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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. starryeyes

    starryeyes Senior Member

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    Bravo Gerwyn!! That post explains the science in a much more understandable way. I am very pleased that you did that for us and I really like the colors. It sure helps delineate the points you are making and helps a foggy brain keep them straight plus they're uplifting.

    Thank you!! :victory::sofa:
  2. Lily

    Lily *Believe*

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    GREAT JOB Gerwyn, THANK YOU MUCH

    I appreciate all the time and effort that must have taken.
  3. Countrygirl

    Countrygirl Senior Member

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    :Sign Good one: GERWYN That you for the time and effort you put into this. It certaily deserves front page status. :Retro smile::Retro smile:
  4. Marco

    Marco Old blackguard

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    Thank you Gerwyn.

    Now THAT I can follow.
  5. Gerwyn

    Gerwyn Guest

    Yes viruses have the genomic equipment to manufacture SAg EBV is a common example .if SAg was causative in ME then anyone who had an ebv would develop the illness.it is not a question of just keeping an open mind but attempting to explain observations

    MMTV is a betaretrovirus which has specific genes thet code for a Sag .A herv does not-fact

    MMRV only infects mice Fact
  6. Gerwyn

    Gerwyn Guest

    Thankyou but you are right.After reviewing the paper and trying to "translate" it iI realise that I slip into science"shorthand" too often.

    When I was talking about cell cell transfer I should have explained it far better

    Basically cells are connected by "molecular corridors" In B cells(where XMRV lives)there are molecules(the restrictive factors in the post you referenced) which could damage or destroy replicating XMRV if it spent too long in the cytoplasm..

    One technique to avoid this is to travel between cells using these corridors actually using the energy of the intrinsic molecules to power the process of movement.These thing are so clever!
  7. valia

    valia Senior Member

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    What everyone else said goes for me too. :thumbsup:


    I would also like to thank you Gerwyn for breaking up and spacing text the way you do,

    you are one of the few who appreciates how difficult it is for some of us to read large blocks of text. :thumbsup:
  8. Cort

    Cort Phoenix Rising Founder

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    I appreciate the time you took to do that. (The colors were really tough for me by the way). I appreciate your pointing out the differences between the Science study and the UK studies.

    It appears that you've answered my question: the WPI did not activate the cells prior to doing the PCR.

    Note that they said the cells for the DNA and RNA extraction were stored as unactivated cells in Trizol. As I noted earlier there is no mention of activation or culturing anywhere in the PCR section. WPI used PBMC's for PCR; they did not break the cells up into their differents - which is where the culturing came in. The WPI later said activation was necessary but there's no evidence I can find that they did that. I want XMRV to work - I'm not trying to bash it - but logic is logic; if they didn't do it they didn't it and nothing that I've yet read convinces me that they did it. I want to be fair to every side - not just the WPI.

  9. Koan

    Koan Be the change.

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    Many thanks, my liege, Wizard of Wales!

    Your grateful subject,
    Koan
  10. Cort

    Cort Phoenix Rising Founder

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    Here's the section where Gerwyn points out that the culturing took place: its titled NEXT Experiment: that's the experiment AFTER the PCR took place. It doesn't appear to me to have anything to do with the PCR test. Note that with regards to the cultured cells they're talking about leukocytes, T-cells and B-cells. They simply used 'PBMC's ' for the PCR. PBMC's also contain monocytes and macrophages and NK cells.

  11. Koan

    Koan Be the change.

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    Hey Cort,

    I wish I understood this better. I keep thinking it's been answered but I'm not at all science savvy! Not at all!

    Anyway, have you thought about asking WPI directly?

    I worry that we will wear out our dear Wizard. Even wizards must have limits - especially wizards with ME!

    Peace out,
    koan
  12. Koan

    Koan Be the change.

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    That's what I thought I understood, Gerwyn!

    I may be learning!
  13. Robin

    Robin Guest

    Cort, the second experiment with culturing you're referring to was prep-work to a nested PCR, and the demonstration of infectiousness.

    I think I understand what Gerwyn is saying. Let me try to reformat the concluding section without the rainbow colors so maybe it's more clear. (Apologies to Gerwyn if I did this wrong.)

    They did three different experiments with PCR. The most successful techniques involved frozen blood that was amplified and activated. The least successful is on fresh blood with no prep.


  14. Hope123

    Hope123 Senior Member

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    I'm not saying that EBV is THE cause or only cause of CFS but rather that the idea of superantigens is interesting.

    Here's the article on MMTV in Wiki. There is ongoing research into this because of a similarities found in human breast cancers (called H (human) MTV).

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

    Also, for some people, EBV is the major factor as there are people I know who have recovered (yes, 100% by their own reckoning, back to work full-time, and exercising/socializing) after treatment for EBV over several months. These folks were diagnosed with CFS, were sick for years, and had EBV IgM (not just IgG) but were not taken seriously. This may be a very small group within CFS but it's there.

    I'm going to stop commenting on this as it is off the topic of the thread.
  15. Gerwyn

    Gerwyn Guest

    i decided my first reply to your post was unfair.You obviously had a great deal of difficulty inreading it because of the colour scheme. While most people have apparently found it to be beneficial I can see that not everyone would

    I will try and keep this post as clear and simple as possible so that it makes it less likely that you form erroneous conclusions because you find the text difficult to understand

    The Paper is devided into THREE DIFFERENT EXPERIMENTS

    Initially 8 ml of FRESH BLOOD was taken from each patient That is FRESH blood.

    The PMBCs were seperated from the blood. to be simple from now one i will call them white blood cells.

