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+====== Presentation - A new approach to treating intraphagocytic CWD bacterial pathogens in sarcoidosis, CFS, Lyme and other inflammatory diseases ======
+{{ youtube>-FzYARFQ9ZE}}
+**Type:** Conference presentation\\
+**Presenter:**  Trevor Marshall PhD\\
+**Conference:**  41st Annual Meeting of AAEM\\
+**Location:**  Hilton Head, SC \\
+**Date:**  Oct. 26-29, 2006\\
+**Additional content:** [[https://autoimmunityresearch.org/transcripts/marshall_aaem_2006.pdf|Transcript and slides]]
+===== Transcript =====
+Thank you John, and thank you to the academy for inviting me to
+come.
+Well the first two slides are just links to some of the papers that
+we've published on these topics. They are there for your references.
+Our FDA coordinator also told me to make sure that I point out
+that there are FDA applications current for both Sarcoidosis,
+designations in the drugs in Sarcoidosis, and also in PTLDS or Post
+Treatment Lyme Disease Syndrome(what most of you would know
+as chronic Lyme) active; and the numbers are all at the bottom
+and you can find a copies of all of those applications on the
+Internet if you want to look in more detail at the regulatory issues.
+The disclaimers. Many of the disease states that I am going to
+cover in this presentation, including neurological states are not
+generally accepted as being caused by pathogens. Much of this
+presentation is based on leading edge science and not on weight
+of evidence. The Phase 2 clinical trial is ongoing.
+Even when shown the science and the microscopy, some experts
+still disagree to the existence of persistent pathogens.
+The FDA has now designated trials for long term use of
+Minocycline and Clindamycin in the treatment of Sarcoidosis but
+not in the other indications I am going to discuss. We are going to
+Phase 3 trials over the next year or two.
+Now in addition to our previous papers, the list of which I've just
+gone through, many of the slides have Pub Med ID numbers on
+them. Those are citations to support the science in any specific
+slide. So if there is something on a specific slide that you feel I'm
+reaching a bit with, make sure you look at the Pub Med ID
+citations first.
+And there are also a few new slides resulting from recently
+published peer review papers which are not in the syllabus and
+which have not been reviewed by AAEM. These have that little red
+logo at the bottom saying "NOT IN SYLLABUS."
+So here is the first one, "Not in Syllabus." Well, actually, the top
+part is in the syllabus but I changed the text on the bottom, as you
+will see on the next slide.
+The quotation at the top, from a gentleman called John Arbuthnot,
+from Sixteen Hundred and Ninety two. Believe it or not, this
+quotation really forms the basis of evidence-based medicine. This
+is the gentleman that wrote the first book on statistics of the laws
+of chance in 1692 and as you would all know, without statistics and
+without the concepts of statistics, we wouldn't have the clinical
+evidence-base that we have at the moment.
+What John said ... [read quote from slide].
+The primary difference between mathematical science, which is
+primarily what I'm going to deal with in this presentation, and
+evidence-based medicine is that one is definitive and one is
+interpretive. Mathematical science tries to define relationships
+between metabolites. Tries to define what's going on. Evidence-
+based medicine tries to interpret the results of observations in a
+reliable manner.
+As we enter the 21st century, the tools to reduce some important
+medical dilemma to mathematical precision are now available in
+Molecular Genomics. Now one thing I want to point out is that
+true science has no concept of weight of evidence. It doesn't
+matter how many times you add up the balance in your checking
+account, you can start at the bottom of the page and work up, you
+can start at the top of the page and work down, the numbers you
+are going to get are exactly the same and any body who has done
+high school arithmetic can sit down and check your calculations
+for you. So therefore, there is no need for weight of evidence.
+As a hypothesis is advanced, it can be tested and it stands until it is
+rejected or improved. E=MC2 has been improved many times
+over the years, but the basic E=MC2 is still there, still in place.
+If the problem is deterministic, then there is a solution.
+This is a new slide, and as an example of how molecular and
+evidence-based knowledge are symbiotic and the need for both
+fields to work in closer cooperation, last week The Lancet
+published my answer to a question which had been posed in July
+(2006) by Dr DS Grimes. Dr Grimes posed the hypothesis, "Are
+statins analogues of Vitamin D?"
+Now that's my paraphrase and I'm being a little bit cruel but I
+wanted to give you a quick encapsulation of what the hypothesis is
+about. This is a hypothesis which cannot be solved by evidence-
+based medicine. It cannot be solved because the variables that are
+involved, the number of variables that are involved is too high and
+they are not appreciated by the investigator.
+When you have too many degrees of freedom, your statistical
+analyses of the epidemiology just break down. However, the
+evidence-based dilemma is easily solved by Molecular Biology and
+I'll show you this later in the presentation. I'm going to be talking
+about statins. That was actually in the syllabus.
+But I wanted to point out that biochemists are also failing. [Read
+slide to 'Vitamins D']. The VDR stands for Vitamin D Receptor.
+[Continue reading slide to knowledge] Yet the insights that are
+resulting from science that the biochemists are producing [continue
+slide].
+First I need to say little bit about Th1 inflammatory disease. [Read
+slide first Paragraph]
+Unfortunately, interferon-gamma is a paracrine cytokine. It exists
+only in the tissues and it's not an endocrine hormone. It doesn't
+circulate through the bloodstream. [Read 2nd Paragraph to tissue]
+if you want to do a biopsy [finish 2nd Paragraph].
+Consequently, a number of other paracrine cytokine have
+historically been measured in order to try and infer whether a
+patient is presenting with a type Th1 inflammation, or a type Th2
+inflammation. A type Th1 inflammation is typically those which
+result from infection, such as Tuberculosis, Leprosy, Syphilis. All
+those are Th1 or they cause Th1 immune responses. Th2 is more
+typically those which result from what we call allergies. However,
+none of these other cytokine are specific and this has led to
+considerable confusion. People think in terms of Th1 and Th2, but
+basically all of the autoimmune diseases are Th1.
+Louis Pasteur once noted that in science, chance favors the
+prepared mind. There are 2 factors: Chance, it's all based on luck
+(chuckle), being in the right place at the right time. But the
+prepared mind is important. You have to be able to observe what
+you see, when you are there. We were lucky, we were able to
+break that particular nexus and realized that all of these diseases
+we were dealing with were in fact, Th1 diseases.
