmune disease, your mortality rate is twice as much compared to one copy, or of course, zero copy, you don’t develop the disease. That is pretty much what has been described so far.

And here is another example of zonulin in inflammatory bowel disease. These are people that have Crohn’s disease on this drug that is a non-steroidal anti-inflammatory drug, and you see the amount of zonulin there. These are in remission. These are the ones that are on the drug, but they are not in remission, so the zonulin is not coming down. And these are the ones that are not treated at all, and you see the highest level of zonulin in the serum of these people with Crohn’s disease.

Now, I told you that zonulin is specific of humans. So animals, including mice, they don’t make zonulin. But you can manipulate the genome of mice by introducing the zonulin gene. This is the normal mouse — how cute is that? That, of course, they have only the Alpha 1 chain, so they only have the Haptoglobin 1. This is a transgenic mouse in which we introduced one copy of the zonulin gene, and this is another one in which we introduced two copies. This guy does not make Haptoglobin 1, but may make the folks, the human folks that have two copies of the zonulin genes.

And when you put these animals under stress; specifically, you treat these animals with a chemical to make their intestines inflamed, and you look at the smaller and large intestine, you see that compared to normal mice, the damage when you expose these animals to these chemicals is much more severe. Look at the colon in these animals compared to this one; it’s a normal mouse, not zonulin, and this is the untreated. The entire column mucous is gone, completely wiped out. I see this kind of damage only in animals that are radiated; they are exposed to radiation, like they are in a Chernobyl kind of situation. It’s that severe.

This brings now to the point, is this leaky gut an epic phenomenon just associated to this condition, or an integral part of how we got sick? There is a lot of evidence that this tight junction dysfunction, alias leaky gut, are involved in this series of problems with some bacterial toxins that can make this intestine to leak. Ischemia/reperfusion injury, this would take a totally different lecture here that is extremely interesting. Many drugs, not only non-steroidal or anti-inflammatory drugs, but many other drugs, and alcohol can make your intestine leak. And infections including bacterial and viral infections can eventually make this leak. But I want to finish up by really focusing on the autoimmune disorders, and what is the meaning of the association received with leaky gut and autoimmune diseases?

What is the evidence right now, going back to zonulin, of this function of this tight junction? Meaning that you produce too much zonulin, therefore, you opened the shortcuts for too long that can create this massive, uncontrolled passage on non-self-antigen that can instigate inflammation and eventually autoimmunity in people that are genetically skewed to develop autoimmunity.

I told you about gliadin, I told you the mechanisms, but the second mechanism that we found that is indisputable, is SIBO, small intestine bacterial overgrowth, approximate bowel contamination. That’s the second strongest. Actually, it’s stronger than the gliadin business. It releases zonulin to make the intestine leak.

These are some experiments that we had done a while ago, published a while ago, in which pretty much you see uninfected cells compared to infected cells, the release of zonulin then increase, and a change in permeability that increase when these cells are contaminated with bacteria. And you see this huge drop in resistance. That means a huge increase in permeability compared to the untreated animals.

And when you block this pathway because we have now a way to do that; i.e., that you can use a synthetic peptide that blocks zonulin, you pretty much reverse the situation. This is under control, meaning that this drop in permeability is totally zonulin-dependent.

What kind of diseases are we talking about? Well, there are many now, at least anecdotally, that have been described to have a leaky gut. And again, we don’t know if there’s a cause and effect relationship, or it’s just an epiphenomenon. And again, it’s not just autoimmune diseases. Look at this; MS, stroke, schizophrenia, asthma, COPD, ARDS, dilated cardiomyopathy…and I can go on and on until you reach tumors and metastatic diseases.

There’s a lot that is now accumulating into the literature, describing this breach of the intestinal barrier. But we’re not clear in understanding which one is the chicken and which one is the egg?

Now, again, we are at an in-house ELISA to measure zonulin in the blood of people. And here, we’re comparing two populations, two autoimmune diseases, celiac disease and Type 1 diabetes, in which we test zonulin levels in patients. Look at the numbers, these are not small numbers — 190 patients here, 340 here. And you see the zonulin levels much higher than in controls that are below this line here.

