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The Race to Retract Lombardi 2009...

Discussion in 'XMRV Research and Replication Studies' started by jace, Oct 10, 2011.

  1. barbc56

    barbc56 Senior Member

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    I have no idea how accurate the statements are from the commenter so it's difficult to know what it means one way or another. It's one of the drawbacks of including comments in articles. I'm not saying comments are a negative thing as they sometimes do make for lively debate.

    Do you have more info. about this situation?

    Interestingly, I was watching the TWIV podcast/video tonight where Trine Tsouderos of the Chicago Tribune was speaking at the ICAAC meeting. She makes some perceptive observations about articles with comments.

    http://www.twiv.tv/2011/09/18/twiv-149-live-at-icaac-in-the-windy-city/

    Let me know if you find more information about this. I'll look, too.

    Thanks. :D
  2. barbc56

    barbc56 Senior Member

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    Lee, You will never change the mind of some here but let me tell you, I am learning a lot from your posts.

    I have been hitting the "report post" button as these attacks on anyone who disagrees with some in the "hgrv's are the only answer" border on or are harassment. We need to speak up and one way to do that is to hit that button!!

    It also shows how they are just digging themselves in deeper and deeper ********** as well as lowering themselves to attacking the messenger instead of the message.

    IMHO!!

    It does get tiring hearing the same mantra over and over and over, doesn't it? Hang in there.
    wdb likes this.
  3. Angela Kennedy

    Angela Kennedy *****

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    I should also say, when people come onto forums and claim scientific expertise in order to impose their beliefs on others while remaining anonymous, a reasonable response is scepticism. That's not personal attack either.
    currer and jace like this.
  4. ikke2001be

    ikke2001be

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    Here are a couple of quotes from the xmrv global advocacy facebook page worth thinking about:

    "Science magazine and John Coffin, the reviewer of paper, were aware of the use of AZA in Lombardi et al. They asked for the labels to be changed on Fig. 2C. and did not ask for AZA to be mentioned in the published paper."




    "A small summary of the issues regarding human gammaretroviruses.



    All the 00 studies are now invalidated because of the use of the VP-62 clone in spiked samples to determine the theoretical limit of detection of their PCR assays using high stringency cycling conditions.



    Firstly ,VP-62 is not related to the gammaretroviral sequences found in Lombardi et al. and Lo et al. and a PCR with reagent concentrations and cycling conditions optimised to detect a free floating synthetic clone is not capable of detecting a methylated provirus integrated into C-G rich regions of host DNA. This would be true even if the sequences were related to VP-62, which they are not.





    The lombardi et al. protocols were not used in the BWG PCR and the RT-PCR assay would have been rendered meaningless by the lack of trizol.



    Lo's samples were not validated as being negative by serology or PCR.



    The blood collection procedures were different for the "WPI" and serology test and the lab donors.



    No PMBC from healthy donors was sent for negative validation.



    MLV viruses are only found in the blood intermiitently after the initial infection period they are impossible to detect in the blood either by PCR or serology without some mechanism which increases B cell transfer from the peripheral tissues into the blood compartment.



    It is time to focus on assays which detect the gammaretroviruses that are replicating in the human population in lymphoid tissues where other members of the genus are known to replicate. Gut biopsies are a good place to start as some 60% of mature preactivated B cells reside there."

    Regards,

    Ikke
  5. Esther12

    Esther12 Senior Member

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    Okay - I've got to go back to PACE and CBT/GET now.

    Surely the claim that PACE showed that 30-40% of CFS patients recovered from CBT/GET is a clear falsification of data? It came from an Esther Crawley paper: She's the chair of BACME, which is involved in educating NHS staff about CFS... do you not understand why CFS patients would be more concerned about that than the possible misuse of 5AZA? The XMRV issue was always going to be sorted out objectively by virologists, and any confusion would be (relatively) quickly resolved and cause little harm. The deceit around CBT/GET for CFS has been going on for decades, and continues to be harming patients right now - there is no particular interest in independent replication, and the standards for psychological research and treatment seem much lower than those for virology, yet the potential for harm is just as great.
    Bob, currer, jace and 3 others like this.
  6. Angela Kennedy

    Angela Kennedy *****

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    Thank you Esther. Exactly.
  7. Mula

    Mula Senior Member

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    5-aza was removed per the request of the editors of Science to facilitate publication.
  8. currer

    currer Senior Member

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    I have heard that 40% of the original paper was edited down prior to publication.

    Does anyone have a link to confirm that, please?
  9. Mula

    Mula Senior Member

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    40% may be a conservative estimate. Dr Ruscetti alluded to a far greater reduction in one presentation, which I don't think is available to the internet anymore.
  10. biophile

    biophile Places I'd rather be.

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    After looking further, I've become more suspicious and await further details

    I was going to quote both [Lee] and [Bob] and make a few comments on my impression of the situation, but I dug deeper instead.

