Polyamines

If it's on your mind and it has to do with multiple sclerosis in any way, post it here.

Postby BioDocFL » Tue Oct 12, 2004 11:00 am

OddDuck,
Sorry if I'm not very real time in responding to your questions. I have alot to describe before I can get into the guts of the hypothesis. Also, I am trying to get a grant application in for an ovarian cancer project, on polyamines of course.
I think for the purposes here epigenetics could be considered reversible modifications to DNA and histones that can alter the expression of the underlying genes. Methylation of DNA and histones can silence the genes but that can be reversed by demethylation. The sequence of the gene and the resulting protein is not alterred.
Genetics, for here, would be irreversible changes to the gene sequence (or its promoter sequence) so that the gene expression or the resulting protein are permanently alterred. The protein may or may not be functional but it is different than the normal protein. Genetic changes can be insertations, deletions, mutations to the DNA code.
X inactivation is an epigenetic mechanism in that genes on the inactive X can be reactivated and express the protein as it should be. When I talk about fragmentation, I am in this case referring to breaks to DNA in individual somatic cells that will not be passed on to children. This fragmentation could occur in only a few cells in the body, not every cell. The genes on the fragments for the most part may be unchanged but the environment that the fragment ends up in may alter the epigenetic control of that fragment. There, that should be even more confusing. I'll give the definitions another try later since it is probably still not real clear. Wesley
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Postby OddDuck » Tue Oct 12, 2004 11:15 am

Actually, I got that, Wesley!! I'm with ya! :wink:

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Postby BioDocFL » Tue Oct 12, 2004 12:59 pm

