Presentation - VDR receptor competence induces recovery from chronic autoimmune disease

Type: Conference presentation
Presenter: Trevor Marshall, PhD
Conference: 6th International Congress on Autoimmunity
Location: Porto, Portugal
Date: September 11, 2008
See also: Transcript and slides; Notes from the 2008 International Congress on Autoimmunity by Amy Proal

Thank you. I am going to talk about the VDR receptor. It’s one of the family of nuclear receptorsIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affect transcription., which includes the glucocorticoid receptor, the thyroid receptors and a number of other very important receptors.

I just want to point out this statement from FDA commissioner Von Eschenbach to congress two years ago, pointing out that “New scientific discoveries are generating an emergent science of safety, where the new science combines an understanding of disease and its origins at the molecular level”. That is what I am going to talk about in this presentation: understanding Autoimmune Disease Pathogenesis at the molecular level.

There are three types of biology in common use today, in-vivo in-vitro and the newer in-silico. The first time I came across in-silico biology was back in 1981. This is a photograph of myself and my colleagues at the Hospital for Sick Kids back in 1981, which was when IBM showed us the in-silico techniques that they had used for the synthesis of the first human insulin. The Humulin, the first human insulin. That was the first time I came across in-silico and realized the power of being able to emulate the operation of the human body at the level of individual atoms.

Since then the in-silico work I think that everybody is most familiar with is the decoding of the human genome and probably more important now, but less well known, is the decoding of around 740 microbial genomes that have been fully decoded to this point in time.

And in fact, NIH has just started a big Human Micobiome Project, with the idea that they wanted to characterize all the DNA that’s available from human sources, all the DNA in the human body. The normal infectious areas that we are aware of: the nasal, oral, skin, GI and urogenital cavities, but also within the cells of the body itself. Because NIH has estimated that around 10 percent of the total cells in the human body are human cells and as many as 90 percent of the cells could be bacterial cells. You will be hearing a lot about the Microbiome project over the next decade.

Why is that important? Well it’s important because the bacterial cells, in many cases, perform functions that are very similar to those of the Homo sapiens itself, of the host. And what I have got here is a slide showing the E.coli Glucose Metabolism. And I know very few of you are going to follow every subtlety of it. Don’t worry; I just wanted to show you that this chart exists. I got this one from Vijay at the Bielefeld University in Germany. But it shows the way that the bacteria E.coli gets from Gluco-6-P substrate down to Pyruvate and produce the Serine Cystein Glycine amino acids, the Purine Nucleotides Adenine, the Tyrosine Phenylalanine, Tryptophan. All of these are produced by bacterial genes. Actually by proteins that are transcribed from bacterial genes – but the bacterium itself, the organism itself is capable of working on exactly the same metabolites as used in the human body for the production of energy and it produces very similar intermediates. You have got Fructose-6-Phosphate here; you’ve got Glycenaldehyde, Glycerade. Many of these intermediate metabolites are very common, very familiar to people who study the human genome. So, if you have got the bacterial genomes working in the same environment within the same cells, in fact infected cells as the human genome and the human body you can imagine the amount of interference between the operation of the two of them.

And that’s exactly what we have found. We have characterized that there is an intra-phagocytic metagenomic microbiotaThe community of bacterial pathogens including those in an intracellular and biofilm state which cause chronic disease.. Metagenomic: many genomes. MicrobiotaThe bacterial community which causes chronic diseases - one which almost certainly includes multiple species and bacterial forms.: community of pathogens. Intra-phagocytic: it does its most of its harm inside the phagocytes, the lymphocytes, the macrophages, monocytes of the immune system. And we have shown it to be the cause of most chronic disease. The genomes accumulate gradually during life, incrementally shutting down the innate immune system. They shut down the innate immune system incrementally during life. Genes from the accumulated metagenome determine the clinical disease symptomology depending on what genes accumulated in the metagenome that determines which effects they are going to have on the body, which of the metabolites of the human body are going to be affected by the pathogenic genomes. The microbiota is located in the cytoplasm of the nucleated cells, where it has access to both the DNA gene transcription and the protein translation machinery of Homo sapiens. In addition the host DNA repair mechanisms are susceptible to modification by junk from the metagenome. That’s very, very important. That, because it’s located in the cytoplasm the microbiota can upset the host DNA repair mechanisms.

