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XMRV and Culturing, HERV's and more

Discussion in 'XMRV Research and Replication Studies' started by kurt, Mar 23, 2010.

  1. kurt

    kurt Senior Member

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    (This thread picked up from the "Dr. Vernon thread on the General News Forum": http://www.forums.aboutmecfs.org/sh...trists-from-the-Wesselly-school-as-Co-Authors)

    So you have no beliefs here? No speculation? I would say you have shown plenty of belief in your posts. I only say I believe something when I do not have the references handy to quote, but most of what I say I can back up with data. Just don't have the time to sort through it all continually, unless I am writing a paper. I could write out all the facts I am working from, probably have mentioned most of them already. But regarding current issues, the most important fact is that I know many people with CFS who are testing negative for XMRV, and they DO have CFS but do not have XMRV, including by WPI, and other outside labs that hopefully will eventually publish their studies.

    Yes, a systems model can really help in a complex situation like this. Systems biology is the study of interactions between parts of a biological pathology. Think of the 'sum of the parts being greater than the whole' but apply that to a disease model. I believe CFS is a perfect match-up with the systems biology paradigm. I believe there is a wiki entry on systems biology.

    [Gerwyn, an aside, you continue to use put-downs, and a condescending attitude in your posts. I can ignore this, and acknowledge that sometimes I may miss some things and over-react myself provoking that (blame the CFS), but I try to avoid direct put-downs and implied insults in disagreement. A disagreement is not an insult. I really think that given your background and working on a doctorate you should be above personal attacks. Can we stick to the discussion without all the insulting innuendo?]

    For the record, I think you are the one who is misreading a study here, namely the WPI study. I realize some parts of that are ambiguous, and some statements after publication about ongoing work with XMRV may be wrongly attributed to the original study. Anyway, what I am saying about HERVs and the WPI methodology is not just my own view, I am also getting this from other researchers back-channel. As I am sure you know, researchers get into factions over issues like this, and if you only talk to one side you only get half the story. So here are some of the points I suspect you may be either missing or ignoring:

    1. WPI did not run any culture on samples prior to their PCR testing in the Science study, and they got positives from a very small blood amount. This has been confirmed by private emails from Mikovitz to outside researchers (I have not read those emails but my source is reliable). Also the order of presentation in the Science article implies they did not culture cells prior to PCR. The paper implies that the DNA was extracted directly from PBMC's for PCR. I realize the wording of the original study is a little difficult to interpret, I had to have some parts explained to me so I can not claim to understand this perfectly, but I believe what I have been told by outside experts. Am I talking with the wrong experts as you suggest? I have no way to know. But some statements WPI has made afterwards about culturing apparently apply to their ongoing work and not the Science article, although you and others on the forum seem to be interpreting those statements as meaning they pre-cultured prior to PCR and so must other labs. I have heard only the opposite from outside researchers. Other labs should not have to pre-culture PCR samples. WPI only cultured for antigen and infectivity tests and microscope images. IF WPI did pre-culture before PCR, then that is a major omission in their Science article that invalidates that article, and it should be amended or retracted. But of course that is not the case because as Mikovitz has apparently told people, they did NOT culture before PCR.

    2. You raise a good point about the peer reviewers and the expert virologists who have made positive comments about the Science study. However, consider also that neither the peer reviewers nor the expert virologists, nor Judy Mikovitz claim to be experts in CFS. The peer reviewers and expert virologists might not have even known about the connection between CFS and HERVs. In fact the peer reviewers apparently wanted the paper to not even mention CFS. But we know that CFS has a host of very strange medical presentations in the brain, viral immune and detox systems, and that HERVs may be activated in CFS. So is it not possible that their assessment would apply more to ordinary retroviral patients and maybe they have not thought through all the special issues related to CFS in making those comments since they are unfamiliar with our disease? Also, if you are going to refer to experts, be sure to look at both sides. Not all experts are happy with the current XMRV testing and results. Dr Kerr for example has apparently believed at least his study and will not take further funding for XMRV research.