+... Suicidal idealation, this is not something I've heard anybody
+speak about, so far at the conference. It's a big problem with us.
+As the immunopathology takes over, the cytokine storm hits.
+Suicidal idealationoccurs in a significant number of these people
+and it's treatable. As a matter of fact, it's avoidable.
+Now those of you that know about the Marshall Protocol and have
+been following what we have been doing over the last five or six
+years would know that we've conducted this on the Internet as an
+open study. Any physician such as yourselves can register and get
+full access to the database of all the reporting from the patients in
+the study going right back to day one, right back to about 2001.
+A year ago, this was heresy. Everybody said you can't do an open
+study. You can't do an adaptive study. You can't change the
+conditions as you are going along. You can't do anything on the
+Internet.
+Well, things change. The drug pipeline is now nearly empty and
+the FDA is getting quite concerned that not only is the drug
+pipeline nearly empty, but the drugs that are there don't work
+properly anyway.
+And so I put a quote in there now which is given by the FDA
+Deputy Commissioner for Medical and Scientific Affairs, who is
+actually in charge of clinical trial evaluation at the FDA, Dr Scott
+Gottlieb, from an actual conference that was held earlier this year
+on adaptive trial design. And basically, I'll save time by saying that
+he says [paraphrase] "we've got to figure out better ways of doing
+these things, guys." And whenever I read the stuff he writes, it
+makes me think of the things we are doing.
+Now we published our initial results in 2004 in the Th1 syndrome
+called Sarcoidosis. Sarcoidosis is a rare disease and it's a disease
+that we chose to investigate first. We were lucky to choose this
+one, it happens to be a prototypical case for all of the Th1
+diseases. And there are a number of things in Sarcoidosis which
+makes the analysis of the disease state very much easier than it is
+in some of the more complex conditions.
+And in particular, by the time the inflammation has advanced to
+the stage where it forms self-perpetuating granuloma, there are
+very few antibodies being formed. So there is no longer the red
+herring of the antibody formation and perhaps the antibodies
+making people sick. We knew the antibodies weren't making
+people sick because there weren't any. We knew there was
+something else that was making people sick, probably a cytokine
+storm. Where was it coming from?
+That was the other Pasteur piece of luck. We happened to be
+studying the correct disease that turned out to be the prototype for
+many, many other Th1 diseases.
+So at Karolinska institutet in Stokholm, at DMM 2006, I gave the
+following figures for the recovery rate in several key autoimmune
+diagnoses. These were extracted from the Phase 2 and 3 reports.
+These are people that have mainly been on the protocol for twelve
+months or so on average.
+As you can see, Rheumatoid Arthritis has very good success. Eight
+have a primary diagnosis of Rheumatoid Arthritis, seven of them
+are reporting improvement in their condition.
+With Hashimoto's Thyroiditis it is a lot easier. Either they need the
+supplements or they don't. There the ratio is twenty out of twenty-
+five are improving.
+Osteo Arthritis is an interesting one. I was surprised how many
+people with Osteo Arthritis we had in the cohort.
+And then the ones that we are known for: CFS/CFIDS/Myalgia
+Encephalmyelitis. Let me stick with Chronic Fatigue Syndrome.
+Seventy-seven with about forty reporting ongoing resolution.
+Cardiac Arrhythmia is an interesting one. This came up in a case
+study yesterday. And I would like to have an opportunity to join this
+group again in about five years time when we have more data and
+a lot more history under our belts. And talking again about
+cardiac arrhythmia, this is a very classic symptom of late stage Th1
+disease.
+Sarcoidosis of course, ninety-two with fifty seven recovering.
+Diabetes, five and three.
+Uveitis, eighteen and twelve.
+/20 in Fibromyalgia and 10/8 in Irritable bowel syndrome.
+It is an open study, we don't put any preselection conditions on the
+patients that want to join, and that's why we have accumulated
+such a wide range of diagnoses. There are about 500 patients at
+the moment that are regularly reporting and about ten times that
+number that we are aware of... of course, that don't report.
+There are a few more diagnoses on this slide. It is in your syllabus
+so I won't go over them in too much detail. Point out Psoriatic
+Arthritis is a new one, also Multiple Sclerosis. We have quite a few
+Multiple Sclerosis patients now, at least it is getting up to statistical
+significance. Most of those are being looked after by Dr Greg
+Blaney in Canada.
+Tinnitus resolves. Usually acute Tinnitus resolves quite quickly and
+then takes quite a lot longer to resolve fully, several years to
+resolve fully. Like peripheral neuropathy, Tinnitus is one of the slow
+symptoms to resolve.
+Juvenile ADHD, I've got two out of two and I've already chatted
+with the autism, the people that are interested in autism. That was
+quite a surprise to us. Both of those patients had primary diagnosis
+in other areas. One had a Lyme disease diagnosis, the other had
+Sarcoidosis and Uveitis diagnosis. The uveitis being key there. The
+patient was going blind. All of the problems have resolved. As the
+primary inflammatory diagnosis resolved, the attention and other
+problems, psychological problems, resolved as well. Resolved
+spectacularly. Both are back at school and doing very well.
+Well, five steps basically. [Read to end of step 2]
+Olmesartan is licensed for sale throughout the world as an
+angiotensin-receptor blocker. But it has significant activity in other
+receptors. One of the receptors it is very active in is the VDR. It
+activates the VDR.
+[Read step 3, to "25"] And when the patients start with 25mg every
+hours it is usually all they can handle. And then increasing to
+mg over about three months.
+[Read step 4] The choice is made based on suicidal ideation.
+Clindamycin tends to exacerbate any suicidal ideation or
+depression that is present and we avoid it in the early stages of the
+protocol with people who have that tendency.
+[Read step 5 to 3 antibiotic combo] "Low dose?", well
+Azythromycin, some of them can handle 1/8 of a tablet every ten
+days when they start. They eventually work up to about half of an
+Azythromycin tablet as ultimately the bacterial load is totally
+removed, and then they can just guzzle the antibiotic like candy,
+like a healthy person can.
