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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. Gerwyn

    Gerwyn Guest

    both amplification(of sample) and activation(ofpmbc) are needed.The concentration of virus needs to be increased and coaxed into replication .Yes amplification of virus concentration is different from PCR amplification cycles.

    If any trial does not include these steps then the chances of finding the bug will be minimal.The WPI method itself would not find XMRV in patients diagnosed according to the Oxford criterea because it is not thought to be associated with people with purely psychological problems
  2. dannybex

    dannybex Senior Member

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    With all due respect, this is not true. The WPI found 3.7 percent of 'healthy' controls to have XMRV...which equals approximately 10 million people without CFS/ME in the US alone.

    ???
  3. Dr. Yes

    Dr. Yes Shame on You

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    Hi Cort,

    I believe what Gerwyn originally meant by amplification was what is referred to in the Science paper as activation; he was originally using the term amplification in a broad sense for any attempt to increase any target (be it whole cells or DNA sequences). For the purposes of these XMRV papers, it may be better to use the term amplification the way they did - to refer only to PCR amplification. All PCR techniques involve amplification of a given DNA sequence; that's the basis of PCR. One thing the WPI did differently was to activate primary cells, i.e. stimulate them to replicate quickly; for this they cultured them with IL-2 (interleukin-2). IL-2 is a cytokine which in our bodies is used as a signal to stimulate the immune response.

    What you mentioned were PCR amplification procedures, but as I noted all PCR techniques use them. The key here is how to increase cell numbers (and therefore viral copy number) to a level that allows for viral detection; this is done by activation of cells taken from patients, i.e getting them to replicate. That is the step missing, as far as I can tell, from the UK and Dutch studies.

    Hope that helps clear things up. It would be impressive if it did; my brain is totally fried.
  4. Gerwyn

    Gerwyn Guest

    the point is that oxford selection criterea can easily select patients who dont have cfs at all but have fatigue caused by psychological factors as per the dutch study for example
  5. ukxmrv

    ukxmrv Senior Member

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    It doesn't really matter if Mikovits et al missed out reporting the step of the culture of the PCR cells using Lncap in the original Science paper. She can answer that question and the record can be put straight.

    It is important for groups planning replications but they could have got the information from her (I've had my own correspondence and am aware of her very helpful attitude toward one researcher). She's been answering my own basic questions and I've been copied into other correspondence so it's not "hearsay" for me.

    It's also important to have as much information in the public domain but this can be covered by questions. Science is going to do this. It appears to be that some people are nit-picking and deliberately making mountains out of molehills.

    The paper doesn't need to be retracted. That's an over reaction. It's just a clarification and everyone wants to see the method explained well.

    In our own UK testing we are seeing many XMRV+ positive people. There is nothing unusual about these patients. Just a group of people with CFS and ME who wanted to be tested and could afford to do so.

    I'm XMRV+ and from a known outbreak of ME, that occurred before CFS was named. I was PCR-, culture +. That doesn't bother me having seen the Japanese abstract and have corresponded with Dr Mikovits.

    Yes, it is important that we determine the XMRV, HERV question. No one, including me wants to go down a blind alley and waste valuable time.

    None of the concerns here invalidate the paper. There are questions to be answered and things may turn out to be different than we think right now.

    Let's see how the Science questions go. I'm not asking any further of Dr M as I know she is busy on the UK study. I have a feeling that some people (not necessarily here) will never be happy with the replies and the nitpicking will continue regardless.
  6. Gerwyn

    Gerwyn Guest

    Yes I mean that the act of activation amplyfied the number of PMBC and thus viral nucleic acid load and ensured that the viruses became replicative producing the cDNA needed if PCR was to work Sorry I can go into shorthand as well
  7. Cort

    Cort Phoenix Rising Founder

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    That was actually very clear Dr. Yes ( as your posts usually are :)) - it did clear some things up for me. I think your brain is doing pretty darn good. But I don't see any evidence of 'activation' as pertains to the PCR section of the paper.

    It could be that I'm missing something. If I'm not then could it be that activation was not required in the early patients (because their viral loads were higher?) but is required in the patients they're seeing now? As I mentioned earlier if this is true then it would provide a good reason why Dr. Vernon has gotten so fixated on that original cohort?

    Because I know everybody loves reading this stuff :rolleyes: - here's the entire methods section leading up to the PCR test

    [FONT=&quot]
    [/FONT] [FONT=&quot] 4 [/FONT]
  8. dannybex

    dannybex Senior Member

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    Very interesting Dr. Yes. My brain is totally fried too, but isn't IL-2 one of the cytokines that has been found to be very low in PWC's? (Or PWME's?) Perhaps XMRV is something that lowers these cytokine levels...I don't know.

