Founder and CEO of Cell Science Systems
Good afternoon. Good evening to everyone. I’d like to welcome you to this Cell Science Systems sponsored webinar. And the topic tonight is understanding the pathogenesis of gluten spectrum disorders. Many people are still joining but we’re going to go ahead.
Okay, I’m Roger Deutsch. I’m the founder of Cell Science Systems and I’m a co-developer of the Alcat technology. I’ve been involved in testing and researching food sensitivities and testing for laboratory based methods of testing for food sensitivities for 30 years so I’m happy to present. This is an area, this is of great interest to me. I became interested in food and health probably about almost 50 years ago and I’ve been more or less a student of it ever since the beginning of the 70s.
What we’re going to cover is celiac versus non-celiac gluten sensitivity. Innate immune disorders in the gut like non-celiac gluten sensitivity and auto-immunity and others, other food triggers besides gluten, some insight into neutrophil – Neutrophil activation since that’s central to the discussion tonight. And then we’ll leave time for Q & A.
This is the, excuse me, this is the paper that inspired us to organize this seminar, which was published just one month ago and as usual, the work that comes from Alessio Fasano and his colleagues is nothing less than brilliant and what he’s presenting here, what the team is presenting here in this paper is information about a new observation that does shed light on the pathogenesis of celiac, excuse me, gluten related disorders. We hear a lot about leaky gut so we’re going to focus a lot of attention on gliadin in gluten and gut barrier integrity and how that relates to other disorders. The main theme is summarizing the abstract or the papers that increase intestinal permeability favors the access of gliadin and other macro molecule antigens from the intestinal lumen into the lamina propia where the host by interpreting them as dangerous signals, will respond with first line defense mechanisms such as neutrophil recruitment at the site of exposure.
The first discussion of this was that I heard was when Professor Fasano presented at one of our Cell Science Systems conferences on gut, brain and autoimmune disease back in 2013 in Los Angeles and he was very enthusiastic about the video, which captures the activation of the neutrophils in response to gluten, triggered in the guts of experimental animals in vivo while they’re still alive. Now I’ll let him explain that.
Alessio Fasano, M.D. (video)
What you see here, is the cross section of this core of the builders, this black stuff here are the capillaries and this green dots that you will see, it will be better, are the soldiers, neutrophils, they are particularly treated in this transgenic animals with this fruit fly gene that makes them to be florescent, okay? So what you’re going to see now is on the left, these are the villi of the mouse that has been exposed to another protein, BSA. This is the mouse that has been enlarged with gluten and this is pretty much what you’re going to see there. So see how many of these soldiers are stuck on the wall of the capillaries here and then eventually, start to move out and get out and got into the external part, what we call the lamina propia. This guy here, they keep traveling and they stay in the lumen. So in animals that were exposed to gluten, gluten is attracting the soldiers outside of the capillary and they start to really get on the battlefield where gluten and God knows, again, what else is sitting in there because it’s been absorbed through this bridge to the barrier. These guys, they keep going. Again, who is close by may see that but even if you’re not that close, you see the mess that’s happening here and this is all due to the simple difference that here you have bovines in lumen and here you have gluten. That’s all.
So that’s kind of a dramatic phenomenon there. Gluten exposed to the lumen of the gut of experimental animals can demonstrate the activation of neutrophils. In a study that we’re talking about today that was published last month, they looked to see if perhaps there was some artifact involved in. They looked at challenges with just phosphate buffered saline and as you can see clearly from this graph, there’s no activation of the cells. They’re minding their own business inside the capillaries. There’s no disruption. So the experimental model does not do it.
Again here, now, the challenge with the gliadin PT means it’s digested, pepsin, trypsin gliadin. Exposed to the gut of the experimental animals shows the activation of the neutrophils very clearly in these transgenic mice where there’s a gene that allows for the fluorescence. They’re called GFP a green fluorescence protein mice to light up when they become activated. And they’re adhering to the walls of the capillaries and then coming out as seen into the tissue as seen in the previous movie that Alessio showed.
The same thing is seen in the murine neutrophil hemostasis in this movie where, again, on the top we have a phosphate buffer saline control. In the lower segment, we have the gluten stimulated neutrophils and in between, they’ve introduced a positive control, which is FMLP, N-Formylmethionine-leucyl-phenylalanin, which is a component of the cell wall of gram negative bacteria and it’s used extensively in research as a neutrophil activator where you can see that the gliadin exposed cells migrate towards the green as much as do those towards the bacterial component and the negative control still for the most part, we don’t see the activation of the cells taking place.
The response is dose dependent. This is the dose of digested gliadin fragments and the control medium where there’s very little – we see very little migration of neutrophils taking place. As we start to increase the concentration of gluten up to 100mg per ml, we start to see that happening and when we increase that tenfold, we see quite a bit. The percentage of migrated neutrophils reaches about 75% of the total population, which is quite interesting because we also see that in our in vitro models, which I’ll get to later on. And the phenomenon of the migration, the speed at which the cells are traveling and the index is comparable for the gliding and the bacterial stimulant.
What does it lead to, gliadin? The term leaky gut is coming into common parlance today. What is leaky gut? Fasano reported on how gliadin causes through this pathway and NYD-88, an activator molecule signals to the nucleus that and code for the production of zonulin, which binds to the receptors on the endothelial cells, which stimulates changes in the structure of the tight junctures and we have of the opening up of the gut and that’s the leaky gut phenomenon and implores gliadin to do whatever it can do there as well as whatever else is hanging around in the gut and we’ll take a closer look at that in a moment. This study is interesting because it shows that whereas the gliadin fragments will bind to CXC receptor 3 to induce these changes in the cell to produce the zonulin, which causes the release of the tight junctures.