At the congress in 2004 in Budapest, I reported that sarcoidosis had succumbed to an antibacterial therapy that we had developed. And over the last six years our cohort of over 500 human subjects has demonstrated reversibility of many auto-immune diagnosis. Reversibility, including Lupus, MS, RA, Type 2 Diabetes and Uveitis. My colleagues will give details of this later in this session.

But surprisingly, as the chronic inflammation receded, CFS (Chronic Fatigue Syndrome), osteoporosis, periodontal disease, cardiovascular disease, cognitive deficiencies, obsessive compulsive disorder, bipolar, memory loss, all of these also disappeared, as the chronic inflammatory condition disappeared.

Well, what do these microbiota look like? Here is a monocyte, an infected monocyte and the cytoplasm has effectively exploded from pressure of the pathogens and it is throwing out these tiny biofilm A structured community of microorganisms encapsulated within a self-developed protective matrix and living together. tubules. I’m not quite sure what, it’s not showing up perfectly but you can see here is probably a dozen tiny biofilm tubules. They are very long and they’re also extremely small. You can compare them with the size of the cell, the standard cell diameter there of 4 - 5 microns or so. And extremely tiny little biofilm parlemor tubules are thrown out as the cell disintegrates. This is untreated blood, this is human blood put between cover slips and allowed to age for six to thirty-six hours. Look at the length of these; this is about 26 cell diameters long. This one is about ten diameters long. It’s amazing and you can see them very easily under light microscopy if you are looking for them.

Under the electron microscope, they look a little bit different of course. Because you can actually look at them in the cells before they become heavily parasitized. This is an image from the Emile Wirostko TEM study in 1989 at Columbia University. This is a Juvenile Rheumatoid Arthritis lymphocyte. And what we have: we have the nucleus area of the lymphocyte. And then the cytoplasmic region outside, the nucleus and in the cytoplasmic region there is a staining artifact which is basically nucleic material, DNA material, inside some form of transparent biofilm type of protection and then a very thin exoskeleton to contain the whole lot. But what’s even more interesting are these tiny little elongated structures – which don’t come up all that well on the projection here, but when you look up the original paper -which was JRA Inflammatory Eye Disease, Parasitization of Ocular Leukocytes by Mollicute-like Organisms from 1989- when you look at the original photographs you can see the incredible detail of these transparent colonies that are living inside these lymphocytes. Of course, the lymphocyte should never allow this to happen. But it does allow it to happen because these microbiota have figured out how to overcome innate immunityThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease.. They have overcome the last line of defense.

Well, how does it do that? In Homo sapiens the VDR nuclear receptorIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affects transcription. transcribes genes for the Cathelicidin Family of antimicrobial peptides found primarily in immune cells and transcribed by the Vitamin D Receptor., beta-Defensin An antimicrobial peptide found primarily in immune cells and transcribed by the Vitamin D Receptor. and anti-microbial peptides. It’s also involved in the expression of alpha-defensins as well. And these are key to the intra-phagocytic innate immune defenses. When these anti-microbial peptides and anti-microbial proteins get knocked out, then the phagocytes can no longer protect themselves from attack by the pathogenic microbiota. The microbiota evades the immune system by blocking DNA-transcription by the VDR. It blocks the VDR which consequently blocks expression of these endogenous anti-microbials. The body cannot produce the anti-microbials because the DNA-transcription, the receptor that would do that –express those anti-microbials- is blocked by the pathogens.

The microbiota changes the expressions of greater than 913 genes. And those are genes which do everything from create a parathyroid hormone precursor, right through to create the Missing in Metastasis protein. MTSS1 is the gene; Metastasis Suppressor number one.

All of these genes, the 913 that have already been confirmed, are transcribed by the VDR. The VDR nuclear receptor is a very key nuclear receptor in Homo sapiens. But homeostasis of the other type 1 nuclear receptors isn’t directly upset by these pathogens. The VDR of course but the PXR, the Pregnane Xenobiotic Receptor, the Glucocorticoid Receptor, Thyroid-alpha-1 and Thyroid-beta-1 are all profoundly affected by the elevated levels of the seco-steroid that are caused by the VDR being knocked out. And obviously, note especially a loss of Glucocorticoid and Thyroid homeostasis leads to the diagnosis of hypothyroidism and adrenal insufficiency. We have demonstrated that both of these are reversible.

