December Blood Prodcuts Advisory Committee Full Transcript

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Thanks for picking out the juicy bits of the document Mark.

I liked this quote from John Coffin - an alternative explanation for the narrow sequence variability found by Hue et al, which seems reasonably consistent with known ME/CFS epidemiology, and a typically fair-minded look at alternative possibilities that might explain a given set of observations...
Or even the contaminated vaccine hypothesis, but I doubt you'd get him to agree with that in the near future.
 

Cort

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DR. COFFIN: I was thinking particularly about the LTR, which might well have been exchanged.
DR. RUSCETTI: We are just beginning to work to look at the LTR. We have been pushed into it by Jonathan, who asked us at every meeting to look at it. So we are now looking at it. But we don't have any results for it.
DR. MIKOVITS: And it could well be a key to the reservoir. The LTR is really a key, maybe, why we can't grow this virus in these cells. We are looking at other cell types right now.
DR. COFFIN: That harks back, in a sense, to the other talks. To my knowledge, nobody has ever grown a virus that has a polytropic or a modified polytropic-type LTR in it, unless somebody in the room has done that recently and I haven't heard about it.
DR. MIKOVITS: Usually these are seen with xenotropic, actually mobilizing the polytropic. The polytropic indeed becomes the pathogenesis. But they need the xenotropic to be --
DR. COFFIN: In mice that seems to be the case.
Does anyone know what the LTR is?
 

Cort

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Of the sequences that have been reported, their similarity to one another and to MLVs is most consistent with very short transmission chains from human to human. Assuming the virus is, in fact, getting into people, it's not impossible to consider that every little localized outbreak starts with a single mouse somewhere, because this virus clearly has very recently come out of the -- all of the sequences must have fairly recently come out of the mouse germline and might be analogous to the hantavirus outbreak, for example, where conditions, for some reason, allow this virus to replicate in some wild mouse and then spread around to humans. The same kinds of things could happen occasionally. One could have these little foci of infection of a virus that worldwide could be exactly identical from one to another, because the continuity has been carried in the mouse germline.

One has to be very careful not to -- although, as you know, I still remain quite skeptical about a lot of the issues, one has to be very careful not to think of this virus in terms of a virus like HIV. You have to sort of put what you think you know about HIV to one side, as far as things like genetic variation, epidemiology, and so on. This could be a completely different situation. We have to keep that in mind.
Fascinating stuff....a very new entrant into humans...(love how ME/CFS is always on the cutting edge...I remember a doctor decades ago telling me..well, you're really on the cutting edge which I thought was mildly uplifting.....at the time.....:rolleyes:)

Rein (?) said the same thing about XMRV and HIV - you cannot look at XMRV through HIV colored glasses - its a very different virus...
 

Cort

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Lo on contamination

Can this be a contamination by murine leukemia viruses commonly used in the laboratory? We also thought this was not quite likely because the sequences are quite distant from all those ecotropic viruses commonly studied in the laboratory. It's very different from the viral vectors normally studied in the laboratory.
The sequences they picked up are not similar to the viruses they look at in the lab; they are not a result of internal contamination...

Mouse DNA was another story...

The third question is the more difficult one: Can this be a contamination by mouse DNA? The mouse DNA genome contains endogenously many copies of the related retrovirus sequences. We thought this would be necessary for us to develop an assay to verify that there is no contamination of mouse DNA in the assay system we use and also in the clinical samples that test positive for MLV-like virus gene sequences. We developed a highly sensitive nucleic acid PCR assay targeting mouse-specific DNA. In this case, it's a mitochondria DNA, because we need a target gene sequence that is very well conserved among all the different species of mouse and also has multiple copies in each of the mouse cells. That's why we selected mitochondria DNA, because each cell carries 200 to close to 2,000 copies of these genes.
They appear to have developed their own assay..to test for mouse DNA..in the assay system itself and the samples.....Some researchers have said theres not enough mtDNA there to test - but he seems fine with it.....

