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Prof Trevor Marshall
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Some months ago we finished our gene-transcriptional description of the Vitamin D metabolism. It was immediately obvious that there was only one mechansim of bacterial action which would give the clinical effects we were observing - and also shut off transcription of the Cathelicidin and beta-Defensin anti-microbial peptides (AMPs).

Shutting down the AMPs is important to the pathogens, for while AMPs are being produced by the phagocytes there will be no persistent pathogens - they will be phagocytosed to fragments of DNA, rather than survive as persistent organisms...

The required mechanism was that the bacteria needed to block the proper functioning of the VDR by producing a ligand which would displace 1,25-D from the VDR binding pocket. In turn, Olmesartan would displace the capnine, at least partially...

I was asked how we could search the bacterial genomes and identify the ligand/chemical which the bacteria might produce to block the action of the VDR, and I responded that it would be like searching for a needle in a haystack.

Well, I was wrong. Pasteur's law struck again, and I now have isolated at least one nanomolar-grade (strong) antagonist which is formed by a special type of bacteria which like to live in biofilms, and which are very poorly studied, and almost impossible to culture.

The breakthrough came when I started to look at a recent paper on some strange bacteria which had been isolated from biofilms on prothestic joints. As you know, I have a complete disease model now, so I understand exactly where prostheses fit in the overall scheme of things, and I understand that healthy folk are just sick folk whose bacterial load has not yet made them very ill :) So it was with that jaundiced eye I read this paper:

"Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and microbiological culture"
http://tinyurl.com/2hqojq  PMID: 17501992

I immediately knew we had something very important here, and when I researched these special "gliding" bacteria, which move by gliding, perhaps like a snail, rather than by whipping flagella like the common blood-borne bacterial species, I realised we had hit mother-lobe.

Associated with the gliding motion is a unique lipid called capnine, a highly charged lipid, and one which is a strong inhibitor (antagonist) of VDR transcriptional activity.



It really doesn't matter whether these gliding bacteria harm the body in any other way, by shutting down the AMPs they create an environment where a plethora of other bacteria and viral pathogens can weave their nasty ways upon the host.

Here is a Real-Format video of my molecular dynamics simulation of capnine in the VDR LBP, showing an exceptionally stable dynamic configuration. Its action as an antagonist is confirmed by the LBP expanding in size during the entire period of the simulation, and losing the configuration in which it can bind to the DRIP205 coactivator and transcribe the AMP genes.

http://autoimmunityresearch.org/models/capnine-vdrh.ram

So now the model is complete.
We know what pathogens can cause Th1 disease, and they were discovered purely by mathematical deduction, and not by looking into petrie dishes... or harming any furry animals:)

So where do we go from here? I had thought of making a big song and dance and trying to persuade a journal to publish this stuff. But that would be a complete waste of our time. At this point we have to start focusing on 'saving the world' not on being ''first to publish.' So there it is .. an almost complete glimpse of the grail...

----------------------------------------

All above content (C)Copyright 2007 Trevor G Marshall, All Rights Reserved.

patrickburke
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Awesome work Prof M!! Congratulations :D:D.

kathleen
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It's totally incredible to be witness to such a huge breakthrough. I can't even think of what to say except for Thank You.

And thank you again for not wasting energy trying to convince tptb, but getting on with learning more about how to help people get better. Like me for instance!!

XXOOO Kathleen

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Wow Trevor ! This is cool !

Now, all we need is a drug that takes care of this capnine and we are home free  :D

But seriously, I understand from what you say, that you have also found which other species can make this capnine?

Am I correct in assuming that capnine is one of the proteins/things bacteria fabricate and we are effectively stopping them from doing that by sabotaging their ribosomes?

Or is it something other than the ribosomes that make capnine?

And going further even, have you perhaps found if the gene(s) that is (are) responsible for this is horizontally transmittable between species?

Just some things that are popping into my mind at this point :)

Sincerely, Frans

Prof Trevor Marshall
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Frans,
The Ribosomes only make proteins. This is a lipid, and a highly processed lipid. Enzymatic activity is what creates it. That is why a full-genome search is futile - gene transcription analysis doesn't take into account the enzymatic actions.

However, enzymes start as complex proteins, and so by blocking the ribosomes we block the ability of the bacteria to form capnine.
 

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Trevor,

Congratulations on an amazing discovery.  I agree that functional genomics would have been a very much slower way of identifying capnine as the VDR anatagonist, but now that you have done the hard work :) I don't think it should take long to identify the genes involved in capnine production.  Fascinating possibilities here to study host-pathogen interactions in human cell lines.  Consider the following information a cursory search shows up in identifying some Lysobacter genes involved in the production of antimicrobials (in a fungal host):

A global regulator was previously identified in Lysobacter enzymogenes C3, which when mutated, resulted in strains that were greatly reduced in the expression of traits associated with fungal antagonism and devoid of biocontrol activity towards bipolaris leaf-spot of tall fescue and pythium damping-off of sugarbeet. A clp gene homologue belonging to the crp gene family was found to globally regulate enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes C3 (Kobayashi et al. 2005).  

From: Kobayashi and Yuen.  2005. The role of clp-regulated factors in antagonism against Magnaporthe poae and biological control of summer patch disease of Kentucky bluegrass by Lysobacter enzymogenes C3.  Can. J. Microbiol. 51:719-723.

I think things will start progressing very quickly (should I say, even more quickly)now on the genetics/genomics front.

One question: capnine seems like a very specialized molecule (or a perhaps quite an ordinary lipid with a very specialized function) (chemists on board care to comment?) - do you think it is more likely one-of-a-kind or one-of-many in its VDR antagonism capbability?

Many thanks,

Ruth  

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Ruth,
I think it is one-of-a-kind, a very special molecule.

I have been alert to the close symbiosis between the retinoids and Vitamins D for some time. For example, either can function to hold together COMP, and both are active in Rhodopsin, but the retinoids have a relatively low Ki for the VDR. When Capnine popped up the similarity in size and shape immediately struck me...

I was particularly intrigued by the "pea-soup" possibilities for the Lysobacters, as they produce toxins targeted at both both fungal and bacterial 'partners.' For example, one study showed that Lysobacter killed e-coli colonies, but not e-coli colonies which had been infected by the T4 bacteriophage.

http://tinyurl.com/2b9pdm PMID: 4927527
 

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I'd also like to say congratulations to you Dr. Marshall.  I see from your response above that capnine seems to be a one-of-a-kind molecule rather than one of many.  Do you also think that the bacteria you identified as producing it is one-of-a-kind or do you think there might be many bacteria that produce it or is that not known yet?

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Russ,
There may indeed be many viable VDR-antagonist-ligands that have evolved with differing species over time, or capnine may be the only one. We just don't know yet.

However, from a scientific point of view, we have one candidate, in a viable bacterial family, and therefore we can move forward exploring those avenues of the pathogenesis in more detail.

I would note that there are more than one species of gliding bacteria, and that capnine seems common to many of the species of gliding bacteria.

http://tinyurl.com/2zac8w  PMID: 6321489
 

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Actually, our good friends the Mycoplasma, long known to be associated with chronic inflammatory conditions are gliding bacteria:

Mycoplasmas are bacteria that lack a cell wall and are parasitic or commensal to many kinds of hosts, including humans, animals, and plants.

In addition, the gliding motility of mycoplasmas is believed to be involved in their pathogenicity and a number of proteins required for adhesion to host cells and gliding have been identified.  I think it has been more or less assumed that the loss of pathogenicity associated with mutations in these proteins is due to the reduced ability of the bacteria to attach to or penetrate the host cells, but maybe these mutants also have reduced abiltiy to produce capnine.

Oh indeed, speculation is good fun ... 

Ruth

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Congratulations. So this is a compound present in bio-film. I wonder if there are databases about chemical structures of bio-films, so they could be tested for vdr affinity.

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Hooray, dear Trevor 
 
Even if you do not want to dance, let me dance in joy and deep admiration for your amazing, breathtaking and pathbreaking work 
 
I celebrate with you 
 
I am overjoyed 
 
Evelin

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Push the button to play the movie

Last edited on Fri Jun 8th, 2007 00:22 by wrotek

Mary
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Dr. Marshall,

Amazing work...

My concern: can patients with prosthetic joints succeed with the MP? Seems a likelihood of another continuous source of infection. Today it is so common to hear of joint replacement surgery.

Any comments appreciated!

Mary

Prof Trevor Marshall
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Mary,
As long as the folk use the MP tp work down their bacterial load to levels of the general population, they will have as much success with prostheses as 'normal' folk do, and a lot less trouble than many folk do :)

The issue here is not prostheses, I am sure that normal joints would have shown similar bacterial populations, it is just that normal joints don't often become available for such a study, whereas prostheses do...

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Dr. Marshall,

This work is brilliant, and I count myself as very fortunate to read it. 

Now my peasant mind is thinking that Benicar is snail bait (LOL).