There are other mechanisms because here are the neutrophils of mice that are knockout mice that don’t express CXC receptor 3 and here again, we see the gliadin causing the migration of the neutrophils. We see that the bacterial stimulant is also causing it. And again, the control, the negative control is not. So it shows that we haven’t completed our study here and we don’t fully understand yet how it all comes to be but we know some of the pathways but it’s still a work in progress.
There’s another receptor and this is one of the main themes of the research called the formyl peptide receptor 1, which of course, is expressed on endothelial cells in neutrophils and when you block that then you block this reaction. Now it’s confirmed that that receptor is involved in that reaction through flow cytometric studies.
Here’s a closer look– a little bit larger of an activated neutrophil. So the neutrophils, this is the migration of neutrophils and you can see that when the cell becomes active, you can clearly see the granules, the primary, the eosinophilic and secondary and tertiary granules there throughout the cells and most contain preformed mediators. So if the cell ever catches up with this most likely bacterium, it will phagocytize it and put it into a vacuole and some of these granules will merge with that vacuole and release toxic mediators and free radicals and kill it, hopefully and that’s the ideal situation, that’s certainly one capability of the neutrophils. So activation of the neutrophils is significant and is showing us something.
So back to the question about gut permeability and how this relates to immune activation, in an earlier paper, Fasano said that classical paradigm of inflammatory pathogenesis involving specific genetic makeup and exposure to environmental triggers has been challenged recently by the addition of the third element, the loss of intestinal barrier function. And the activation of the zonulin pathway by food-derived triggers or changes in gut microbiota all seem to be key ingredients involving the pathogenesis of an inflammation also auto-immunity and cancer. So what we’re seeing is the full cycle back to the ancient wisdom of Hippocrates. Health starts in the gut. It’s very critical.
A new theory implies that once the pathological process is activated, it is not auto-perpetuating so it can’t be stopped and of course, how would one stop it is take away the insighting factors or improve the environment in the gut, improve the balance of micro flora in the gut.
What does it mean to look, how does this leaky gut scenario play out? Who has leaky gut? Does everybody have leaky gut or all food sensitivities occurring only because people have leaky gut? Well, an interesting study from Fasano and his group earlier looked at celiac patients and patients with gluten sensitivity versus a group of controls. And the controls were people who had dyspepsia but not gluten sensitivity. Gluten sensitivity was defined as having symptoms that went away when a person removed the gluten from the diet and then came back when the gluten was reintroduced. So we have the three groups and we’re looking at a standard test for measurement of intestinal permeability. It’s a lactulose mannitol challenge and a glass containing solutions of lactual mannitol, which are the small sugar and a big sugar, lactulose and mannitol a small sugar respectively are consumed and then excreted and measured over after five-hour period. Now, the concept here is that the lactulose is too big to pass through the gut, through the trans-cellular pathway. It can only enter through a paracellular pathway, which denotes the process of intestinal permeability, which is like the sugars that are being used today commercially in a lot of beverages and foods that say they have zero calories. Nothing has zero calories. It has zero calories because it doesn’t pass through the gut normally but having these complex sugars accumulate in the gut and not pass through may not be the best idea so maybe there’s other alternatives. Anyway, looking at these findings, these are the amounts of the respective sugars in the urine after five hours and these are the means and interquartile ranges. So we see, when lactulose is passing through certainly in the patients with celiac and the whiskers here report on individual findings so it was some people have very high levels of lactulose coming through, high degrees of intestinal permeability. The control group had certainly less. What’s interesting is that the people with gluten sensitivity had tighter intestinal barrier function than the control group did and the inverse of that is seen more or less with the mannitose except some of the CD patients also had large amounts of that coming through and again, here’s the ratio. In the gluten sensitivity group, it’s very low. It’s modest in the control group and it’s much higher amongst the people with celiac disease. So you can say that celiac disease is associated with leaky gut. Gluten sensitivity, really not so much or really almost not at all.
Elsewhere, Fasano again, found that gluten sensitivity is mediated by the innate immune system. Sorry, this is actually the same study report. And among these findings, TLRs, which are toll-like receptors, which are expressed on myeloid cells like neutrophils and macrophages and recognize repeat patterns on pathogens in PAMPs: pathogen associated molecular patterns. We represent a family of evolutionary conserved receptors able to detect microbial invasion via pattern recognition and mediate a rapid inflammatory response, which may or may not progress into an antigen dependent adaptive response. Now an antigen dependent adaptive response would be celiac. There needs to be the two diversions of gut permeability and glucosal immune gene expression to gluten associated conditions or celiac disease, which is an antigen dependent adaptive response and gluten sensitivity, which is not, which is mediated by the innate immune system.
And in, again, the same populations the expression of toll-like receptors, toll-like receptor one, two and four, which were studied was fairly low in the controls across the board relative to the people with gluten sensitivity and of kind of in between was celiac so that makes sense. The celiac patients, of course, have an adaptive immune response but does that mean that they do not have an innate response? It does not. The innate immune responses we see from the study that’s published last month is the first step. That’s the first line of the defense. The neutrophils are reacting against the substance.