+But initially, when the immune system is working, those antibiotics
+do an incredible job of killing the bacteria.
+So, this was a warning that my FDA liaison — contact — said
+"please put in."
+[Read warning to apoptosis] programmed cell death [continue
+warning].
+Immunopathology. That is the word that is being used instead of
+"Jarisch-Herxheimer." Jarisch-Herxheimer typically refers to the
+cytokine storm that occurs in acute episodes. "Immunopathology," I
+first heard Rolf Zinkernagle use it at Karolinska and it is becoming
+more and more commonly used. It is basically what happens to
+the body when the immune system is doing its job. It includes
+apoptosis, cytokene storm, all of the other effects on the body from
+the immune system doing its job. Immunopathology.
+[Read Steps needed].
+The moment pathogens start being recognized and start being
+killed, your white and red blood cell counts are going to go all
+over the place.
+[Read Step 1 to receptors], which is rather handy to reduces
+fibriotic tissue deposition.
+Now we use special antibiotics. You might not think they are
+special, but they are special in their interaction. The antibiotics
+have a direct action on a part of the bacterial genome which is
+called the ribosome. The ribosome, if you cast your mind back to
+molecular biology, is where the messenger RNA from gene
+transcription, are turned into protein.
+In bacteria, they only have one ribosome. The human body has
+two ribosomes. Bacteria only have one, called the 70S ribosome,
+and the antibiotics that we use block the action of the 70S
+ribosome. They block the ability of the bacteria to produce protein.
+Particularly to use the protein that they use to evade the immune
+system. Because clearly, the bacteria wouldn't be capable of living
+within the phagocytes unless they had a mechanism evolved
+probably over centuries that allows them to evade phagocytosis.
+So the rate of bacterial death is controlled by inhibiting the protein
+synthesis, using only intermittent low doses of bacteriostatic
+antibiotics.
+The other advantage of going after the ribosome is a small
+amount of antibiotics kills a small number of bacteria. In fact, one
+molecule binds into one ribosome. So it is not quite linear, but it is
+a proportional response.
+So why? Why does this intervention work when all the previous
+antibiotic therapies used for these diseases didn't induce recovery?
+[Read 1]
+In other words, there is a model. We can fit the disease that we
+observe to a model. So when I say that five MS patients is a good
+sized sample, its because they are behaving exactly as the model
+predicts. So we have the extra statistical guidance from the model
+as well as from the data.
+It works because [Read 2].
+[Read 3] The VDR, which was long thought to be 'just' associated
+with 'vitamin D', is now known to be at the heart of innate
+immunity. And I'll talk a LOT more about this as we go forward.
+[emphasis repeat] Vitamin D is at the heart of innate immunity.
+[Read 4]. That is part of the model. We have to know how the
+people get sick, and how the sickness progresses over the years.
+[Reference 5]. For example, sequencing the bacterial genomes
+have shown that many of the common bacteria, even Staph
+aurious, has got plasmids. Plasmids are little self-replicating loops
+or sticks of DNA that can exist and replicate on their own without
+the main chromosome. And many bacteria have these plasmids.
+The plasmids can persist and if they are not destroyed by the
+immune system, they can interact with other species of bacteria.
+Toward the end, I will show you exactly how that happens with a
+bacterium species called antrhax.
+So the plasmids are persistent and prolific. So when you have a
+chronic disease which is accumulating over a lifetime, certainly
+over many years, you're not going to have just one species that is
+active. You are going to have a number of species that are active.
+You are going to have a number of plasmids that are active. You
+are going to have a number of viruses that are active.
+The ubiquitous EBV. I think I can find a paper for just about any
+disease you like name which blames EBV as the cause of that
+disease. No. But EBV is very hard to kill off. Its plasmids is just two
+genomes long; something like a 160,000 base pairs long; it's very,
+very small; very, very hard for the immune system to recognize and
+kill; and it persists. And it interacts with any other species of
+pathogens in exactly the same way as it interacts with the human
+genome. And it will cause mutations. The bacteria cause
+mutations.
+And the last reason the intervention works is [reference 6] because
+the MODEL recognizes that neo-natal pathogens persist in the
+brain. [Repeat intended] Neo-natal pathogens persist in the brain.
+Well, now we get into some pretty pictures.
+These are pictures of a receptor, it is a G-Protein Coupled
+Receptor that goes through a membrane. And each of the little
+colored balls are atoms. Each of the similarly colored atoms form
+part of an amino acid, which together make the protein which is
+folded into the receptor.
+Well it's not very easy analyzing that sort of diagram, so
+biochemists tend to draw everything in terms of spirals and loops.
+Each one of these spirals is a cascade of amino acids. It is shown
+diagramatically as a spiral so that we can see more easily what it
+is actually doing.
+We also have a more precise 2-dimensional representation that
+you will find in some of my papers. And here we have the actual
+amino acid of the protein (see lower right "Leu 293" highlighted in
+video) and it shows which are the atoms or ligand of the chemical.
+In this case it is olmesartan but it could be any of the vitamins D.
+It shows which of the atoms interact with the ammino acid residues
+(see lower right O1 red atom highlighted in video), and it also
+shows the hydrogen bonds when they exist as well. Making it very
+easy to try and figure out whether we are dealing with an agonist,
+or an ANtagonist. Much harder to do that in 3-dimensions.
+I want to talk about the nuclear receptor type one family because it
+turns out that this family is most closely related to the operation of
+the immune system — both the innate immune system, and the
+adaptive immune system.
+The key receptors for which we have known structure models. In
+other words, we not only know that the bacterial genome looks like
+this but we also know in 3-dimensional space that x-ray
+spectroscopy has told us exactly where each atom is in the
+molecule. In this case, we're talking about the human genome. But
+it's the same theory, we've got the VDR (Vitamin D Receptor), the
+Progesterone Receptor, PPAR-alpha (which is one of the lipid and
+immune system mediators), PPAR-gamma (which is also lipid and
+immune system), Androgen Receptor, the Estrogen Receptor,
+glucocorticoid receptor, Thyroid-alpha-1, Thyroid-beta-1, and the
+mineralcorticoid receptor.
