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Japanese study detects XMRV using Whittemore Peterson Institute techniques

Discussion in 'XMRV Research and Replication Studies' started by VillageLife, Mar 16, 2010.

  1. VillageLife

    VillageLife Senior Member

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    United Kingdom
    Saw a link to this today on the WPI's website. http://www.wpinstitute.org/news/news_current.html

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    THE PREVALENCE OF XMRV IN HEALTHY BLOOD DONORS IN JAPAN

    XMRV Research


    THE PREVALENCE OF XENOTROPIC MURINE LEUKEMIA VIRUS-RELATED VIRUS IN
    HEALTHY BLOOD DONORS IN JAPAN

    Rika A. Furuta1, Takayuki Miyazawa2, Takeki Sugiyama3, Takafumi
    Kimura1, Fumiya Hirayama1, Yoshihiko Tani1 and Hirotoshi Shibata1

    1 Department of Research, Japanese Red Cross Osaka Blood Center, 2
    Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto
    University, 3 Department of Urology, Nishiwaki Municipal Hospital.


    To estimate the impacts of infection with xenotropic murine leukemia
    virus-related virus (XMRV) on the blood service, we investigated the
    prevalence of this virus in both prostate cancer patients and healthy
    blood donors in Japan. All specimens from the prostate cancer patients
    were collected after obtaining their written informed consent. The
    ethical committee of the Japanese Red Cross Society approved the
    examination of XMRV antibodies, but not nucleic acids, in random donor
    sera.

    All serum samples of healthy blood donors tested negative for HIV-1,
    HIV-2, HTLV-1, hepatitis B virus, hepatitis C virus and human
    parvovirus B19.

    To make a recombinant virus as test antigens for the antibody
    screening, 293T cells were transfected with an expression vector
    carrying an XMRV provirus clone, namely, VP62 (kindly gifted by Dr. R.
    H. Silverman). We used an env-defective mutant of HIV-1 derived from
    pNL4-3 (kindly gifted by Dr. A. Adachi) as a negative control.

    Two days after transfection, the culture supernatants of the
    transfected cells were collected and concentrated 20 times by
    centrifugation. We implemented western blotting assay to screen
    antibodies against XMRV in sera because a high background was observed
    if we performed enzyme-linked immunosorbent assay. In the western
    blotting, the blot strips were incubated with the serum samples
    diluted 1:100 with 5 % skim milk in Tris-buffered saline overnight at
    4C.

    Two of 32 serum samples collected from the prostate cancer patients
    and 5 of 300 serum samples collected from healthy blood donors tested
    positive for antibodies against XMRV Gag protein. We did not observe
    any specific signals against Env proteins in the western blotting. Of
    the 2 serum samples that were obtained from prostate cancer patient
    and tested positive for anti-XMRV antibodies, the XMRV specific
    nucleic acid sequence was detected in only one sample (patient #24) by
    using nested RT-PCR.

    In addition, we collected 7 mL of whole blood cells from the patient
    #24 and cultured the peripheral blood mononuclear cells (PBMCs) in the
    presence of recombinant interleukin 2 and concanavalin A.

    The PBMCs were harvested after 10 days of culture, the virus was
    isolated from the cells by performing a LacZ marker rescue assay and
    RNA and genomic DNA were extracted. The nested PCR performed to detect
    XMRV yielded positive results for both genomic and RT PCR, although
    the virus was successfully isolated in only 1 of 3 independent
    experiments. To examine the susceptibility of PBMCs derived from
    healthy individuals to XMRV, we inoculated activated PBMCs from 3
    healthy volunteers with the culture supernatant of the PBMCs obtained
    from patient #24. By using the nested PCR, we detected the
    XMRV-specific nucleic acid sequence in the genomic DNA of the PBMCs
    obtained from 2 of the 3 healthy volunteers.

    We conclude that XMRV infection is prevalent among both prostate
    cancer patients and healthy individuals in Japan. Although our study
    had a limited sample size, the prevalence among blood donors as
    determined by identifying XMRV-specific antibodies was found to be
    1.7%, while that among prostate cancer patients was found to be 6.3%
    (P<0.05, one-sided Mann-Whitney U-test).