With the identification of capnine, is it possible that a better drug might be found?  Is capnine found in any specific foods?  I consulted the USDA Nutritional database, but found nothing.

With deep thanks,

Sherry

Prof Trevor Marshall
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Sherry,
Capnine is uniquely made by gliding bacteria.

If capnine turns out to be the only bacterial product which blocks the VDR, you really don't want to try and target this with a drug. If you did neutralize the capnine, you would end up with an immune system that is suddenly freed to attack ALL of the pathogenic load at once.

Think about it - you are using just enough antibiotic to kill a few bugs every day. Imagine if suddenly your immune system was returned to 'normal' and started attacking all the bugs, all at once. You would become very ill indeed. Probably you would die.

So we need to be careful what we wish for :):):)

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Remember this?

http://en.wikipedia.org/wiki/TGN1412

What a disaster. And they were "healthy" volunteers. Imagine the effect on a th1 patient :shock:.

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Pat,

That is quite telling when the study says that it caused catastrophic system failure in spite being used at "some 500 times lower than the dose found safe in animals".

No doubt most animals did not have a latent pathogen load anywhere near what long-lived human subject would have had. (Especially of course Mice -- here today -- gone next year).

Maybe some day TGN1412 will be harnessed for good.  That would be something.

jim

 

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Dr Trevor Marshall wrote: Mary,
As long as the folk use the MP tp work down their bacterial load to levels of the general population, they will have as much success with prostheses as 'normal' folk do, and a lot less trouble than many folk do :)


This reminded me of a question I have had for a while.  Is it primarily the bacterial load, i.e. the quantity of bacteria, that separates "normal", "healthy" individuals from those of us that are "sick"?  Or are those of us who are sick infected with especially nasty species/strains of bugs that are integrated into our bacterial pea-soup but absent from the bacterial population of "normal", "healthy" folks?

Last edited on Fri Jun 8th, 2007 11:06 by Russ

Prof Trevor Marshall
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No doubt most animals did not have a latent pathogen load anywhere near what long-lived human subject would have had.

Jim,
My presentation last week at DMM2007 tells you the answer - the VDR of animals is nowhere near that of man. Even the apes' VDR are not close in homology to man. The VDR in rats and mice does not even transcribe the antimicrobial peptides.

http://www.marshallprotocol.com/forum39/9219.html

Animal models did not allow us to discover the secrets of immune disease - mathematics did, mathematics that very few scientists can even comprehend, let alone practice...

Prof Trevor Marshall
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Is it primarily the bacterial load, i.e. the quantity of bacteria, that separates "normal", "healthy" individuals from those of us that are "sick"?  Or are those of us who are sick infected with especially nasty species/strains of bugs that are integrated into our bacterial pea-soup but absent from the bacterial population of "normal", "healthy" folks?


Russ,
There is no simple answer. One certainly needs to have enough bacterial load to shut down the VDR. Most healthy folk still have a partly functional VDR. But then, folk with a heavy load of gliding bacteria are apparently more likely to have a non-functional VDR than folk with a heavy load of, for example, strep or e-coli.

As we collect more data it will be interesting to see if there are any particularly nasty species. At the moment I don't really see it, as folks with diagnoses of RA, Lyme or Sarcoidosis can be debilitated by CFS (for example) to similar degrees. So it will be interesting to see how things pan-out as we gather more data and understanding.

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Jimbbb,

My thoughts are "catastrophic system failure" = "gobbledygook".

In other words all they know is "it went wrong" :X plus lots of other scientific definitions that other great people before them have defined.

The sooner these mediocre scientists open their eyes and start listening to the ground breakers like our Prof M the better.

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I wish I had the background and functioning brain to understand this science and to ask a discerning question.

As it is, I can only ask if this has anything to do with the hip joint pain that I and many others have experienced as IPR. I'm inferring that MPers will be less likely to need hip replacements?             .............Sharon

Last edited on Fri Jun 8th, 2007 21:41 by ShrnHml

wrotek
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Maybe we could take just a tiny tiny dose of anti-capnine :)
Sherry, Capnine is uniquely made by gliding bacteria. If capnine turns out to be the only bacterial product which blocks the VDR, you really don't want to try and target this with a drug. If you did neutralize the capnine, you would end up with an immune system that is suddenly freed to attack ALL of the pathogenic load at once. Think about it - you are using just enough antibiotic to kill a few bugs every day. Imagine if suddenly your immune system was returned to 'normal' and started attacking all the bugs, all at once. You would become very ill indeed. Probably you would die. So we need to be careful what we wish for :):):)

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Maybe we could take just a tiny tiny dose of anti-capnine
It's called 'Benicar.'
 

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I hope this is not seen as unrelated, but the joint replacement study has made me want to ask a question that I've been having for some time.  What is the role of bacteria in the rejection of organ transplants (for both the donor and donee)?  It seems to me that the field of organ transplants has so much money flowing into it that there should be a natural interest in Dr. Marshall's work.  Claire

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And I've been wondering for a while about the various CWD / mycobacteria / L-forms / and now gliding bacteria... of which I understand we all have a whole menagerie. Do they all interfere with the VDR or do some of them just take advantage of the situation and set up housekeeping in our cells because the more potent bacterial bretheren have done the dirty work? It seems some intracellular bacteria - in other, far-flung tissue, just "exist" but don't necessarily affect us directly/systemically.

Before this thread was posted, I was thinking of it mostly in terms of those pathogens infecting the phagocytes doing most of the work and those pathogens living in other tissues getting an advantage. But now there is this lipid, capnine being pumped out by biofilm bacteria. Which brings up a secondary question: are the gliding bacteria "complete" i.e. having normal cell walls? If so, that brings up the next logical question: what happens when you treat gliding bacteria / biofilms with ordinary antibiotics? That led me to look into biofilms, which it turns out, are really very neat little colonies of various organisms, not necessarily exclusively bacteria! Thence to reports of bacterial "persister cells" and I can't figure out if they are L-forms or "spores" or ??? But anyway, this fellow's research on that topic was interesting: http://www.biology.neu.edu/faculty03/lewis03.html 
Good luck to him, as he is trying to develop materials with medical applications which bio-films won't grow on.  Also, he makes the observation that plants - having been at it longer than humans - are "smart" enough to use synergistic interaction among different compounds in producing their natural antibiotics. 

Medical researchers need to stop looking for the (singular) magic bullet because you need to tie those little suckers up before you shoot 'em or they just glide away!  Oh dear, I think I'm suffering from a cascading-metaphor-storm...


:P Claudia

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Trevor

As a personal experience, I have 8 screws in my right tibia which are palpable through the skin. I have had them since 1990. They always were sensitive and periodically felt swollen. I considered several times to go through the trauma of removal but resisted. Over the last year, the inflammation and sensitivity have waned to an almost non-existent state.

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Claudia,

Along the lines of your comments concerning the many types of bacteria that could possible cause problems with VDR (or do they all do so).

Science News (May 19) had a very interesting article about microbial populations inhabiting the human body and discussed a few specific cases of their disease effects.

But here are two quotes i found fascinating:

"In fact, in every person's body, there are 10 times as many microbial cells as there are human cells. "The microbial part of ourselves is highly evolved," says Jeffrey Gordon, a microbiologist at Washington University in St. Louis. "These organisms have learned to adapt to life with us."  "

and this one:

"In any one human, there are 100 times as many microbial genes as there are human genes"  -- David Relman, Stanford University

This article is available in full at:

http://www.sciencenews.org/articles/20070519/bob9.asp

And as always Science News articles are very readable (but of course they are only articles about the much more detailed research studies).

jim
 

Last edited on Sun Jun 10th, 2007 18:07 by Jimbbb

Prof Trevor Marshall
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Thanks, Jim, your link led me to

"Fat people harbour 'fat' microbes"
http://www.bioedonline.org/news/news.cfm?art=3017

and
"An obesity-associated gut microbiome with increased capacity for energy harvest"
http://tinyurl.com/2rfrzp   PMID: 17183312

which was a very nice find indeed :)
 

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My girlfriend is certified in clinical nutrition. She's all about "diet" and "exercise" being the biggest factors of obesity. I do agree that scarfing down Dr. Pepper and Cheeto's all day long sitting on your couch influence obesity, however sometimes I wonder how much of the problem is diet and exercise and how much is microbial. Besides the two divisions of bacteria detailed in the links above, how much more involved is it? I mean, surely obesity is a much more of a system wide pea-soup aggregation of L-forms and good old regular bacteria? I guess what i'm getting at is do anyone think L-forms play a role in obesity? if so how much of the overall picture do they play?

~Greg

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Greg,
Yes, the bacterial family simplification that they made during that study is clearly only scratching the surface. It's value to me is in firming up my model, which predicts that all the Th1 diseases should be exponentiating together, and obesity now certainly can be placed into that model. Yesterday we found that Alzheimers does too (quadrupling by 2050). Joining diabetes, arthritis, asthma, and all the rest...
 