+Now surprisingly, if you look at the pictures of each of these drawn
+as spirals (I guess maybe I should have done a slide of it for you),
+but they are all very, very similar. And that is why they are the type
+nuclear receptors.
+And you know molecules that will go into the VDR usually only go
+into PPAR-gamma and some of the other type 1 receptors as well.
+They affect more than one function of the body.
+So nuclear receptors are responsible for the transcription of the
+DNA genes to strands of RNA, which are then translated into
+proteins in the ribosomes. And they are very important.
+I've put a link there to a simplified set of flash animations for those
+of you that want a crash course on how this all works. It is a very
+simple crash course. I really recommend you have a look at it.
+(https://www.johnkyrk.com/)
+Now we're going to look at some simplified 3-D animations of the
+transcription molecules, just to give us an overview.
+What we have here, we have a strand or double strand of DNA
+(highlight blue/grey/red/pink balls base right of diagram). These
+are the base pairs that run across (highlight on video shows
+vertical ball lines at bottom center of diagram) the two backbones
+of the DNA. Across the center you have the hydrogen bonds that
+hold the DNA together (highlight moves L-R across center of
+vertical ball lines). Hydrogen bonds that are peeled apart is when
+the RNA is produced. The RNA is basically ½ of the DNA double
+spiral.
+So when the nuclear receptors do their job and transcribe a gene,
+what they are actually doing is breaking the hydrogen bonds and
+producing the RNA in a very complex and clever fashion which
+doesn't occur very fast because it requires a lot of things, the
+statistically drift around and come together in exactly the right way.
+Nuclear receptors typically take days to effect their action.
+Key to nuclear receptors are the zinc fingers here (video highlights
+upper left center large yellow/green balls). The green atoms are
+zinc, the yellow, sulpher-disulfides. Actually cystines to be honest.
+And they are a key part to positioning the helixes which go through
+the (video highlights lower left solid light grey spiral) DNA and find
+the exact chain that needs to be translated based on the exact
+mapping of attractive and repulsive forces and then do the DNA
+transcription.
+It is an amazing system. I've shown here that there two receptors
+because usually one nuclear receptor doesn't do it by itself. It can
+form a homodimer but it usually forms a Heterodimer. So you have
+a VDR coupled with a retinoid-X receptor and then the two of them
+transcribe genes. There is also a cofacter that gets involved as
+well. Quite complex and we really don't know a lot about it yet.
+I mean we now have the genes to transcribe, but we don't know
+exactly which co-factors go with which receptors.
+The biggest surprise we got when we started doing this modeling,
+was the high affinity of the ARBs and Statins for VDR and PPAR-
+gamma, Nuclear Receptors which are both key to the immune
+system.
+Now these are very flexible, highly polar ligands. And we thought
+they might have an affinity for other G-protein coupled receptor
+membrane receptors, in other words receptors that go through a
+membrane rather than exist in the nucleus of the atom.
+But their affinity for the Nuclear receptors was a real surprise.
+Here's a big table which represents months of computing.
+But what we have done here is used special software that
+computes the interaction between thousands of atoms and figures
+out for each of the (video highlights drug names in the sartan
+group) ARBs and each of the Statins (video highlights drug names
+in the statin group), which receptors they are going to have an
+affinity for — if any — and which receptors they are going to
+switch on or switch off.
+And if we just look at the statins first, thinking back to the question
+posed by Grimes in The Lancet: "Are statins analogues of vitamin
+D?" Well the first thing that pops out is that the VDR here (video
+highlights VDR column head), there is a huge difference between
+the statins.
+Simvastatin (video highlights 4 at base of the VDR column) hs got
+quite a low number which means quite a high affinity, but
+Rosuvastatin, fluvastatin, and particularly Atorvastatin (Lipitor) don't
+have any affinity for the VDR at all. Pravastatin that is effectively no
+affinity. Lovastatin would have a little affinity if a low dose is used
+in vivo.
+So there is a huge difference between the statins. Just thinking of
+the statins as a group is a concept that is dangerous clinically, let
+alone to try and extrapolate that to talking about the statins
+functioning as a hormone would.
+But you can also see if you take Simvastatin, not only does it have
+a high affinity for the VDR, it's got 10x higher affinity for the PPAR-
+gamma, same for PPAR-alpha as the VDR. The Gluco-corticoid
+receptor -the home for cortisole- it has a high affinity for, also the
+MCR. The Progesterone receptor, the Alpha-Thyroid and the Beta-
+Thyroid. Simvastatin is a very, very active statin.
+Whereas Atorvastatin for example, primarily hits PPAR-gamma and
+alpha and incidently hits the Gluco-cortisole axis for the GCR.
+However, some of the ARBs are a little bit better behaved than that.
+Telmisartan is very poorly behaved. It totally shuts off the VDR the
+moment you give it to somebody. This is NOT good.
+Olmesartan, here (video highlights 10 in the VDR column) has got
+a moderately low affinity for the VDR, and that is one of the
+reasons why we have to use a higher dose when we are using it to
+modify the VDR than when we are modifying the Angiotensen-
+receptor where it has a much higher affinity. But it is nicely
+controlled in the PPAR-gamma and -alpha (video highlight across
+Olmesartan Estimated Ki row) and GCR. It hits the progestrone
+receptor but it doesn't really hit Alpha-Thyroid.
+Beta-Thyroid it hits but we're not really sure what Beta-Thyroiod
+does yet. What we know about Beta Thyroid is that if you breed
+mice that don't have the beta thyroid receptor, then they are born
+deaf. Otherwise they seem to be fine. (Audience chuckles) So we're
+still trying to figure out what the Beta Thyroid does.
+The ability to breed mice with gene knock-outs without these
+receptors is a PRIMARY new tool that has become available to the
+biochemist. It is invaluable. Unfortunately with the GCR, if you
+knock out the GCR the mice never survive past gestation. So we
+don't know a heck of a lot about the GCR either except it is at the
+root of the Cortisole axis of the adrenal.
+But anyway, it is a big table and I have it printed on a sheet of
+paper and I have to refer to it all the time, so I'm not going to
+expect any of you to commit that to memory.