    3. My opinion of the European studies is tempered by the fact that WPI did not pre-culture their PCR tests, and if there were ANY real CFS patients in the three cohorts used, then some of those studies should have had some positives. The fact that one UK study did include an antibody test and had some hits (more on the controls) suggests that the MuLV antibody study worked, as it hit something, such as an activated HERV perhaps, or some other antigen it cross-reacts with. If any significant number of those patients had XMRV, there would have been many, many more hits from the MuLV antibody. So why are these studies all negative? Either the cohorts were completely non-specific for CFS, in which case there should have been 2-4% positives, or the tests were incompetent, but they did get hits on control positives so even if they were diluted in strength there should have been some positives, or there was simply no XMRV in the patient population. Remember, WPI got hits on PCR without culturing. So that is why I am not making a big deal of the European studies except what I have already said, the UK studies had low sample volumes and one did not fraction out the Lymphocites further lowering the resolution, and the Dutch study may have had a very weak cohort (we can not know for certain without sample-by-sample background checks since in any study like this the samples may always exceed the diagnostic criteria). But some of those tests were RT-PCR and very sensitive and should have gotten some hits, even without culturing, given the WPI claims.

    4. The situation in the UK is awful and I realize that XMRV, if it is true for CFS, could make some change happen there. That is great, but I don't think that issue has any place in the discussion of the science of XMRV. The lack of careful analysis of the WPI claims suggests to me that a bias is at work here on the forum. I want answers as much as anyone, but only correct answers. A false finding that is over-promoted could do tremendous harm to our cause, so I think it is best to wait for more studies before turning XMRV into an advocacy campaign, trying to force CAA to change their cautious stance towards XMRV, and raising patient hopes and expectations. Honestly I wish everyone would just wait, put this on hold, at least until after the CDC study and a few others are released (hopefully by mid summer). Anyway, CFS certainly DOES have a biological root cause, regardless of the outcome of XMRV, this is not the only angle for a solution to the political ME problem in the UK, I believe there is already more than enough evidence that CFS is biological, maybe the CDC and CAA should send a letter of protest to the NHS, and UK ME patients should find some way to have a voice. I would certainly sign on to any petition as a 'friend of the UK ME community' to advocate for change in the NHS position on ME, as I believe most here would, you really are among friends... You have to tackle political problems with better politics.

    5. You say I speculate, and sometimes I do, just as you and many others here. So here is my connecting the dots, I think WPI may have inadvertently demonstrated the huge involvement of HERVs in CFS. After all, their 98% positive rate (or whatever it is) is using an antibody type (MuLV) that can sometimes be used to test the presence of HERV activation! That would be huge, and in a political sense would be better for us to have a HERV I think than an exogenous and communicable retrovirus (but that is a separate political issue, of course).

    6. You have mentioned repeatedly that without culture the virus can not be found. I question that, particularly given one study that I quoted a few weeks ago that showed dozens of copies of XMRV in every infected cell (sorry have lost that ref, it is back on one of my prior posts). The virus should be found by PCR if it is present in the blood as WPI claims.

    Yes, that is an obvious problem with the 'quick and dirty' European studies (anyway that is what some researchers here call that type of study). It shows the low value of ME/CFS to those establishments. Regardless of my other views on the facts, this point is more clear now. To really check the XMRV results the outside labs need to take ME/CFS seriously. That means plan a clean study, carefully build a validation or replication assay and test it thoroughly before even collecting data, collect fresh blood samples, write a balanced report, draw only supportable conclusions, go through hostile peer review, and then give us something useful to talk about. (and yes, I do value hostile peer review, that is the best kind, a good scientist is as happy to be proven wrong as proven right, as both build knowledge, and anyway the important point is to get to the proof, not to be right).

    Sorry, I am missing your point here.

    HERVs are not completely understood yet, I know there is an issue with their being more active when methylation is failing, as LTR methylation is required to keep them dormant. Also, that HHV is known to activate them, but my point was that WPI used an MuLV antibody for the final step in this test, which is known to cross-react with some HERV classes. Therefore, the transfection may simply be movement of HERV activation triggers between the samples.
  2. kurt

    kurt Senior Member

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    Not sure what you mean by that, but what I am sharing is not just my own opinion, I am also summarizing inputs I receive regularly from other labs.