And here’s a summary of other laboratory markers amongst these three groups: the controls, the dyspeptic controls, the patients with celiac and those with gluten sensitivity and there’s the demographic factor. And start with the symptoms, the dyspeptic control group, the controls didn’t really have any other extra intestinal symptoms. And in fact, I don’t even know if the dyspepsia is really intestinal, which is why they’re good for controls. The celiac patients had quite a few other processes going on, including tooth discoloration, thyroiditis, infertility, which I want to touch on a bit later on. Menstrual problems, cramps, joint pain, behavioral issues. Behavioral issues are significant – people have been inquiring about whether or not gluten is associated with psychological disorders and attention deficit and even autism. The gluten sensitive group also had some comorbidities. Let’s look at their labs. Endomysial antibodies, the dyspeptic controls all negative, gluten sensitives: all negative. The celiac patients 34 out of 37 that were tested were positive so that would be a pretty sensitive marker for the presence of celiac disease. Even though you have patients who have a sensitivity to gluten, you can rule out inactive celiac disease pretty well because these tests are so sensitive by using a test for endomysial antibodies. But what’s even more sensitive is the newer test, which is antibodies against tissue transglutaminase. We include that testing in our Gut Health Profile, both the IgA antibodies and the IgG antibodies against tissue transglutaminase because that today is the most sensitive marker to indicate an active disease process of celiac, anti-gliadin antibodies. What’s interesting, the dyspeptic controls, they’re negative. It’s a fairly sensitive marker, not as sensitive. For the celiac patients, we’re going to kind of hone in on that a little bit more and so-so for the gluten sensitive group. Now, of course, the adaptive immune response – in order for an adaptive, the next marker is MHC, which stands for major histocompatibility genes and what has been shown for patients with celiac is there are some genes expressed on a chromosome six that encode for HLA or human lymphocyte antigen, major histocompatibility molecules that recognize gliadin peptides. And in order for the adaptive immune response to occur, there needs to be a presentation of the inciting antigen to the T-cells and the antigen processing that takes place requires that the antigen presenting cells have the right molecular structure that they can recognize the gluten peptides and without that, there’s no chance of it progressing to celiac. So what we find is that all of the celiac patients that were tested, 22 out of 22 were positive. 100% have the genes for celiac and those genes are necessary to develop celiac. So the question, there was some preseminar/webinar questions. One was, are there markers for celiac? And this is certainly one of them. Does it mean that a person who doesn’t have this genetic predisposition can eat gluten at will? Well, probably not. Amongst the gluten sensitive patients, a little more than half of them did have these genes but a number of them did not. Now, allergy – another specific immune response is mediated by IGE and it also has to be antigen recognition and there’s T cell involvement and there’s T cell a T helper too. Profile would show instruct B cells to make antibodies of the IgE isotype and they bind to mast cells in the tissue and basophils on blood and gets cross-linked. There’s a degranulation of mass cells and basophils and a release of histamine and other mediators. We have what we call allergy or type 1 hypersensitivity according to Galen Kuhn’s classification. Does it play a role? No. One of the control groups had IgE antibodies to wheat, none of the patients with celiac had IgE antibodies to wheat and none of the 22 of the 26 gluten sensitive patients had antibodies to wheat. So people who – a lot of people have been doing this for years, testing for IgE for antibodies for wheat amongst patients who have GI complaints. That needs to change. That needs to be upgraded.
So we see leaky gut is not as big a deal. I’m going to bring Alessio back here for a minute.
Alessio Fasano, M.D. (video)
The second element that just came into the picture – again, I will briefly mention today but we can go in large, more deep details tomorrow is this leaky gut. Now, again guys, I’m pretty sure that you’re familiar with this leaky gut syndrome and so on and so forth. And again, unfortunately there is a very few facts because this is a brand new field – a lot of fantasies, to the point in which, again, if you go Google leaky gut syndrome, it looks like that everything that goes wrong in the world is due to leaky gut. So if we had your was because of leaky gut. If the election would not go the way that you like, it’s going to be leaky gut and so on.
Okay, okay. So leaky gut happens when you have celiac. In order to have celiac, you need to have the genes as I mentioned. The MHC class 2 genes, by the way, all cells, all sematic cells in the body express MHC molecules, class 1 in order to let the immune system know that it’s us, not to attack and class 2 help with antigen recognition.
And let’s look a little more deeply into this process. We have gluten causing, it’s binding through certain receptors. It causes the reproduction of zonulin, which causes the breakdown of the tight junctions and the barriers breached and gluten can come in. When gluten comes in, it can bind with tissue transglutaminase. Recall that from just a moment ago. Tissue transglutaminase is very sensitive serological marker for the process of celiac disease so I think it’s important to look at it in these steps of how the disease progresses. Where does it come from? It comes from damaged enterocytes so unless the process is taking place, we don’t produce this tissue transglutaminase. But what happens to kind of progress what really is a vicious cycle is the tTG binds with gliadin peptides and gets recognized by antigen presenting cells in the lamina propria that again, will have the right molecular configuration, the right MHC 2 molecule expression on its surface, which again is coded in the genes and those genes are the DQ8, the HLADQ8 and DQ2. So again, if we don’t have those, we don’t have this. No HLA DQ8, no activation of the antigen presenting cell processing the antigen. And then expressing on its surface where it can then activate a T-cell, which without this activation of T-cells its not going to produce cytokines, send signals to B cells, start to eat up the cells themselves. So this is the process right here. Without this gene, we don’t have celiac. But we can have all the stuff that comes before that. We can have various symptoms of gluten sensitivity, irritable bowel disease. Depending on other factors, we might have Crohn’s disease depending on the factors, which we’ll cover in a little bit, in a few minutes. So it’s a complex thing that’s taking place. The understanding of it is unfolding. Fortunately, we’ve discovered this when you have good leverage with a marker but again, it relates to the question, should someone asked the question should any of us really be eating gluten? And of course, after the T cell gets activated and we see the process a bit blown up here, the MHC molecule binds with the peptides of the antigen and latches on and transports it to the surface and that’s a blow up of that.