+Now I'm going to look at a very simple hormonal system. The
+adrenal axis. And up here (video highlights top word
+"GlucoCortocoid Receptor"), I'm just looking at part of it and I'm
+looking at the GlucoCortocoid Receptor, the nuclear receptor, with
+a promotor which is "c" AMP (cAMP). Promotor binds to the
+receptors, and then the promotor and the receptor together bind
+to the DNA and transcribe the gene.
+The GlucoCortocoid Receptor is responsible for producing
+Corticotrophin Releasing Hormone (CRH highlighted), and that in
+turn produces the Pro-Opeo-Milleno Cortin and when that is
+metabolized, you get Beta-endorphin and also an ACTH
+produced. ACTH of course you know, that catalyzes the conversion
+of Cholesterol into Cortisol. Then Cortisol goes up and docs into
+the GlucoCortocoid Receptor (highlight shows arrow line path left
+and up to GCR) and you have a feedback loop, which keeps the
+Cortisol levels of the body at roughly constant levels.
+That is a very simple, straight forward hormone loop.
+Unfortunately, with 'vitamin' D, it is a little bit more complex. As you
+would expect for something that is at the heart of the immune
+system. It has adapted over many, many millennia.
+The "not in Syllabus" flag indicates some of the enzymes here have
+shifted from what is printed in your syllabus. Also, since that
+syllabus was published, there has been new studies showing that
+the Androgens (video highlights lower left corner word
+"Androgens"), particularly testosterone, reduce the breakdown of
+,25-D by the enzyme CYP24.
+But basically, the body starts off with 7-dehydro-Cholesterol, which
+is part of the sterol subsystem. It is a cholesterol derivative (video
+highlights upper left 7-dehydro-Cholesterol), and that is converted
+to pre-Vitamin D by energy. Conventionally thought to be UVB, but
+the biochemists will easily tell you it doesn't have to be UVB and it
+can even be an enzyme. Enzyme at this point, we have no idea
+what that enzyme would be if it is an enzyme. But it certainly
+doesn't have to be UVB to convert to Pre-Vitamin D (Pre-Vitamin D
+highlighted). All you need is a contra-rotatory electrocyclic reaction
+which occurs in other substances and which is pretty well
+characterized.
+(Vitamin D highlighted) Then, Pre-Vitamin D is formed into Vitamin
+D by a Sigmatrophic shift. A very small shift of the electrons (????).
+Vitamin D is converted by the enzyme CYP27A1 -or- CYP2R1 and
+both of these are P450 digestive system enzymes into 25-
+hydroxyvitamin-D (highlight drops to 25-hydroxyvitamin-D) and this
+is the one that most of you are measuring when you measure
+somebody's 'vitamin' D levels, you are almost certainly measuring
+the 25-hydroxyvitamin-D.
+(Highlight drops to 1,25-dihydroxyvitamin-D) That then is
+converted to 1,25-dihydroxyvitamin-D, hydroxillation by enzyme
+either CYP27A1 or CYP27B1, both of them will hydroxyllate. And
+that 1,25-dihydroxillate with activate (highlight moves to
+*activate*) the VDR and allow the VDR to do the gene transcription
+that is at the heart of innate immunity.
+And then finally, the 1,25-D is inactivated (highlight opens on
+lower left "Inactive 24,25 & 25,26") by the VDR up regulating this
+CYP24 enzyme so that you don't end up with too much of it in the
+body.
+And that is what goes wrong in Th1 disease. You end up with too
+much 1,25-D in the body because the VDR inactivation path —
+something goes wrong with it.
+Well, OK. Here we have some pretty pictures. We have atoms and
+bonds. The grey atoms (highlight upper left picture, left grey atom
+selected highlight in video) are carbons, the red ones are oxygen,
+that green is a fluorine, That's a hydroxy OH there (highlight
+moving from green to lower center red/grey center bottom), the
+light blue is the H of the hydroxy. And this molecule is
+Dexamethasone in exactly the configuration where it binds into the
+Gluco-Corticoid Receptor (GCR binding pocket), you know the
+adrenal axis, the Gluco-corticoid receptor.
+And here (video highlights lower right figure center), we see 1,25-
+Dihydroxyvitamin D competing with Dexamethasone for the
+binding pocket of the GlucoCortocoid receptor.
+,25-D has a very high affinity for the adrenal axis. This is new, it
+is not in the syllabus, it's taken from a paper which was accepted
+for the nuclear receptors conference next week, actually.
+So what does that mean? If we go back to our nice simple adrenal
+axis, what does that mean?
+Well it means that (highlight focuses on top black box 25-D and
+,25-D) 25-D and 1,25-D can both bind into the GlucoCortocoid
+Receptor and displace Cortisol from doing that.
+When it does that, the amount of CRH and particularly ACTH is
+down regulated, and you end up with Adrenal Insufficiency
+(highlight on lower black box "Adrenal Insufficiency").
+OK, the vitamins D also compete for the Thyroid binding pocket.
+I'm talking about the alpha thyroid here, the important one.
+There we have T3 as it docs into the alpha thyroid to activate the
+alpha thyroid (highlight shows green end molecules on the top left
+illustration). These are the iodine molecules, three of them of
+course.
+Here we have 1,25-D competing (highlight center of lower R
+figure) with T3 for the alpha thyroid Binding Pocket. It wants to
+dock in exactly the same binding pockets as the T3 would. And by
+doing that in high enough concentration, it will stop the T3 from
+binding. It will stop the alpha thyroid from working properly.
+We end up with, among other things, hypothyroidism.
+So I've done a summary slide here of the operation of the VDR. It
+is key to both the endocrine and immune systems.
+The VDR is responsible for decreasing parathyroid Hormone
+Transcription. [Reference slide to TLR4], and therefore the
+bacterial response.
+Toll-like Receptor 4 is responsible for sensing
+lipopolysaccarhride. Toll-like Receptor 2 is responsible for
+sensing the presence of other bacteria protein. When the VDR is
+not working, because you have administered vitamin D and are
+experiencing the immuno suppressive action of the vitamin D,
+when the VDR is not working, you do not have a functioning TLR2
+and TLR4 system.
