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Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice

Discussion in 'XMRV Research and Replication Studies' started by Jemal, Jul 10, 2012.

  1. anciendaze

    anciendaze Senior Member

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  2. Bob

    Bob

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    Hello Dusty,

    Yes, there have been many negative studies, but there has been a positive prostate cancer study published, subsequent to the original:

    XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors
    Robert Schlaberga, Daniel J. Choeb, Kristy R. Browna, Harshwardhan M. Thakerb and Ila R. Singh,​
    September 8, 2009​
    PNAS​
    (Results: XMRV in prostate cancer tissue by IHC = 23%. XMRV in benign tissue controls by IHC = 4%)​


     
  3. Dusty Miller

    Dusty Miller

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    Thank you for being alert and asking good questions. The paper by Schlaberg et al. is fatally flawed and should not have been published. If there was a place on the PNAS website to post comments on papers, I would have done so long ago. These authors make a classic mistake of using polyclonal antibodies in an attempt to detect particular proteins, in this case, XMRV proteins in prostate cancer samples. The problem is that these mixtures of antibodies (polyclonal antibodies) raised against particular proteins often cross-react with other proteins, giving false-positive results. Worse yet, in the case of this paper, was how the authors generated their antibodies. They grew the XMRV virus (derived from the VP62 clone) in LNCaP prostate cancer cells. They then harvested the culture medium from these cells, which contains fetal bovine serum, proteins secreted by the human prostate cancer cells, and XMRV, and filtered this mix to remove things larger than 0.22 micrometers. What they did next is uncertain, but it appears they centrifuged the virus, lysed it using detergent, and injected this preparation into rabbits to make polyclonal antibodies. The problem here is that viruses acquire proteins from the cells in which they are grown, in this case, PROSTATE CANCER CELLS. So, they were making antibodies against XMRV and prostate cancer cell proteins. See the problem? They are then using these mixtures of antibodies in an attempt to detect XMRV in prostate cancer cells! No wonder so many of their patient cancer samples stained positive using these antibodies (23%). And, oh, only 6% of these samples were PCR-positive for XMRV. What, no XMRV DNA and yet the XMRV proteins are being made? Whoops. In short, this paper provides no convincing evidence that XMRV is present in humans.

    You may be asking how someone with only a passing knowledge of virology could figure this out. It would be difficult. In this case, the scientists that reviewed the paper appear to have missed the problems! Anyway, suffice it to say that there are problems with all of the papers claiming the presence of XMRV in humans, most less obvious than in this high profile paper published in a top journal, PNAS.
     
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  4. Bob

    Bob

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    Thanks for the helpful info. You seem to have studied the paper in great detail!
    I think we would probably all agree that the peer review process should never be relied upon!
     
  5. Dusty Miller

    Dusty Miller

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    When the paper came out, I was impressed, until I looked into the details.
     
  6. barbc56

    barbc56 Senior Member

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    Yes, it seems it's always in the details, eh? Thanks Dr. Miller. Your information is much appreciated.

    Barb C.:>)
     
  7. currer

    currer Senior Member

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    "But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen"

    Dr Miller, the authors of the Zhang paper disagree with you as they warn that

    "Laboratories working with xenograft derived human cultures should be aware of the risk of contamination with potentially biohazardous human -tropic mouse viruses....
    "Infected cultures usually release large numbers of infectious virions, and intra-laboratory spread of MLV virus to other cell lines maintained in the same facilities may occur, confirming the highly infectious nature of MLV virus. Retroviruses have been associated with multiple diseases including solid and hematologic malignancies, AIDS as well as with non-malignant diseases. The high susceptibility of human cells to infection with XMLV, the high levels of reverse transcriptase activity present in culture supernatant fluids and the demonstrated infectivity of the shed virions suggest that such viruses may present potential biohazards to laboratory personnel involved in cell culture facilities or to those handling human xenografts. In addition, the effects of the integrated provirus or the released virions on the biology of infected tumor cells are unknown. Provirus integration into the genome is not random, and occurs preferentially at transcription start sites, CpG islands, DNase-hypersensitive sites and gene-dense regions, suggesting that provirus integration may influence transcription in the host cell.43 Thus laboratories handling or culturing human xenografts should monitor for the presence of MLV, and should consider monitoring personnel for viral antigens or antibodies to them"
     
  8. currer

    currer Senior Member

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    The Zhang paper also remarks that most scientists working with xenograft cultures were unaware of the contamination problem although it agrees that initial reports about this were published in the seventies.

    "However, there are virtually no reports of contamination of xenograft cultures published during the past 15 years and most scientists appear unaware of the contamination problem (authors' observations)"
     
  9. currer

    currer Senior Member

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    With all due respect, Dr Miller, I do not believe that reports of MLV-like viruses detected in human tissues can be dismissed so readily. There are many ways an infection COULD spread into the population and this possiblitiy needs to be properly researched, (not dismissed ) by the scientific community. ( And particularly if MLVs are still being considered as gene vectors for therapeutic purposes.)
     
    natasa778 likes this.
  10. jace

    jace Off the fence

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    The discussion here seems to be focussing on XMRV, which as we know means a xenotropic virus. But, what was found in patients with ME/CFS were polytropic viruses, both in the October 2009 Science paper and in Alter and Lo's paper, the following summer. I am well aware that these papers have been retracted, but I have yet to be given a convincing reason that relates to the scientific issues rather than political issues.