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Dr. Marshall, Are there stats that might show this exponential increase of chronic illness increasing in countries that are supplementing with Vit D (and/or using vaccinations) or does the condition of countries that do not supplement (e.g., food, sanitation, etc.) sort of cancel out any stats of this kind?  Claire

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Claire,
There is a general trend that US health is poor by comparison with other Western nations, but little solid data. I think Michael Moore's 'Sicko' film does go into the statistics a little.

The problem is that Vitamin D is pervasive throughout the world. Indeed, baby food with Vitamin D was introduced (by Nestle corporation) into the third world back in the 1950's. I know that I was given plenty of cod liver oil by my mother in Australia (also in the 1950's).

So you can see, the epidemiological problem is not simple.

However, countries which do not have fortified milk generally seem to have a lower rate of Th1 disease than here in the USA.
 

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A formula called "lebertran" which is a fish liver oil was very popular in Germany when I was a child. Just did some research and looks like it was in use from early last century and at one stage kids were forced to drink it daily at school. I can still taste the revolting taste of it. It came in a bottle and you took a tablespoon full. So....probably several generations raised on massive daily doses of vitamin D. Inge.

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Think about it - you are using just enough antibiotic to kill a few bugs every day. Imagine if suddenly your immune system was returned to 'normal' and started attacking all the bugs, all at once. You would become very ill indeed. Probably you would die.

So we need to be careful what we wish for :):):)

Dr. Marshall,

Your comment has reminded me of a question about bacteriophages:

http://en.wikipedia.org/wiki/Bacteriophage

Is that the same reason that bacteriophages couldn't be used to treat Th1 illnesses? I am thinking so, but wondered what you think of bacteriophage therapy.

Thanks,
Elizabeth

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Elizabeth,
Phages form part of the "pea-soup" and I am therefore very loathe to consider them as part of any therapy.

For example, these gliding bacteria produce a toxin, myxin, which kills nearby colonies of e-Coli. yet when those colonies have been infected with the T4 bacteriophage, the e-Coli are no longer killed by that toxin.

"Effect of myxin on deoxyribonucleic acid synthesis in Escherichia coli infected with T4 bacteriophage"
http://tinyurl.com/2b9pdm  PMID: 4927527

The pea-soup is a very complex mix, and is dictated by history. My tendency is to always try to return health by drawing down the size of the communities which contribute to that soup, including all viral, phage and bacterial components.
 

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If L-forms are 0.01microns (1X10^-8m) and bacteriophages are 20-200nm (2X10^-8m ->20X10-8m), that would mean even bacteriophages are at least twice, if not 20 times bigger than CWD bacterial form. However, that's not that much bigger when one considers microscopes. I'm curious as to what bacteriologists think when they encounter L-forms. Surely they can see them. I'm under the impression L-forms can't be cultured easy but they should be easily seen.

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Greg,
When pathologists see L-forms in their microscopes they ignore them because they know that something that small couldn't possibly harm a human being:)

That might sound like sarcasm, but Alan Cantwell tells me he used to be given a plethora of similar platitudes..
 

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If they aren't thinking they can hurt us, then i'm assuming either pathologists know they're absolutely alien and playing some 'benign' role or a 'beneficial' role. Either of which I would like to still insite some kind of question as to what exactly they do. After all no organism in our bodies has a 'benign' role, other wise it wouldn't be there. Anyway, I guess the trick would be to find evidence of any such role instead of just a visual with no dynamic understanding. How about nanotechnology? Perhaps a tiny nano-syringe to extract nucleic acid. Anybody want to donate an electron microscope and some crazy nano technology tool making system? I've got the garage. We'll do it Bill Gates style.

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Gordon's ending comment on video was something to the effect of taking your bacteria to lunch or bringing them to the table because there are some things we use microbes break down to provide our energy.

The discussion on variety or not enough variety to establish phylo/heritance/relationship groupings (not just regrouping from scratch) is interesting and may have implications that good/bad bacteria might be a blurry line to draw, especially with regard to rather crude single and accute attack antibiotic use dominance in medicine. Made me think on the pulsed, long term combo abx effort of the MP and really appreciate enabling my immune system to figure out and handle what even antibiotic combos cannot.

Maybe a dumb question, but thinking about receptors and docking, if capnine, 25D and Benicar all arrive at the VDR at roughly the same moment in time, which would dominate and win the docking opportunity? If one is docked already in the VDR, would another displace? Paper, rock, scissors... single dominator effect or might 25D give capnine the competitive edge? Ki?

The required mechanism was that the bacteria needed to block the proper functioning of the VDR by producing a ligand which would displace 1,25-D from the VDR binding pocket. In turn, Olmesartan would displace the capnine, at least partially...

We can curtail 25D with food/light choices, capnine is stopped only by benicar... (at least partially) and ????

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Maybe a dumb question, but thinking about receptors and docking, if capnine, 25D and Benicar all arrive at the VDR at roughly the same moment in time, which would dominate and win the docking opportunity?

Here is a graph from my Visiting Professor presentation to the FDA last year.



It depicts the concentration-dependent displacement of one ligand from a receptor by another. You might like to review my FDA presentation, or Google for "homologous binding" for for the details :)
 

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Trevor,

I was looking over a paper on capnine and have a question.

PMID: 2992489

I am not a chemist, so perhaps I am reading this the wrong way, but it seems that cysteine is necessary to form capnine, perhaps leading to lower cysteine and glutathione levels? Might that be why some people have positive effects from taking cysteine or glutathione, where in fact, they are helping the bacteria form more capnine?

Sincerely, Frans

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No Frans, a single cysteine amino acid will make no difference when compare to the ton of them in your body. Even enough cysteines to block every VDR molecule will not be noticed. You have to be very careful drawing conclusions from science, many experienced investigators make the same mistake - not looking closely enough at phsyiological concentrations before drawing a conclusion.

And you can forget about glutathione. That substance is so far downstream from the cause of the disease it is 'in the noise'.

..Trevor..

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Trevor, I already was afraid I was wrong, maybe it is best to delete my post so as not to add to the noise.

Sincerely, Frans

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Dr Trevor Marshall wrote: I have been alert to the close symbiosis between the retinoids and Vitamins D for some time. For example, either can function to hold together COMP, and both are active in Rhodopsin, but the retinoids have a relatively low Ki for the VDR.  

More support published today (admittedly from mouse work) for Trevor's ideas that retinoids are important in inflammation, even if they don't exert their influence through the VDR. The following article (published on-line in Science) indicates that (at least in mice) retinoids may influence inflammatory response through interactions with the cytokine TGF-beta.

Reciprocal Th-17 and Regulatory T Cell Differentiation Mediated by Retinoic Acid

Daniel Mucida, Yunji Park, Gisen Kim, Olga Turovskaya, Iain Scott, Mitchell Kronenberg, Hilde Cheroutre 

Ruth

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Ruth,
Those working with mice get side-tracked to NF-kappaB as the murine VDR doesn't even  transcribe the antimicrobial peptides, and operates totally different from that of man.

In Homo sapiens the key to the inflammatory response is not NF-kappaB, it is the type 1 nuclear receptors, pretty well all of them, interacting with 25-D, 1,25-D and the VDR.
 
NF-kappaB has a role, but just a role...
 

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Hmmm........ interesting.

The study below would seem to indicate that the promoter elements in rat and mouse AMP genes also contain binding sites for nuclear hormone receptors - do you think that a different NHR plays the role of the VDR in rodents?

Interestingly, NHRs were more prominent than immune cell-specific TFs in the analyzed AMPcg families [22 othologous families across rats, mice and humans].  Experimental evidence showed the involvement of NHRs in various immunomodulatory pathways. However little is known about their direct involvement in innate immunity.  Recently, there has been evidence that VDR plays a direct role in the induction of antimicrobial innate immune response. The results of the computational analysis which took a bird's eye view of the transcriptional regulators involved in multiple AMPcg families, concur with this evidence and revealed a number nuclear hormone receptor as candidates.  For example, GR, RXR-alpha, AR, VDR and T3R-alpha, seem to be involved in control of 20, 18, 17, 16 and 15 families respectively, out of 22 analyzed. I n addition we discovered 102 new motifs as candidate TFBS with a role in antimicrobial innate immunity.


From: Brahmachary et al.  Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes.  Bioinformatics. 2006; 7(Suppl 5): S8

Obviously, the authors are a little behind the Marshall curve in their understanding of the role of NHR in transcritional control of AMPs in humans but their results also highlight some interesting differences between AMP TF binding sites in rodents and primates that will probably lead other researchers to recognize the species-specific aspect of transcriptional control.