    As to disagreements over whether XMRV is a valid finding, well, nobody knows for certain yet, so we shall see who was better to listen to ...
  3. starryeyes

    starryeyes Senior Member

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    Kurt, unless you are willing to divulge who these other labs are that you are reporting opinions from, we have to take everything you write as your own opinion. Please tell us who these other labs are that you are getting back-channeled info from.
  4. kurt

    kurt Senior Member

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    You may take this as my opinion if you like but I do not have to reveal my sources. Besides, all will be revealed in time. Anyway my point is not to promote any 'leaked' information. You can forget I said that if you like. I only mentioned the back channel information in passing because I am frustrated that so few members here are taking seriously or discussing possible problems with the WPI study. This issue of the credibility of the original study IS a serious issue. Simply stating that some retroviral experts liked the WPI study is not a good defense of the study. Consider that members here are picking apart the UK and Dutch study rather than relying on retroviral experts. So where is the balanced treatment in that?

    As for my part, I am taking what I have heard back-channel and trying to work out if it makes sense. I am genuinely interested in inputs from other knowledgeable posters here, evaluation of these possibilities, and instead I get scornful posts and attempts to discredit me personally (which is pointless, because regardless of my past life, all I have for a credential right now is a CFS brain and an interest in finding the truth).

    If you look seriously at the possible confounds to the WPI study, they are significant for at least part of the study (the antibody, culture and antigen findings all could be due to use of HERV cross-reactive MuLV species). I have not found a good alternate explanation for the PCR testing except that controls may have been handled differently. But that is a long shot, more likely there would be some type of systematic false positive. The sequencing study was conducted at a lab studying that exact same XMRV sequence so contamination there is a possible alternate explanation.

    So my goal is simply to raise these issues. I am not the expert to determine if they have merit. The attitudes on this forum are well known now in the CFS community. Probably most of the outside researchers are reading these threads, to gauge how people react. I believe they are more likely to tell what they know sooner if people are open to accept the information, and not taking a hostile position, not shooting the messenger.
  5. kurt

    kurt Senior Member

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    Gerwyn,
    I/O psych - see http://en.wikipedia.org/wiki/Industrial_and_organizational_psychology

    Systems science - see http://en.wikipedia.org/wiki/Systems_science

    My post-doc at the mil research lab was as a research psychologist. I assume you know research teams often pull in allied professionals, I was in an experimental psych lab that was working on advanced training problems. So that was my post, not my credential, sorry if that was not clear. I am a systems scientist in the technical sense, but work cross-discipline so wear many hats. My job was as a research psychologist. I was well qualified for my work, ran several large-scale studies, published, etc. I have also taught ed. psychology at graduate level so they must have thought my background qualified...

    Expertise is one of my 'topics', and I have used cognitive task analysis and designed training systems using that method. An example of my work, I meet with experts, could be doctors, lawyers, engineers, business experts, educators, whatever. Then work through various protocols to elicit expert performance models from them. What I do with the models varies by project, in the medical case I helped analyze how Cardiologists read EKG for purposes of training GPs to do a better job at that. So I worked with the subject-matter experts. Sometimes I help clients produce new training technologies, sometimes just produce new performance models or guidelines, or help solve professional performance problems. You might say I am a type of facilitator, certainly not the expert, but I have to learn everything in the expert process I am analyzing.

    So given that pre-CFS background I have a kind of mindset to analyze things, including what is happening now with XMRV but also the larger picture of research into CFS.

    And I do respect what you say about lab experience, there is no substitute. In my case there is no substitute for knowing the back-room part of the research cycle. I known how research studies can be subconsciously biased, sometimes for political or business reasons. So I know how important a full group of studies is before reaching global conclusions, particularly about something like a retrovirus causing CFS.
  6. Koan

    Koan Be the change.

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    Hey Kurt,

    I'm with Teejkay on this. I really don't like rumours. The analysis that's being done on the forum is relying on available facts and the intrusion of rumour helps no one. So, if it means anything to you, I would appreciate no rumours and only substantiated information. I am assuming that you are still in touch with your friends at Co-op Diagnostics and that the information is coming from them. If so, that information would be quite revealing and would help us to put the rumours in context.