Perhaps because the test is so darn sensitive - 1,000 times (!) more sensitive than the PCR assay for the gag XMRV sequences...

Importantly they show significant differences in the XMLV's they found in the old samples and the new ones...as would be expected over time - the sequences changed.

The gag gene sequence showed a quite noticeable variation in the blood obtained from most of these patients 15 years later. The details of that comparative analysis will be reported separately.
This is how crazy the science is....Dr. Coffin notes that he actually suggested to Dr. Lo that they use the mtDNA test....now he's reconsidering.....in fact he worries 'very much' about it. Its amazing how little is nailed down in this field.

Second, although I think I may have originally suggested mitochondrial DNA to you as a way to test for contamination, I worry very much about it, for a couple of reasons. One is that, of course, if there is contamination of DNA from some source -- you point out that there is a wide variation in the number of mitochondria per cell -- you have no idea what the cells are giving rise to that contamination, much less any idea of how many mitochondria you might have. PBMCs, for example, have relatively small amounts; muscle cells or oocytes have huge numbers of mitochondria. You have no idea.
 

Cort

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Later it was interesting that Dr. Klimas asked about freezing and rethawing the original samples - something Dr. Mikovits suggested in her Swedish talk could disrupt the virus - making it impossible to find. Dr. Lo stated the samples had been frozen or over a decade and never thawed before. Dr. Mikovits noted that only in the WPI's and Lo's study was the sample frozen and then thawed once.

DR. KLIMAS: The samples that were in the freezer all these many years, had they been manipulated -- frozen/thawed or in any way --
DR. LO: No. Regional samples in this study have been kept frozen for all this period of time. They have never been thawed out to use for different studies.
 

CBS

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Does anyone know what the LTR is?
[FONT=Verdana, Arial, Helvetica, sans-serif]http://www.stanford.edu/group/nolan/tutorials/retcl_3_ltrs.html

[/FONT][FONT=Verdana, Arial, Helvetica, sans-serif]3. Long Terminal Repeats: The Retroviral Promoter

[/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif]The long terminal repeat (LTR) is the control center for gene expression.


[/FONT]
 

Mark

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Dr. Mikovits noted that only in the WPI's and Lo's study was the sample frozen and then thawed once.
I'd love to see that confirmed but that's not precisely what that quote said; I don't see justification for the word 'only' in that quote. If it could be confirmed that all the other studies did refreeze, well that would really be something...

Exactly what the Long Terminal Repeat is, is perhaps a bit wider question than CBS' summary suggests...my short description would be 'a long string of repetitive sequences at the beginning and end of the chromosome' but it's mentioned on wiki that HIV does indeed use the LTR for viral insertion.

I've just had a quick look on wiki to find out if "LTRs" and "telomeres" are the same thing, and I can't see any difference between the descriptions so they seem to be...but maybe LTRs are always at the ends and telomeres can flank any encoding sequence?...or the other way round?...anyway they sound like the same thing to me...

Telomeres seem to be like a kind of 'buffer zone' around the real genes, and they seem to shorten with age (with replication)...as the string of nothings gets whittled away, the risk of cancer increases...though over-long telomeres may bring other risks too...and different people have different lengths of telomeres to start out with...or so I've read...just another fascinating subject I'd love to have more time to explore...
 

Deatheye

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I'd love to see that confirmed but that's not precisely what that quote said; I don't see justification for the word 'only' in that quote. If it could be confirmed that all the other studies did refreeze, well that would really be something...

Exactly what the Long Terminal Repeat is, is perhaps a bit wider question than CBS' summary suggests...my short description would be 'a long string of repetitive sequences at the beginning and end of the chromosome' but it's mentioned on wiki that HIV does indeed use the LTR for viral insertion.