Ruth

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Ruth,
I am pretty busy at the moment, but I would be appreciative if you could spare the time to look over supplementary table 5 of Wang, et al, and make sure it correlates with the citation you posted:

Wang TT, Tavera-Mendoza LE, Laperriere D, Libby E, MacLeod NB, Nagai Y, Bourdeau V, Konstorum A, Lallemant B, Zhang R, Mader S, White JH. 2005. Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes. Mol Endocrinol. 19(11):2685-95 PMID 16002434

Yours is  a valuable citation for me, for some reason I had missed the Brahmachary paper.



The following research group failed to induce cathelicidin in the murine VDR:

Gombart AF, Borregaard N, Koeffler HP. 2005. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.  FASEB J. 19(9):1067-77 PMID 15985530

Again, if you could take a quick look and let me know if there are any significant differences in the outlook of the two groups.

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Can do.  I'll get access to the papers at work tomorrow.
Ruth

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Wow! Congratulations, Dr. Marshall, and thanks for sharing the news!
 
I hope IDSA and ILADS quit arguing and take a look at what you are accomplishing.
 
Also, thank you for posting and answering what some members call “dumb” questions. I learn from both the questions and the answers.
 
[Kindly edit out if the following is irrelevant:]
 
Since I have neuroborreliosis, I checked if Borrelia is able to produce capnine (not that it needs to…just curious). According to Das et al, “ …the lipid biosynthesis pathway is absent from the spirochetes.” Source:  http://papers.gersteinlab.org/e-print/spirochete-jmmb/text.pdf.


 
Benk

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Good eye. I remember Dr Marshall mentioned on 30th Lyme Anniversary video Presentation that borrelia lacks any lipid synthesis.

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To Wrotek:
 
Thanks. Obviously, not as good as your eye.   (I try not to miss any of your postings; they are particularly informative…and humbling: I don’t understand things as well as you do.)
 
Benk

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Oh :) I think If more we understand than more question we have.
For example I wonder if disease severity depends from bacterial ligand VDR affinity.

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Trevor, I have a question regarding homologous binding etc.

I had an interesting discussion with a friend of an acqaintance, who turned out to work for a vitamin manufacturer.......

He was adamant that the last paper about vit. D being protective (which we have discussed in another thread) was game set and match.

I disagreed of course and after some heavy debating, we finally arrived where I wanted to be.

He understands that 25D etc. do NOT activate VDR.

However: he still believes that D has nowhere else to go but the VDR, after being hydroxylated to 1,25D.

To make a long discussion short, we ended at the following conclusion:

The main thing that decides if 25D displaces 1,25D from the receptor is the concentration in the cell itself. I got him wavering in his believes...

Now, I know this must be something that's incorporated in Ligplot, but I am just not sure.

Can you set me straight? How high will the cellular concentration be at which plasmaconcentration? Is there some ratio by which that happens ? It would be interesting to see what he can do with this information. (perhaps even without him being kicked out of his company :))

Sincerely, Frans

Last edited on Sun Jun 24th, 2007 10:45 by Frans

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Frans,
I guess we might expect broad economic repercussions when the real light dawns.

This realization may play a part of the overt or inadvertant denial process now and ahead.

Did your friend see the FDA visiting professor illustration above?--Janet

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Janet, I am not sure if that depicts the plasma concentration or the cellular concentration and how these two relate to each other.

Maybe there is a constant, that cellular concentration is always a certain percentage of plasmaconcentration or something like that?

I know from earlier posts that there is a definite difference in these two.

I know it is a detail, but it is an important one and I never would have thought I would need to go into such detail with anyone, this guy really knows something, but still, not enough   :D

Sincerely, Frans

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Janet, Trevor,

I am looking over the presentation at the FDA again and see that cellular concentration is indeed in the graph.

I don't understand it yet, but will keep on looking at it till the quarter drops...

One thing I understand from the presentation is that the cellular concentration of Simvastatin and Telmisartan has been found in-vitro.

Has the same been done for 25D? Or is it simply a fact of the Ki value deciding how much of the drug will enter the cell?

Sincerely, Frans

PS  Janet, I think I will indeed have to show him this graph. Thanks for that !

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Frans,
The concentrations at which the ligands will become active depends both on their affinity for the receptor, and on the concentrations and affinities of any competitors for the LBP (ligand binding pocket). That is what the graph depicts - competitive displacement of ligands.

The concentration of 1,25-D in the macrophage cytoplasm is about 2 nanomolar. We know that since its Kd (disassociation constant) is about 8.48 and also from a couple of in-vitro experiments.

However, 1,25-D has a Kd for the Glucocorticoid receptor of 8.12, for the Androgen receptor of 8.05 and for the alpha-Thyroid receptor of 8.41. There is little doubt it is also active in these receptors, as they form a subset of type 1 nuclear receptors which plays a key role in the immune system (see, for example, this paper).

And that info will boggle the mind of your vitamin-savvy associate...
 

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I was looking up "biofilms" and now realise that we have biofilm on our teeth - associated with dental plaque.  Might this start to explain the connection between poor dental health and heart disease & other problems?

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Trevor said:

"I am pretty busy at the moment, but I would be appreciative if you could spare the time to look over supplementary table 5 of Wang, et al, and make sure it correlates with the citation you posted:


The following research group failed to induce cathelicidin in the murine VDR:

Gombart AF, Borregaard N, Koeffler HP. 2005. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.  FASEB J. 19(9):1067-77 PMID 15985530

Again, if you could take a quick look and let me know if there are any significant differences in the outlook of the two groups."

 

Sorry to be slow on this but I did take a look at the results from the three studies and conclude the following:



I think all three studies are consistent with each other and with the idea that, in primates, the VDR is a major transcription factor (TF) involved in the up-regulation of CAMP (cathelicidin antimicrobial peptide), whereas in mice, rats and dogs, the VDR is not a TF for CAMP.  

 However, the general picture emerging is that even in mice and rats, the NHRs, including the VDR,  are major TFs for other classes of AMPs.  In addition, the murine CAMP gene is regulated by one or more of the NHRs (just not directly by the VDR).

 Here is a summary of findings:

Gombart et al.:

Simple and elegant study demonstrating that in a variety of human cells and cell lines, 1,25 D (and some analogues) induce transcription of the CAMP gene and production of the active peptide.  Used DNA sequence analysis to identify a VDR response element (VDRE) in a SINE about 600 base pairs upstream of the VDR transcription start site (TSS).  Cloned the DR3 VDRE into a reporter plasmid and demonstrated its ability to induce expression in cells treated with 1,25 D. 

 Also demonstrated that 1,25 D did not induce transcription of the orthologous C(R)AMP gene nor production of the active peptide in murine bone marrow cells.  Found that there was ‘considerable’ homology between the murine and human DNA sequences upstream of the VDR TSS, but that mice (and rats and dogs) lacked the SINE containing the VDRE.

Did not look for other NHR response elements in the mouse CAMP promoter regions.

Wang et al.:

Performed ‘genome-wide’ DNA sequence scan (humans and mice) for VDREs (both types, DR3 and ER6).  Also did some expression analysis in a human microarray to confirm that putative VDREs were indeed associated with up- or down-regulation of associated genes. 

Microarray analysis revealed 913 genes that were up– or down-regulated by 1,25 D on the human Hu133A microarray (but not CAMP).   However, the authors claim that they showed that 1,25D induced expression of both human CAMP and defensin beta 2 (defb4) genes in SCC25 cells (human squamous carcinoma cell line) in a previous study (Wang et al. 2004).

 Genome sequencing identified a DR3 VDRE upstream of the human CAMP gene (as far as I can tell, this is the same one identified by Gombart et al).  (N.B.  Wang et al. searched further (10 kb upstream of the gene compared to a 1 kb search by Gombart et al.) but apparently identified only the same VDRE).  

Screening of the mouse genome for VDRE sequences, revealed about 3000 orthologous mice and human genes that both had VDREs.  However, the CAMP gene was not one of them (according to Supplementary Table 5 and to the following statement from the study):  

We identified consensus promoter-proximal DR3-type VDREs in the promoters of the [human] defB2 and camp antimicrobial peptide genes, which mediated their induction by 1,25 D.  However, neither the [response] elements nor the regulation by 1,25D appear to be conserved in mouse. 

 OK – now things start to get interesting ….

 Brahmachary et al.

These researchers did no expression work whatsoever – but did do a large-scale genome sequence search for many TF response elements (NHRs as well as others) in the promoter regions of 22 classes of AMPs for which they could identify orthologous genes in human, rats and mice.  They identified 3 classes of TFs (NHRs, liver-specific, and neuron system-specific) that were consistently associated with AMP genes.  NHRs were in the top-ranked TFs for eleven of the 22 AMP families (including cathelicidin).  GR, RXR-alpha, AR, VDR and T3R-alpha were involved in transcriptional control of 20, 18, 17, 16 and 15 of the 22 AMP families (in one or more species).  