    Here's a study that found undetectable IL-2 levels in the spinal fluid of PWC's:

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC540195/

    d.

    p.s. thanks Gerwyn. I knew that was what you meant...makes sense...but one would think if WPI had done the testing, they would find at least some with XMRV.
  9. dannybex

    dannybex Senior Member

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    MY EYES...MY EYES!!!..:eek:

    :Retro smile:
  10. kurt

    kurt Senior Member

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    I have been told the importance of amplification varies with the sensitivity of the test. For a less sensitive PCR more amplification prior to testing may be critical, as Gerwyn says, getting the cells to replicate the virus. However, some of the other studies after WPI used more sensitive real-time PCR tests, I believe one of the UK studies mentioned their test could detect a single virus. Therefore amplification might be less critical, as long as DNA was properly extracted.

    No doubt more of the pending studies will be using more sensitive tests than the WPI version, which was a standard PCR. Certainly the researchers involved all understand the concentration/sensitivity issue, I don't think that is the only problem here.

    Yes, time will tell. Meanwhile the WPI hypothesis is also still unconfirmed information. Just pointing that out, the caution about getting people's hopes up or dashing them goes both ways.

    If you want to know about the HERV K18 superantigen in CFS, you might read some of Dr Bridgette Huber's work. That can be activated apparently by HHV, and I have read that HERVs are also more like to activate when methylation is failing (a methylation stage is involved in keeping HERVs quiet).


    This is what I was trying to say above. The European study authors probably thought their tests were sensitive enough, at the level that allows for viral detection. They had the count levels and sensitivity and sample volume information from Science. Figuring if their test might work was basic math, and they obviously thought the tests should have worked. And they DID work on positive controls. But they got no PCR hits, so AT THAT RESOLUTION nothing was detected.

    Maybe it does not matter to you but I think that would matter a lot to the many labs who have spent hundreds of thousands of dollars of their budgets running tests based on the Science paper. What is published peer review like this must be adequate information. These labs, particularly private ones but also University, are all competitors. We do not know their real relationships of these labs behind the scenes. Anyway, finding a specified virus in a specified population is a very generic finding, labs should be able to validate that on their own given the information in a journal article. If Mikovitzs has to give 'extra' information behind the scenes, then the Lombardi et al article was incomplete.
  11. kurt

    kurt Senior Member

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    Yes of course, any cohort definition part of their failure was the fault of the labs. I was only speaking to the possibility of techniques that may have been missing from the article. That would be on WPI and is a serious mistake. At this point we do not have enough facts to draw well-supported conclusions in either direction.
  12. julius

    julius Watchoo lookin' at?

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    Hey Kurt. I am wondering how the HERV theory squares with the statement by Dr Mikovits that they sequenced the entire XMRV genome two and a half times. It seems to me that with the whole genome you would know for certain if you were looking at a HERV or XMRV.
  13. Gerwyn

    Gerwyn Guest

    yes you would I have looked at the sequencing a herve would stand out like a sore thumb
  14. Gerwyn

    Gerwyn Guest

    yes you would I have looked at the sequencing a herv would stand out like a sore thumb
  15. Gerwyn

    Gerwyn Guest

    This following comes from the Imperial college study

    Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness

    Ok what does medical screening mean ---In this context patient history and physical examination by a competent physician .Detectable by blood tests OR patient self reporting This is what taking a patients history means.

    Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness

    Ok what does medical screening mean ---In this context patient history and physical examination by a competent physician . So Detectable by blood tests OR patient self reporting ..This means that the patients had none of the symptoms reported by the patients in The WPI cohort OR any of the biochemical or immunological annormalities. By McCure,s own introduction they are an entirely different cohort to those investigated by the WPI


    DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR

    Now this seems an innocuous statement does it not?

    The point to realise is that she knew about Mulv and the fact that it was closely related to XMRV.

    As a virologist surely she would know that Mulv does not replicate when in the bloodstream.It only replicates in lymphoid tissue,after replication it quickly becomes latent..

    So knowing this she attempts to isolate the XMRV DNA directly from the blood by PCR even though she knows that this would not be possible with Mulv

    She knows that Mulv lives in lymphoid cells but chose not to examine lymphoid cells for XMRV content at all

    To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

    Ok the concentration of Beta globulin gene is many orders of magnitude higher than the concentration of cDNA would be This proves nothing at all



    Received: December 1, 2009; Accepted: December 4, 2009; Published: January 6,

    2010


    Peer review in 4 days could be as little as one .They invite you to believe that underwent peer review in the period between the two dates.How can anyone anyone review a paper in this time.The Science peer review took months
  16. Gerwyn

    Gerwyn Guest

    can anyone get me these papers please
  17. Knackered

    Knackered Guest

    Before August I'd have thought.
  18. Gerwyn

    Gerwyn Guest

  19. Gerwyn

    Gerwyn Guest

    We have more than enough information to draw conclusions about the cohort issues
  20. Gerwyn

    Gerwyn Guest

    Just words Kurt no relevant facts at all.You have no time to"dig out" the data which you claim to have to support your arguments-as yet unseen-but you have time to write a convoluted letter of such length!!

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