But I want to actually go back for a second and mention one other thing. In our schematic here, we have that interaction but also here you don’t see a label on it but this is a secondary signal from the antigen presenting cell to the T-cell and it’s usually mediated by a B cell molecule on the antigen presenting cell or APC and a CD 28 receptor on the T-cell. And the reason I mention that is because it’s known now that if that secondary signal does not occur, there’s no deal. There’s no activation of the T-cell and the whole sequence of inflammatory events just doesn’t occur. In fact, the T-cell becomes anergic and that’s actually the mechanism that the body employs in early life to develop tolerance to self proteins as they pass through the developing T-cells in the thymus. So you can see it’s kind of a complex process. It’s tightly regulated. There’s a number of different steps. There are different pathologies taking place that we can call gluten spectrum disorders. How we’re wired and what our exposures are and now the next item, which Fasano mentioned as well, in which you hear about everyday is the microbiota.
So back to the main thrust of this paper – the conclusion of this study published last month and this is the last sentence verbatim from that paper, is this question – actually a statement: “To what extent neutrophil function adds to or protects against gluten intolerance is currently under vigorous investigation.” So we see that there’s a phenomenon. There’s gluten exposure. There’s the activation of the innate immune defenses pretty quickly. Do symptoms happen quickly? Maybe, maybe not. Maybe it’s the first step in trying to neutralize something that could be harmful to another pathway. We know for sure, and this is where it gets kind of, I think, fascinating is that neutrophils and I’ll show some studies on this, tend to generate a lot of free radicals.
The largest source of free radicals are activated neutrophils. That’s how we age. Neutrophil, excuse me, free radicals can damage anything – microchondrial DNA, nuclear DNA, proteins, so forth. Neutrophils also release preformed mediators that are toxic and can cause tissue damage as well. So on the one hand, we know that neutrophils can cause damage. Can they also be signaling or trying to neutralize something in such a way that they don’t cause damage? Maybe, and I think sometimes they do because neutrophils as I’ll show in a moment have different repertoires. Even though they’re called innate immune cells and they’re innate – they’re not specialized. They’re not adaptive. Everybody has more neutrophils by a factor of five than they have lymphocytes. Neutrophils are the most common white blood cells. They’re kind of like the linemen on the defensive football team. There’s more lineman and then you get half backs and defensive backs and there’s one tailback – that’s like the T-cells so they’re more specialized. But even they bend the rule. All neutrophils are more or less the same as one another where T cells are all different from one another but even they have different repertoires. Now, what comes to my mind is something – it’s anecdotal but it’s something I experienced myself whereas in my position, I take the Alcat Test fairly regularly and a couple of surprising things would come up all the time and one of them was cucumber and I didn’t think too much about it but I just started to avoid cucumber. One day, inadvertently, I got a veggie juice from one of the local restaurants and a wrap and I brought it back to my office for my lunch and I was kind of involved in different things and I was eating in a mindless manner and I glanced over at the – I asked the people to make me a green veggie juice but leave out the cucumber and when I got it back it settled down in the cup and I glanced at it and I thought, “Hmm, that looks too translucent to not have any cucumber.” But I didn’t think about it again. I drank the whole thing. That night, I didn’t have any symptoms for a long time but about 10 hours later, I woke up with an immense intestinal cramping. Now, I don’t know – it seems to me that – I don’t know the mechanism. There was some chemical in the food that threw a pharmacologic mechanism of some sort causes that intense muscle spasm in me and my neutrophils tried to neutralize it and it probably did to a great extent but not enough. So I’m jumping ahead. We’re going to fill in some of the gaps but I believe that regardless of whether food induced activation neutrophils is protective or pathogenic. We have a question, do other foods besides gluten cause intestinal permeability and activation of neutrophils? And of course, is it pathogenic or not? So let’s look at that question.
A group from Scandinavia developed another method – now Alessio Fasano’s method of testing the gluten induced activation in neutrophils in vivo was kind of tough on the experimental animal. I wouldn’t want to be an experimental animal but we learn a lot. These guys developed a method whereby they analyze the gases that come from the rectum and they can determine what cytokines were being expressed and if nitric oxide is being expressed and they found, not surprising, that in 18 out of 20 patients challenged with gluten in this faction, this is a rectal challenge, there was neutrophil activation defined as an increased release of myeloperoxidase, which come from neutrophils and increased nitric oxide synthesis. However, 10 of these patients also showed a similar inflammatory reaction to cow’s milk challenge so it’s not as common but yet, it exists. A mucosal response similar to that elicited by gluten was produced in cow’s milk protein in about half the patients with celiac disease. I forgot to mention – these are celiac patients. Casein was the most involved.
Prior to that, the same group using the same technique had found the same thing – activation of neutrophils and eosinophils which precedes the enhanced production of mucosal or endothelial nitric oxide following rectal wheat gluten challenge. They also found it occurring with several other patients after corn gluten challenge. So that would tend to confirm other foods besides gluten maybe through different receptors can cause activation of innate immune cells upon exposure.
Another method, we have a laboratory in northern Germany outside Berlin and nearby there’s a hospital that tests very ill patients when food sensitivities suspected. And this is a short video that’s only going to be a couple of minutes abridged to show the highlights of what other challenges can take place in another form of a rectal challenge and another method of measuring intestinal permeability and what the results are when looking at different foods. And this is pretty – I’d hate to have to go this route but if you have to, you do it. This particular patient did it. That’s him today or at the time of the video and he’s looking at pictures of himself before he underwent this testing so he had GI symptoms but he had other symptoms. He’s speaking about that. He had serological tests for IgG and IgE antibodies and he excluded the foods. He went scratch testing for a number of common food allergens. He was still sick so they took him into this hospital where they do this rectal challenge and they put him out. They gave him anesthesia. They used a microscope that’s illuminated a thousand times and they actually gavage with different test foods. In this case it’s milk, soy, vitamins, hefe is yeast. For their technique is they need to use a contrast media, which they’ve injected and they can see after the gavage, whether or not the light is permeating through the tight junctures and they didn’t see it with many of the allergens. Here they’re testing milk and we saw those results but in their final test, see the milk doesn’t do much to the internal structures – when they challenged with yeast, immediately the light shines through abundantly, which is indicative of a strong breach of the tight junctures and this man, all he had to do was take away yeast and his symptoms resolved.
