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home:publications:marshall_bio21_2006 [04.24.2009] neldawhitehome:publications:marshall_bio21_2006 [11.02.2009] paulalbert
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 + ====== Presentation - Molecular mechanisms driving the current epidemic of chronic disease ======
 +
 +**Type:** Conference presentation\\
 +**Presenter:**  Trevor Marshall, PhD\\
 +**Conference:**  Bio21\\
 +**Location:**  University of Melbourne\\
 +**Date:**  May 22-24, 2006\\
 +**Additional content:** [[http://autoimmunityresearch.org/transcripts/marshall_bio21_2006.pdf|transcript and slides]]; [[http://autoimmunityresearch.org/bio21a.ram|streaming video (.ram file)]]
 +
 +===== Transcript =====
 +
 +This presentation is available on the DVD "The Science —
 +Marshall Protocol" available upon request from the [[http://www.AutoimuunityResearch.org|Autoimmunity
 +Research Foundation]].
 +A lower quality real-time streaming version may be found at [[http://autoimmunityresearch.org/bio21a.ram|this URL]].
 +
 +
 +I've put some citations and disclosures and things on there. We
 +have a number of applications current with the Food and Drug
 +Administration in the USA.
 +
 +We've had [phase 3 testing designation] approvals for two
 +antibiotics, clindamycin and minocycline in sarcoidosis. We're also
 +talking with them about chronic fatigue syndrome and a number of
 +other studies that we're trying to get underway to move as quickly
 +as possible and consolidate on the Phase 2 studies that we
 +ourselves did.
 +
 +And there's a list of the FDA application numbers at the bottom
 +and any of you can look up those numbers and look at it in more
 +detail at the science that I'm going to cover in the presentation.
 +2
 +
 +I usually start with a quote from John Arbuthnot, 1692. John
 +Abiernot wrote the first book on statistics of the Laws of Chance
 +and of course it was about cards, playing cards, but it was the first
 +formal text on statistics. But he wrote this wonder passage that he
 +says: There are very few things which we know which are not
 +capable of being reduced to a mathematical reasoning. And
 +where a mathematical reasoning can be had, it's as great a folly to
 +make use of any other, as to grope for a thing in the dark when
 +you have a candle standing by you.
 +
 +The primary difference between mathematical science and
 +evidence-based medicine is that one is definitive and one is
 +interpretive. Mathematical science, biomedical science tries to
 +define relationships between entities. And evidence-based
 +medicine tries to place reliable interpretations on observational
 +data. Primarily, I mean, there's obviously some fluidity there.
 +True science, the primary difference between them is that true
 +science has really no concept of weight of evidence, that the
 +hypothesis is advanced. E equals MC squared, for example, it's
 +tested and it stands until it's improved or rejected. And if the
 +problem is deterministic, it's solvable.
 +
 +Now of course in medicine most of the problems are not
 +deterministic — yet, but as our knowledge advances more and
 +more of the medical mysteries, if you like, clinical mysteries, start
 +to succumb to molecular biology and that's what I'm going to talk
 +about today.
 +
 +It's very important that both the molecular biology and the
 +clinical sides of medicine work together in close concert. And this
 +is the example that I'm going to discuss. It's from the Lancet. Dr.
 +D.S. Grimes wrote a hypothesis in July of this year [2006] which
 +was titled: "Are Statins Analogues of Vitamin D?" And Dr. Grimes
 +had derived his hypothesis from the following observations:
 +Vitamin D is good for you, statins are good for you, therefore,
 +statins must be similiar to Vitamin D. Now I'm being very harsh
 +there and it's a much better hypothesis than that. You need to look
 +in detail at it, but that's the essence that I pulled out of it.
 +
 +The problem that Dr. Grimes has in analyzing this particular
 +question using evidence-based medicine is that there are far too
 +many degrees of freedom. You have the degrees of freedom
 +related as to how the statins act — and clinical medicine has no
 +idea how the statins act, how Vitamin D acts — and clinical
 +medicine has very little idea of how Vitamin D acts. And then of
 +course you've got the experimental degrees of freedom as well that
 +are going to come into the data and make it very difficult to draw
 +a good conclusion. And of course Dr. Grimes really wasn't able to
 +do so.
 +
 +However, my letter was published which did solve the dilemma
 +because, by recourse, the molecular biology, I do know what the
 +statins do and I've also got a pretty good idea what the Vitamine D
 +does. And because I can reduce that to mathematics with the
 +molecular biology it's very easily communicable to people that can
 +understand molecular biology. And later in the presentation I'll go
 +over the statins.
 +3
 +So clearly that's a gap in evidence-based medicine, but you
 +know the biochemists are also failing because a search at PubMed
 +shows an average of one paper a day is currently being published
 +about the VDR, the Vitamin D receptor. And if Dr. Grimes had
 +been following this literature it would have helped him alot in
 +understanding at least half of his conceptual problems.
 +
 +Yet, the insight resulting from the knowledge about the nuclear
 +receptor, the VDR, are not being communicated through to the
 +physicians who are on the front lines.
 +
 +One-paper-a-day, this tremendous weight of evidence.
 +Somebody the other day wrote that I am alone in saying that
 +Vitamin D is a steroid. Well, I'm sorry. You cannot read these one-
 +paper-a-day on the VDR and the nuclear receptors in PubMed
 +without understanding that everybody that's working in this field
 +has come to that same conclusion.
 +
 +But the biochemists, the molecular biologists, are not getting
 +that information across to the clinicians. It's very important that
 +both phases of medicine work in concert. And one objective of this
 +presentation is to share what we now know about the Vitamins D
 +and how they affect both the immune system and the endocrine
 +homeostasis.