    Also, I don't think that legitimate scientists working in the field of retrovirology would be cowed by people discussing these issues on an ME/CFS forum no matter how passionately or intelligently they are discussed. I think they would not join the discussion for a host of other reasons, though: they don't have as much time on their hands as we do, for instance, or they are working on studies which would be harmed by any revelations they make before time... and so on. I just don't think we are all that intimidating, frankly.

    I would ask you to consider if your sources would appreciate your leaking information they are not able or willing to openly reveal. I really don't think they are reluctant to share because our "attitudes" precede us.

    Peace out,
    Koan
  7. Esther12

    Esther12 Senior Member

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    I think we're all a bit desperate to have something solid for CFS research to get going on, and XMRV looked like it was going to be it - I don't think we can expect a forum like this to be truly balanced on this subject.

    It's great to have Kurt providing a different viewpoint here - especially because it may not be what we want to hear.

    Until there's independent replication of the WPI's work, we should see their Science paper as just pointing future research in an interesting direction. Hopefully it will result in something significant, but I think our hunger for some solid science is leading a lot of us to believe This Is It! The key hase been found!

    The lack of positive news from alternative sources has calmed my initial excitement, but I'm still hopeful the WPI's work will lead on to something. Emotionally, it's going to be a pretty heavy blow if it doesn't - but we should still try to stay ready for that possibility.
  8. V99

    V99 *****

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    Kurt

    Just want to challenge the idea that there is a "bias is at work here on the forum".

    I realise that you suggest there may be a bias, but I haven't seen that. I have seen varying opinions, some questioning the Science study, some supporting it, and the majority waiting for the truth either way. Patients only want the truth, so that appropriate treatments can be sort.

    I do however agree, that the UK's position "why bother with biomedical research", is a separate issue, and regardless of XMRV, now or in the future, it should be corrected. Yet XMRV may start a real change. If it is only found in a small percentage of US patients, they are still patients that were once told it was all in their mind, and were denied what I consider to be their basic medical entitlement.
  9. Cort

    Cort Phoenix Rising Founder

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    I don't know how there could but be a bit of bias here. Just about everyone is yearning for XMRV to work out (myself included). People are using everything they can - basically throwing the kitchen sink at any paper that discounts it. The CAA is raked over the coals for their cautious stance. Yes, there are good and reasoned arguments pro and con but there is clearly bias for XMRV to work out. How could there not be? CFS patients have been starving for news like this for 20 years.

    The important thing for me is that Kurt has delivered an absolute wealth of information. Below is an astonishing bit of news. If its true that the WPI did not culture their sample first then its a real problem. I imagine the the Science makeup article will concentrate on this. How could they not?

    See the section below from the Science papers supporting materials. I don't see any culturing there - none! I'm surprised because I know there is stuff on culturing elsewhere in the study but I'm kicking myself for not looking here.

    You realize that if this is true then Dr. Mikovits has changed the rules in midstream - apparently because she could no longer find the virus using her initial methods. It also means we can stop yelling at all those researchers who didn't culture XMRV first since if they had cultured then they would have been not been following the WPI's protocol. By not culturing they were actually following the WPI's methods - a strange turn of events.

    I don't think this is the end of XMRV. If Dr. Mikovits now has to culture for it to find it then she's still finding it - its still there. But if the process of culturing does increase the possibility of endogenous retroviruses being found then she may be finding an endogenous retrovirus.

    That would seem disappointing but think of this - she's still finding something in CFS patients and NOT in healthy controls. She's still finding something and that something could very well turn into something important.

    I fail to see why learning the name of the lab would help. Are you going to check their credentials? Give them a call and try to confirm Kurt's email? I doubt it. Kurt has given us alot of information; it seems very credible; he's laying out a possibility - we'll see how it checks out over time.

    Here's from the Supporting Materials of the paper:


    .
  10. Cort

    Cort Phoenix Rising Founder

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    Culturing XMRV

    Here is where the culturing took place. They took mononuclear leukocytes (monocytes and lymphocytes) from both the patients and the healthy controls. They culture for 72 hours, subcultured every 3-5 days apparently to refresh them. Then they isolated them into their separate types; T helper cells, Cytotoxic T cells, NK cells, etc.

    Then what happened?