I've just had a quick look on wiki to find out if "LTRs" and "telomeres" are the same thing, and I can't see any difference between the descriptions so they seem to be...but maybe LTRs are always at the ends and telomeres can flank any encoding sequence?...or the other way round?...anyway they sound like the same thing to me...

Telomeres seem to be like a kind of 'buffer zone' around the real genes, and they seem to shorten with age (with replication)...as the string of nothings gets whittled away, the risk of cancer increases...though over-long telomeres may bring other risks too...and different people have different lengths of telomeres to start out with...or so I've read...just another fascinating subject I'd love to have more time to explore...
If I remember right they found out years ago how to repair Telomeres in mices. It was sad that this could be the key to eternal live. It seemed that without any telomeres left the cell died or started to mutate (cancer).
The mices lived a lot longer with that. This was long ago when I've read this. So I'm not that sure anymore I'm right with it. And propably there was more science about it later on which I didn't follow.
Seems that it's a topic in cancer research.

I've tried to get the difference of LTR and telomerase.. but I don't really get it. ACcording to the Information I got my above statment would be wrong. It seems that relomeres is that thing that repairs LTR. And LTR is the added Buffer Information. So that would mean that they propably found out how to make more telomeres to repair LTR in mices...
Then again in other explanations it seems that telomeres are the end and start buffer information... I really don't get it...


Explanation what telomerase does:
http://www.youtube.com/watch?v=AJNoTmWsE0s
NHI consequences of telmoere dysfunction:
http://www.youtube.com/watch?v=CqnavdHw-K8&feature=related
 

free at last

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The famous speech became famous, in the only form we had it - which was hastily liveblogged summary notes provided to the community by someone who attended the meeting - because it was HARVEY ALTER, standing up and directly rebutting all the accusations about contamination, and then saying that he's learned a lot about this disease, it looks like a viral disease, and if it isn't XMRV we need to find out what it is.

So not only was an elder statesman of virology forecefully defending his research, but he was going even further and saying the hunt for the cause of ME/CFS must not stop, even if XMRV doesn't pan out. I think those were very powerful words of hope for our community - that the reality of our disease is being validated, that the validation doesn't have to depend on this one particular theory of causation working out, and that we will NOT BE ABANDONED if XMRV doesn't pan out.

May it be true.
Maybe in America but a lot of GPs and decision makers in the uk, will use the demise of xmrv against us, it will signal to them the whole lets treat it seriously argument, is and always was a lost cause, a waste of time. and money. Back to work as usual guys. nothing has changed about this silly ME/CFS lets not waste anymore money time and effort, as we all know it always was a somatizing disorder. Thats my view. Alter in the uk will be ignored. Hell Towers is ignoring him already, and XMRV MLV. has even properly been discredited yet. theres a clue right there of what infact will happen. i think we will be back to the dark ages. and the retro virus failing will be all the evidence they need that its just not, and never was a real disease. can you imagine the press storys if both lines of research ( wpi lo/Alter ) truely fail with a contamination proof. God the mind boggles. what a scary thought that is for us all. Lets hope it doesnt fail. If it does im moving to mars, where they havent already decided im nuts
 

free at last

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The gag gene sequence showed a quite noticeable variation in the blood obtained from most of these patients 15 years later. The details of that comparative analysis will be reported separately.

I wonder if Judy has any such samples either from the states or the uk, that span 15 years
I wonder if kings college has my blood samples still stored from way back ? maybe i should ask them ? wonder if any variation would show ?
 

ukxmrv

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Jonathan Kerr had a big collection of old ME/CFS blood samples but I don't know what has happened with these now. He would, for example, have my blood from the gene expression study and that could be compared to the latest sample given to VIP. That's not very long though.

He would have other old blood samples from before then. We did talk about this briefly at the Invest in ME Conference. Sadly, he has lost his job so couldn't follow this up.

Dr Mikovits commented to the BWG though about the repeated freezing/thawing of samples and this could be a potential problem though. Also for the sample kept by Kings?