 +
 +Th1 inflammatory disease is defined as disease which results
 +from a significant increase in the expression activity of interferon
 +gamma. It's a paracine cytokine, very hard to measure and it
 +doesn't circulate in the bloodstream, therefore, most clinicians tend
 +to look at other cytokines and try to draw an inference about what
 +interferon gamma probably is.
 +
 +Well, unfortunately that doesn't work very well and it's led to
 +considerable confusion in trying to determine whether patients are
 +exhibiting inflammation of type Th1 or Th2. Th1 being innate
 +immunity. Th2 being adaptive immunity, broadly speaking.
 +Now in 2001, we noted that interferon gamma catalyzed by a
 +large amount thirty times, up to thirty times the formation of a
 +secosteroid which did circulate as a hormone: 1,25
 +dihydroxyvitamin D. And we've used that marker in our ongoing
 +research.
 +
 +Most diagnoses are normally thought to come from an
 +autoimmune pathogenesis or of type Th1. These range from
 +anorexia nervosa through diabetes and rheumatoid arthritis to
 +sarcoidosis, MS, and ALS. But physicians treating these Th1
 +conditions with our antibacterial protocol in the Phase 2 trials have
 +also observed that the following neurologic manifestations resolve
 +as the patient recovers: The inflammation disappears, excessive
 +aggression starts to disappear, mild paranoia, mild obsession, and
 +compulsion — we actually have classic obsession compulsion
 +disorder amongst our cohort, loss of memory, loss of cognitive
 +ability, and attention disorders, bipolar disorders, and even
 +suicidal ideation.
 +
 +
 +So over the last five years from early 2002, we have conducted
 +an observational, adaptive, open-label Phase 2 study of an
 +antibacterial therapy in a variety of Th1 diagnoses. I've given a
 +quote there from the FDA Deputy Commissioner for Medical and
 +Scientific Affairs. It's a good quote and the paper, the complete
 +paper at the bottom I recommend to any of you who are involved
 +in clinical study design. This represents a major change in the
 +thinking of the regulators about how trials should be designed.
 +They should be designed to be successful and actually produce
 +results is what the FDA is starting to say now. I'll just refer you to
 +his paper and move onto our next slide.
 +
 +One of the first big breakthroughs we came across was to
 +understand that antibodies themselves were not capable of making
 +the patients as sick as they were presenting.
 +
 +Now Pasteur said that "in science chance favors the prepared
 +mind." So there's two elements for success in science: Sheer luck
 +— chance, and when it's in front of you, you can see it and
 +understand what it is, that's the prepared mind. And you need
 +both those elements to make discoveries. And we were lucky. We
 +noted, we had started with the disease sarcoidosis which is really
 +an end-stage Th1 disease similar to MS and ALS. It kills pretty
 +quickly after diagnosis. And we noted that the number of
 +sarcoidosis patients had case histories involving phases where
 +antibodies had been clinically recorded, but where the antibodies
 +have disappeared as the disease progressed to sarcoidosis.
 +
 +So I've shown time one here health and time 'n' here, sarcoidosis, and in the middle we had, and I'm specifically looking
 +at lupus SLE and rheumatoid arthritis diagnoses, both of which
 +involve specific antibody presentations in order for the diagnosis to
 +have been made, and the patients later progressed from those
 +diseases to the end-state of sarcoidosis.
 +
 +The interesting thing was that sarcoidosis there are usually no
 +antibodies. There is usually a very low sedimentation rate
 +indicating that there's no real obvious pathogens, the immune
 +system is not feeling anything. And that's one of the reasons it's
 +such an idiopathic and has been such a puzzling disease.
 +Yet, when you look at the malaise suffered by the patients, the
 +malaise is a straight line. There is no peaking of the malaise that
 +was coincident with the peaking of the antibodies. In fact, the
 +antibodies just seem to be bystanders, if you like, of the main
 +disease process. When we understood that, that was about in late
 +2003 – early 2004, we opened up the Phase 2 study to allow
 +different inflammatory diagnoses to become part of the study. And
 +in particular that was when we started taking in the chronic fatigue
 +syndrome patients, the lyme disease, chronic lyme disease
 +patients, the rheumatoid arthritis, Hashomoto thyroiditis, all the
 +rest of them.
 +
 +And this is what we found. In 2004, we published our initial
 +results which were basically just of the sarcoidosis subset of the cohort in Autoimmunity Reviews. But I travelled to Karolinska
 +Institute in Stockholm in May [2006] and I gave the following
 +figures for the recovery rate of key autoimmune diagnoses which
 +were extracted from our Phase 2/3 reports as of May 2006.
 +Now these are people who have been on the protocol typically
 +eighteen months and are well advanced on their road to recovery,
 +and I have two numbers here, for example rheumatoid arthritis, we
 +have the total number in the cohort, that have been in the cohort
 +eighteen months which is relatively low at that point, eight. And
 +then the number that we're reporting recovery in this case it's very
 +high. It is very high in this disease which is seven. Hashimoto is
 +twenty out of twenty-five. Osteoarthritis we're at a five. CFS, we've
 +been enrolling for quite a long time so we have a large number in
 +the cohort. And forty out of seventy-seven were reporting recovery
 +there after eighteen months. Cardiac arrythmia. Sarcoidosis has
 +the largest numbers of all because that's how we started the study
 +rolling. The diabetes, three out of five. Uveitis, twelve out of
 +eighteen. Fibromyglia syndrome twenty out of thirty-four. And
 +irritable bowel syndrome, eight out of ten. And that's just the
 +subset of the major diagnoses of the subjects in the cohort.