    The results of genomic PCR performing on the PBMCs indicate that XMRV
    is sustained in a few fractions of blood cells and can spread through
    blood even though the virus replication rate appears to be very low.


    Source: 1 Department of Research, Japanese Red Cross Osaka Blood Center, 2
    Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto
    University, 3 Department of Urology, Nishiwaki Municipal Hospital.
     
  2. VillageLife

    VillageLife Senior Member

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    This is good news then!!
     
  3. V99

    V99 *****

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    Excellent. They should look at cfs patients next.
     
  4. dsdmom

    dsdmom Senior Member

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  5. Esther12

    Esther12 Senior Member

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    Has this been properly published anywhere yet?

    I was hoping this would be a CFS study...
     
  6. dsdmom

    dsdmom Senior Member

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    Esther, I agree, but I guess the exciting thing about this is that they were able to replicate WPI's findings using their methods. Where the other studies to date have not even attempted to replicate them and were therefore unable to find XMRV.
     
  7. Lesley

    Lesley Senior Member

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    This is a Japanese study that was never published, but presented at a conference and cited in XMRV papers. It is still not a full paper published in a peer-reviewed journal, but just the abstract on a website. What is new is that the WPI has linked to it and said that the Japanese researchers used WPI methods.

    This has been discussed on this thread: http://www.forums.aboutmecfs.org/showthread.php?3238-Who-found-XMRV-in-Japan
     
  8. Cloud

    Cloud Guest

    Exactly!! This study suggests faulty technique with the other (so called) replication studies. It also also suggests to me that faulty technique was more the problem than cohort/selection, or even geographics.
     
  9. Hope123

    Hope123 Senior Member

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    WPI shouldn't have put this as breaking news although it might be new? to them. This was the Japanese study presented 5/2009 that was discussed on the forum recently and for which a member (ukxmrv) e-mailed Cold Spring Harbor and got the abstract. The part about it using WPI methods is also unclear to me. It's similar in that the Japanese team used a variety of methods (PCR, culture, Western Blot, antibody) to find XMRV but I don't know if the exact methods are those used by the WPI.
     
  10. kurt

    kurt Senior Member

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    USA.Earth
    This is not a peer-reviewed study so I fail to see how it matches up with the four published XMRV CFS studies, let alone illustrate a problem with the failed validation attempts. In fact isn't this just the Japanese study that has been talked about for months? Hardly 'breaking news' except that it now appears on a new XMRV website.

    Unfortunately, there is not enough in this abstract to tell if they really did use the WPI method. Given that this is actually a new report of an older study by the Japanese Red Cross, I seriously doubt they had contact with WPI. But perhaps they used the same type of culture as WPI, that might be worth reporting. Or is there some undisclosed relationship between this group and WPI?

    Still, they may have really found 1.7% XMRV positives in blood donors. And the problems in the study do support the notion that PCR testing does not always work right for XMRV, it appears their PCR could not confirm all the antibody results, something WPI has been saying now for awhile. Interesting....
     
  11. gracenote

    gracenote All shall be well . . .

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    Santa Rosa, CA
    This thread really ought to be combined with this one:

    Who found XMRV in Japan?

    This is the first reference I found to this study and it was from the first UK paper. This has been discussed previously in the other thread.

     
  12. Gerwyn

    Gerwyn Guest

    pcr does not detect latent virus full stop. Have a look at the Plos one peer review criterea when you have the time. See what you think of the process and the time it takes.

    The criterea for publishing make intersting reading too .Why did the BMJ break their own peer review rules when publishing the dutch study/
     
  13. justinreilly

    justinreilly Stop the IoM & P2P! Adopt CCC!

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    NYC (& RI)
    I didn't know about the Japanese study. Didn't register in my foggy brain when I read the first UK study.

    The fact they found xmrv in healthy controls reinforces for me just how arrogant and out of touch McClure, Wessely, the Dutch study people and BMJ are- trying to trash WPI when the evidence points to the conclusion that they are the ones who are probably not doing the science well enough to pick up xmrv cases.
     

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