In the case of CAMP, the following TFs were identified in the upstream sequences in a composite of three species (mouse, rat, human):

  1. The TF associated with binding of PEA3, c-Ets1, PU.1, LyF-1, c-Ets-2, and NF-AT1, AT2 and AT4
  2. Nkx2-1
  3. Unknown (newly-identified) TF
Overall, NHRs were the most frequent TFs associated with 6 of the 22 AMP families in humans, rats and mice:   alpha defensin, cathelicidin, Dbi, lactoferrin, Spyy and ZAP.

The VDRE was detected in 16 families of AMPs (but not in all species) including the  eight listed in the previous sentence and apoa2, beta defensin, bpi, calgranulin, spli, granulin, hepcidin, mbp, secretogranin and vasostatin.  The VDRE is conserved among humans, rats and mice in some AMP families (e.g. ZAP).

So, I think that everyone is agreement that the VDR does directly induce CAMP expression in humans (and other primates) and does not directly induce CAMP in rats, mice and dogs.  However, other NHRs such as RXR-alpha are important TFs for CAMP in mice.

 
  Ruth

 

Last edited on Tue Jun 26th, 2007 13:20 by Prof Trevor Marshall

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Excellent summary Ruth. Almost exactly what I concluded for the review paper I just finished:) I did edit the bit about RXR as RXR always heterodimerizes with VDR when working on human CAMP (I think:))

So here is the really, really interesting bits:
If you look at my data to the FDA talk you can see that 1,25-D goes into all the Brahmachary receptors with high affinity - GCR, AR, PR, and alpha-Thyroid. There has to be a very good chance that they all work together in the innate immune system, each with slightly different distribution of tasks. So here are the pretty pictures. First, 1,25-D docked into the GCR pocket alongside Dexamethasone for comparison



and into the Thyroid-alpha alongside T3



Enjoy!
..Trevor..
 

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At the risk of being labeled a dolt, I admit:  I don't understand what these pictures mean.  I've read the whole thread...

Yes, D125 or D25 or VDR is making my immune system malfunction.  But I don't understand the pictures.

With honesty,

Sherry

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Sherry,
The pictures show that a high level of 1,25-D is capable of displacing cortisol and T3 from the active ligand binding pockets of a portion of those receptors, and inactivating those receptors. Hence those receptors can't make those nice antimicrobial peptides (and other things). Nor can the Thyroid alpha or beta receptors respond to normal quantities of T3 (T3 is made in the body from T4, Thyroxine).

1,25-D would only displace the natural ligands when its concentration rises well above where it would normally be controlled by the VDR. Since the bugs cripple the VDR, the 1,25-D has few limits on its strength, and we see (clinically) the results of it affecting these other receptors.

So although the microbes only evade 16 families of AMP by disabling the VDR, the consequential rise in 1,25-D can disable a bonus 67 families of AMP... Unfortunately, it also makes the host sick...
 

 

Last edited on Tue Jun 26th, 2007 14:55 by Prof Trevor Marshall

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So bacteria disable VDR which contributes to the rise of 1,25-D which disables other receptors ? This is pretty twisted :) And these other receptors are responsible for immune system proper function too ?  I wonder why nature made 1,25-D being able fit other receptors.

Last edited on Tue Jun 26th, 2007 15:02 by wrotek

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That's real interesting.  The model is getting clearer and clearer all the time.  Eventually it will be so clear that even the skeptics will be able to see it.

It sounds like the immune system would work better if 1,25D did not have any affinity for those other receptors and only binded to VDR.  Do you think there is an evolutionary reason why 1,25D has affinities for these other receptors?  Or is it just the nature of ligands and receptors that at a high enough concentration a ligand is going to bind to a lot of different receptors?

Thanks!


Edit: Wrotek, just saw your post.  I guess great minds think alike.  Or maybe that should be brain-fogged minds think alike. :)

Last edited on Tue Jun 26th, 2007 15:11 by Russ

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Trevor,
In your answer to Sherry, I think you answered my question ... which was:

When 1,25 D is docked into the other nuclear hormone receptors, is it an agonist or antagonist?

Ruth

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Ruth,
I can't be 100% sure 1,25-D functions as an antagonist, as we really don't know very much about the GCR (GCR-null mice do not survive gestation), or the residues which are required to activate the receptors, or whether there are multiple modes of activation.

However, there appears to be sufficient divergence between the residues which are 'contacted' and the mode of contact, to make me think that antagonism is far more likely than agonism.
 

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http://winmlm.neostrada.pl/mp/affinities/1.jpg

Interestingly all D metabolites have higher affinities to Thyroid alpha 1 and Thyroid beta 1, than they have to VDR. I thought that metabolite is named after the receptor it binds best. And from the table above it would seem that 1,25-D is more like a thyroid ligand than VDR ligand. But than it comes that it is antagonist so... It would apper it has something like emergency shut down function there.

From the table above 1,25-dihydroxy has 5 times higher affinity for Thyroid alpha 1 receptor (0.03/0.006=5) than to VDR
and 1,25-D has 15 times higher affinity to Thyroid beta 1 (0.03/0.006=15) that for VDR, right ?

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Well, not really. Those values were obtained from Autodock. A better package, XSCORE, estimates Kd values of 8.48, 8.12, 8.41, 8.05 into the VDR, GCR, Thyroid-alpha1 and Androgen receptor.
( Ki = 10 **-Kd )

Remember that concentrations are also a factor in homologous displacement , and 1,25-D is usually very low in concentration compared with the other hormones.
 

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Wrotek,

I did a little googling for "evolution of NHR" and came up with this fascinating document in a Biochem Projects 2000 folder at Stanford -- not a pub-med doc which means it is fairly READABLE!

http://cmgm.stanford.edu/biochem218/Projects%202000/suchorolski.pdf

Just a few lines within the first pages:

"Considering that the pathways for catabolism of each ligand is relatively more complex than the single transcript necessary for each nuclear receptor,  some authors have claimed that the co-evolution of the hormonal pathway lies mostly with the receptor evolving around a set of fairly stable ligands."

(and goes on to mention many orphan receptors for which no ligands have been identified and other things).

Fascinating stuff!

jim

 

 

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Is this ok ? What about units ?

Last edited on Wed Jun 27th, 2007 08:44 by wrotek

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8.05 not 8.95
Ki is molar, VDR is therefore 3.31 nanomolar, for example
 

Last edited on Wed Jun 27th, 2007 09:33 by Prof Trevor Marshall

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:) OK ?
VDR                         Kd =8.48 ,   Ki=0.000000003311311215  mol  =  3.31 nmol
GCR                          Kd =8,12 ,  Ki=0.00000000758577575    mol  =  7,58 nmol
Thyroid-alpha1         Kd =8,41 ,  Ki=0,00000000389045145    mol  =  3,89 nmol
Androgen receptor.  Kd =8.05 ,  Ki=0,000000008912509381  mol  =  8.91 nmol  

So the new additional variables actually make ligands less compatible ?

Last edited on Wed Jun 27th, 2007 09:54 by wrotek

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Dr. Marshall,

Thank you for the lucid reply.  I've mulled this over many times in my mind, and it reminds me of "stress corrosion cracking"  or SCC of metals.  For the non-mettalurgical reader, "stress corrosion cracking" occurs when a major stress leads to the electrochemcial process we know as corrosion.  The corrosion is the result of an energy effect, and the chemical process runs along grain boundaries  and then a crack occurs in the metal.  Such failures can be disasterous; i.e.,  bridges collapse.  Steam turbines go down.  Engines die.

The analogy between SCC and biology runs like this:   the VDR is getting stressed with  CWD bacteria, and over time, leads to the excess D125 from the fortified food supply going to othr ligands and receptors (corrosion?) and then our bodies fail (i.e., crack). 

Stress is the driving force in SCC; CWD bacteria are the driving force in chronic disease and amplified by the unsafe food supply (i.e., foods fortified in D25).


Does the analogy work? 

Sherry


Last edited on Fri Jun 29th, 2007 08:10 by scooker48

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Simulations unravel outer membrane transport mechanism

http://www.life.uiuc.edu/emad/TonB-BtuB/btub-2.5Ans.mpg

Not that this specifically pertains to anything much relevant other than gram-negtive bacteria. It's just nice to see that others are practicing the same sort of molecular modelling as Dr. Marshall and in my mind at least, it lets me know Trevor isn't a quack-pot! :D LOL

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Greg,
"Any sufficiently advanced technology is indistinguishable from magic" - Arthur C. Clarke.

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Reminds me of a german poem that my father used to quote to me:

The dove said to the eagle "beyond knowledge comes belief" and the eagle replied "yes, but where you already believe, I still know".

Flying higher the eagle had a much wider horizon. It's just such a pity that the doves don't often listen to the eagles! Inge.

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curious as to the doctor's thoughts on this article on science daily. The title had to do with bacteria no longer being able to hide from testing with some new device or method. Also mentioned biofilms etc... link is:

http://www.sciencedaily.com/releases/2007/08/070802095344.htm


, does this apply to the cwd bacteria in th-1 as well?