 +So how complex a Therapy is needed to address alll these
 +different Th1 Diagnoses?
 +
 +So how complex a therapy was needed to address all of these
 +different Th1 diagnoses and induce a response from these
 +disparate, apparently disparate, groups of patients?
 +  * Step one was to remove all sources of exogenous Vitamin D.
 +  * Step two was to activate the VDR nuclear receptor with
 +Olmesartan. Olmesartan is a relatively new sartin drug. It's a very
 +active ligand, very polar ligand, and it targets a number of
 +receptors. And I'll talk more about that as we move on.
 +  * Now for the first three months we administer a tetracycline,
 +preferably demeclocycline or minocycline every 48 hours starting
 +at an extremely low dose of 25mg every 48 hours working up to a
 +100mg over the three months, typically.
 +  * Then for the next nine months we add a second antibiotic, either
 +a low dose of azithromycin or clindamycin, whatever the patients
 +can handle. And the patients at this stage of the therapy after the
 +first three months have great deal of difficulty tolerating one-eighth
 +of the tablet of azithromycin every ten days added to the antibiotic
 +cocktail that they're taking, which at that point is minocycline. By
 +taking out the exogenuous Vitamin D and activating the innate
 +immunity via VDR, the sensitivity of the antibiotics is increased
 +many, many times over.
 +  * And thereafter until complete recovery we administer pulsed, low
 +dose, three-antibiotic combo: minocycline, azithromycin, and
 +clindamycin.
 +
 +And this is the warning which I was told to put in by my FDA
 +liaison guy: Any one of the above steps may cause cell
 +apoptosis with an intensity requiring emergency room
 +care. Immunopathology must be respected.
 +Immunopathology is the lastest keyword for the damage done
 +to the body by the immune system doing its job.
 +6
 +So the immune system kills the bacteria and in this case you'll
 +see the bacteria intracellular. You lose cells, the cells die when the
 +bacteria are killed inside them. The cytokine storm and just
 +cleaning out the dead cells profoundly affects the malaise and
 +general health of the patient. So steps are needed to reduce
 +systemic damage as the intraphacytic pathogens are killed by the
 +innate immune system. And I've got the steps there and you can
 +look them up from the PDF on the web.
 +
 +The key thing is that the rate of bacterial death is controlled by
 +inhibiting protein synthesis in the pathogens using intermittent low
 +doses of bacteriostatic antibiotics. And particularly bacteriostatic
 +antibiotics that target the translation of mRNA into proteins, target
 +the ribosome, the 70S bacterial ribosome. One bacterium is
 +weakened it's just one abx molecule is bound into one bacterial
 +ribosome. Low doses proportinately control the rates of bacterial
 +death.
 +
 +So why? Why does this intervention appear to work when
 +previous antibiotic therapies didn't induce recovery?
 +
 +The intervention works because, one, it's based on knowledge
 +derived from a rigorous theoretical model which is based on
 +molecular genomics.
 +
 +The intervention recognizes that autoimmune disease is caused
 +by a defect in innate immunity and not by antibodies themselves.
 +It works because it recognizes that the VDR, long thought to be
 +just associated with Vitamin D, is actually at the heart of innate
 +immunity.
 +
 +And it works because sequencing the genomes of the bacteria
 +and viruses has led us to an understanding of how these species
 +interact in a chronic environment — how they interact building up
 +slowly in a person's body during the course of a lifetime, and how
 +the horizontal transfer of DNA occurs between the species, blurring
 +the distinction between species and allowing one species to
 +acquire the ability to invade the immune system from another
 +species.
 +
 +And finally we recognize that the neonatal pathogens can
 +persist in the brain.
 +7
 +So let's go through those points one at a time. I just got a
 +picture there of one way of visualizing a large protein. In this case
 +a G-Protein coupled receptor GPCR. And we've got different
 +atoms there with force field radiuses and they were pretty colors
 +and they look nice and stuff but it's really hard to analyze much
 +from an image like this.
 +
 +
 +So biochemists tend to prefer to draw the large proteins in terms
 +of helical structures, loops, and the other structures that are
 +associated with the larger proteins, and the folds are visible. There
 +is more detail in that on the PDF.
 +
 +When you want to look at interatomic reactions to try and figure
 +out whether you've got agonists or antagonists, then you have to
 +sit down a little bit more carefully and these 2-D plots, I prefer you
 +use the program called Ligplot, L-i-g-p-l-o-t, which produces this
 +type of diagram. Makes it extremely easy to see how any of the
 +amino residues such as this one exactly interacts with the ligand.
 +Each particular force is a significant, interatomic force is significant
 +is plotted on a graph like this.
 +8
 +So what can you do with all this technology?
 +
 +This is a protein, SAR0276 which was taken from the genome
 +of a staph bug called MRSA252. And as you know MRSA252 is
 +one of the super bugs. This is a particularly nasty beast indeed.
 +But its genome is being fully sequenced by the Sanger Institute and
 +is one of the 363 fully sequenced bacterial pathogens that's now
 +available in the gene bank.
 +
 +And when you go to the DNA and start looking for proteins
 +within that genome which are likely to have some pathogenic
 +intent, this is the first one that I came up with. I went looking for a
 +GPCR that was similar to CCR2B and CCR4, CXCR4. For
 +example, CXCR4 is where HIV enters the phagocytes through those
 +receptors. And I was looking for some way that these organisms
 +facilitate egress and ingress to and from the phagocytes.