+But you also lose the antimicrobial peptides. It transcribes the
+cathelicidin antimicrobial peptide (cAMP) and it's also responsible
+for beta defensins. The beta defensins are absolutely key to the
+proper operation of small intestine. The small intestine, in fact,
+the whole GI tract is critically dependent on the defensins and
+especially the beta defensins and the cathelicidins to work
+properly. When you knock out the VDR with immunosupression
+— either a steroid or 'vitamin' D — you lose that ability.
+And finally, the VDR is responsible for binding the interluken 2
+(IL2) promoter. Now you all probably know that you measure
+interluken 2 when you want to measure a Th1 reaction. Well, that
+is why, because the VDR actually produces the interluken 2, even
+if you can't measure it.
+I've got some other functions there, I guess the main one there is
+it regulates the TACO gene, which is responsible for
+mycobacterium tuberculosis intraphagocytic survival, and it
+promotes transcription of Insulin Receptors.
+Let's skip the rest because we're running a little late bit short in
+time.
+The current status of the VDR is that the VDR is responsible for
+TLR2 and TLR4 expression, as well as cAMP... hey, I just said that
+on the previous slide! Sorry.
+What we've got here is two pictures and we have all the vitamin
+D's (video highlights left image) superimposed. We've got 1,25-
+Dihydroxyvitamin-D, that's the one that has the activating hydroxyl
+(highlight arrow and red atom in lower right of left image) here,
+the only one that can activate the VDR. Various other atoms can
+activate the GCR or de-activate the GCR and the thyroid, but the
+only one that will activate the VDR is this one (highlight again on
+arrow and red atom) on the 1,25-Dihydroxy. But you can see that
+all the vitamins D fit into the receptor in almost identical
+positions. Whether it is 24,25-D, 25,26-D or 1,25-D. Even
+vitamin D itself fits in there as well and they are all competitive.
+If you're giving people 40mg, if you're giving people vitamin D
+such as such as to get the blood concentration at 25-D to 40
+nanograms per ml, then you're sitting with 25-D in most of your
+VDRs and they are NOT activated, it is immuno-suppressive. The
+patient will feel better short term because the inflammation is
+suppressed. Long term, with the bacterial pathogens running right,
+it's a different situation.
+The picture on the right hand side just shows olmesartan binding
+into the VDR pocket and you can see if you look at it carefully, you
+can see that the olmesartan hydroxyl here (video highlights upper
+right atoms O1 and O2 in lower right of right image) are actual
+capable of activating the VDR in the same way as 1,25-D does.
+So, we've got some little pictures here. We have 2 molecules.
+What we have here is the steroid rings (video highlights right
+image, upper right rings of molecule) of prednisone. This is
+actually prednisone.
+We have the steroid rings of prednisone, and over here (video
+highlights middle two rings on left figure) we have the steroid rings
+of "vitamin" D. What I wanted to show you was that you've got the
+steroid rings.... I want to show you the only difference between the
+secosteroid and the steroid is one bond. It's this carbon to carbon
+bond across here (highlight shows critical top left ring closure of
+right image, in 2nd ring from left). And it is missing across here
+(highlight now shows open ring in left image in the 2nd ring from
+left) in the seco-steroid. And that is why it is a secosteroid rather
+than a steroid.
+It is a very subtle change, it actually gives the [vitamin D] [not VDR]
+more flexibility to move in a socket and that is one of the reasons
+why it binds into so many of the nuclear receptors. The structural
+similarity (video highlights cortisone bond again) between
+prednisone and vitamin D basically come down to that one carbon
+bond. And that is why it is a secosteroid and not a corticosteroid.
+So I have a slide here on Vitamin D in Bone Remodeling because I
+know all of you are going to say "well, If I withdraw vitamin D from
+my patients, their bones are going to get weaker." This gives you
+some up to date citations you can go away and look at, which
+show that actually "vitamin" D is not really responsible for calcium
+at all. It's the Calcium Sensing Receptor and the Parathyroid
+hormone that are responsible for Calcium homeostasis. And
+secondly, even the cannabinoid receptors play a key role in bone
+remodeling and bone mass. That paper is only a few weeks old.
+The sex hormones are also players in bone density, I think you
+know that one.
+And that elevated levels of 1,25-dihydroxyvitamin-D, such as we
+have found to be associated with Th1 immune disease, actually
+encourage osteoclastic actions and breakdown of bone and
+deposition of bone into soft tissue. The last thing you want in these
+patients is high levels of 1,25-Dihydroxyvitamin-D.
+So this has got a "not in syllabus" picture because the text that I
+had in the syllabus was a lot more caustic. This is diluted.
+Public-Health Consequences of Regarding 'vitamin D' as a
+'Vitamin.' Sometime during the 20th century, we began to view
+"Cholecarciferol' as a 'Vitamin' rather than recognizing its steroidal
+and hormonal activity. And we put the seco-steroid
+'Cholecarciferol' into the food chain, in a futile attempt to eliminate
+the rare disease, Rickets. And as physicians, you would know the
+side effects from administration of steroids.
+So the CDC now says we are heading towards half of all US
+seniors being Diabetic by the year 2050.
+And, Oh, steroids often induce obesity. I'll leave you to connect the
+dots.
+I haven't said anything about bacteria yet. When I was trying to
+figure out how Sarcoidosis patients could be made sick in the
+absence of antibodies, this paper was published back in late 2001
+on the Internet, and what it showed was Rickettsia Helvetica inside
+Phagocytes (video highlights upper right image E at two arrows).
+Persistent inside phagocytes, not broken down by phagocytosis but
+actually living in the phagocytes of Sarcoidosis patients.
+That was the Eureka moment for me. I should have known it, there
+is plenty of other literature talking about mycobacterium being
+intraphagocytic, but that was the actual paper.
+And then the real studies that you all will want to see if you are
+interested in the actual pathogens, were the Wirostko Studies from
+Columbia University in the late 80's. There are bout 30 or 40
+electron transmisional microscopy photographs showing stained
+bacteria. Tiny, tiny colonies (video highlight shows lower left arrow
+circle of bacteria, then moves to second to left arrow colony, to
+middle arrow pointing to colony and then to upper right arrow) of
+stained bacteria in the phagocytes. This is a quarter of a
+macrophage.
+And they were very thorough, they did macrophages, monocytes,
+lymphocytes and neutrophils. And they found that in the
+Sarcoidosis patient, they found the intraphagocytic bacteria
+persistent in all of them.