    They also cultured B-cells

    And they cultured the LnCap cells they grew the virus in (This is how I got so confused...there seemed to be culturing going on everywhere....actually ironically except the PCR).

    Its truly amazing how complex the processes are. All the culturing done appears to have been done for experiments after the PCR.

    I'm interested in getting Gerwyns take on this.
  11. Koan

    Koan Be the change.

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    Nice posts, Cort! I'm intrigued!

    (Koan rubs hands together!)
  12. rebecca1995

    rebecca1995 Apple, anyone?

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    Kurt wrote:

    Dr. Mikovits reported in the ProHealth talk 1/22 that Ila Singh has developed an XMRV antibody. See transcript.

    Kurt wrote:

    Retract the Science paper! That's quite a strong statement, Kurt. Why do you suppose that John Coffin didn't point out this possible problem in his companion piece in Science? And, on the subject of retractions, do you feel that there are any grounds for Dr. Vernon to retract or amend her response to the Dutch study, in light of the information about its cohort uncovered by Parvofighter?

    Cort wrote:

    Cort, is it your opinion that the electron micrograph, printed in the Science article, that depicts a virus budding off a CFS patient cell and infecting a cell line, is in fact an endogenous retrovirus? And do you feel that Silverman, the discoverer of XMRV, and Ruscetti, the co-discoverer of HTLV, have erroneously confirmed it as XMRV?

    Furthermore, do you feel that Dr. Mikovits deserves to be referred to by the same phrase you used to describe Dr. Vernon in another thread today--"a serious and respectable researcher"?

    If so, I find your below statement disrepectful and more than a little presumptious.

  13. Koan

    Koan Be the change.

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    Fantastic rebuttal, Rebecca!

    I, too, wonder what people make of Coffin, Silverman, Ruscetti, et al, if they think they can (collectively, no less!) be bamboozled by Dr Mikovits. Seems highly unlikely to me! Also, Dr Mikovits simply doesn't strike me as anything other than a very straight-up woman.
  14. Esther12

    Esther12 Senior Member

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    I really don't think anyone's accusing Mikovits of deliberatly misleading people. Not that I've seen here anyway.

    re Coffin not being concerned about them not mentioning culturing prior to PCR testing: maybe he didn't know they had done so? It seems unclear whether they did, or whether they've only started doing so since then, or what.

    re the electron micrograph: from my understanding, this is not a great way of identifying XMRV. It's another bit of evidence, but not one which adds a whole lot of weight to the XMRV/CFS argument.

    (I'm quite aware I'm a bit out of my depth here, so could well be wrong about all of the above - but I thought I'd post my understandings anyway).
  15. Esther12

    Esther12 Senior Member

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    Which is true - but I don't think it allows us to be sure the virus pictured is XMRV. I don't think electron micrography is used as a diagnostic test for anything, because it's too easily misinterpreted. I seem to remember virologists saying this at the time, when they were generally being very positive about the paper.
  16. kurt

    kurt Senior Member

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    This information goes beyond that source, just so you know, although yes, I am still in contact with them as well. They have stopped testing for XMRV due to the testing controversy (as they say anyway) and moved on, they are no longer running new XMRV tests for CFS and are focused now on getting low-cost AIDS testing to Africa (and succeeding on a large scale fortunately).

    The last person to see a bias is the one with the bias. I think your last few sentences are the basis for the bias I see here. Yes, the lack of a biomedical research focus in the UK is a huge problem for ALL of us, particularly given the strong research capability and the innovation that often comes from the UK. Imagine if more of those resources would turn to solving CFS, particularly given the philosophy of medical care for everyone there. I would be first in line to sign petitions or whatever to help get your situation there changed, as I think everyone would benefit. And we already have the data required, the CDC has already started that shift. But can you honestly say that you think the UK posters here supporting XMRV are not being driven by the idea that a retrovirus might force the NHS to change their stance on CFS? That is bias in that it can interfere with objective analysis of both sides of a scientific debate like XMRV. If I am wrong and there are UK posters here tearing apart the WPI study, then I apologize, please point me to those posts...