    The reason they did this was TO CONCENTRATE the sample of white blood cells

    these cells were devided into two equal amounts

    One lot was frozen for later use and the other lot was broken up and the DNA AND the RNA extracted

    They Ran nested PCR on the DNA sample and found that they had a 99.9% match to XMRV as first identified and genotyped by its original discoverers.They also found that there was not any DNA belonging to any other MuLV gamma retrovirus present. this is important because XMRV is a member of that family of viruses so any future tests on the sample would be difficult if there was DNA belonging to another member of the family present

    The exact same experiment was carried out at the cleveland clinic with exactly the same results ruling out contamination

    The RNA was also investigated by RT-PCR.Now this is a different kind of PCR .The reason that virologists do this when trying to isolate a RNA virus is that this technique can detect an RNA virus when it is not replicating at all.Normal PCR cannot do this

    Now just to make it clear PCR cant isolate viral DNA unless the virus is replicating. RT-PCR can

    The results of the RT-PCR confirmed the presence of XMRV .NOW THAT IS THE END OF THE FIRST EXPERIMENT THERE IS IS GOING TO BE SOME MORE PCR LATER SO KEEP READING

    How does this compare with the English studies.

    THIS EXPERIMENT DOES NOT COMPARE AT ALL because they Used FROZEN WHITE CELLS in whole blood

    So lets look at the experiment that was carried out in the science study which looked at extracting XMRV from FROZEN WHITE BLOOD CELLS

    THE first difference is the concentration of white blood cells at the start.The Science researchers used white blood cells which had been isolated from blood.

    The English studies did not.The English sudies began with a much lower concentration of white blood cells to begin with

    The science researchers activated the white blood cells.The English researchers did not.Activation stimulates and induces replication now remember that PCR only works if a virus is replicating

    The science researchers massively increased the number of activated white blood cells by repeated culturing.The English researchers did not

    The science researchers ran their PCR on massively amplified numbers of white blood cells which had been activated.The English researchers did not

    The science researchers detected XMRV.The english researchers did not.

    In summary the English researchers used frozen diluted white blood cells. In one experiment the Science researchers also used frozen white blood cells which were concentrated

    The English researchers performed their PCR without any attempt at concentrating their sample .They did not activate the white blood cells or attempt to amplify their numbers.The Science researchers did

    The English researchers could not detect xmrv and did not The science researchers could and did

    I hope that this is clear now
  16. Gerwyn

    Gerwyn Guest

    if you read it properly it may" appear" otherwise
  17. Gerwyn

    Gerwyn Guest

    There seems to be some confusion about the PCR run by the Science researchers on the pmbcs that were frozen after extraction thawed activated and amplyfied by culture. here it is

    Genotyping. The rs486907 R462Q SNP was genotyped using Applied
    Biosystems’ Taqman 5' nucleotidase assays, Taqman Universal PCR Master
    Mix: No AmpErase UNG, and 5 ng of genomic DNA. The thermal cycling
    conditions consisted of an initial hold at 95o C for 10 minutes followed by 50
    cycles of a 15 second 95o C denaturation step and a one minute 60o C annealing
    and extension step. A 7900HT instrument was used to detect fluorescent
    probes, and Sequence Detection Software (SDS) v2.2 was used to discriminate
    alleles and call genotypes (Applied Biosystems, Foster City, CA

    It is sometimes called allelic PCR and confirmed the presence of whole genome XMRV in the activated highly concentrated white blood cells originating from the blood of patients properly diagnosed with ME/cfs
  18. Gerwyn

    Gerwyn Guest

  19. Cort

    Cort Phoenix Rising Founder

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    OK they used RT-PCR without activating the cell to look for XMRV. They then did PCR on activated cells even tho they never explicitly said they did.

    What they should have said here - simply they apparently spelled everything else out elsewhere - was

    Cells where activated with X,Y, Z and then

    In the culturing section

    They should have noted the cells were refrozen again (ie. Cells were refrozen for PCR analysis) because PCR section states they used frozen cells . I assume that freezing/refreezing cycles can have an effect on viability.

    Its messy to me. To me you have to infer that the cells were activated prior to either PCR. I can see how people would wonder. Its very detailed account of the process. Its designed to demonstrate exactly how to replicate it -yet it doesn't explicitly spell out some key steps.

    Later they explicitly say the T-cell cultures were used in the viral transmission experiment:

    Its odd they would note the culturing and activation in the viral transmission section but not in the PCR section.

    Maybe its time to move on- we'll have new studies soon I hear to dig into. What they did or didn't do in the first study will eventually be moot.

    One of the reasons I've stuck with those is not to get at the WPI but point out how people like Dr. Vernon, might, based on these finding legitimately wonder if the patient cohort was very different from those in England. I know that you noted that section of the paper and they appear to be very different but then WPI personnel have repeated stated that they were 'typical patients'.

    In any case, based on our 'data' from the Light/Bateman study my sense is that the three CFS studies (really two CFS studies) made a critical error somewhere - as you have surmised throughout - and we're going to get very different results as better studies come out.
  20. Gerwyn

    Gerwyn Guest

    cort a competent virologist could easily follow those instructions they were not constructed for laymen but proffessional retrovirologists

    it may well "seem" messy to a layman but to the science peer reviewers it was clear enough as indeed it is to me.Now Mclure and Groom have many o rders of magnitude more knowledge in this area than me.You tell me why the methods and proceedures were not clear to them.

    the WPi said that their patients were typical of patients with ME/cfs using their diagnostic criterea NOT typical of the patients labelled with CFS by the Oxford mythodology.The two patient cohorts are clearly different and Dr Vernon should be repeatedly stating that

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