As I mentioned, I’ve been working for almost 30 years with an in vitro method for looking at cellular responses to various substances, ingestants being primary but also food additives and colorings, naturally occurring pharmacologically active substances – it means it occurs naturally in foods or chemicals that are added to foods, medicinal herbs and things that people can breathe in from the environment or contact environmental chemicals and molds. And what we do is an ex vivo challenge and this I think is far more convenient. We withdraw the blood cells and then we culture the cells. We separate the white cells from the blood, add back or autologous plasma and then culture the cells individual allaquive so the cells and plasma with the whole battery of substances and there can be as many as almost 400 different items assayed and quite early on in my history of this involvement here, I got together with some folks and in London, London University, College and I think it’s London, it’s the School of Medicine at London University, working with a former director of a drug company who was a clinical pharmacologist who got dicentra cromoglycate through the FDA, which is believed to block allergies: nasal chrome, intall so forth – the products that are based on dicentra cromoglycate and he approached them both from a neurological point of view and a pharmacological point of view and wanted to verify whether or not the test in fact was valid so the way to determine whether an in vitro test is reliable or efficacious is to compare against a known standard, which in this case is a double blinded challenge. And when this was done, the sensitivity of the test was determined to be 80% or just under 80%. The specificity was a little over 85% so the overall correlation between the double blind challenge was just a little north of 83% so that was good and we continue and I continue my involvement here.
This is how the Alcat method works and I will just mention before I start the video that we, I’m actually quite proud to say that we’re the only laboratory which is also a medical device manufacturer and we manufacture the device to do the Alcat Test exactly according to our required specifications so the test does exactly what it is that we want to measure and what we want to measure is the activation of the cells and there are many methods extant for measuring cell size and particles and activations so forth. The simplest, most economical and when I say economical, we charge $1.50 per determination andthe most accurate is the impedance method and we’re now on our fifth generation of instrument that we’re close to releasing, which should be in the next calendar year. And the next instrument was funded partially from grants from the European Union and also out of our own operations and we probably dedicate – we have a huge full time R&D contingent and a staff of engineers and programmers and we love to have people come into our laboratory. Since everyone’s scattered around the country and the world, it’s not feasible for many people so we produced a video recently that highlights what the inside of our laboratory looks like and is on our new website at cellsciencesystems.com and I encourage people who are interested in this field to look at it and to really understand a little bit about this technology because although we’ve been doing it for 30 years and we got the ideas from people who are looking at cell activation since the 1930s following food challenge, it’s still the best method. It’s a biological test. It shows a biological response to a challenge and instead of having to do what was seen in the previous video, you can just have some blood drawn so it’s pretty simple. Now, this is a blow up and I’ll start this – of where the rubber meets the road in our test. These are blood cells, patient’s blood cells that are either in a controlled solution or have been exposed and cultured with a test reagent passing through an electromagnetic field. And as they pass through, there’s a current going across this field and as they pass through those cells, impede the electrical current in direct proportion to their mass and from that analysis, cells one at a time, we are able to generate a sized distribution curve.
Now we create a curve of the controls, which are the blood cells with no substance and then a control with the substance and we do a comparison. So if the cells have degranulated, we see that. The peak is lower. If they’ve swollen up in a process of vacuolization, the cells have shifted to the right so some cells – what’s interesting is that the total population of granulocyte, which is what Fasano saw is that a huge number when you have the right dose of sufficient dose of gliadin. 75% of the granulocytes were actually migrating, became activated. That’s exactly what we see so these are gross changes in cells that are pretty easy to see. Do we know what’s following on after this? No. We only know that the body doesn’t like it and the innate immune system is reacting against it.
We did some work comparing our graphs to what you see under the microscope looking at cells and what’s seen through flow cytometry. We’ve run a number of control and so, again, we have a control curve here and test curve here but since it’s controlled, there’s no substance, you expect them to line up and they do. And this is the smaller cells – these are larger cells, these are the lymphocytes and these are the granulocytes. It’s a bit exaggerated but it’s similar to this in most patients. There’s far more granulocytes, which are almost all neutrophils than there are the specialized lymphocytes. Monocytes are here and then mature into phagocytes, macrophages. So that’s what a control looks like and that’s what a cell looks like from this sample. It’s nice and round. It’s happy. It’s not like the one we saw chasing around the bacteria a bit earlier.
Here we see a response to barley. This patient had a response to barley and we see some swelling of the cell and some loss in cell number.
And here we see a strong degranulation that cells are gone. And this cell has really kind of degranulating and these are granules coming out that are seen. Innate will cause some problems but they’re trying to neutralize so whether they cause strong problems or whether they succeed in neutralizing something that’s harmful, I’m not so sure.
But when people follow a diet based on these results, there’s a significant improvement. This was a study done some years ago at Baylor Medical College and the experimental group showed improvement in symptoms versus the control group. These were obese patients, overweight patients I should say and the only parameter where there wasn’t a statistically significant differential between the two groups was with the overeating. Of course the control group was asked not to overeat to see if an Alcat diet improved body composition more so than it did caloric restriction.