Interestingly enough, first-degree family members, also, they have slightly increased zonulin compared to controls, but lower than the patients’ counterparts. Meaning that if the zonulin-dependent breaches on the intestinal barrier, it’s important for the pathogenesis of autoimmune diseases like celiac disease and Type 1 diabetes is necessary, but not sufficient. And now we know why, because when you breach the barrier and stuff comes through, the destiny, as I mentioned to you before, depends on who you are. If you have an immune system that is functioning correctly, the immune system would take care of that, and we would not even know that it happened. But if we have an immune system that is skewed towards food allergy, towards cancer, towards autoimmune diseases, then that uncontrolled passage of antigen can put us over the edge and create the problem.

I think that contrary to what was 10 years ago, there is a general agreement, the recipe of autoimmunity called for these three readings: genes, environment, and this dysfunctional intestinal barrier that allows these two physically to interact. If this is true, it’s also true that if you take any of the three elements out of there and you can stop or prevent autoimmunity. Eliminating the genes is a no-proposition. There are too many we don’t know. For most of the autoimmune diseases, which genes are we talking about? That’s not a proposition.

Eliminating the environmental trigger of autoimmunity is a proposition that is valid and available only for specific diseases because we don’t know. We’ll make people sick with MS or rheumatoid arthritis. What about eliminating this third element? Because that was the key passage to prove that this is not an epiphenomenon, but is an integral part of the pathogenesis of autoimmunity.

And that’s where we developed this synthetic peptide that I was mentioning to you before that the way that works, binds to this EGFR receptor, but does not communicate to the cell to open the gate. So when the real deal, zonulin, comes it cannot communicate to the cell to open these tight junctions. And what I will show you is a proof of concept that zonulin, inflammation, and autoimmunity pathogenesis in animal models and specifically, in Type 1 diabetes, and acute lung injury.

This RAG model; it’s a RAG model of diabetes that’s very close to the real deal; i.e., diabetes in humans. These are animals that develop diabetes around age 60-65 days that is correspondent of age 30-40 in humans. And they are insulin-dependent. If you don’t give insulin to these animals, they die because of hyperglycemia.

What you see on the left is one of these animals that…this is a group of animals that developed diabetes and there are two parameters that we follow here. One, the permeability in magenta, and the other one, the serum glucose in blue. This is when these animals are weaned. In other words, food is introduced. They are breastfed by their mums until age 20-25, and then food is introduced. At the time of food introduction, the intestine permeability is normal, glucose level is normal.

After roughly one or two weeks they introduce baby food, they start to leak. This is the normal threshold of permeability of the gut, and you see this across the line. But the glucose level is still normal. After one more week, they start to produce auto antibodies, the one that attacked the outlets that produce insulin. Still, no diabetes. It’s only after two, three weeks that all of this happened — leaky gut and autoimmune production of antibodies — that they start to lose tolerance of glucose, and eventually, they develop diabetes. It looks like that the leaky gut precedes the onset of the glucose intolerance.

Now, the same animals that were given on drinking water, this blocker, you see that the permeability did not change, and the glucose level stayed normal. This was the first time that we proved that this leaky gut is not an epiphenomenon, but an integral part of the pathogenesis of autoimmunity.

And when we looked at the outlets; i.e., where insulin is produced and we do the immuno-staining, we had the confirmation that we did the trick. These, on the top, are the outlets of animals that developed diabetes, they were untreated. The outlets are like a balloon in which the air, i.e., the content of this balloon is made by these beta cells that produce insulin, and the wall of the balloon is made by these cells that produce glucagon.

In animals that develop diabetes, you punch the balloon that it’s deflated. So the air, i.e., the insulin-making beta cells, are gone. As a matter of fact, you don’t see anything here. Why…if you stain for glucagon, you see the balloon deflated that has collapsed on itself. And that’s the classical picture of a pancreas of a diabetic individual; animal or humans, it doesn’t matter.

 

The same animals that were treated with zonulin inhibitor, you see that the air, i.e., insulin-producing cells, are still there. And of course, the glucagon are still at the edge of the balloon. This is a present islet compared to a destroyed islet.

Now, this shows mechanistically that indeed, the prevention of diabetes in these animals was due to the protection of these outlets. Why? Because this uncontrolled instigator, we don’t know what, that was coming through, that instigates the immune cells to produce an attack against these beta cells was prevented.