 +
 +Here is what I found, but what's particularly interesting is that
 +I've got this Olmesartan, this little ligand, the angiotensin receptor
 +blocker in here, and it binds very, very firmly into an active or an
 +inactiving position in that GPCR. It's a very highly polar ligand
 +and bascially any GPCR you find in a body, these new sartans and
 +statins will have some affinity for. Olmesartan happens to have a
 +high affinity for this particular bacterial protein, GPCR.
 +
 +But you know what really surprised us was that the ARBs and
 +statins also had a very high affinity for the nuclear receptors,
 +particularly for VDR, PPAR gamma, and the other nuclear receptors
 +which are key to the immune system. So we were surprised
 +because while it was reasonable that these very active ligands
 +would bind into transmembrane receptors — the GPCR is a
 +transmembrane receptors — for them to bind into nuclear
 +receptors and into the ligand binding pocket in agonistic and
 +antagonistic locations was total surprise.
 +
 +The nuclear receptive family I have on this slide I'm sure most of
 +you would be familiar with what we have there: The type 1 nuclear
 +receptors are the VDR — the Vitamin D receptor, the PPAR alpha
 +receptor, the gamma, the glucocorticoid receptor — that's the
 +home for cortisol, mineralcorticoid recceptor, progesterone
 +receptor, androgen receptor, estrogen receptor, thyroid alpha-1,
 +thyroid beta-1.
 +
 +Now we have good x-ray structures of all of these nuclear
 +receptors so they're quite easy to study how drugs actually affect
 +these nuclear receptors because we know what the nuclear
 +receptors look like, very precisely.
 +9
 +So when we look at the Vitamin D Receptor, at this point each
 +one of those one-paper-a-day that's coming out on the VDR is
 +increasing our knowledge on what the VDR does.
 +But the key things that I want to point out at this point is that VDR is
 +the key to innate immunity. It's responsible for the expression of
 +toll-like receptor 2, toll-like receptor 4, the cathelicidin anti-
 +microbial peptides and the beta-Defensins.
 +
 +The beta-Defensins are key to the way that the gut, the immune
 +system in the gut, handles flora and handles pathogens which
 +appear in the gut. Beta-Defensins and alpha-Defensins are very
 +active there.
 +
 +The cathelicidin anti-microbial peptides typically tend to be
 +more active within the phagocytes themselves.
 +TLR4 is the receptor that is sensitive to lipopolysaccharide, the
 +prototypical bacterial marker. TLR4 is sensitive to
 +lipopolysaccharides. TLR2 is sensitive to other bacterial lipo
 +proteins.
 +
 +So if you're not expressing the TLR2, TLR4, and the antimicrobial
 +peptides very well, then the immune system, and particularly the
 +innate immune system, is not going to be functioning very well.
 +And what I did was to take the various Vitamins D, there are a
 +number of metabolites. I took 1,25 dihydroxy-vitamin D, this is the
 +active metabolite, versus 25 hydroxy-vitamin D. That's the form
 +that the Vitamin D metabolite the sterol is stored in the body with
 +the D-bonding protein in the body fat. And it's stored for very long
 +times. Half life is a month, in some people a year for the storage
 +of 25-D. The body is very good at storing 25-D in between
 +manufacture of it. And then 24,25-D, 25,26-D are just
 +metabolites that occur when 1,25-D is deactivated. And they all
 +lie in the binding pocket. You can see they're essentially
 +coincident, one on top of the other. The tails are a little bit
 +different, but the tails don't have very much to do with the
 +activation of the VDR, certainly with the transcription of the
 +immune mediators that we spoke about.
 +
 +The only difference between all of these metabolites really is just
 +one alpha hydroxylation and that stabilizes helix 12 and that's
 +known to be essential for the binding to the promoters which allow
 +VDR to do its immune functions.
 +
 +On the right-hand side for those of you that are more
 +technically inclined, I've got a molecule of Olmesartan as it's
 +bound into the VDR ligand binding pocket alongside 1,25-D, and
 +you can have a much closer look at the partial agonism which is
 +produced by that drug in the VDR.
 +
 +So I'll just do a little bit of basic work here. We've got--on the
 +left we've got Vitamin D which is a secosteroid. And on the right
 +we've got corticosteroid. In this case it's prednisone. (And I've got
 +it upside down. And around the front. Okay, try the side. There
 +we are. Okay.)
 +
 +So we've got the steroid rings here. You've got the five-member
 +ring, the six-member ring with the methane. Here you've got the
 +five-member ring, six-member ring with methane — it's
 +characteristic of all the sterols. And the only difference is that the bound between these two carbon which is present here on
 +prednisone and the corticosteriod has in fact been cleaved by an
 +electrocylic reaction, and that makes the Vitamin D a secosteroid
 +rather than a steroid. The interesting thing is that it actually gives it
 +a higher affinity for some of the nuclear receptors because there
 +are more degrees of freedom for this bottom ring because of the
 +inherent flexibility of these two double bonds and the single bond
 +down here. But that's the difference between Vitamin D and
 +prednisone, that one bond.
 +
 +I'm going to skip this slide. The main thing to know about the
 +nuclear receptors is that we don't know a heck of a lot about them.
 +We're learning more as every month goes by. But the key two
 +words that I would use to describe how the nuclear receptors work
 +is redundancy and complexity. It's very, very complex.
 +
 +The receptors combine as homodimers, they combine
 +heterodimers, they can bind with the Retinoid-X receptors and
 +even with the Retinolic acid receptors, and all of these combine to
 +promoter proteins, which then will transcribe different genes from
 +the DNA. Because as you would all know the job of the nuclear
 +receptors is to transcribe the DNA into messenger RNA and then
 +into protein.