So why haven’t we seen this microbiota before? There has been so much study of pathogens in mankind. Well there are a few reasons. The first one is that the VDR homology, the shape, the amino acids that go together to make up the VDR, is a little bit different in Homo sapiens to what it is in all of the other mammals and all of the other fish etc. as well which have VDR. And it transcribes different genes from the VDR of the mammals. And you know how much of our work we have been doing in animal models. Well, a very key-function of the bacteria that have evaded the human immune system do not appear in mammals because the VDR homology is so unique to Homo sapiens. The VDR from the murine and canine genomes for example doesn’t transcribe Cathelicidin or the Defensins, at all. So a human metagenomic microbiota won’t survive if it was transfected into, for example, a mouse. Because different species and different mutations would be necessary if the microbiota was to knock out the different gene pathways needed for survival in a mouse.

Further, the microbiota is only stable in-vivo. It defies extraction using standard techniques. You saw how that cell had disintegrated in about six hours of aging. You can imagine what it does under centrifuge. Further, most of these species in the biofilm microbiota defy attempts in-vitro (culture). This is a study from Dempsey et al, which was a study of biofilm from prosthetic hip-joints, which were removed during revision arthroplasties. And they did gene sequencing and tried to match up the 740 known genomes that we have for bacteria against what they found, the DNA that they found in the biofilm. And this is what they found. Lysobacter. Lysobacter was about 44 percent of the clones that were sequenced. Proteobacterium, Methylobacterium, Staphylococcus. Well Staph, Staphorus, you would actually expect to find that in a human biofilm, that’s not unusual. But its size is small, only about 4.2 percent. That is unusual. Unidentified clones… But look at this: Hydrothermal venteubacterium. This was a eubacterium that was first located in hydrothermal vents under the ocean and here it is, its DNA is popping up in man. And, at high concentration: 5.1 percent of the clones that were sequenced which is higher than Staphococcus genus. There are some other genus here, you can look up the paper and go into more detail. This is what the Human MicrobiomeThe bacterial community in the human body. Many species in the microbiota contribute to the development of chronic disease. project is aimed at setting out. Exactly what species, what genes exist in Man.

Until the genome was cracked, we only had the postulates of Koch as a guide. They caused us to search for Koch’s singular pathogenic species. We kept looking for one pathogenic species. As a result you can find a paper that will blame poor old EBV for just about every disease known to man. And CMV and HHV as well. Because Koch’s singular pathogenic species in this age of the genome really means very little. It sidetracks science from understanding horizontal transfer of DNA within the microbiota. Sharing of genes between the organisms, which we now know occurs very, very much faster than we ever could have dreamed to be the case. Science became fixated on the co-infections, those things we could see, like the EBV and missed the primary disease mechanism: the ability of the pathogens to knock out the innate immune system.

Another reason is that Vitamin D is the primary ligand that activates the VDR receptor. And at some stage during the twentieth century mankind decided that Vitamin D was nutrient. Well, Vitamin D is not a nutrient. It is a seco-steroid transcriptional activator. And its concentrations are very closely controlled by a very complex control system which involves not only the VDR but also the Pregnane X Receptor, the Pregnane Antibiotics Receptor, the P300/CBP PKA pathways and feedback via a number of enzymes. CYP24, 27, A1, 27B1. There is transrepression from VDR activation, feedback path, there is transrepression or actually antagonism, receptor antagonism from the metabolites. And there is also feed-forward pathways. Quite a complex mechanism… If Vitamin D was a nutrient we would see a simple first order mass action metabolism. We do not see that. We see the complex control system of a hormone. Of a seco-steroid transcriptional activator which is what it is. When we concentrate on the concentration of this intermediate substrate, 25-hydroxy-vitamin-D, that’s the one that Medicine has been measuring as an indication of Vitamin D status. That is down-regulated in disease. When the VDR is knocked out, the production of the CYP27A1 is down-regulated and the production of 25-hydroxy-vitamin-D in the body is down-regulated. Down-regulation is not ‘deficiency’. It is the body regulating the concentration of the metabolite. What is happening is, because the VDR is knocked out, no longer can these genes degrade the 1,25-DPrimary biologically active vitamin D hormone. Activates the vitamin D nuclear receptor. Produced by hydroxylation of 25-D. Also known as 1,25-dihydroxycholecalciferol, 1,25-hydroxyvitamin D and calcitirol. and the 1,25-D is becoming very high in concentration, affecting the other receptors and so it tries to block the metabolism of this pathway and in so-doing down-regulates the level of the thing that we’re measuring, thinking that it is a meaningful measure of Vitamin D homeostasis. It’s not. It’s down-regulated in disease. My paper “Vitamin D discovery outpaces FDA decision making” in Bio-essays last February has contained the diagram and all the associated description.