So what happens if there is a break in the X chromosome? Not necessarily during embryogenesis, but perhaps later in pre-adolescence. Now we can finally get into the hypothesis. Think about where the breaks could occur: between Xq13 (XIC) and the telomere of the long arm; between Xq13 and the centromere; somewhere in the short arm separating it from the centromere and the XIC. Here are two items to consider and I will give some references.
Chromosomes have fragile sites. These are not just single base pairs that can break but stretches of chromatin up to 4 million bases long in which at any of numerous points there could be breaks, single strand or double strand. So you would not see a specific point where a break occurs but a range, ie. not a single mutation point in an 'MS' gene. Each cell in which damage occurs might break at a slightly different spot in a fragile site but this could lead to differences in the genes that are on the fragment and genes that remain with the rest of the chromosome. (Blair HJ, et al. Positioning of five genes (CASK, ART, SAT, IMAGE cDNAs 24828 and 253949) from the human X chromosome short arm with respect to evolutionary breakpoints on the mouse X chromosome. Mamm Genome 2000; 11:710-712. I'll have to explain later how these breaks might occur in a fragile site.
Next, consider that there have been reports of chromosome abnormalities in MS (and RA). It is only a small percentage of cells showing abnormalities but it differs from normal humans. (D'Alessandro E, et al. Nonrandom chromosome changes in multiple sclerosis. Am J Med Genet 1990; 37:406-411.) (Kinne RW, et al. Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases. Arthritis Res 2001; 3:319-330.) In the MS study, the authors found an increased number of cells with chromosomal abnormalities in approximately 50% of MS patients versus 2% of controls. If I remember correctly, they looked at ~1000 cells in blood samples and typically found 1-6 cells with abnormalities. In the MS patients with abnormalities, about 50% of the abnormalities were related to the X chromosome (duplication, fragments, redundant sections). It is only a small percentage of cells (0.1-0.6%) showing the abnormalities. My thought is that cells with abnormalities could have lost some of their epigenetic control over genes in the extra chromatin. It might be that there are clusters of these cells that have replicated at one site inheriting the abnormality from one cell that originally suffered the damage. If this cluster of cells all have the same loss of control, the local site could have disruption of the specific functions of that cell type, such as chondrocytes that are supposed to make collagen for connective tissue or oligodendrocytes that are supposed to make myelin.
So if there is fragmentation of the X chromosome, the fragment may no longer be contiguous with the XIC on the long arm. In that cell or perhaps later in daughter or granddaughter cells there could be a loss of X inactivation since the XIST RNA may not spread to the fragment anymore. Then the X-linked genes on that fragment could become reactivated. There could then be overexpression (loss of dosage compensation) since the fragment and the active X could now be expressing the same genes.
In mitosis, the two centrosomes in the cytoplasm have migrated to opposite poles of the cell and fibers form connecting the centrosome to the centromere of one copy of a chromosome, the other centrosome does the same attaching to the other set of chromosomes. Then one set of chromosomes is pulled towards its centrosome and the other set of chromosomes is pulled towards the other centrosome. After each set of chromosomes has been pulled to opposite sides of the cell, the cell splits into the two daughter cells. So the arms of a chromosome are pulled along with the centromere to which it is attached. But the fragments are not attached. So they could potentially wind up in a different daughter cell than the rest of that Xi. The two daughter cells may inherit different amounts of chromatin. One daughter might get both fragments along with the active X. That daughter may then conceivably have three active copies of X-linked genes whereas it should only have one active copy. The other daughter cell gets short-changed.
There may be variations on the fragments that occur and other chromosomes may be fragmenting also. However, the occurrence of fragmentation in the short arm of the Xi is the best example for this problem.
So how could this affect males? Certainly Klinefelter's syndrome males (XXY) could have a problem with their Barr bodies. But in XY males there could still be a problem if their only X, the active X, fragments. The fragment could go to a different daughter cell so that one daughter cell gets a complete set of chromosomes plus the fragment and the other daughter cell gets short-changed. The first daughter cell then has the potential to overexpress X-linked genes, similar to the female scenario I described. Now, you can start to see the ratio of females to males in autoimmune diseases. Add to that swings that might be attributable to estrogen. (Estrogen can stimulate polyamine synthesis.)
It is difficult to say whether the imbalance of chromatin is immediately lethal to the cell or not. Certainly tissue cultures of cells can grow with varied amounts of chromosomes and can have a range in one tissue dish. For example, XXXXX human female fibroblasts that are sold for research are (if I remember correctly, I've worked with them) ~70% XXXXX and ~25% XXXX, and some 5% other. Of course tissue cultured cells can rapidly develop abnormalities and eventually a culture will fizzle after ~30 passages (ie. replenishing and splitting to new containers). If the cell in the MS patient can survive with its abnormalities, perhaps it is only a problem when the cell is stressed and there is an induction of some of the genes on the extra fragment, particularly if those genes are somehow involved in a stress response. I can suggest some genes that might be of interest.
At Xp22.1 there are two genes involved in polyamine synthesis and recycling. These genes are spermine synthase (SpmS) and spermidine/spermine N1-acetyltransferase (SSAT). Both of these genes are normally inactive on the Xi and active on the Xa. And they are on the short arm (about in the middle) at a distance from the XIC on the long arm. Polyamine synthesis is usually tightly controlled in cells but it is often induced as part of a stress response. So I guess I need to describe the polyamines.
Later.
In the meantime, draw two bow ties vertically one over the other. Color one in dark to represent the Xi, leave the other light for the Xa. Then duplicate these with two more bow ties to the right of the first two. Then draw a dot (for the centrosomes) at the left side of the page and a dot at the right side of the page. Connect the left centrosome with a line (fibers) to the centromere of each of the left most Xi and Xa. Do the same from the right centrosome to the right Xa and Xi centromeres. This is how the chromosomes will be pulled to the daughter cells. Now duplicate the whole picture but cut the short arms of the Xi off from the centromere and leave the fragment light, as if reactivated. Think of the combinations that can emerge when the chromosomes are pulled to the centrosomes. Remember the arms go with the centromeres if they are attached, the fragments aren't attached.
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Postby BioDocFL » Tue Oct 12, 2004 1:51 pm

This is where I starting thinking of tangents. From the scenario I just described, some daughter cells get extra chromatin, other daughter cells get less than the normal amount of chromatin. So which ones are the bigger problem? Seems both could be a problem, the ones with less chromatin may be dying off too soon and the others with extra chromatin are overreacting to stresses. I'll be rambling on about polyamine genes and their potential overexpression, but keep this in mind. It really gets complicated in Wesley world!
Later.
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Postby raven » Tue Oct 12, 2004 1:58 pm

Sorry to butt in

I'm following this with great interest and learning a hell of a lot at the same time. I can understand the explanation and how expression of the X chromosome can explain the male/female ratio within MS. However there is another auto-immune condition, Wegener's granulomatosis which affects males more than females in a roughly 3:2 ratio. It may be a red-herring but I can't see how it would fit into the scenario being described.
Don't let my question detract from the thread, I just wondered if the current scenario caters for this ratio as well.