    I was never under much doubt from the beginning the slides were essentially the same, I had assumed a sloppy mislabeling error before Ruscetti confirmed they were indeed the same. I just had another look at the slide labeling and also read this Nature article (http://www.nature.com/news/2011/111005/full/news.2011.574.html). I don't care that the numerical patient codes were different, that has been adequately explained (numbers are changed to protect ID, she was in a rush and made a sloppy mistake). However, lanes 2 and 5 were labeled as "normal" PMBCs (peripheral blood mononuclear cells) in the original 2009 Science paper but then labeled with patient codes in the recent 2011 Toronto presentation.

    Nature states: "The corresponding slide presented at the meeting was said to show activation of the protein's expression following treatment by a compound called azacytidine." This detail is something that shouldn't have been omitted but is somewhat understandable why it wasn't included in the original paper if it was supposedly their choice for what they believed is a routine procedure and wasn't "germane" enough to bother mentioning. However, perhaps it should have at least been mentioned in the supporting material or the 2nd paper they published to clarify on their methods? I think so, but not necessarily indicative of fraud in itself.

    Moving on to the issues which Lee et al are banging on about. From the Nature article cited above:

    I don't like the idea of using the gelslide as more of a stage prop, and Mikovits doesn't seem to be claiming that she accidentally used the wrong slide (correct?), so at first I considered the possibility that whatever she did at the presentation was somehow understandable out of convenience or some other detail I wasn't yet understanding at face value. However, after re-examining Figure 2C in the original 2009 Science paper to see how these patients were possibly obscured under the label of "normal", I found the following explanation: "Lysates of activated PBMCs from healthy donors (lanes 1, 2, 4, 5, and 7) or from CFS patients (lanes 3 and 6) ...". So guess what? Lanes 2 and 5 are given patient codes in the recent presentation, but in the original paper these same lanes are not just labeled "normal" but are actually described as from "healthy donors"!!! Unless I've misunderstood something here this does seem questionable.

    Frankly I don't fully understand all the technical details and hearsay to comment with much confidence here. I wasn't at the conference like Lee claims to have been so I don't know what context the slide was presented in. Is there a video or transcript available online yet? An investigation should reveal more details. I don't really care about the failure to mention azacytidine, or different patient codes, or even labeling patient lanes at the conference as "normal" lanes in the original paper if "normal" meant more than one thing, however to me the labeling of multiple lanes as "patients" at a conference but "healthy donors" in the original paper, assuming I have this correct, is the most suspicious aspect of this whole saga.

    [EDIT: After Bob later pointed out evidence that "activation" in the original paper meant PHA and IL-2 rather than "activation" by azacytidine as in the recent presentation, I removed two confusing sentences.]
  11. Bob

    Bob

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    Hi biophile,

    I think it's fair to say that we have established that the use of the word 'activated' does not refer to the use of 5AZA.

    This subject has been discussed at length in other threads, and so may i refer you to this thread which discusses all of the issues:
    http://phoenixrising.me/forums/showthread.php?14060-Why-does-5AZA-matter

    And also specifically to Mark's helpful post in that thread:
    http://phoenixrising.me/forums/show...es-5AZA-matter&p=210834&viewfull=1#post210834

    Lee's opening post is helpful, however please be careful to separate his facts from his opinions.
    Some of his opinions do not stand up to scrutiny based on the known facts, in my opinion.


    The Western Blot images used in the published paper and the conference slide are the same, and the labelling of the image was clearly changed for publication. Specifically, any mention of the use of 5AZA was omitted, and the negative lanes were relabelled to indicate a different Western Blot test which also had negative results.

    What we don't know is exactly why they were changed, or who changed them, and how significant the 5AZA issue is.
    If the 5AZA issue purely relates to one Western Blot test that was added in a last minute rush to satisfy the peer reviewers, or the editors didn't want to include the details, then it could be purely an editorial issue. In any case, as that specific Western Blot test was a small part in an excessively complex study, the occasional mistake or omission does not necessarily amount to misconduct. We'll have to wait for the official verdict.
  12. currer

    currer Senior Member

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  13. biophile

    biophile Places I'd rather be.