Last edited on Mon Aug 6th, 2007 10:57 by kess3881

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Dr. Steve Fry at Airpark Medical Center in Scottsdale, Ariz., is hoping the Lateral Flow Assay will help him diagnose his patients' medical problems faster. "This new device will allow doctors to test drugs and materials simultaneously and will save time and money," Fry said.

Well, if it worked as the article advertises it does, then Dr Fry would be finding bacteria in every one of his patients, and trying to puzzle out why. So, based on the above quote, I therefore suspect that this attempt to use antibodies to definitively diagnose disease has failed, as have all attempts before it.
 

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I've wondered myself the difference or similarity of biofilm populations on surgical devices and other such "popular" news stories. What is the difference between CWD form biofilms and those such as these?

"The device identifies the presence of biofilm-specific antibodies in patients by allowing the antibodies to bind to biofilm-specific proteins on the device."

I'm also curious about this because i'm under the impression that very few antibodies occur due to successful parasitization of CWD forms, except that early on some pea-soups exhibit some antibody phenomenon ("autoimmune antibodies".)

I wonder what is the "biofilm-specific protein" they are talking about.

~greg

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Best to ask them, Greg. It doesn't sound to me as though they have any idea what they are doing.
 

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:D I've heard that one before.....and they were talking about you! Anyhows, I emailed Dr. Leid. This paper of his is interesting, but still leaves me questioning what connects the dots between big biofilms and tiny coccoid films. I like his description of the contents of the biofilm being "extremely porous hydrogel" :)

weird....

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Greg,

As I understand it, we have both biofilms (which may include CWD and non CWD bacteria) and then we also have CWD throughout the body, independently of biofilms.

That paper you linked to had an interesting quote.  They conclude that:

The mechanisms behind biofilm resistance, especially with regard to leukocytes, are not clear and have been explained by the paradigm of a lack of penetration by the leukocytes and a decreased ability of phagocytes to actively kill the bacteria, a process termed "frustrated phagocytosis"

I suppose we may conclude that capnine or similar VDR blockers are causing the frustrated phagocytosis?

Joyce Waterhouse

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There are different types of evasion techniques used by intraphagocytic infection. The 'biofilms' photographed by Emil Wirostko's team use one, Mycobacterium leprae and Mycobacterium tuberculosis use a different technique, and yet another is used by the Rickettsias. The subinhibitory dosing of the MP seems to work differentially on Mycobacteria and the 'Biofilm' species. Although ultimately the restored immune system gets them all, we are still trying to gathering data which describes this differential...
 
 

Last edited on Mon Aug 6th, 2007 19:33 by Prof Trevor Marshall

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Dr. Marshall,

What does subinhibitory dosing mean?

Well, Dr. Leid came back with a couple of interesting papers.

Intracellular Bacterial Biofilm-Like Pods in Urinary Tract Infections 


Maturation of Intracellular Escherichia coli Communities Requires SurA


He wanted to know a little more about what the heck i was doing. So he may reply again to my explanation and talk more about what he knows. I asked him what he knew of Capnine and the VDR.


It's quite confusing with the HGT cascading mess and the pea-soup conundrum. So many ways to evade the immune system and so many ways to kill the little critters.


~G

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Bump...

Well Dr. Leid never got back to me :) Interesting papers though. I know someone on the MP who had Interstitial cystitis disappear within the first year.............interesting hugh?

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Greg, try this Pubmed search for 'subinhibitory clindamycin':
http://tinyurl.com/27f8wm

you will find a lot about 'subinhibitory' there :)

 

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Dr Trevor Marshall wrote: There are different types of evasion techniques used by intraphagocytic infection. The 'biofilms' photographed by Emil Wirostko's team use one, Mycobacterium leprae and Mycobacterium tuberculosis use a different technique, and yet another is used by the Rickettsias. The subinhibitory dosing of the MP seems to work differentially on Mycobacteria and the 'Biofilm' species. Although ultimately the restored immune system gets them all, we are still trying to gathering data which describes this differential...
 
 


Are we essentially saying that biofilm species aren't the only "main" culprits in Th1 disease?

I was under the impression that biofilm species were the main problem, but in light of other intraphagocytic pathogens being successful with other maneuvers, how might the VDR become the king pin otherwise? 

what are the similarities between biofilm species and the others?

~Greg

 

 

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The species which shut down the VDR, and thus the generation of antimicrobial peptides, are the cause of Th1 immune disease. At this point I do not believe Mycobacteria or Rickettsia persist via that pathway.

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Trevor, not sure if I understand your comment (previous post). Are you saying that at this point you do not believe Rickettsia are directly involved in causing Th1?
And expanding on that question....will the MP get rid of the Rickettsia?


Thanks. Inge.

 

Last edited on Thu Aug 30th, 2007 23:57 by IngeD

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Inge,
One of the most difficult dilemma I had to resolve, and even more difficult for some patients to come to grips with, is that the infections thought to be causing them to be ill are likely to actually be co-infections, unwitting bystanders at the scene of the crime. These infections are easily observed, but are not necessarily contributing to the illness...

You see, when the innate immune system becomes weakened by the Th1 pathogens which attack the VDR, it can no longer clear viruses, and other infections. Rickettsia and Mycobacteria can be very nasty pathogens indeed, but their mode of action is different from the Th1 pathogens, and they evade the immune system differently. As the body heals, and its anti-microbial peptides start to be produced again, the immune system eventually takes care of fungal, viral and bacterial co-infections.
 

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So is it fair to say that whatever mode of action it is that Rickettsia and Mycobacteria use to avoid the immune system and persist, that mode of action is only effective against a weakened immune system and not a fully-functioning one?

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Russ,
It is safe to say that these pathogens are more effective against a weakened immune system than a fully functioning immune system.

One paper exploring the mechanisms used by Mycobacteria can be found at
http://tinyurl.com/2wrtpr
with commentary at Nature.com (free access, registration required)
http://www.nature.com/nrmicro/journal/v5/n8/full/nrmicro1728.html
 

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Thank you Trevor. Yes. Hard to accept, but it has been dawning on me gradually since doing the MP and reading up on this site that my illness goes beyond when I thought I succumbed to Rickettsiosis. The whole thing is such a muddle because there appeared to be a point in time when things got really bad. 2002 when I was living in Malaysia and I contracted what the locals called a 90 day chinese cough. Only in my case the 90 days stretched to indefinate.

Now I understand that I already had chronic conditions which go back a lot further than 2002 but which were things I simply had learnt to put up with and put down to all sorts of things! So what must have happened is that I already had Th1 and this made me succumb to the Rickettsia which my immune system was not able to fight off. For 2 years I battled on with steroid inhalors (occasional use) when asthmetic coughs made it hard to breathe. And then I started on antibiotic treatment which did not eliminate the Rickettsia but kept secondary (now understand them to be tertiary???) infections at bay so that I was reasonably comfortable and kept out of emergency rooms. But beneath it all the Th1 was progressing as is evidenced by new chronic diseases becoming apparent (e.g. Peripheral Neuropathy, Hypertension, Insulin Resistance etc) during the last 4 years.

So....the pre-MP antibiotics were killing opportunistic bacteria that my compromised immune system no longer was able to fight off. The MP is killing CWD bacteria and restoring the health of my immune system i.e. fighting the Th1 condition. I then have to hope that this restored immune system will eventually kill the Rickettsia for me to regain full health. Because there doesn't seem to be any other treatment around that can eradicate Rickettsia!

I think my doctor thinks the Rickettsia are to blame for all of my health problems and I had incorrectly assumed Rickettsia to be CWD bacteria. So they are in effect not part of the pea soup? A colleague in Malaysia told me after I was diagnosed with Rickettsiosis: "You sound like a walking biological laboratory".  She obviously knew what she was talking about :( ! So in my case it's pea/ham and whatever soup. Probably some Mycobacteria as well considering the number of times I have had Pneumonia in recent years. Which makes sense considering I have probably had a compromised immune system for many years. Peas soup is hard on the digestion. With the other bits it is giving me a stomach ache :( But...nothing for it but to continue on the fight! There are others here much worse off so together we will beat the buggers! Inge.

 

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Agreed, with the one exception of peripheral neuropathy, which may be due to a secondary infection by Mycobacterium leprae. These are known to be nourished with the epinephrine present at peripheral nerve synapses, where they congregate. So I am not sure about the neuropathy. It might be secondary, and clear up after the Th1 pathogens have been dealt with.

Rickettsia and Mycobacteria are something like the Th1 CWD in that they are obligate intraphagocytic pathogens. But they might not form part of the biofilm-protected intraphagocytic colonies, I just don't know yet.
 

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see Will the Marshall Protocol treat co-infections?