This is what I alluded to earlier. This is the last sentence of, its not the last slide, I’m sorry, this is the last sentence of Fasano’s paper from last month. “To what extent neutrophil function adds to or protects against gluten intolerance is currently under vigorous investigation.” And I can’t wait to see what he has to say but this is what I found just looking recently. I’ve spoken about my anecdotal report with cucumber. I have other reactions to other substances, other foods and I have immediate symptom onset so it’s a whole repertoire. The immune system is complex. Complex it is.
The editors of Science Magazine felt, in 2013, to run a special edition about inflammation and they say its complex. That’s what’s implied by the cover graphic. We have, which many people would be familiar with this yin yang symbol. And yang, of course, in Taoism and yin are the two polar opposites, male and female that are constantly in motion, constantly transforming from one to the other and they express the yang expresses heat, hardness. You think of inflammation, activity and yin is non-activity, more nurturing and cool. And so, I think what they were sensing was that the immune cells can promote inflammation. They can promote resolution of inflammation, type 1 microphages, promote inflammation. Type 2 macrophages promote resolution of the inflammation and then let’s see what else we can find here.
With respect to GI symptoms, there’s a study just a few years ago reported from the University of Pavia looking at Alcat and avoidance of foods that activated cells resulted in very important proven 71% of the study population. Only two people, sorry, 2%, 4 out of the 48 subjects did not have any improvement. The remainder had some improvement so looks like that in activation of cells means something.
In the opening, I mentioned that Fasano said there’s three parts to this thing: there’s the gut microbiome. How we react or what’s our intestinal flora look like? What’s the micro environment look like? It’s pretty important. I want to expand on that but we’re seeing an increase in – it seems to be updated but we’re seeing pretty sharp increases that go along with the increased incidence of reports of intestinal problems. That’s associated with increases of autoimmune diseases, of course, a whole spectrum.
The yang portion, oxidative lesions are the hallmark of Alzheimer’s and Parkinson’s and circulating neutrophils are the most significant sources of reactive oxygen species and they’re seen to be increased in patients with dementia.
Disregulation of phagocytosis so here’s where the ideal plan with the neutrophil or the innate immune system neutralizes the bad guy and if it can, grab it and produce antibodies and we get this adaptive immune system coming in. Here’s where it may go array. If there’s a disregulation of the phagocytosis, and again, if you recall back to the movie, we didn’t see phagocytosis but we saw that the preview to that where the cell is chasing the pathogen. If it goes array, there can be the development of autoimmune diseases and it’s been related to lupus and arthritis. Why?
Well, what these guys thought about 10 years ago was that, well of course, leukocytes mainly neutrophils infiltrate the tissue and then when they, after they’re done vacuolizing or they spew out toxic mediators to kill something externally or after they’re done phagocytizing something internally, they become apoptotic or necrotic. They’re then cleaned up by the microphages, hopefully. But what happens if you overload the system?
Okay, I’m going to tell you the answer to that question before I go onto a nuanced explanation of that. The answer is that you have a lot of nuclear material that’s been released from the neutrophils that’s not being cleaned up and it becomes immunogenic and another way in which neutrophils fight pathogens is to produce nets and the neutrophil first gets activated and produces oxygen radicals and we have the merger of the nuclei with some of the granules and we have this material, which contains nucleic genetic material and mediators spewing out to form an actual net outside the cell. So even though the cell might be dead it produces a net.
And this what a normal net looks like and this is the net from one neutrophil and look what it can do in terms of trapping a number of bacteria. Not only does it trap it, the chromotin in the nucleus, the genetic material is inflammatory and it kills the pathogens.
And here we see an early stage of it and there we see a really blown up picture with nets and trapping Klebsiella. So it’s a very intricate thing but if we don’t – what happens after the job is done? We have all that material floating around and it’s inflammatory and it’s immunogenic and it can initiate autoimmune diseases. It’s broken down by enzymes but if we have a deficiency in the enzyme system such as DNAs 1 deficiency, it becomes immunogenic. So maybe there’s a connection with this increased prevalence of autoimmunity and changes in our food supply and how we eat and our lifestyles and the fact that more people have symptoms that are vague such as the ones that we saw amongst the gluten sensitive patients or people that may not have bowel disorders or intestinal complaints, they have other vague things. There’s fatigue, central nervous system issues, dermatologic, respiratory, musculoskeletal – these people feel better after they go on an exclusion diet or they avoid foods that are shown to be activating their cells.
When the activation in the immune system occurs, this is a very interesting meta-analysis of a number of studies that was one of the first to identify that chronic activation of the innate immune system probably underlies metabolic syndrome, which we’ve seen increasing. Why? Well, it produces proinflammatory cytokines and that stimulates cortisol production to counter that. And that, in turn, causes disruption in the pituitary hypothalamic pituitary adrenal access and suppression of cortisol. Excuse me, suppression of sex hormones, which cortisol competes with for production.
Pick an organ system. Here I’m going to pick one that is before we have organ systems. In the sperm, there are neutrophils and macrophages. In men that are infertile, they have three times higher a level of the macrophage mediator, neopterin than fertile men. Go figure. Again, inflammation kills sperm cells, it looks like. We’re looking to do a prospective study on female infertility as well. We’ve seen a lot of that result and I think normalization of hormones through normalization of immune function, which is connected, plays a role.
Inside the nuclei, inflammation causes shortening of telomeres and you have premature cell senescence. Each cell takes care of telomere length is associated with a three-fold increased risk of heart attack. They speculate it comes about from inflammation. Also, there’s a significant inverse relationship between telomere length and Interleukin 6, inflammatory mediator and metabolic features glucose, insulin, diabetes and a plaque buildup.