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Postby BioDocFL » Tue Oct 12, 2004 2:09 pm

Raven,
I'm not familiar with that particular disease, however, there is a disease (don't know if they are related) X-linked chronic granulomatous disease (XCGD) which is attributed to a site at Xp21.2. Males get the disease because they have only one copy (the bad one) of the gene (don't even remember the gene name) but females can use their good copy and inactivate the X with the bad copy. However, sometimes mothers of boys with XCGD have lupus-like symptoms (Cordoba-Guijarro S, et al. Lupus erythematosus-like lesions in a carrier of X-linked chronic granulomatous disease. J Eur Acad Dermatol Venereol 2000; 14:409). And a patient (male) with the disease also had lupus-like lesions when there was duplication of that region (Ortiz-Romero P, et al. Lupus like lesions in a patient with X-linked chronic granulomatous disease and recombinant X chromosome. Dermatology 1997; 195:280-3). My thought is that perhaps the abnormality in XCGD (and there are different genetic aberrations that have been found in the gene) at Xp21.2 could be disrupting the X inactivation process in the mothers since it lies between the XIC and Xp22.1. Perhaps it gives more fragility to the chromosome at that site.
Again, it is just hypothesizing on my part but there seems to be some logic to it.
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Postby raven » Tue Oct 12, 2004 2:23 pm

Thanks Wesley
That does make sense.

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Postby OddDuck » Tue Oct 12, 2004 2:36 pm

Hi, Robin!

Good point! And of course, since I'm usually coming from the "back end" of MS (re: therapy - pharmacology), I'm following along and waiting to see what therapy is suggested in order to correct this possible dysfunctional chromosomal fragmentation. Or should I say I am thinking about how or what might possibly be the treatment(s) as we go along?

Wesley, forgive me, but I CAN see the direct applications of this as it pertains to cancer, and even though I myself see many overlaps between MS and cancer and have many probing questions about it, I still am having trouble correlating your hypotheses with what IS known about MS directly. But I know it's early yet. Please don't think I'm not interested. I TOTALLY am!!

I myself am of the opinion that classifying MS as a purely "autoimmune" disease is misleading. That's an argument (or should I say discussion) that my previous neuro and I had quite a few times. I don't disagree that the immune system may kick into the mix and cause further disruption, but that's not always the case in MS. I, also, am of the opinion that there is a genetic component involved in MS, but I'm not sure what. Of course, who is? LOL

As Robin pointed out (in a way), are you not possibly eventually going to come to the conclusion that since MS as a disease entity in and of itself is so hard to determine (unlike RA and lupus, even), that proving epigenesis as causative in MS would be next to impossible? I mean, I have no idea, and I am very interested in knowing more from you, but as it stands right now, I can't fathom how a connection can be drawn.

I do know that Aventis Pharmaceuticals was working with another computer science company (whose name escapes me at the moment) in developing an in-silico model of CNS disease (MS), but until that happens, what methodology can you employ that can provide any semblance of substantiation? That's what I'm wondering if you aren't coming up against with the "establishment", as I jokingly call the MS clinicians and researchers.

I believe epigenetics is totally valid, but as I believe you have already noticed, the MS "world" is pretty old school at this time. There are many who ARE blazing new trails, but.......it's slow going still. It's hard to teach old dogs new tricks! (Gosh, did I say that? :oops: Well, I speak from personal experience.) Anyway, and as I think you noticed, MSers don't always (nor is there a prevalence, I believe) have more than one classically defined "autoimmune" disease. I could be wrong, but I just haven't seen it very often in my small world. But then again, perhaps that is part of what you wanted to provide evidence of....that the autoimmune diseases do NOT go hand in hand that often.

But, please..........DO go on!!! As you can see, we are VERY interested!! We just might be your toughest critics, though. Don't take it to heart. We're just a curious bunch!