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ok...after one hour of reading (thanks Barb for the link that I seem to understand better now) I am still in a bit of shock re the following:

Dr Trevor Marshall wrote: Agreed, with the one exception of peripheral neuropathy, which may be due to a secondary infection by Mycobacterium leprae. These are known to be nourished with the epinephrine present at peripheral nerve synapses, where they congregate. So I am not sure about the neuropathy. It might be secondary, and clear up after the Th1 pathogens have been dealt with.

Are you say that it is possible to be infected with Mycobacterium leprae without developing full-blown leprosy? I found on the internet that it is now thought that the peripheral neuropathy that people suffering from leprocy experience, and the resulting loss in limbs etc is due to the mycobcactberium leprae. But I did not find a suggestion so far that other forms of peripheral neuropathy are caused by the same bacteria. In 1981 and 1982 we were living in an area where leporsy is still a problem (Northwest Australia). I also read somewhere that it can take up to 20 years incubation for this disease.

Going back to the link Barb provided, couple of comments:

1. I almost want to thank my co-infections because they are what drove me to get medical help. I arrived at the MP missing a gallbladder, 2 ovaries and with a sick liver amongst other things. And had no idea what was the underlying cause.  If it were not for the co-infections being the straw that broke the camels back so to speak, it seems to me that the Th1 disease in its stealthy way could have done a lot more harm to my remaining organs!
2. I also read that fungal infections will be healed by a restored immune system. For years now I had a wart on the palm of my left hand. After 7 months on the MP there is absolutely no trace of it left. Can I then say that my immune system is already more active and that it is the healing immune system that eradicated the wart? Cause for celebration! (the wart may only be a minute issue but if it is a sign that my immune system is already starting to recover..then that is HUGE).

Inge. 

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Inge,
Truth is, I cannot answer any of your questions about leprosy. Too little is known about it, or why it, and tuberculosis, are currently enjoying a resurgance. In some US states 'latent tuberculosis' may be as high as 10% of the so-called "cured" cases. Is the resurgance due to the Th1 epidemic? Who knows - my recent paper (still in press) did cite a study showing that recovery from Tuberculosis was directly dependent on which VDR gene haplotype a patient has :):)
 
We do have a little data from the SARS epidemic, however. It took out the elderly and infirm, particularly diabetics (a Th1 disease).

I remember clearly one of the diabetic patients I studied in Perth, ca. 1981. He had a foot amputated because it had sepsis. 'Diabetes foot' was the diagnosis, but a young intern from Pakistan differed. He said that he recognized it as "Leper's foot" from his experience in treating the condition in Pakistan. Hmmmm...
 

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Now there is another epidemic I was bang in the middle of. I was living in Malaysia during the SARS epidemic. Travelled through Singapore airport then Hong Kong where everyone was wearing masks. Coincidentally (??????) this was the time my 90 day cough emerged.....!!!!!!  So I can be thankful that despite my Th1 illness I survived that epidemic!

Re diabetes and peripheral neuropathy. My father had diabetes and very shortly after diagnosis developed peripheral neuropathy. I was prediabetic with peripheral neuropathy. Both peripheral  neuropathy incidences were labelled "diabetic peripheral neuropathy". I now understand the connection. Diabetes is a Th1 illness and many people with Th1 present with peripheral neuropathy. Hence the connection. Not that diabetes CAUSES the peripheral neuropathy. That interpretation is due to an incorrect interpretation of the statistics imo.

Thanks Trevor for your explanations. Hopefully things will get a lot clearer over the next few years! Inge.

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Just a note about the peripheral neuropathy.  I developed a wee bit of it in both feet and one hand in the year (or two, can't remember) before starting the MP.  All but a smidge of it is gone (and I think that is a bit unusual so early in the MP--about 3 to 4 months in at the time).  (My brother also had this and my mom just asked me the other day what could be causing the numbness in her hands--she has Th1 illness and if she had a biopsy for the lumps in her lungs, little doubt that the diagnosis would be sarc.)  Hoping that the neuropathy is not leprosy related!  And even if it is that a body without Th1 illness will be able to take care of it.  Claire

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No sign of leprosy in our cohort to this point Claire. You can relax :):)

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Where does Borrelia fit into the picture?  Is it's persistance dependent on the VDR not functioning properly or do you suspect that like Mycobacteria and Rickettsia it has other ways of persisting?

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see Post Treatment Lyme Disease Syndrome Definition, symptoms, transmission, testing, treatment

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Dr Trevor Marshall wrote: Here is a graph from my Visiting Professor presentation to the FDA last year.



It depicts the concentration-dependent displacement of one ligand from a receptor by another. You might like to review my FDA presentation, or Google for "homologous binding" for for the details :)
 


Trevor, I hope I am not asking too much, but would it be possible for you to post the same graph, but one that shows the homologous binding of 25D vs 1,25D to the VDR? That should wrap things up :D

Sincerely, Frans

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Er, um, I give the formulae there. Can one of our maths buffs crunch the numbers for me? This took me several hours when I first did it, and right now every minute of my time is scheduled for the next several weeks :)
 

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Anyone up to it? :D

Best, Frans

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Dr Marshall. First I want to say how appreciative I am that your science is real and it works. My gradually elevated level of quality of life, and that I am living is proof of that.

Secondly I had a question. Since the beginning of this thread was devoted to the lipid generation of certain bacterial microbes found in joint transplant replacement surfaces (Capnine), and since I have RA along with Sarc, I was wondering if salinity might play a roll in microbial affinity or propagation of lipids. 

Has anyone looked at a possible correlation between location/ concentration of microbes and or the ability of these microbes to produce Capnine with the levels of serum and cellular salinity?

The basis of my question is that I have noticed that any added intake of table salt from what occurs naturally in the food that I eat will cause a corresponding increase in inflammation and pain at the RA affected areas. This can be a pre-prepared meal or a can of soup. Unless I prepare it myself, there is always this risk. Once this salinity level is decreased to it’s normal level, the inflammation and pain subsides. I should also note that potassium chloride does not affect me the same way.

Last edited on Mon Apr 21st, 2008 14:33 by bookdad

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My lady can crunch the numbers for you. Is all the data that she needs in the graph? I will give it to her tonight.:dude:

Last edited on Mon Apr 21st, 2008 14:32 by bookdad

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dr. m, is there a relationship between surgical hip repair of torn labrum, reshaping and re-attaching of head of femur, and repair of ligamentum terres, as in hip prosthesis surgery; i.e. surgical tools, sutures,loosening of area by centimeters to allow for "repair work", and antibiotics.hank you for considering this thought. smsinger

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This paper http://jb.asm.org/cgi/reprint/JB.01444-07v1.pdf  contains another example of a bacterial ligand (3OC12-HSL) that interacts with at least two host nuclear hormone receptors – PPARbeta/delta and PPARgamma. Possibly also with RXRalpha which, of course, heterodimerizes with both PPAR and VDR.  Interstingly, the bacterial ligands are also signalling molecules in the bacteria themselves – quorum sensing molecules during biofilm development.  The bacteria involved is an important lung pathogen for cystic fibrosis patients.
 
The authors of the paper were astute enough to realize that the host inflammatory responses that were induced in the presence of 3OC12-HSL were likely mediated by one or more Nuclear Hormone Receptors and tested 3OC12-HSL against a panel of the of NHRs (but not the VDR) in mouse and human cell lines.   They found:
 
J Bacteriol. 2008 Jan 4; : 18178738 (P,S,E,B)
Peroxisome Proliferator Activated Receptors Mediate Host Cell Pro-inflammatory Responses to P. aeruginosa Autoinducer.
[My paper] Aruna Jahoor, [url=http://lib.bioinfo.pl/auth:Patel,R]Rashila Patel[/url], Amanda Bryan, [url=http://lib.bioinfo.pl/auth:Do,C]Catherine Do[/url], Jay Krier, Chase Watters, Walter Wahli, Guigen Li, Simon C Williams, Kendra P Rumbaugh
The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing (QS). 3OC12-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This pro-inflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC12-HSL influences host responses are unclear, and no mammalian receptors for 3OC12-HSL have been identified to date. Here, we report that 3OC12-HSL increases mRNA levels of a common panel of pro-inflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC12-HSL receptors, we examined the expression pattern of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC12-HSL functioned as an agonist of PPARbeta/delta transcriptional activity, an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The pro-inflammatory effect of 3OC12-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC12-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC12-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics in P. aeruginosa infections.
 
To my untutored eye, the structure of 3OC12-HSL (shown in this publication: http://www.mrc-lmb.cam.ac.uk/genomes/awuster/wecb/Shiner_et_al_2005.pdf ) looks a lot like retinoic acid but perhaps Trevor, in his spare time, will find that it is also an agonist or antagonist of the VDR.
 
Ruth

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Ruth, good work !

Since PPARgamma is a type 1 nuclear receptor, just like the VDR, meaning they look structurally alike, I suspect you have, indeed, found another capnine.

I hope Trevor finds time to start the models to see if it does bind into the VDR with enough affinity and perhaps if it is actually an antagonist.

It is extremely interesting that it actually plays a role in biofilms.