B, phospholipids, one of the things is we can get into a chronic stage, especially we eat a lot of –Barry Sears teaches about this for a long time now – we eat a lot of foods like corn and soy that contain a lot of omega 6. The breakdown products, those like casonizer proinflammatory. We probably eat more than we should and we if we balance it more with omega 3s, we probably have less chronic inflammation going on and those phospholipid bed themselves in cell membranes and you can change that through diet and they’re in a in the blood and when you trigger inflammation, you have plenty of fuel for the fire if you eat that kind of a diet. Never mind the fact that we had to improve upon those things to make them sit on the shelf for years, preserve their colors and be able to resist tense amounts of dosings with herbicides. I kind of wish, like a lot of people do that those guys would knock it off but it’s not helping.
And low and behold, from Harvard, Ridker reported a long time ago that the then – now we have some advanced markers but c-reactive protein, when that goes up along with cholesterol so does the risk of myocardio infarction. So it’s not just the cholesterol and that’s what Vershov said back in the 1890s when he did biopsies on little lab animals. They had these fatty streaks if they had inflammation going on.
So other researchers have found that MPO, again from neutrophils, is a better marker than inflammation of the local plaques than LDL and it’s probably that MPO is inducing transformation of LDL to oxidized LDL. And of course, neutrophils – neutrophils are evolved in damaging everything when they want to. They damage after a stroke and reperfusion of the blood, the neutrophils that kill the heart muscles.
We’re going to skip over this because I think it’s a question of time.
We see a big increase right now in diabetes type 1. Diabetes type 1, not the adult acquired but diabetes type 1, which supposedly is genetic, right? Now, this brings to mind just like the yin yang symbol kind of coming to mind and I know back when I was first getting interested in this stuff, there was a guy named Fritjof Capra wrote a book in the 70s about the “Tao of Physics” and how so many of the guys involved in quantum physics were actually students of eastern philosophy like Delbert started Bhagavad Gita and some of the others studied Taoism because when you look deeply into science, you kind of come to a point where you see that it’s really hard to say things are black and white. And I think with genetics, it’s kind of like that and it brings to mind one of the things I’ve got interested in a little bit is how the philosophers interpret karma. So you all know from lots of traditions, what goes around comes around or what you sow, so shall you reap. And they dove into that a bit more in the beta class when they described the three types of karma. The sanchita karma, the parabda karma and the kriyamana karma and the sanchita karma is like the total genome, okay? And I say that’s the total amount of aggregate karma that a soul carries. Now, the parabda karma is what the person is going through in this particular time and that’s what genes can express. And the Kriyamana karma is what you’re doing right now we’re saying interplays with your other karmas and promotes the future karma. That reminds me a lot of what we understand about genetics. We have our genome but if you have a certain SNP, you’re definitely going to get a disease? Of course not. It depends on lifestyle. The government says that 70% of cancers are preventable by lifestyle so it’s not just the genes. But diabetes 1 is the genetic type, right, or it used to be but now it’s increasing so who’s it increasing the most? The medium risk? Yeah, it’s going up, it’s doubling. The low risk genotype, it’s tripling. The very low risk, it’s going up seven times so there’s got to be some kriyamana karma going on in the lifestyle so we need to remedy the lifestyle. I think that all the technology pails in significance in terms of treating people with lifestyle.
Again, back to the yin yang portion, remedic patients have two to eight times as much free radical production.
Okay, let’s wrap it all up. An interesting review article from Journal of Immunology, toll-like receptors signaling in the gut in health and disease. A number of things to contemplate: the secretion of various antimicrobial peptides like anocells, which are cells that are in the lumen of the gut, which are in the crypts in between the villi and enterocytes’ are likely to be regulated by TLR mediated recognition of PAMPs. So those cells secrete antimicrobial peptides for TLR mediation. Exposure of colonic epithelial cells to bacteria results in sealing tight junctures so there’s your paradoxical reaction that the tight junctures increase in exposure in the colon, in exposure to certain bacteria. Depending on the scenario, toll-like receptor signaling may aid in certain aspects of barrier function but not others. Toll like signaling and the response to the presence of bacteria in the lamina propria following epithelial injury is critical for healing of the gut. So the immune system there is healing the gut. It’s not just swollen out of control beast causing damage all over the place. The question is balance. Intestinal bacteria are essential for the normal development of the gut-associated infloititial. We know that. That’s why they’re called commensal bacterial. In the absence of luminal bacteria, B cell and T cells do not cling to the laminal propia and IgA, secondary IgA is not secreted, which excludes antigens from coming into the blood.
The gut environment uses signals from luminal bacteria to develop tolerance by mechanism involving regulatory T cells. T regs, they shut down the inflammation, definitely the end part of the sign. Polymorphisms in toll-like receptor 9 has been linked to Crohn’s disease. We also test for antibodies against saccharomyces cerevisiae in our Gut Health Profile because that’s been a factor. So maybe it’s a factor for people who have some polymorphisms in their innate immune function, specifically toll-like receptor 9s, maybe others. It’s a big world in there. Mice with myeloid cell, again innate immune cells are myeloid cells, depletion of stat 3, which is an activator molecule, which is needed for certain activation are deficient in IL-10 production, which is, again, part of the yin component there. IL-10 calms down the inflammation and then they develop into enterocolitis. And lastly, experimental animals that lack toll-liks receptor 4 and MYD-88, remember MYD-88 is an activator platform that other dock onto and send signals down to the nuclei, produce things like zonulin. Well, and Dr. Fasano said that this release of cytokines and activation of neutrophils is MYD-88 dependent. Mice who lack that can develop pathology when they’re challenged with toxins and have fewer neutrophils.