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    OK thanks Bob, I did skim through those links you gave. It is clear that activation with 5-AZA in the presentation is not the same as "activation" in the original paper, despite being the same WB. However we still have the other issue I highlighted, the labeling of patient lanes at the presentation as healthy donor lanes in the original paper. This has to be explained, to me this is the most important issue, and Mikovits doesn't seem to be saying she used the wrong WB in the presentation slide. If she did, then there wouldn't really need to be an explanation about the change of patient codes. I've been drafting too many posts lately so I'm spent and probably missing something, right now I'm not really sure what she saying exactly in her justification, I will comment in more detail later after a break. Like you said we don't really know what's going on yet and need further details.
  14. Bob

    Bob

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    Yes, the negative lanes were relabelled, probably as a short cut to save space in the final paper.
    A negative result is a negative result, so it's not exactly misrepresenting the results.
    Lee says that this is a massive breach of trust, but I'm not convinced about that.
    I think it's a sloppy short cut in the presentation of the data, but I'm not convinced it's a massive breach of trust.
    And we still don't know if the editors were involved in changing the way the data was presented.
    Lee says that the reader should be able see the actual results and interpret the results for him/herself, which I agree with.
  15. Mula

    Mula Senior Member

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    It was Dr Coffin/Science/editors who requested the label switch, as you say for editorial and ethical reasons. Is it possible that in moving to the use of the term "normal" they inadvertently stated they were healthy. Dr Coffin will have viewed the original label, so no doubt he would have known the status of those lanes post decoding. Dr Blomberg last work suffered from an editorial error with another journal only recently.
  16. Bob

    Bob

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    I'm not sure that the peer reviewers necessarily study all of the original information so closely Mula.
    They might have done, but we don't know this.
  17. kurt

    kurt Senior Member

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    Isn't it possible that the false labeling in the Science article was to boost confidence in the overall thesis of the paper? If JM relabeled negative patients as normal controls, that is a serious issue in my opinion, because that increases confidence in the overall finding. Had she showed on that original graphic that there were in fact many negative patient results, that would have created less confidence. I realize it is a bit of a balancing act, to decide what to show in a given graphic, when you are publishing a scientific paper. The researcher wants to show their interpretation of things, while at the same time presenting the data authentically. The reason to even show graphics like that is to make a point about the data. So what point was she trying to make? This seems like a bit of 'marketing' by JM. Technically I would not agree it was falsification of data, the overall data numbers do not change. However, it does seem to be a bit of spin-doctoring of her charts, and that does make me want to more carefully evaluate other things she has said. So I am saying, I agree with those here who are saying JM has given us reason to doubt, when she says something is it marketing spin or real data? But I also agree with those here who say this is not equal to scientific fraud. I suppose that puts me in the middle.

    Anyway, IMHO the whole gel issue is not relevant to the reasons the Lombardi article should be retracted. And I do believe it should and will be retracted. But not because of the gel graphic, really that is unfortunate trivia here. There is a far greater reason for retraction, and that is 1) the inability of WPI to replicate their own experimental data in the BWG; 2) the many 0/0 papers, many of which are very solid science (there may have been a few with problem cohorts, but most used real ME/CFS patients). Neither of these reasons would have changed had the gels been properly labeled in the Science study.

    WPI has had two years to conduct blinded studies that would prove they could replicate the experiment and they failed. So the greater question I have for all the pro-JM forum members is this. How did WPI get their original PCR test results? 67% positive among ME/CFS patients and only a few % positive in the control group. At one point JM did say in a presentation (I believe in 2010) that they often had to run patient samples several times in order to get positive results. There are indications the control groups used in the Science study may have been tested at different times or in some other way differently than the patient samples. That is what I expect the Science investigation is most interested in, because realistically, if WPI could not replicate with the BWG study, there has to be an alternate explanation for the original PCR results. If the patient and control samples were run differently, then the whole paper is likely invalidated and should be retracted. That is my opinion anyway, I don't expect everyone here to agree, but I think in time the issue of how the original results were obtained will become more important. Were the patient samples run many times, until most were positive? In nested PCR testing every additional run increases risk of contamination...so if patient samples were run more often than the matched control samples, there would be a high risk of false results. I'm not saying that happened, just pointing out there has to be some explanation for the original PCR testing. And in the face of the BWG study, that explanation may not be favorable to JM/WPI.

    None of this takes away from the importance of continuing research into all types of pathogens in ME/CFS, including any type of virus for which there is solid evidence. The possible retraction of the Lombardi paper is really not an all-or-nothing situation for us. But I can understand people who have supported JM and WPI don't want to see their hope and financial support evaporate and contribute nothing to ME/CFS. But to those of us who have experience in the research world, a retraction will come as no surprise, research is a very expensive, high risk activity.
    wdb likes this.
  18. currer

    currer Senior Member

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  19. kurt

    kurt Senior Member

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    Thanks, I had not seen that thread.

    IF there are HGRVs in ME, they are unlikely in the MLV family as that has been thoroughly tested. So that might require novel sequences be discovered. But that still would require a transmission vector that defies the epidemiology of ME/CFS, unless the virus is nearly ubiquitous. Will be interesting to see if anybody finds reproducible evidence.
  20. alex3619

    alex3619 Senior Member

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    Hi Kurt, I have said this many times, on many threads, the retroviral theory EXACTLY matches the epidemiology, its a PERFECT match. Its why the hypothesis is so compelling. I am not going to bother reiterating, again, why this is so. Claims to the contrary are based on simplistic viral, not retroviral, models. Bye, Alex

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