 +
 +And here we have a closeup of the glucocorticoid receptor
 +Homodimer zinc fingers. That means there are two of these
 +glucocorticoid receptors bound together with the correct
 +promoters, and these zinc fingers are key to the location of the
 +parts of the receptor that actually target the gene and transcribes
 +the gene from the DNA spiral.
 +
 +You can see here the backbone of the DNA spiral. The base
 +appears across the center. And you can also see the hydrogen
 +bond across the center too that holds the whole structure together.
 +That's a structure that you can download and play on your
 +computer at home, if you're interested.
 +11
 +
 +The estimated affinity for the ARBs and statins into the nuclear
 +receptors was what surprised me. And I've got it tabulated here
 +and I'll just take a few data points off this and show you what we're
 +talking about.
 +
 +Firstly, to answer Dr. Grimes' question in the Lancet, he said that
 +statins, "Are statins analogues of Vitamin D?" he asks.
 +Well, here's the VDR and if there are going to be analogues of
 +Vitamin D they would have at least have to activate the VDR. And
 +Atorvastatin doesn't have any affinity at all for the VDR, neither
 +does Fluvastatin. Lovastatin has a moderate affinity for the VDR.
 +Pravastatin at normal doses, that is essentially no affinity.
 +Rosuvastatin, no affinity. And Simvastatin a very high affinity. Four
 +nmol Ki indicates that at normal doses Simvastatin would be
 +targeting the VDR.
 +
 +So the first answer is you can't even talk about the statins as a
 +group because they're very different. Each of the statins is different.
 +And if you take another key receptor, the alpha-Thyroid receptor
 +over here, once again you see Atorvastatin, Fluvastatin has little
 +affect. Lovastatin has a high affect, a very high affinity on the
 +alpha thyroid. Pravastatin and Rosuvastatin is essentially inactive.
 +Simvastatin again has activity in the thyroid subsystem.
 +
 +So it's not a simple question are statins analogues of Vitamin D.
 +We've got to break down the degrees of freedom. We've got to talk
 +about which particular drug are we talking about. And then we've
 +got to look at does it have an affinity with Vitamin D. Simvastatin
 +does. So that's a good start.
 +
 +In the case of the sartans, you've got a similar picture, except
 +the sartans tend to be more homogenous as a group with
 +Telmisartan being the only one that's out there on a limb with an
 +extremely high affinity for the VDR, but it acts as an antagonist for
 +the VDR. It also acts as an antagonist of PPAR-gamma and PPAR-
 +alpha. We wrote a paper last December that goes into that action
 +in more detail.
 +
 +The Olmesartan that we are particularly interested in, has a
 +wide range of affect across all of the receptors, except the alpha
 +thyroid. It doesn't really target the alpha thyroids very much which
 +is probably a good thing.
 +
 +I've got similar charts of steroid activity in the key nuclear
 +receptors. I'll just leave that to the PDF and skip that at this point.
 +Hypothalamic-pituitary-adrenal axis
 +
 +I'm going to look briefly at the hormonal control system in the
 +human body. It's pretty well characterized. This is the cortisol
 +access where you've basically got the glucocorticoid receptor with
 +a cAMP promoter. The promoter is known, the GCR as being the
 +transcriber of the gene is gone, and the first metabolite is the
 +corticotropin releasing hormone which is produced from the DNA
 +by the GCR with the cAMP promoter. But then changes to or is
 +converted to Pro-opiomelanocortin, which breaks down into beta-
 +endorphin and ACTH. ACTH then catalyzes the conversion of
 +cholesterol into cortisol and then the cortisol goes around to the
 +top and binds into the glutocorticoid receptor. It also affects the
 +decomposition of corticotropin-releasing hormone, but you have a
 +basic feedback control system there. And the body tends to
 +control all of its hormones, all of its steroids very, very closely.
 +12
 +If you give a person cortisol and exogenously as an injection,
 +you upset the whole of this balance because you try and bypass
 +the feedback loop. That means you generally have to use much
 +higher doses than are present endogenously within the body itself
 +and you don't get the results you necessarily expected.
 +
 +Now slightly more complex metabolism is the Vitamin D steroid
 +metabolism and this is pretty well up to date, I think as of October
 +the 23rd [2006], a compendium of everything that's being
 +published and is reliable on the way that the body regulates the
 +hormone called Vitamin D, 1,25-dihydroxy-D, which is what
 +activates the VDR, what allows the transcription of the
 +antimicrobial peptides and the toll-like receptors.
 +
 +And we start at the top from 7-dehyro-Cholesterol. All of the
 +Vitamins D are produced endogenously in the body from 7-
 +dehydro-Cholesterol. Typically, we think of a vitamin as being
 +something that you have to ingest in order for the body to work
 +properly. But that's not the case with Vitamin D. All of the Vitamins
 +D can be produced from 7-dehydro-Cholesterol and that seems to
 +be the normal way that the body produces it.
 +
 +The first step to pre-vitamin D is typically thought to involve UVB
 +energy. However, the electrocyclic reaction — conrotatory
 +electrocyclic reaction — is found elsewhere in nature, catalyzed by
 +enzymes. So there's probably some question marks there that we
 +still have to go looking for. Do we need UVB or do we need
 +enzymes? Big question mark over the whole concept of whether
 +Vitamin D is a vitamin or whether it is just a hormone precursor.
 +
 +And then pre-vitamin D the electrons rearrange slightly in a
 +sigmatropic shift to create vitamin D which then is hydroxilated to
 +25-hydroxyvitamin, the single hydroxy by CYP27A1— That's one of
 +the p-450 enzymes or CYP2R1. And then that, monohydroxy, is
 +converted to the dihydroxy by CYP27A1 again — a heme-related
 +enzyme — or by CYP27B1. And B1 is primarily expressed in the
 +macrophages, in the monocytes, in the phagocytes and it converts
 +25-D to 1,25-D, but it doesn't go all the way back to the sterol
 +system and that's the one which is most active in the immune
 +system.