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Postby OddDuck » Tue Oct 12, 2004 2:42 pm

Males get the disease because they have only one copy (the bad one) of the gene (don't even remember the gene name) but females can use their good copy and inactivate the X with the bad copy.


How?

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Postby raven » Tue Oct 12, 2004 3:55 pm

Hi Deb
I think we're still on the genetic predisposition at the moment, casual factors have yet to come. Wesley did allude to this at the very start of this thread.

My thought on the efficacy of antibiotics is that the pathogen is stimulating a heat shock-type/stress response in the cells


My own thoughts are that MS is a loose term to describe a number of heterogeneous conditions that have distinct phenotypes. It would explain why treatments appear to be more effective in some cases than others. It would also explain why in some cases neuron / oligodendrocyte apoptosis appears to precede the immune response, yet not in others.

Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H.

Department of Neurology, Mayo Clinic, Rochester, MN, USA.

Multiple sclerosis (MS) is a disease with profound heterogeneity in clinical course, neuroradiological appearance of the lesions, involvement of susceptibility gene loci, and response to therapy. These features are supported by experimental evidence, which demonstrates that fundamentally different processes, such as autoimmunity or virus infection, may induce MS-like inflammatory demyelinating plaques and suggest that MS may be a disease with heterogeneous pathogenetic mechanisms. From a large pathology sample of MS, collected in three international centers, we selected 51 biopsies and 32 autopsies that contained actively demyelinating lesions defined by stringent criteria. The pathology of the lesions was analyzed using a broad spectrum of immunological and neurobiological markers. Four fundamentally different patterns of demyelination were found, defined on the basis of myelin protein loss, the geography and extension of plaques, the patterns of oligodendrocyte destruction, and the immunopathological evidence of complement activation. Two patterns (I and II) showed close similarities to T-cell-mediated or T-cell plus antibody-mediated autoimmune encephalomyelitis, respectively. The other patterns (III and IV) were highly suggestive of a primary oligodendrocyte dystrophy, reminiscent of virus- or toxin-induced demyelination rather than autoimmunity. At a given time point of the disease--as reflected in autopsy cases--the patterns of demyelination were heterogeneous between patients, but were homogenous within multiple active lesions from the same patient. This pathogenetic heterogeneity of plaques from different MS patients may have fundamental implications for the diagnosis and therapy of this disease.


If we could sub-classify MS more accurately then perhaps it would be easier to target specific treatment regimens. I'm not a big fan of the one size fits all theory.

But I think we can all agree that genetics has a large part to play in the picture. So lead on Wesley.

Robin

Whoops quick edit...
Deb, I've just read your response to the Loader thread. We're obviously on the same wavelength regarding one size fits all....
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Postby BioDocFL » Tue Oct 12, 2004 4:44 pm

I certainly agree that MS and the other diseases can each be a heterogeneous group of sub? diseases. There can be different mechanisms leading to similar consequences, such as the heavy metals short-circuiting mitochondria or a virus invading a cell and causing it to go into apoptosis. The end result can be loss of a key oligodendrocyte or cluster of them, with the problems that that can cause. I am just putting polyamines out there because I haven't heard enough consideration of them as having a role but there are alot of previously unrelated research reports that I think I can tie together. So polyamine and X inactivation abnormalities may only be a minor subset of the bigger diseases.
Even though I have tried to understand polyamines, and have worked in labs on basic chromatin structure, and on X inactivation, in each of these there is still alot to be learned and still some very contradictory reports. I am trying to see the plot and whodunit perhaps even before all the characters have been introduced and developed. Unfortunately I think some researchers won't consider an idea unless it is in a textbook, which means it is at least 5-10 years old. I guess that's the safe route to take. There's always the gnawing feeling in research on new ideas that you may have taken the wrong path on one of the contradictions before it could be resolved. That's why I keep saying that this is only hypothesis on my part. Hypotheses always need further refinement (read: correcting).