Best, Frans

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Hey folks not to sound dumb, but my lady is willing to take a crack at your equation. Since she is a mathematician and not a medical person, we need to know what Ki is in the graph. Since Kd is based upon Ki, we need to know what that is. Also there is no reference for 25 d in the equation.

Thanks

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Dr Trevor Marshall wrote: Thanks, Jim, your link led me to

"Fat people harbour 'fat' microbes"
http://www.bioedonline.org/news/news.cfm?art=3017

and
"An obesity-associated gut microbiome with increased capacity for energy harvest"
http://tinyurl.com/2rfrzp   PMID: 17183312

which was a very nice find indeed :)


I am not a scientist or anything close to it......But after reading this, I wondered if a reverse hypothesis might be relevant...... we know that losing weight cannot cause chronic inflammation......but could losing weight help expose Th1 more quickly?    Weight gain is common for those who have hypothyroidism.   My daughter was counselled by her doctor to reduce her weight and by the time she had lost about 30 lbs, she got uveitis.  Have you found any correlation between weight loss and the appearance of Th1 symptoms?   Just uneducated speculation.  My mom had uveitis in the 30's or 40's--has never had a relapse.  At some point in her life she lost a lot of weight, but I don't know the timeline.  For being in her 80's now, she is fairly healthy, but all my siblings have multiple symptoms of Th1 and symptoms of Th1 have mushroomed in her grandchildren:( Thanks, Trevor, a lot, for all your work   Carol

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And here is another one.  Bacterial-Modulated Signaling Pathways in Gut Homeostasis.  Lee, Won-Jae.

Sci. Signal., 27 May 2008
Vol. 1, Issue 21, p. pe24
[DOI: 10.1126/stke.121pe24]

You can see the abstract at :

http://stke.sciencemag.org/cgi/content/abstract/stke.121pe24

Ruth

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Bless-You and your Knowledge.          Lori

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'Clingy' bacteria causes infection

http://www.sciencealert.com.au/news/20100903-20692.html?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+sciencealert-latestnews+%28ScienceAlert-Latest+Stories%29

 

Impact of Nanoscale Roughness of Titanium Thin Film Surfaces on Bacterial Retention

http://pubs.acs.org/doi/abs/10.1021/la902623c

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I think when i lost 30 pounds in 30 days it was more like a month long IP mega herx.
 the little bugs and time of year gave me stomach flu symptoms cranked up to 11.

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Biofilm Production Aids Campylobacter Survival

http://www.sciencedaily.com/releases/2010/03/100324184602.htm

Campylobacter cannot survive in the oxygen levels in the air, which forces the bacterium to adapt for survival in the food chain. One such a survival strategy is to form a biofilm, whereby the bacteria stick to a surface and encase themselves in a sticky 'slime' which protects them.

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Can bacteria create biofilm inside white blood cells and use it to disable nuclear receptors ?

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Intracellular bacteria certainly can. Whether they exist as a biofilm or not, I am unsure.

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Yes, I am especially interested in the biofilm inside cells, I wonder if the biofilm structure can grow so big that it will disrupt cell machinery - organella, or perhaps even nuclear receptors immobilizing them, somehow.

Last edited on Sat Jun 18th, 2011 08:38 by wrotek

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The biofilm-like masses called intracellular bacterial communities are in 'Yellow' in this image.

http://iai.asm.org/content/vol78/issue3/images/large/zii9990985010006.jpeg

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4+ and TLR4 mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.


Full Text Here:  http://iai.asm.org/cgi/content/full/78/3/963

Last edited on Sat Jun 18th, 2011 11:53 by Phillyguy

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Widespread occurrence of an intranuclear bacterial parasite in vent and seep bathymodiolin mussels

http://onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2008.01847.x/full

SummaryMany parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite “Candidatus Endonucleobacter bathymodioli” that invades the nuclei of deep-sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur- and methane-oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep-sea ecosystems. We first discovered the intranuclear parasite “Ca. E. bathymodioli” in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid-Atlantic Ridge. Using primers and probes specific to “Ca. E. bathymodioli” we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of “Ca. E. bathymodioli” showed that the infection of a nucleus begins with a single rod-shaped bacterium which grows to an unseptated filament of up to 20 μm length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by “Ca. E. bathymodioli” were those of the gill bacteriocytes. These cells contain the symbiotic sulfur- and methane-oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the “Ca. E. bathymodioli” belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates.

Last edited on Sat Jun 18th, 2011 11:54 by Phillyguy

wrotek
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from
Physiology and evolution of spirochetes.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC413998/

Possible Relationship with Gliding Bacteria Some recent reports suggest that the phylo-genetic relationship between spirochetes and gliding bacteria may be closer than previously believed. Greenberg and Canale-Parola (67) de-termined the molecular structure of the major carotenoid pigments of the facultative anaer-obes S. aurantia and spirochete RS1 (Fig. 4), the only known pigmented spirochetes. Except for their presence in the spirochetes, and in an unidentified microorganism (4), these carote-noids have been detected only in gliding bacte-ria (see section, Carotenoid pigments). Another characteristic common to the facul-tatively anaerobic spirochetes and gliding bac-teria is that their growth is strongly inhibited by actinomycin D (49, 67). Since gliders and spirochetes are both gram negative, this is an unusual response, inasmuch as gram-negative bacteria generally are not appreciably in-hibited by actinomycin D (49). Possibly, the sensitivity of gliding bacteria and spirochetes to this antibiotic reflects similarities in cell surface composition or in specific physiological processes. Studies of motility mechanisms may reveal additional similarities between spirochetes and gliding bacteria. As previously mentioned, spi-rochetes not only swim free-floating in liquids, but also "creep" or "crawl" on solid surfaces (23, 45). The mechanism responsible for the latter type of movement may prove to be identical or similar to that which propels gliding bacteria.


Carotenoid Pigments S. aurantia and spirochete RS1, the only known free-living, facultatively anaerobic spi-rochetes, produce carotenoid pigments (27, 67). Aerobically grown colonies of S. aurantia are yellow-orange, whereas those of spirochete RS1 are red. Anaerobically grown colonies are white. The molecular structure of the major pig-ments of spirochete RS1 and S. aurantia strain J1 was determined by analytical procedures in-volving mass spectrometry, infrared spectros-copy, chromatographic analysis, hydride reduc-tion, and acetylation and silylation experi-ments (67). It was found that the major pigment of spirochete RS1 was 4-keto-1',2'-dihydro-1'-hydroxytorulene, also called deoxyflexixanthin (Fig. 4). This pigment accounted for at least 90% of the total pigment content of spirochete RS1. The major pigment from S. aurantia was 1',2'-dihydro-1'-hydroxytorulene (Fig. 4), dif-fering from deoxyflexixanthin only in a substi-tution in the cyclohexene ring. Chromato-graphic and spectrophotometric evidence indi-cated that 1',2'-dihydro-1'-hydroxytorulene was also present, as a minor carotenoid compo-nent, in spirochete RS1. The two major carotenoid pigments from S. aurantia and spirochete RS1 (67) had been pre-viously detected almost exclusively in gliding bacteria, such as species ofFlexibacter (2), Stig-matella (112), and Myxococcus (142). The possi-ble evolutionary significance of the occurrence of these pigments in both spirochetes and glid-ing bacteria is discussed in a subsequent sec-tion of this review. Saproxanthin, a carotenoid pigment remarkably similar in chemical struc-ture (Fig. 4) to the identified pigments of spiro-chete RS1 and S. aurantia, is the major carote-noid of Saprospira grandis (1). It is noteworthy that S. grandis is a gliding bacterium previ-ously believed to be a spirochete (25, 33).
Capnine



deoxyflexixanthin (picture below)

http://ctdbase.org/detail.go?type=chem&acc=C010907





Last edited on Fri Mar 1st, 2013 07:06 by wrotek

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Salmonella and its biofilm

http://www.vtnews.vt.edu/articles/2013/04/041013-fralin-biofilmponder.html

In moist conditions, Salmonella thrive and reproduce abundantly. If thrust into a dry environment, they cease to reproduce, but turn on genes which produce a biofilm, protecting them from the detrimental environment.  

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Oh, and this:   Clinging to crevices, E. coli thrive

https://www.seas.harvard.edu/news-events/press-releases/flagella

 E. coli are equipped with two types of appendages: pili, which are short, sticky hairs, and the whip-like flagella, which are often twice as long as the bacterium itself. Pili had previously been recognized as playing a critical role in the formation of biofilms. These short hairs, up to only a micron in length in E. coli, can stick to surfaces temporarily, while the bacteria secrete a thick slime that holds them permanently in place.


Flagella, on the other hand, typically play a propulsive role, helping bacteria to swim and steer in liquid environments. As it turns out, though, when it’s time to settle in one place, flagella also contribute to adhesion on rough surfaces, where the pili would have access to fewer attachment points.



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