 +
 +Then finally, 25-D is degraded into an inactive form by CYP24
 +and CYP3A4 which are up regulated by the VDR. So if there's too
 +much 1,25-D, the VDR upregulates the inactivation pathway and
 +that upregulation is cancelled or offset by androgen activity:
 +testoterone, estrogen, interferon gamma; and that's why interferon
 +gamma and 1,25-D are so closely coupled together because
 +interferon gamma stops 1,25-D from being degraded. It stops the
 +enzymatic feedback there and also by PXR. And when the VDR is
 +activated you have innate immunity and you have down-regulation
 +of the parathyroid hormone by the 1,25 dihydroxy-D — 1,25
 +dihydroxy-vitamin D — and then the parathyroid hormone in
 +conjunction with the calcium sensing receptor which is in the
 +kidneys sorts out the body's calcium homeostatis and then the
 +body's calcium homeostatis feedbacks into the expression of
 +CYP27A1. And there's your feedback path for the Vitamin D
 +steroid metabolism.
 +
 +Not simple by any means, and actually not unreasonable if you
 +consider that the immune system would have had to adapt through
 +evolution in many stages and that's really what we're seeing as we
 +dig further and further into the way that the Vitamin D metabolism
 +works in man.
 +13
 +Well, up on the top left here I've got a configuration of
 +dexamethasone as it stops into maximum affinity and to the
 +glucocorticoid receptor binding pocket — ligand binding pocket.
 +And the backbone is in purple. Here is the fluorine atom [green]
 +and the oxygen is obviously in red. And on the lower right I've
 +got 1,25D superimposed on that, with a green backbone,
 +showing how it binds into the GCR and it binds into exactly the
 +same ligand binding pocket. There is no doubt that 1,25D
 +displaces cortisol and dexamethasone from the GCR in a
 +concentration-dependent manner and vice versa of course.
 +
 +So when you factor this knowledge in, what happens when the
 +immune system run haywire and 1,25D levels build as we see in
 +clinical practice?
 +
 +25-D and 1,25-D go into the glucocorticoid receptor and
 +intercede — make this whole hormonal feedback system break
 +down — and you end up with patients who have adrenal
 +insufficiency which is very, very common in these Th1 diseases.
 +However, I point out that increasing the dose of cortisol to try
 +and cope with the adrenal insufficiency is the incorrect way to go
 +about treating the problem. It has to be attacked at the actual
 +cause by lowering the 25-D, the ingested Vitamin D, and the
 +1,25-D, the inflammatory load.
 +
 +However, the Vitamin D also competes for the alpha-1 Thyroid
 +binding pocket, and hypothyroidism is very common in these Th1
 +diseases. Here's T3. You can see the three atoms here in green.
 +T3 as it binds into the alpha-1 Thyroid receptor. And here we
 +have 1,25D competing with T3 for the ligand binding pocket of
 +the alpha-1 thyroid receptor. And as you can see they will
 +displace each other from the receptor in concentration and
 +affinity-dependent manners.
 +14
 +
 +Vitamin D in bone remodeling I'll skip in this group because I'm
 +running behind time.
 +
 +But I do want to briefly consider the public health consequences
 +regarding Vitamin D as a vitamin.
 +
 +If we look back to the 1923 nobel oration the 1923 nobel
 +prizes given for the elucidation of the function of the sterols in
 +particularly what we know as Vitamin D in the body and if you look
 +at the nobel oration, you'll find that there really no concept of
 +vitamin in it. Originally, the concept was this is a sterol. This is a
 +sterol, a secosteroid, not a vitamin.
 +
 +Sometime during the 20th Century we began to view
 +Cholecalciferol as a vitamin rather than focus on its steroidal and
 +hormonal activity. And when we started putting this in the food
 +chain, in a futile attempt to eliminate the rare disease rickets —
 +rickets now turned out to be in the latest, very comprehensive
 +studies to be eliminated only by calcium and phosphorous in the
 +diet. But we put it into the food chain back starting about 1950s to
 +try and eliminate rickets and we haven't eliminated rickets,
 +although the incidence has dropped.
 +
 +As physicians, many of you who are physicians would certainly
 +know the side effects from the administration of steroids. The
 +Centers for Disease Control in the USA says that the US is heading
 +towards half of all US seniors being diabetic by the year 2050.
 +That is exactly the track that the US population is on right now.
 +And we all know about obesity.
 +
 +Here's an article from earlier this week. "U.S. Children grow
 +bigger bellies," from Reuters. Startling data.
 +
 +Actually over here [left column under picture] it's interesting.
 +Canadians getting fatter but more slowly. But what they found was
 +that this particular study that Rochester School of Medicine found
 +was that ten and a half percent of boys and girls had too much
 +abdominal fat in1999, as measured by waist circumference. This
 +included seventeen and a half percent of boys and girls in 2004.
 +In just five years, the obesity rose sixty-five percent.
 +
 +Now I don't think McDonalds sales rose sixty-five percent in
 +those same five years from 1999 to 2004. There were other
 +factors at play. Obviously I have my theories as to what those other
 +factors are.
 +15
 +Here they are. These are the US experience and I've put this
 +slide together because in Australia you've all been pretty well
 +shielded at this point from supplementation of the diet with
 +exogenous Vitamin D, artificial Vitamin D.
 +
 +In America, milk and yogurts are fully supplemented with
 +Vitamin D. If you have two glasses of milk a day you get the entire
 +supposed RDA of Vitamin D, and we find that that is more than
 +enough to take the levels of 25-D in the body into
 +immunosuppressive range.