XMRV+
 

currer

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Having read the entire BWG transcript- and suffering with brain fog fron the effort - I noticed that concerns were raised in it about the low replication of XMRV. (As has also happened in the Retrovirology papers)
My thoughts were that given the features of CFS/ME low replication would be expected. Some of us are ill for decades but do not necessarily get worse or die, instead we may have remissions. If an infectious agent were multiplying rapidly in the body surely we would rapidly deteriorate or die. But as this does not happen you would expect to look for an agent in CFS which does not replicate much and remains as a latent infection of some sort.
So I do not see the low replication of XMRV as a problem. What do others think?
 

Esther12

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I think that for a virus to cause CFS, it would have to be a funny virus.

Others have mentioned XMRV seeming to lack that ability to overcome some of our bodily defenses, and using this as a reason that XMRV is unlikely to be a human pathogen. Could this provide one explanation for low levesl of variance found? What if XMRV is a rubbish human retro-virus - barely able to do it's job, so it's much less able to sustain any mutations that occur? Could that be possible?

I guess it doesn't really matter at this point. If we get evidence that XMRV is related to CFS I expect the 'how' will come long after.
 

Mark

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Retroviruses are indeed funny, in all the right ways, of course, hence all the interest...

I can only guess that the lack of ability to overcome some of the defences refers to evidence of successful inhibition of XMRV by APOBEC3. However, XMRV has also been seen inhibiting APOBEC3 and also resistant to its mutations through homology - that is to say: APOBEC3 swaps over the Cs As Gs and Ts but the changed sequence is homologous (there is some such redundancy known in the translation) and so the same proteins are produced. Roughly speaking... ;-)

in this sense XMRV seems small but perfectly formed: resistant to mutation in fundamental ways, possibly. Only rubbish in the sense that it's easily mutated, perhaps, lacking the defences to stop the gene switching...but maybe that's also part of its stealthy nature, because what gets produced after the switching happens, is the same proteins...could even be another explanation for Hue's observations here maybe...

Another possibility that's been around for ages is that immune challenges such as APOBEC3 genetic-related deficiencies being the difference between healthy controls with XMRV and ME/CFS etc patients with XMRV...

I think the weird thing is, we have loads and loads about the how already, the how is well under way, with much to be published - already the Spanish findings re: B and T cells are unpicking the role in immune dysfunction and co-infection; the monkey studies are going to show more detail about infection (persistent infection of multiple systems already demonstrated in macaques) and disease progression, role of co-infections etc; understanding of the 'how' seems to be progressing rather well, I'd say, it's proving the whether that seems elusive...
 

Megan

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From Dr. Alter:
An extremely sensitive mouse mitochondrial DNA has always been negative in the Lo laboratory. Lo has done the IPA assay that Dr. Coffin recommended.
As others have observed this is very interesting, though it's hard to know if they tested just a fer samples or all of them. Taken together with the information that was in the contamination papers on the mtDNA test, I think the "contamination paper" data could equally be used used to show the WPI and Lo/Alter studies were not contaminated:

1) Table one in the Oakes paper shows the mtDNA test picked up 90% of contaminated samples and the IAP picked up 100%. We know that both the WPI and Lo/Alter papers were entirely negative on the mtDNA test and at least some Lo/Alter samples were negative on the IAP as well. Hard not to see that this data can easily be used to support the case that Lo/Alter and the WPI cases are not contaminated while the ones in the Oakes paper were.

2) The Robinson paper also states in the abstract that many of the contaminated samples tested positive on the mtDNA test, which none of the WPI or Lo/Alters did.

Amazing how the data was 'spun' in the above papers (your had to go out of your way to find the mtDNA figures in both papers as they just focussed on the IAP).
 