Another disease, quite interesting, that again was totally unpredictable, is the acute lung injury. It was already known that you develop acute lung injury, for example, also in the COPD and so on and so forth, due to the breach of the barrier of the epithelium of the airways. And there is an animal model in which you can do that, in which these animals are treated in a certain way. So they go from this lung permeability that is very low, to this lung permeability that is in black that is very high. And if you block this process by increasing the amount of the zonulin blocker, you see that this comes down gradually. And again, you see this also in terms of different ways that you do this subcutaneous versus local; in other words, you put this in the airways, and this is the activity in terms of the inflammation that comes down, the cytokines that come down, and so on and so forth.

And if you look at the staining of the lungs of these animals, this is a control animal. This is…in animals, it goes through the process of lung injury because of this process that is artificially done. And the animals in which zonulin was blocked, it looks much closer to this than this. Even acute lung injury, it seems that the zonulin process is important.

I’m going to go straight to this. This is in humans now. And you see that this is the lactulose/mannitol ratio; i.e., the gut permeability of individual control versus asthma. The people with asthma, they have a leaky gut. And you see now that zonulin in the blood of these folks controls compared to the asthma people, it goes up. Interestingly enough, even if you have allergies, multiple food allergies, that zonulin levels are up very similar to the guys with asthma.

 

Let me wrap it up with…there are so many studies in the animal model that doesn’t translate in humans. Does this apply to humans as well? Forty-five minutes? I’m going to slow down. No, again, I will take another 20 minutes or so, so that you guys…we can chat or go to lunch earlier. It’s a beautiful day outside.

I don’t think that I have to spend too much time; this is self-explanatory here. I’ve been showing this forever, I still don’t understand it. Anyhow, these are all the steps that we now know that are involved in the pathogenesis of celiac disease. And again, one, you eat gluten. Two, you have this leaky gut. Three, gluten comes through and it’s deamidated and therefore, we interact with dendritic cells that will present this to mature dendritic cells to immune cells. And these immune cells, because they are genetically skewed, rather than to get rid of the enemy, will start to produce chemokynes. They are very offensive; i.e., they will kill cells because there are TNF-alpha interfering on them, and destroy the intestine. This leads to the autoimmune process. Then there are the B cells that produce all the antibodies including the TtG antibodies that we use for diagnostic, and maybe they also have a role in this destruction.

Theoretically, any of the 10 points that are outlined in yellow here could be a point of attack to stop this process. What do we do right now? We are here. We do not eat gluten, so that all the other steps cannot happen. And definitely is the best way to go.

The question is if we go to step number two; i.e., we stop this process, are we going to obtain the same results; i.e., prevent all the downstream process? In other words, if we block this gut permeability with zonulin inhibitor, can we do that?

Now, this is probably the longest clinical trials of alternative treatment to the gluten-free diet that is out there. Starting in 2006, almost 800 people have been through these trials. And the bottom line is in terms of safety of this molecule, this zone inhibitor is a pussycat, it doesn’t do anything. That’s good news.

Now, the very first trial that we did was a single dose, double blind simple controlled trial. You have now people with celiac disease on a gluten-free diet, so they are in remission. And they eventually, blindly, either they got gluten alone knowing that this will give them trouble, or gluten plus a zonulin inhibitor. The one that got gluten alone, they got an increase of permeability of the gut that interestingly…this was a single dose of gluten. In other words, a single meal.

Interestingly enough, they stay elevated for almost a week. That this really reiterated another concept; sure, gluten does this leaky gut to everybody. But in people with celiac disease and Type 1 diabetes, we have some they did in MS as well. This leakiness is much more sustained compared to normal people. Normal people will go up and down. But in terms of…people with celiac disease will go up and will stay up for almost a week.

The folks that they got the gluten plus this zonulin inhibitor and no leakiness to the gut, and consequently, compared to the ones that got to gluten alone, they had this amount of symptoms in blue here…they were talking about GI symptoms in general, they had almost 60% less symptoms.

Now, this is the summary of many trials. There are three different trials again in which we look at placebo, eating gluten alone, or gluten plus the zone inhibitor in blue; you see how the symptoms are abated in any kind of trials that they went through.

