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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.
+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.
+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
+– 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
+mg 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.
+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.
+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.
+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.
+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.
+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.
+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
+-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
+-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.
+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?
+-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
+,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.
+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.
+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.
+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.
+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,
+, 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.
+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 Bio21 2007}}

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