So, again, it’s balance. However the balance between the requirement of toll-like receptor 4 and other toll-like receptors to initiate and sustain acute and chronic inflammation versus its role in healing of the epithelium is probably contextual and depends on other host factors, no doubt gut bacteria. So we want to feed our gut micro flora the right stuff for sure. They like fiber, don’t like so much refined stuff. Conclusion: in response to recognition of pathogen associated molecular patterns through toll-like receptors, both inflammatory and homeostatic pathways are activated. Okay, so again, the balance of the yin and the yang. In this way, there is clearance of bacteria and resolution of normalcy.
My closing comments are that manifestations of GI disease depends on genetics, microbial and host interactions, environmental exposures and other psychophysiological factors. The molecular and cellular pathways associated with GI disorders are now being elucidating. We’ve talked about some of those. I’m sure we’ll find a lot more. The immune system normally confers balance and homeostasis but it may tip into the imbalance and promote disease. Acute activation of the innate immune system precedes adaptive immune responses or not. It can also cause disease without adaptive immune response. Regardless of whether the innate immune system is protective or pathogenic, it may be a reliable indicator of pathogenic food and environmental triggers and that’s what we were doing here.
So I’d encourage anybody who’s intrigued by this to visit our website. There’s a lot of research studies there that I didn’t get a chance to touch upon and we’ll open it up to any further questions that anyone might have. Just put those through, type those in right now. While those are coming through, there were some of the things that we’ll look forward to is that we’re winding up a research project with scientists and doctors from Yale School of Medicine and we’re looking at Alcat perspective of immunological mechanistic studies as well as clinical outcomes with patients with IBS and other pathologies and that information or those studies – they’re almost done and they’ll be reported fairly soon and we’ll look forward to communicating that information when that becomes available.
Someone asked if I suspect a patient is reacting to gluten, what are the best tests genetic to see if a SNP has been activated? And that’s the DQ2- DQ-8 genes and that can be done with buccal swab and I don’t know of any others at the moment. I’m sure we’ll find others when we hone in more on these pathways. But for now, those are very good but again, the question is, should we have gluten if we have symptoms regardless of the genes? If you have the genes, I think it’s a no brainer.
Someone else is asking, my patients that choose to go gluten free, seem to become more aware and become more involved with the world around them. I believe this is because they are no longer producing gluteomorphins, which was drugging their brains. I don’t know about that because I’ve done exclusion diets for many years and I experienced the increase in awareness and I don’t feel that there’s a drugging of the brain. It’s a nice feeling and doesn’t desensitize me. It’s not just a euphoria – I don’t know if that’s there. I apologize if there’s some information that’s been researched already that I’m not aware of. But just anecdotally, I don’t know if I agree with that. I think that it’s probably a positive thing.
Can an anti-inflammatory nutrient or even simply low dose antigen reduce an innate reaction? Yeah, an innate reaction in gluten or gliadin or food component – yeah, absolutely! That’s what we saw. That’s what Fasano reported. There was a clear dose response, which is probably why the last time I ate a whole pizza by myself, I really felt bad. However, if I have one slice or even two slices, I’m okay with that but I know. I’ve tested my genes so I wouldn’t do that otherwise but I’m negative for that genotype.
What gut restoration suggestions do you have? I’m not a physician so the ones I have would be do an Alcat Test first. I strongly believe in that. I would want to look at probiotics. I’d want to look at a lot of fiber. Fiber feeds good bacteria like bacterial faculae, prosnitziae. I think I got the first part of the wrong so don’t quote me on that but they need fiber and that’s been shown when they look at village kids in Africa that eat a high fiber diet, their gut micro biome profile reflects that of a lean phenotype and they have lean phenotypes and they look at children eating diets that have a lot of processed components, they have a different microbiome profile that is associated with metabolic disorders so I would definitely clean up the diet. Glutamine supplementation has been used for years. I can’t comment on the dose because there are protocols for that the IFM publicizes widely so one might look there.
And I think maybe we have time for one further question. Is there anything else? Okay, thanks very much for a great presentation. You’re welcome. It was my pleasure. Thanks to everybody for helping making this possible. I look forward to reviewing the website. Okay. Is there a question?
How often should you redo the Alcat? That’s a really good question. It’s not a hard and fast answer there. I think if finances were not a consideration, I would repeat it every time the season changes because we do see dose responses and there are definitely changes that occur physiologically in our adaptations and changes in the seasons and there are changes in our exposures. For example, people who have reactions to apple, raw apple, may be able to tolerate them except when the birch trees are pollinating in Sweden and then they can’t do it because there’s a molecular similarity there so there’s course reactivity and you exceed that load and symptoms break out. So it’s definitely continuum. When you’re talking about innate immunity, it’s on a continuum. Unlike classical allergy where IgE, it’s really interesting, very, very, very low doses of substances that follow an IgE pathway, which is kind of rare can be sufficient to inducing anaphylaxis. In fact, there was an uproar back in 1987 when Jacques Benviste, the head of the allergy department in INSERM, which is the institution in France, made a report that very, very, very high dilution of IgE, which was even so high there was not even a single IgE molecule present in the solution was able to degranulate basophils and the Editors of Nature, a very prestigious international journal published it and it created a huge uproar because it tended to prove that the possibility of such things are homeopathy, being real and who knows what and they sent Randy the Magician down to his lab to try and debunk it and all that. And in the end, the jury was out on that but even that aside, someone kisses their girlfriend, their girlfriend gets an anaphylactic shock and turns out he ate a handful of peanuts 10 minutes earlier so depends on the pathway but we’re looking with Alcat at an innate immune response that may lead to adaptive immune responses in some instances and I think they may in many cases but there, when we’re looking at innate immunity, we’re looking at a dose response. So again, it’s not black and white. It’s on a continuum.
Live well, educate, do the right thing. They’re your patients and thanks to all for their participation. Good night.