But, there is another problem too. Only 1,25-dihydroxy-vitamin-D, the doubly hydroxylated version can activate VDR transcription, can actually activate/transcribe the genes. Well, Vitamin D that we ingest and 25-hydroxy-vitamin-D that is hydroxylated from that, both inhibit transcription. Here we have in-silico data showing how each of these Vitamin D metabolites fit into the VDR receptor. And you can see that only one of them has the 1-alpha-hydroxylation which is necessary to actually activate the receptor so it transcribes genes. And yet all of them occupy similar space inside the VDR and they all have very similar values of Kd as well. So if you are getting a lot of Vitamin D supplementation, it is actually tending to displace, on a concentration dependant basis, the active metabolite from the VDR.

Well, luckily there is an agonist that works in-vivo. It’s a drug called Olmesartan. Here we have a molecular dynamics emulation of the human VDR with Olmesartan sitting in the binding pocket in an activated position. As you know, all proteins are in motion, at all times.

And, here we have the same thing in rat (Rattus Novegicus). It’s a very, very similar VDR and a very, very similar ligand, positioned, but they are not quite the same. And when we put them side by side you can really see the difference. In particular: look at this Tetrasol. The tetrasol-ring is a totally different orientation because it’s binding to totally different amino acids in the VDR of the rat and the VDR of the human being.

It’s only by getting down to the level of the molecules that we can really understand the difference between our animal models and Homo sapiens.

Then, once the innate immune system has been activated again, we can use very low-dose bacteriostatic antibiotics to block protein synthesis. Here I’ve got Azithromycin blocking the 70S-ribosome which translates RNA into proteins in the bacterial organisms. They are being blocked by Azithromycin.

The rate of bacterial death when you are using bacteriostatic antibiotics is controlled by inhibiting the protein synthesis and we can use sub-inhibitory, low doses of bacteriostatic antibiotics. Later on Dr. Blaney will talk a little bit about the dosing issues.

But just remember that one bacterium is weakened if just one antibiotic molecule is bound into one ribosome (because these antibiotics actually block the functioning of protein generation). So intermit low doses can proportionally control the rate of bacterial death. And that’s very fortunate. Because recovery isn’t easy. There’s a huge bacterial cellular load, whether it’s ninety percent of the body or not I would not know but it is a huge load. As the intra-cellular bacteria are killed, some of the infected cells undergo apoptosis, some even disintegrate.

The loss of cells -both white and red cells- and the cytokine storm which is concomitant with that, has to be controlled so it doesn’t become life-threatening. The damage is called immunopathology. And people who are seriously ill carrying a heavy bacterial load (which isn’t about every individual with an autoimmune diagnosis), they need to spread the therapy over many years if the immunopathology is to be kept at a tolerable level.

You can’t just give the patient the anti-biotics, kill the bacteria and send the patient home feeling well. The problem is, there are just too many bacteria to kill, the load is too high, and just like you have with for example Anthrax, the patient dies from the cytokine storm. You have got to be very, very careful.

Finally, I want to point out that what we have done is evolutionary. It’s not revolutionary. We have built on the work of people like Wirostko, Yahooda from back over the last couple of decades… Many of you, I’m sure, too in the audience… Our model, the molecular in-silico model of the disease fits your data! Please seek out my colleagues over the next couple of days. Talk with us about your data, your studies, especially if you think that our model does not stand up to scrutiny. We would love to discuss it with you. It does fit your data!

And, finally, we’ll end this presentation contemplating Newton. Thank you.