 +
 +Cheese, particular cheese slices that are intended for kids'
 +meals, for kids' lunches, are fortified with Vitamin D in the US.
 +Kraft Macaroni and Cheese and all of the cereals, all of the
 +breakfast cereals for kids, they're all fortified with Vitamin D. The
 +kids can't get away from this stuff. And now of course orange juice
 +is fortified with Vitamin D to make it really good for you.
 +
 +That's the experience in the United States. It's a very sad
 +experience, but that's what's happening at the moment. And I put
 +at the bottom right there a bottle just to remind us, but the calcium
 +for Vitamin D is so often prescribed to patients who present with
 +these inflammatory disease presentations, diagnoses, and who
 +also have osteopenia. And that's counterproductive because the
 +people that need the Vitamin D least are the ones that get
 +supplemented and that's purely because clinical medicine is not
 +measuring the correct metabolite. They need to measure 1,25
 +dihydroxy, the active metabolite, as well as the inactive metabolite.
 +
 +So let's go on to talk about the pathogens which is the other
 +half of the disease equation.
 +
 +Now we've known for sometime that there are bacterial
 +pathogens that are capable of living in the phagocytes. This is
 +from a study "Presence of Rickettsia Helvetica in granulomatous
 +tissue from Patients with Sarcoidosis," 2001 — late 2001 this
 +came out. And it showed bacteria staining inside the phagocytes.
 +The authors inferred that these bacteria were rickettsia and they
 +may very well have been, but the point was bacteria can survive
 +inside the phagocytes. That is a mind boggling concept. The
 +phagocyte is supposed to phagocytose they are supposed to
 +breakdown the bacterial DNA and render the bacteria harmless.
 +Protection from Phagocytosis — Biofilms
 +
 +This is a photo from the paper, the review paper, The Microbial
 +Resistome. This talks a lot about horiozontal transfer of DNA.
 +Search for it PubMed or in Google, The Microbial Resistome. It's
 +brillant.
 +
 +The reason I put it on there is to show biofilms. This is from the
 +Centers for Disease Control and it shows how Staph aureus
 +produce biofilms which are a sticky looking substance. They
 +secrete sticky looking substances called biofilms.
 +
 +And that's what we see associated with these tiny bacteria that
 +are found in the inflammatory diseases.
 +16
 +The Wirostko Studies were performed in the late 1980s at
 +Columbia University in New York by the Wirostkos, the family of
 +Wirostkos and another collaborator. And what they did was quite
 +unique. They took patients who had Crohn's disease, juvenile
 +rheumatoid arthritis, and sarcoidosis and they used transmission
 +electron microscopy to examine the phagocytes and they stained
 +the bacteria.
 +
 +They were particularly interested in seeing whether it was
 +possible for phagocytes to become infected. And they found that
 +all of the phagocytes — monocytes, macrophages, lymphocytes,
 +and neutrophils — were all infected by these tiny little L-form
 +bacteria that were first described in 1934 by ME Kleinberger
 +Nobel of the Lister Institute.
 +
 +And we have here a single isolated coccoid about .01.015
 +microns diameter. What am I saying, microns? Nanometers in
 +diameter. Ten to fifteen nanometers diameter.
 +
 +And then we have a kidney-shaped colony which in itself I haven't
 +counted these but they must well be over a hundred individual
 +coccoids in there, string of about a dozen coccoids in here, and
 +another gaggle of coccoids there, and another artifact of stained
 +bacterium there. These are tiny little bacterium.
 +
 +And what was particularly interesting about this photo was this
 +crenation in the nucleus which doesn't show too well here, but the
 +nucleus, part of the nucleus is actually breaking off as shown by
 +this dark arrow. It's shown by the big arrow. And that nuclear
 +activity was close to the largest colony of bacteria in this case.
 +Every one of these photographs that they took showed infection
 +and every one of the photographs that they took show slightly
 +different physical manifestations of an infection in some case vastly
 +different. But the two things that were common throughout them all
 +was a lot of transparent and semi-transparent material like this, the
 +biofilm, and also the tiny, tiny coccoids.
 +
 +Now is a phase contrast optical microscopy. This is done with a
 +Bradford Microscope. It has also been done with quite inexpensive
 +microscope, microscopic equipment. I'll just stop it there, pause it.
 +And what we've got is these tiny little threads, thread-like
 +appendages, which are made of a translucent tubular material
 +which stains with fluorescent antibodies for bacterial RNA. (How
 +did I get back there?) And these are exiting a crenating red cell in
 +this case.
 +
 +This is a technique which was devised by Andrew Wright in
 +Northern UK where he takes pinpricked blood, puts it between a
 +slide, seals air out with vaseline and then allows the blood to
 +degrade a period of six to thirty-six hours. And as you can see this
 +is a monocyte that has disintegrated. You can see these tiny long
 +forms that have no analogue in pathology. There is no explanation
 +in pathology for what these are, except of course that they are the
 +bacterial L-forms.
 +
 +(Video continues of biofilm moving very different from Brownian
 +elements moving in background, and static cells.) This particular
 +picture there is a bacterial L-form here which is going to do a
 +linear movement toward the bottom, just to show those of you that
 +think it's all Brownian in motion. It is not. It's going to gradually work to the bottom, as I speak, but this equipment, the equipment
 +
 +that was used to produce this photograph, was bought off E-Bay,
 +very, very simple analogue microscrope x400 and a standard
 +camera with computer software to amplify up the size of the
 +image. So this little fellow will go off the screen and I'll just allow it
 +to refocus to show you that it keeps going down and eventually
 +disappears totally from the field of view. There is Brownian motion
 +visible and something like Brownian motion in the other particles
 +there, but this thing seems to know where it's going.