I’m going to finish with this that I think is quite interesting. This is a brand-new field, guys, this just came along — “The Controversial Questions About the Microbiome, the Leaky Gut, and Disease Status; Obesity, Non-Alcoholic Fatty Liver Disease, and Diabetes.”

What is this all about? What’s going on? The overall rationale for these studies is that a leaky gut is considered to be associated with an inflammation tone that can lead to obesity, fatty liver, and eventually Type 1 diabetes; very bold proposition, working hypothesis.

Now, I want you to take a look at these numbers. Because we’re talking about, in the last three years, there was nothing in the literature before two years ago. We start to see this coming. And again, these are not the “wackos” that publish this stuff. These are the ones that were considered to be the conservative folks. Gut microbiota, intestinal permeability, obesity-induced inflammation, and liver injury; very interesting paper that, again, links all these phenomenon together.

 

“Intestinal permeability parameters in obese patients are correlated with metabolic syndrome risk factors”, i.e. Type 2 diabetes. Again, this was published in March, 2012. “Nutrition, intestinal permeability, and blood ethanol levels are altered in patients with nonalcoholic fatty liver disease”, 2012 again.

And this was one of the last one that was published, “Circulating Zonulin, a marker of intestinal permeability is increased in association with obesity-associated insulin resistance.” In other words, it looks like that now, we are accumulating data, this human data in which a breached intestine barrier can lead to the uncontrolled passage of substances; mainly, bacterial components that will eventually find as a first station, the liver. Because all the blood from the mesentery circulation will, first off, be delivered. And when this bacteria component got there that can create the inflammation that characterize this nonalcoholic fatty liver disease, and with that, the metabolic consequences that we discuss; i.e., Type 2 diabetes.

I don’t want to leave you with the misconception that now we have the solution of obesity and Type 2 diabetes, because this is only part of it. Lifestyle is important. If you eat 5000 calories or behave like this, of course you will be obese. But there is now the possibility of opening a new paradigm in which even if you have a regular lifestyle, if you, for example, have SIBO and you have genetically skewed towards this metabolic imbalance, you may eventually develop Type 2 diabetes and obesity just simply because of this dysbiosis. There are now studies from Wash U in which they found a specific microbiome composition that makes you fat. And they showed this by taking this enterotype kind of macrobiota and transplanted it in different animals, and make lean animals fat.

And again, stay tuned because this an extremely dynamic field in which now, we start to see unbelievable stuff. The most skeptical individual that leaky gut exists really starts to rethink about it when the human genome, and therefore, epigenetic came into the picture because now, we start to see the many other autoimmune diseases they have as a genetic component. Genes, they are mutated; they control the permeability of the intestine.

All this to say, guys, that we are on the verge of a revolution, if you wish, that was totally unintended as far as I was concerned because I told you how the story started. And this is a typical example of how science advances, and the fact that it takes a village to do that, including the fact that that you have to have an open mind and be ready to say, “I was wrong.”

I want to finish by saying that I don’t want to leave you with the misconception that this is a single-man operation. I didn’t do that. I make the mistakes. The ones that fix the mistakes are these folks here. These are the people that are dedicated to science for the good of knowledge. These are people that work really, extremely hard to try to achieve the goal that we signed for; i.e., to increase the quality of life for the people that come to us. Thank you so much, guys, for being here.

Dr. Blyweiss

You’ve just listened to Dr. Alessio Fasano’s second lecture, and his research results on “Zonulin, Intestinal Permeability & Immune Mediated Disorders,” differentiating between fact and fantasy.

Cell Science Systems has several assays relevant to this lecture series. The first being markers which determine genetic predisposition for celiac disease. This includes the HLA DQ2 and DQ8 genes. Also available are various antibody markers such as tissue transglutaminase, TTG, and deamidated gliadin peptide, DGP. As well, ASCA Anti-Saccharomyces cerevisiae antibody, found primarily in inflammatory bowel disease patients with Crohn’s disease. Additionally, cellular analysis for gluten and other food sensitivities using the Alcat methodology is available.

These essays are available to providers in various single and multiple essay configurations. To inquire for additional literature or research, you can contact the lab at info@alcat.com.