+And in fact, the nucleus here (video highlights heavy bolded arrow
+left of center pointing to nucleus) is breaking away a little bit near
+this larger colony of bacteria (video highlights second lower left
+small arrow). That's why it has the big arrow on it.
+Fascinating photographs. Very important if you want to understand
+the actual bacterial pathogens, the L-forms that cause these
+diseases (cell wall deficient bacteria called L-forms for the Lister
+institute where they were first identified). They are tiny pathogens.
+They are so small. They are hundredths the diameter of the
+macrophage. I've got 200 nanometers here (video highlights lower
+right slide legend). We're talking about cocciodes, tiny little dots
+that are about 10-15 nanometers in diameter.
+These are pictures that show the diagrammatically show the
+survival of bacteria in the phagocyte. I've covered these in my 30th
+anniversary of Lyme meeting and with your permission, I'll skip over
+this. You can find it in any book. (because we're running late on
+time).
+Bacterial protein synthesis, and remember I talked about the 70S
+Ribosome, the bacterial Ribosome. Here we actually have the
+structure of the bacterial Ribosome, it is primarily made up of RNA,
+unlike the human ribosomes which are primarily protein based.
+But the 16S RNA (video highlights the lower left 16S rRNA on
+figure), which you would all recognize as being the PCR, the
+substance which is used as the target for most PCR tests. 16S rRNA
+forms the 30S side of the ribosome (video highlights upper left 30S
+figure subhead). The top of this 30S we have a heliacal structure
+(video highlights top left ribbons) which is responsible for translate
+but actually for ... translating the mRNA into a protein.
+The actual translation is done on the 50S side, you can see a
+protein (video highlights lower right quadrant) coming out the
+bottom here and it's in yellow. The protein transferase center
+(video highlights middle, slightly right) is right in the center which
+you see, and there we have tRNA (video highlights blue grape
+cluster mid top) which is donating the amino acid into the growing
+protein.
+And this is an Xray structure, which was produced from the Max
+Planck group in Germany, and this x-ray structure shows a
+number of antibiotic molecules found in, you can look at them in
+your syllabus', but the one's we're interested in primarily are the
+tetracycline. And in fact, the tetracycline binds right at the top
+(video highlights top of 30S Sub-unit, see Orange color), and
+inhibits RNA translation.
+On the 50S side, you can see how the protein is assembled and
+exits the exit tube a little more easily. And in particular, the
+important thing is here, you will notice that Clindamycin (video
+highlights center formation), just the light orange one, sits right at
+the PTC. And the Azythromycin sits a little bit below it, so they are
+actually symbiotic antibiotics.
+All three antibiotics that we use are symbiotic. That means that
+each of them progressively reduces the function of the [bacterial]
+ribosome. That means that when you give them together, they are
+not competing with each other, they're complementary. It is
+symbiotic. Very important. Something that the genome, the
+bacterial genome, has told us quite clearly.
+So now we talk about the species.
+Which species of bugs is it?
+Well, it is not a single species. We're dealing with a polymicrobial
+disease. If you look at the various diseases, and the bacteria that
+have been reported with them all, the common ones like Staph-
+aureus and Propionibacterium-acnes, Propionibacterium-
+granulosum, seem to be the ones that crop up most of all, with the
+nasty bugs, micobactera cropping up far less frequently. And
+certainly less than 100%.
+So we're almost certainly dealing with a polymicrobial disease.
+And depending on the exact mix of pathogens, determines which
+of the Th1 diseases, which of the Th1 symptom syndromes the
+patient will progress to. Whether it turns into Rheumatoid Arthritis,
+Multiple Sclerosis, or Anorexia Nervosa.
+It's become obvious that most bacteria species are not
+homogenous. They've got not only the chromosome, but also the
+self-replicating plasmids which carry DNA and genes. Borrelia
+burgdorferi has got a large number of plasmids, in fact, nearly half
+of its genome exists on 21 self-replicating plasmids sub-units.
+Now I'm not saying that makes it any more strong of a pathogen,
+but it certainly adds quite a lot to the "Pea Soup" which I like to
+think of as mix of DNA which accumulates in the chronic diseases.
+Yet even Staph. epidermidis gives rise to plasmids. It's got about
+% of its DNA spread over six self-replicating plasmids.
+These can be shared between species. But the plasmids are not
+targeted by antibiotics. Antibiotics don't target plasmids. Only the
+innate immune system can go after the plasmids. So unless the
+plasmids are destroyed by the immune system, they will persist in
+chronic intra-cellular infections.
+And I think of it as a "DNA Pea-Soup."
+Plasmids do transmit DNA horizontally. Notice the citation here, it
+was published in the Proceedings of the National Academy of
+Sciences in 2004. And what you had was, you had a patient that
+got very, very ill with a species of Bacillus cereus. Now, Bacillus
+cereus, we all know is a [relatively] harmless bacteria. It's a
+Bacillus that doesn't cause the body [much] trouble, it clears it
+(video highlights B.cereus yellow circle on figure, upper right of
+center).
+Whereas just a little bit further around on the phylogeny here, we
+have Bacillus-anthracus (video highlights red spike circle far upper
+right). And what distinguishes Bacillus-anthracus is 2 plasmids.
+One plasmids contains the ability to evade the immune system, the
+proteins that give it the ability to evade the immune system, the
+other plasmids carries the ability to produce a cytokine storm and
+kill the host.
+Now what happened in this case was the two plasmids from
+anthracus were transferred into cereus and formed an extremely
+toxic combination.
+And you can see that the mice that were just challenged with
+cereus, they all survived (video highlights upper center double red
+circles on figure 4).
+Those that were challenged with anthrax (video moves down to
+yellow circle dropping line) -which color is anthrax? yellow- most
+of them died.
+BUT they died a lot quicker if they were the hybrid (video highlights
+green circle line) of the anthrax plasmids and the cereus genome.
+This is a big, big problem!
+Why is it a problem?
+Well, (video returns to first horizontally slide) if you look further
+around on this phylogeny (video highlights B thuringiensis
+israeliensis, mid far left green triangle), you'll find here Bacillus
+thuringiensis Israelensis. That one is sprayed onto agricultural
+crops as a herbicide. Sorry, it is an insecticide.