    It is very confusing, for us here on the sidelines, when so many 'definite' statements are made by people on both sides of the argument. For instance, Dr Miller, I wonder what you think of this web page?

    It begins:

    The page goes on into more detail, with graphics, data and analysis. I would be grateful for your take on this.
    The following argument is presented to give you a chance at rebuttal, as well.


     
  11. natasa778

    natasa778 Senior Member

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    Huh, thanks for adding your opinions to debate, but a word of caution here as whatever you say will be preserved for posterity. Absolutist statements have throughout history often turned into big sources of embarrassment.
     
  12. currer

    currer Senior Member

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    "But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen"
    MLVs have been tested as gene vectors because of their ability to infect every human cell via the XPR1 receptor. The gene vector is presumably injected, thus bypassing the body's natural defences and the fragility of the retroviral envelope.
    If contamination with XMLVs is as widespread and unsuspected in laboratories as the Zhang paper indicates, is there a theoretical possibility of such human cell tropic MLVs contaminating cell lines used for the development of vaccines?

    Should the scientific community be concerned about this theoretical possibility?

    (sorry if this is off-topic for this thread)
     
  13. ikke2001be

    ikke2001be

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    Hi,

    I just ask myself why nobody is talking about anti bodies? I was found pos for these antibodies, and as far as I know, nobody is making antibodies against a lab contaminant. I wonder what's the deal here. Do I, and those few others positive for antibodies against XMRV deserve a place in the guiness book of records then?

    Regards,
    Ikke
     
  14. barbc56

    barbc56 Senior Member

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    If I remember correctly, it's because of cross reactivity. The antibodies weren't necessarily reacting to XMRV. This is a good example of the importance of the details that we lay people often miss.


    http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-C/cross-reactivity.html

    Barb C.:>)
     
  15. Dusty Miller

    Dusty Miller

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    A quote from the last paragraph of a paper from my lab (Knouf et al., J Virol 83:7353, 2009):

    "Finally, production of XMRV by 22Rv1 and potentially other prostate cancer cell lines should be carefully considered
    from the standpoint of possible virus transmission to laboratory personnel, to other cells cultured in parallel, and as a confounding factor in the interpretation of experimental results."

    I agree that scientists should be careful when working with viruses, especially those that can infect human cells. When we first found XMRV in 22Rv1 cells, I emailed American Type Culture Collection (ATCC) personnel and asked them to increase the biosafety level for this cell line, which they did, due to the potential hazard associated with this virus. The ATCC provides characterized cell lines to scientists worldwide. When I work with XMRV I am careful, but not as careful as I would be when working with HIV, a known human pathogen. But, potential hazard is not a known hazard. One can't call XMRV a human pathogen without data that it causes disease in humans. So far, such data is lacking.
     
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  16. Dusty Miller

    Dusty Miller

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    With all due respect, the scientific community has not ignored the possibility that retroviruses like XMRV might spread in the human population, and much research has been done to detect such retroviruses in humans. When retroviruses were first found to cause cancer in animals, it was obvious to look for such associations in humans. Part of the excitement generated by the Urisman et al. paper on a potential role for XMRV in prostate cancer was because it would vindicate the huge effort spent looking for such viruses with little success.

    With regard to the use of retroviruses in gene therapy, a large amount of effort has been devoted to testing the safety of these vectors. So far, the appearance of replication-competent recombinant retroviruses has not been a problem, but cancer resulting from insertion of the therapeutic vector near cellular oncogenes has been detected in multiple gene therapy recipients.
     
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  17. Dusty Miller

    Dusty Miller

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    "But, because human cells can be infected by a virus in animals or in cell culture doesn't imply that the virus is a human pathogen."

    I am simply making a logical statement here. A threat of rain doesn't imply that it will rain.
     
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  18. natasa778

    natasa778 Senior Member

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    And would most of them be willing to discuss it openly, in case theoretical possibility is misinterpreted ;-)
     
  19. ikke2001be

    ikke2001be

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    OK, cross reactivity, but with what? As far as I have read about antigens, they are pretty specific to the pathogen. So if there is cross reaction, doed this mean that the pathogen is "of the same family" as xmrv, or can this happen with completly different pathogens as well?

    Thanks for your clarification,

    Ikke
     
  20. Dusty Miller

    Dusty Miller

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    Regarding the apparent lack of an env gene in the 22Rv1 virus sequence, this is simply a problem with the annotation of this sequence. That is, someone left out a description of the Env protein, even though an open reading frame from which the Env protein is translated is clearly present in the sequence. Do you really think that all of the retrovirologists working on XMRV would have missed the absence of an env gene in their flagship virus?

    Regarding the CWR-R1 designation, this is another name, as is 22Rv1, for cells derived from a particular human prostate tumor. Cells derived from this tumor were initially called CWR22, but as derivatives with different properties were isolated, they were given different names. The replication-competent retrovirus found in both CWR-R1 and 22Rv1 are identical, and thus, both names are included in the sequence description. The viruses from both cell lines have been sequenced multiple times, and their sequences match, therefore the viruses from these two cell lines are considered to be the same.
     
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