OddDuck,
As far as therapies, of course that would be the big interest to MSers. I think the area of polyamine inhibitors is receiving more interest, but primarily for cancer research. I chose to get into cancer research because I felt it was where God was leading me and because I felt there was not enough interest in new ideas for autoimmune research. Also, the projects are further along towards polyamine research. But again there are restrictions. My impression is that we are to only be focused on cancer (ie. seek funding for cancer projects and angles) since it is the biggie. But I see the parallels and believe that there will be synergy and hopefully new drugs that can be used in both cancer and MS et al. My background is very unique so I think I am very fortunate to be in the situation where I am and intend to make it count, both for cancer and autoimmune diseases.
I've got to figure out where I left off. Still have alot of interesting things to say relating this to particularly lupus. I have some other work I have to do now though.
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Postby BioDocFL » Tue Oct 12, 2004 4:53 pm

OddDuck,
Just remembered your question about XCGD and how could the female cell inactivate the X with the bad copy of the gene. I don't know that it is more than a 50/50 shot of inactivating it. There have not been any discoveries yet that I am aware of how a cell could determine the better of two chromosomes but there may be a mechanism. Also, depending on the gene involved, the overall level of the gene product in a tissue may be all that matters, compensating for cells that have a bad copy. As long as other genes that must be tightly controlled do not lose their restraints, maybe some cells that have the bad copy active don't matter.
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Postby OddDuck » Wed Oct 13, 2004 2:44 am

Wesley: Ok....I'm with ya. Sorry if I got a little off track with my questions.

Robin: Yes, I think you and I are on the same wavelength. I refer to Claudia Lucchinetti's work quite often.

Lead on, Wesley!!

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Postby BioDocFL » Wed Oct 13, 2004 4:22 am

I might not be able to write much today because I have alot of work to do at the office on my projects. I have alot left to discuss though on this hypothesis, like the double-strand breaks in fragile sites, the activation of previously sequestered genes (like the reverse transcriptase HERV-W and the possible ones in LINE1 sequences), and how lupus autoantigen groups might be related.
Explaining the hypothesis and getting your questions though has been really useful. I am thinking now that fragmentation of either X (Xa or Xi) could lead to an uneven distribution of the chromatin. Probably the Xi might be damaged and unrepaired more often but both could be targets. The daughter cell that gets too little chromatin may be missing important genes, such as SpmS, so that the cell dies giving to apoptotic activity in the disease with the influx of calcium (possibly because there is not enough spermine being created to control the channels?) The other daughter cell with too much chromatin may overexpress some genes, such as SpmS and thereby reduce SAM for DNA methylation and stimulate histone acetylation so that there is an opening of previously sequestered genes, such as HERV-W. This could generate some of the autoantigens. Still have to consider fragmentation in other chromosomes besides the X since they could have fragile sites and important genes. This could give more heterogeneity to MS. Still, the inactive X is the best scenario for describing the hypothesis.
Anyway, like I have been saying, hypotheses always need refinement.
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Postby OddDuck » Wed Oct 13, 2004 6:39 am

Wesley,

Not to get too far off topic, but something occurred to me this morning. If you are REALLY serious about trying to get someone in MS research to take a serious look at your theory, Samuel F. Hunter, M.D., PhD. (owner of Advanced Neurosciences Institute here in Nashville) will be in Tampa on October 18 (next week) giving a presentation at Maggino's in Tampa. (I used to live in Clearwater, by the way.) He'll then be in Lakeland on the 19th.

As a reminder, Sam is basically a researcher and has been in MS research all his life. He also treats MS patients, though. He did his residency with Dr. Moses Rodriquez and worked with Dr. Claudia Lucchinetti at the Mayo. He then worked as an Associate Professor at Vanderbilt with Drs. Sriram and Moses. He has partnered in research and published a paper with Dr. David Hafler (whom I have mentioned before.) Sam now is on his own and is developing high profile MS clinical studies and research.

Two things about Sam, though. His motto is "keep it simple". The second thing is IF you can show a DIRECT link between your hypothesis and MHCII, Sam will listen. But you gotta get there "quick". Sam's mind runs fast, and he's always thinking of several things at one time. He's a bit high strung, but he's a really nice guy, and as I say he WILL listen, but it takes an "in person" meeting most times to get his attention. (Tell him I sent you. :wink: )

Besides, it might be interesting to the rest of us, too, if you could go (it's not open to the general public, but someone with your background, etc. would be welcome, I'm sure), and report back to us on anything interesting.

Just a thought.

Deb

P.S. We look forward to your next installment, Wesley! :P
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