Megan

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[FONT=Verdana, Arial, Helvetica, sans-serif]
[/FONT][FONT=Verdana, Arial, Helvetica, sans-serif]3. Long Terminal Repeats: The Retroviral Promoter

[/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif]The long terminal repeat (LTR) is the control center for gene expression.
[/FONT]
From Judy:
In the assay that we used in the Lombardi study, shown in the top line here, we take plasma or activated -- this is dividing peripheral blood and mononuclear cells -- from the patients, and we co-culture them on the prostate cancer cell line, which was responsive to androgens and inflammatory cytokines. This is important because we know we have characterized the LTR, in Steve Goff's lab and Bob Silverman's lab, and we know that there are hormone-responsive elements there that would be an on switch to make the virus replicate more in the cells that were responsive to androgens.
Not sure how to interpret this but as I understand it the LTR contains the part of XMRV that responds to addrogens and cytokines. It sounds to me from the above quote that they might be using such cytokines and androgens to culture the virus (nor entirely sure here?).

This seems interesting as I am sure I saw somewhere along the line (I think from Sandra Ruscetti) that the hormone resonsive elements were not typical of MLV's but were on XMRV, though I dont know if that applied to the Lo/Alter strains. In a recent lecture MIkovits gave in Sweeden she said that the culture was expressing XMRV preferentially over MLV. I wonder if this is the reason why, though you think she would have said that in the talk?

Then there's the other quote from Mikovits that was posted earlier by Mark:
DR. MIKOVITS: Usually these are seen with xenotropic, actually mobilizing the polytropic. The polytropic indeed becomes the pathogenesis. But they need the xenotropic to be ---
The connection between the xenotropic and polytropic versions seems to be one of the great unanswered questions in all this. Seems like we might have got some hints here.
 

August59

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There was mention about the cell line being used for xmrv will not grow the mlv's and that they were trying to identify a new cell line for mlv's. I think it was in an article Cort did within the last couple of weeks. I'll see if I can dig it up.
 
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I think the "contamination paper" data could equally be used used to show the WPI and Lo/Alter studies were not contaminated:

1) Table one in the Oakes paper shows the mtDNA test picked up 90% of contaminated samples and the IAP picked up 100%. We know that both the WPI and Lo/Alter papers were entirely negative on the mtDNA test and at least some Lo/Alter samples were negative on the IAP as well. Hard not to see that this data can easily be used to support the case that Lo/Alter and the WPI cases are not contaminated while the ones in the Oakes paper were.

2) The Robinson paper also states in the abstract that many of the contaminated samples tested positive on the mtDNA test, which none of the WPI or Lo/Alters did.

Amazing how the data was 'spun' in the above papers (your had to go out of your way to find the mtDNA figures in both papers as they just focussed on the IAP).
Well put, Megan
 

Jemal

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My thoughts were that given the features of CFS/ME low replication would be expected. Some of us are ill for decades but do not necessarily get worse or die, instead we may have remissions. If an infectious agent were multiplying rapidly in the body surely we would rapidly deteriorate or die. But as this does not happen you would expect to look for an agent in CFS which does not replicate much and remains as a latent infection of some sort.
So I do not see the low replication of XMRV as a problem. What do others think?
It depends if the infectious agent is causing problems in the host, I guess. If it doesn't, it could happily spread all over the place. We have many viruses or debris of viruses in our DNA and they aren't all causing problems.

At the moment I still subscribe to the theory that XMRV is an infectious agent, but not necessarily causing problems on its own. The immune system might be causing the problems, because it's trying to eliminate this foreign agent, but it's isn't able to do so. Many of our symptoms can be explained by an immune reaction: the fatigue, the muscle or joint pain, the throat aches, the feeling of malaise, etc. It would be more like an autoimmune disorder this way.

We could also be the "lucky" ones. There's also a theory that people with XMRV, but not CFS, die earlier of cancer for example... (yes even earlier than people with CFS). So XMRV could still be a nasty virus, but our bodies recognize it and because of the immune reaction, we get to live longer. The immune reaction might be causing all sorts of problems, though which results in CFS.