 +
 +Okay. So what do our antibiotics do? Our antibiotics go after
 +the bacterial ribosomes. The 70S bacterial ribosome is in two
 +halves, the 30S, 50S subunits. The 30S, the MRNA is translated by
 +the helical structures at the top. You've got the tRNA here which
 +donates the amino acids to the growing protein chain. And then
 +the protein leaves the bottom of the 50S section. And the
 +antibiotics we use target different areas of the ribosomes.
 +
 +If we look up at just the 30S subunit, what we've got is up top
 +these helical structures which involve with advancing the mRNA as
 +it is decoded.
 +
 +And right sitting there are the molecules of tetracycline in the
 +various locations that they bind with quite strong affinity.
 +
 +This work is from the Max Plank Group in Hamburg, Germany.
 +Max Plank Ribosome Group.
 +
 +And so minocycline, demeclocycline, the first of the antibiotics we
 +use inhibits — it doesn't stop — it inhibits, it slows down the
 +translation of the mRNA at that point, the top of the 30S ribosome.
 +
 +Then the 50S ribosome, you've got the protein exit channel here
 +at the bottom and in red here, that's where the macrolides bind the
 +erythromycin and the azithromycin that we use. And just above
 +that is where the clindamycin binds. If you'll remember that's the
 +third antibiotic we use in the longer term cocktail to totally get rid
 +of the bacterial load that the patients are carrying.
 +
 +And the important thing to note from the animation is that each
 +of these antibiotics is symbiotic. None of them interfere with each
 +other. The addition is linear and it's symbiotic.
 +18
 +Now what about the actual pathogens? Still everybody wants to
 +know well what species is it; what species causes these infections?
 +The answer is: You're dealing with a chronic infection building
 +over a lifetime. You're not going to be dealing with a species.
 +You're going to be dealing with massive amounts of horizontal
 +transfer of DNA, particularly of plasmids.
 +
 +And now that we know what the genomes look like we've got
 +some bacteria, for example, borrelia burgdorferi, that has gotten
 +nearly half its genome on 21 self-replicating plasmid subunits. It is
 +most definitely going to be sharing plasmids. And even common
 +species like staph epidermidis have ten percent of the DNA split
 +over six self-replicating plasmids. Those plasmids can easily be
 +shared. They are not targeted by antibiotics and they are only
 +targeted by the innate immune system and then only weekly. They
 +are going to accumulate over time unless cleared by the immune
 +system and you're going to be dealing not with UNI-microbial
 +disease but with a POLY-microbial disease. A combination of the
 +infectious history of the individual including both viral and
 +bacterial pathogenic challenges.
 +
 +And here is a paper from the National Academy of Sciences,
 +2004, where two plasmids from bacillus anthracis were transferred
 +to the genome of bacillus cereus and a patient died very quickly as
 +a result of that. They actually were wondering why the patient had
 +died from bacillus cereus until they noticed that, that particular
 +genome had also acquired at some point the two plasmids from
 +bacillus anthracis. And those two plasmids, one conveys the ability
 +to evade phagocytosis, the other conveys the ability to kill people.
 +And particularly interesting over here is bacillus thuringiensis.
 +That's used as an insecticide. That's sprayed onto our crops as an
 +insecticide. That's very close in phylogeny.
 +
 +Now this is survival of mice challenge with that particular hybrid
 +of cereus anthracis plasmids. And normally it's harmless. That's
 +anthracis itself [refer to chart] whereas the hybrid kills more quickly
 +than anthracis itself. The horizontal transfer of DNA is absolutely
 +key to understanding the pathogens in these diseases.
 +19
 +My final slide is work from Rolf Zinkernagel who together with
 +Peter Daughtery at John Curtain did a lot of study on antibody
 +presentation and got the 1996 Nobel prize for it.
 +Now Rolf has dedicated his life subsequent to the Nobel to
 +showing that autoimmune disease is not due to antibodies
 +themselves. And I had a very good long discussions with him at
 +Karolinska [DMM 2006].
 +
 +And this particular experiment which is a recent publication by
 +his, 2006, they injected lymphocyctic choriomeningitis virus into
 +newborn mice, into the brain of newborn mice. And what they
 +found was that the virus appeared to be cleared and it couldn't be
 +detected by normal techniques, but it was clearly persistent in the
 +brain. Because when those mice were challenged later in life with
 +either a slightly different virus or with the same virus, then there
 +was a very energetic immune reaction and the mice died.
 +Whereas, if the mice had been initially challenged later in life after
 +the adaptive immune system had started to work properly then they
 +were able to cope with both the initial and subsequent challenges
 +from the virus.
 +
 +Because, you see, mice are just like human beings. When
 +they're born they only have innate immunity. They don't have
 +adaptive immunity running. In the case of mice you can see the T
 +cells starting to become active here at about day seven in their
 +lifespan. In humans, it's a little bit longer than that. It's in weeks.
 +But it takes a while. And during that time the infant is only
 +protected by the innate immune system. And what happens in that
 +time is quite critical.
 +
 +And what Rolf's group has shown is that you can have an
 +occult virus which is persisting in the brain through life which is
 +later activated into an extremely energetic and pathogenic state.
 +That is a model for what we're also seeing with the bacterial
 +pathogens in these chronic diseases.
 +
 +So there we are. Thank you very much for listening to the talk.
 +And we'll see if there are any questions.
 +
 +{{tag>presentations Trevor_Marshall_PhD 2007}}
  
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