+Anyway, you might have noticed, for example, Garth Nicholson
+found a higher incidence of autism in the agricultural areas. This
+fellow (video highlights B thuringiensis israeliensis) is sprayed in the
+agricultural areas.
+If the chronic infection allows the plasmids to spread, these people
+will get very ill.
+The final thing that I want to show is that the pathogens actually
+persist in the brain.
+This [slide] is taken from a paper by Rolf Zinkernagle, whom I was
+lucky to meet at Karolinska, the home of Team Nobel. Rolf gave
+the keynote speech there back in May [2006]. And what they
+found they've done, is they injected the brains of mice at a very
+young age (video highlights A - neonate mice figure upper right
+figure), neonatal in fact, with a virus. And this is injected before the
+mice have an adaptive immune system.
+As you all know, the adaptive immune system in humans and mice
+takes some time to kick in (video highlights top time line center,
+showing Time after rLCMV/INDG). I think these are helper cells
+coming up to indicate the adaptive immune system has kicked in
+after about 7 days.
+But when the mice are born, the only thing they have to protect
+themselves is the innate immune system. You know, the thing we're
+knocking out with 'vitamin' D.
+Anyway, what Rolf did was, he showed that if they injected this virus
+during this period, it remained dormant in the mouse. And later,
+when a similar virus was used to challenge the mouse, It killed
+them (video highlights upper right dead mouse figure).
+Whereas mice that were challenged in adulthood by the same
+virus (video highlights lower left time line, far left), injected by the
+same pathway were not killed when they were re-challenged, in
+fact, they survived primarily.
+This is a fascinating paper, it is quite detailed with citations there at
+the bottom [of the slide]. And Rolf is working on dispelling a myth
+of autoimmunity — or response to self — by showing occult
+viruses can, in fact, can exist and they can indeed persist in the
+brain. Very important when we are trying to understand the
+symptoms of our patients.
+And here is the slide from Professor Bach, Jean-Francois Bach,
+which was presented in Budapest in 2004. Jean-Francois Bach
+was the person who proposed the hygiene synthesis.
+But what he found was that bacteria, the particular strain of mice
+called non-obese diabetic mice (NOD), where typically 60% of
+them died (video highlights upper right dark dot line) from
+diabetes — type 1 diabetes during their lifetime — that if he
+injected them early enough with a mix of common bacterial
+proteins, that they survived (video highlights cyan and yellow end
+point line).
+And the ones that survived best were the ones that got the
+bacterial proteins earlier in life than later in life (video highlights
+magenta square dotted line lower right).
+Again, another fascinating insight into exactly how these diseases
+persist and why some people get ill, and other people carry the
+bacteria and don't get ill.
+Because it is no secret that the NIH/NHLBI did a study in 2001
+where they tested with PCR, they tested the blood of controls. No,
+they didn't use PCR, they cultured the L-form using Wayne State
+Lyda Mattman's technology. And they found 60% of their controls
+were carrying L-forms in the blood. Supposedly a sterile
+compartment.
+This has since been confirmed by the Relman Lab, Dave Relman
+up at Stanford published a paper in the last year, where he too has
+noted that 60-80% of the population are carrying around bacterial
+RNA in their blood. It should not be there. Many of them, most of
+them in fact, are not ill or certainly not diagnosably ill. Some
+however, regress to really serious illness. It all depends on the DNA
+mix and how the mutations progress in life.
+So just a few quick final thoughts that my colleagues at FDA felt I
+had to put in because I am talking to clinicians here, [are] just the
+simple stuff again.
+Final thoughts, if you are thinking if your patient might in fact be
+suffering from Th1 diseases, then the therapeutic probe is really
+the gold standard. That's because it is greater than 95% response
+rate. Most of the patients will immediately feel immunopathology.
+They'll immediately feel more ill when they start taking the
+antibiotics. At that point you know that you are killing bugs. You
+know that these people are responding.
+The only thing I would say is 25-D levels above about 20ng/ml
+you will be getting immuno-suppression there and it will be
+suppressing the therapeutic probe. So you need to be somewhat
+careful about the 25-D level before initiating a therapeutic probe.
+Hypotension is not a concern, even though we are using an
+angiotensin-receptor blocker. These are the graphs from the FDA
+describing insert, the package insert [see slide figure]. As you can
+see, the maximum drop is about 12 millimeters of mercury [12mm
+Hg], and that value is independent of dose. You can see the dose
+along the bottom here the response tops at 40mg. It is not a very
+good hypotensive. Benicar is not a very good hypotensive. Luckily,
+it does other things as well like protect the kidneys, and protect the
+eyes and all the other things that you are reading about in the
+clinical literature right now.
+Hypotension is not a concern.
+Dizziness. When the patients get dizziness, especially in the first
+month, it's usually a sign of the disease process. It is not
+hypotension. It's actually better to increase the Benicar to give
+them a better blockade than to back off the Benicar.
+There is no way you can start with a low dose and work up. You've
+got to have those receptors being blocked, all the receptors being
+blocked, or it doesn't work properly. You will increase the
+immunopathology with only a low dose. That means the patient
+will get, "Herxheimer" if you wish, they will get ill from only a low
+dose of Benicar, but they won't get the palliative effects, they won't
+get the renal-protective effects, until the dose goes up.
+So it is very important to give them the correct dose so they are
+actually going to make it through the therapy and kill those bugs
+off.
+The only thing I didn't mention was how long it would take: three
+to five years for the seriously-ill patients to totally get their lives
+back. Most of them are happy with some form of recovery. They
+see the light at the end of the tunnel after about six months. Very
+few have not seen the light by twelve months but actually, they are
+symptom free or say they are symptom free at about two years. But
+all that means is they are getting along from day to day very nicely
+and much better than they were before. There is still a lot of
+disease present, they are still getting a lot of reaction to the
+antibiotics.
+It takes about another two years to where they can just guzzle the
+antibiotics like candy. At that point, the bacterial load is low
+enough for them to pronounce recovery.
+Thank you.
+{{tag>presentations videos Trevor_Marshall_PhD AAEM 2006}}