    Thanks Rebecca, I did not know Singh had reported an XMRV specific antibody, will look into that. Yes of course, retract or ammend would be required if a major error like failure to report pre-culturing of part of a test occurred. That is not a strong statement, just business as usual, happens all the time in scientific research. As for Dr Vernon, she had very little time to study out that issue and people here have taken apart the Dutch study and tracked down references, etc., over a few week's time. I can not speak for her, but if I were in her shoes, probably I would amend my response to the Dutch study, knowing what I know now (thanks to Gerwyn's research and some others).

    Regarding your comment to cort, the EM study shows a particle the size of a gammaretrovirus. Well, some HERV types are classified as MuLV type gammaretroviruses, and are in that same size range. And budding particles have been observed by EM. This is the type of important fact that needs to be tracked down.

    As for Silverman, etc., I don't know which statements you refer to, but the WPI study was very complicated, and I believe Silverman's tests were involved so that is not really an outside endorsement. But anyway, as I said earlier, relying on endorsements for WPI but then having members conduct their own investigations of the follow-on studies is a differential standard, reflecting bias.

    All I am asking is that people here evaluate the WPI study with the same level of detail as all the others. I have added the information I can. IF you want to rely on the endorsements made, be sure to look also at those experts who are not endorsing or have expressed concerns. This is still preliminary research, and as stated in the article, is only showing a correlation not a cause, and whether or not XMRV is just a passenger is still to be determined. I would add that whether it was really XMRV in all of the tests is also yet to be proven.
  17. kurt

    kurt Senior Member

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    I receive inputs from other labs (and also individuals). That is how I learn how retrovirologists outside the WPI circle interpret the XMRV tests. This has been educational and I am trying to pass along what I am learning. This is not rumor, but information sharing about issues to look at regarding risk factors in testing for XMRV.

    None of the information I have mentioned about: HERV cross-reaction with MuLV; problems with many prior research efforts to find retroviral explanation for diseases; issues regarding whether or not culture was performed before PCR; or issues of systems biology and models for CFS are rumors. Those are all factual discussions based on what I have learned from outside experts via back-channel. If you want more information, just look up some of the references I have given. There is no 'context' for this, other than the fact that I am talking to researcher scientists with experience in the research and testing world who are looking at the WHOLE body of evidence and not just hunting down the information that supports their personal viewpoint.

    As for the XMRV studies themselves, they are out there, many more than I know about personally, and in time I expect we will hear report after report.
  18. Cort

    Cort Phoenix Rising Founder

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    This is really interesting stuff. I want to point out that finding a biomarker for this illness is one of the holy grails of research. Maybe it is XMRV, maybe its not but the fact that the WPI was finding something and still is finding something that's present in CFS patients but not controls is extremely interesting just on the face of it.

    Honestly I think the great likelihood is that the WPI didn't culture the cells beforehand. I would be 'shocked, just shocked!' :Retro smile: if they somehow forgot it and then it was not caught by their own reviewers.

    What an amazing turn of events this would be: WPI thinks they have XMRV - they don't; maybe the original samples were somehow contaminated. When they culture the cells to check on their finding they find that CFS patients have something that looks just like XMRV but its actually something else. What are the odds of that happening?

    Throw this in there as well. US researchers find that XMRV is associated with an immune defect (RNaseL) similar to the one that CFS patients appear to have. Noting this Dr. Mikovits examines CFS patients samples in her repository for XMRV and finds it. Shortly after that research suggests that XMRV infection is NOT associated with RNase L.

    If Mikovits waits another year perhaps she never tests those samples and appears to find XMRV but actually doesn't but instead finds something else very close. How strange that would be.

    After Kurt's analysis that upcoming Science article just got that much more interesting.
  19. kurt

    kurt Senior Member

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    Yes, this could be good serendipity for us if they really found HERV activation with the antibody, culture, antigen and EM studies. They ARE finding something and finding HERV fits well with their virachip study and the new MS research showing HERV activation. I guess we will have to wait to find out more answers, this should be an angle that will be explored by researchers eventually.
  20. cfs since 1998

    cfs since 1998 *****

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    You keep saying this over and over again and each time it does not get more convincing. This hypothesis is illogical and far-fetched. How far are you going to go, was the virus found in prostate cancer an HERV too? Is XMRV a figment of our imaginations? Geez.

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