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XMRV Replication Studies

Discussion in 'XMRV Research and Replication Studies' started by Cort, Dec 5, 2009.

  1. usedtobeperkytina

    usedtobeperkytina Senior Member

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    Most interesting part: XMRV is hard to find in testing.

    That tells me the UK study likely didn't find it because of method. Also, false negatives are very probable, according to the assay being used now.

    Tina
  2. Esther12

    Esther12 Senior Member

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    I really don't understand this.

    All the virologists I've been reading who are not directly connected to XMRV do not understand why XMRV would be especially difficult to find. But the varying XMRV results, both for CFS and prostate cancer would, do my ignorant mind, seem to show otherwise. It could show the WPI results were affected by being unblinded, and they looked harder for XMRV in CFS patients? I've not heard anyone try to explain why XMRV would be especially difficult to find. If they've got a good idea why this would be the case, the WPI should start talking about it imo. (Maybe they want to keep quiet for commercial reasons?) More replication studies which performs normal PCRs and fail to find XMRV are going to seriously undermine the WPI research. From my reading of blogs etc, scientists do not seem unimpressed by the methods used in the london study.


    Scientists can sometimes give the impression they know more than they do, but I'm so poorly informed about these matter I'd still tend to give them the benefit of hte doubt. It's all really strange.
  3. Eric Johnson from I&I

    Eric Johnson from I&I Senior Member

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    Low number of virions and provirions -- possibly. Serology should probably remain pretty sensitive/easy in the face of that. Less so PCR, viral culture, viral protein detection.
  4. Esther12

    Esther12 Senior Member

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    I've not understood any explanation as to why XMRV might be hard to find....

    I think my problem might be trying to use my own judgement in an area where I'm totally out of my depth! It would probably be sensible to just forget about this stuff for six months and then come back when the dust has settled, but I'm too involved. Hopefully if there are difficulties with identifying XMRV, those involved with the replication studies will understand this and have the patience to work arround it.
  5. usedtobeperkytina

    usedtobeperkytina Senior Member

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    I agree.

    I agree, seems like you just get a few cells, color them, then put them under the microscope. If it's there, then it is positive. If it isn't, then you aren't. But clearly, it is not that simple.

    For example, I think the WPI tested samples at least three different ways. In the CFS cohort, only after the third type of testing, did they find 98% positive. And it isn't just the difference of active or antibodies. If you want to understand, you can see Peterson's presentation to the CFSAC. Although I don't understand all he said, it is clear that it isn't just as simple as looking for the virus and looking for antibodies in the cells.

    Even if it were that simple, then what are the chances that the sample they look at has the virus or antibodies. How many microscope slides do they have to look at from each sample before they say, "nope, can't find it, so it isn't there." Is it 10, is it 30, is it 50? I read that some of this is like looking for a needle in a haystack. That brings up an image that might explain why it is so difficult. Does the virus infect one in 10 immune system cells, one in 50, one in 1,000 or how many? So how many do they have to look through?

    And yet, it is even more complex than that. The test involves primers that make the virus grow, thus showing the sample has the virus. But what primers? That is a matter of difference between the WPI study and UK study. The Imperial folks said they used same primers in water and it grew, therefore it should have grown in the blood samples using the same primers. WPI says no. Until you can use primers and find it in a true blood sample, then you can't say your primers work in the blood samples of the patients.

    Plus, what part of the virus genetic material are you looking for?

    Another factor is freezing. DeFreitas said the virus she saw disappears from the sample after six days. "It's there, it's there, it's there. And then it is gone," she said. And freezing can corrupt the DNA to the point that it is fragments. So, how well will frozen samples respond to the primers?

    So these factors may explain why it isn't so easy to find the virus.

    Tina
  6. Human retroviruses differ greatly in their rate of replication once they have infected a host genome they can quickly produce dna on entering a cell which becomes rapidly integrated into the hosts DNA.The aids virus replicates quite quickly and can be assayed using antibody titres.If the replication rate is slower however then the viroids have such a low titre that they are very difficult to detect other by DNA sequence identification -a horribly slow process hideously expensive and still hit and miss.Fast insertion and slow replication are thought to be evolutionary adaptations to avoid the human immune system as much as possible

    I hope it helps -there were 4 existing human exogenous retroviruses before xmrv and now there seems to be 5
  7. usedtobeperkytina

    usedtobeperkytina Senior Member

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    And yet, false positives are also possible with the current, FDA approved, HIV assay. A person has to be tested twice before it is known if they have the virus.

    Reminds me of pap smears. If a person shows positive for abnormal cells, then you wait six months and then test again. If positive the second time, they do a freeze. If you don't, no action taken. So false positives are possible there also.

    Tina
  8. Rita

    Rita Senior Member

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    Summary of XMRV Research Studies
    XMRV studies done in Germany, Ireland, UK, Japan and the USA.



    German prostate cancer study #2

    0/589 German prostate cancer patients XMRV 'Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients'

    Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, Miller K, Kurth R, Bannert N. Retrovirology. 2009 Oct 16;6:92. http://www.ncbi.nlm.nih.gov/pubmed/19835577


    Irish prostate cancer study

    0/139 Irish prostate cancer patients XMRV 'NO EVIDENCE OF XMRV IN IRISH PROSTATE CANCER PATIENTS WITH THE R462Q MUTATION'

    D'Arcy F., Foley R., Perry A., Marignol L., Lawler M., Gaffney E.,Watson R.G.W., Fitzpatrick J.M., Lynch T.H. European Urology Supplements Volume 7, Issue 3, Page 271 (March 2008) http://www.europeanurology-supplement.com/article/S1569-9056(08)60798-8/abstract


    PLOS XMRV/CFS study

    0/186 UK CFS patients XMRV 'Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome'

    Otto Erlwein, Steve Kaye, Myra O. McClure, Jonathan Weber,Gillian Wills, David Collier, Simon Wessely, Anthony Cleare PLoS ONE 5(1): e8519 - Published: January 6, 2010


    Original XMRV/prostate cancer study(US)

    8/20(40%) prostate cancer patients XMRV+ and 1/66(1.5%) different kind
    of prostate cancer patients XMRV+ 'Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant'

    Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL. PLoS Pathog. 2006 Mar;2(3):e25. Epub 2006 Mar 31. http://www.ncbi.nlm.nih.gov/pubmed/16609730



    Replication XMRV/prostate cancer study(US)

    334 consecutive prostate resection specimens- 62/233(27%) w/prostate cancer XMRV (XMRV DNA found in 6% and XMRV protein expression in 23%) vs. 6/101(6%) of cancer free controls XMRV , 'XMRV is present in malignant prostatic epithelium and is associated
    with prostate cancer, especially high-grade tumors'.

    Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR. Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16351-6. http://www.ncbi.nlm.nih.gov/pubmed/19805305



    5/300(1.7%) of healthy Japanese blood donors positive for antibodies to
    XMRV 'The Prevalence of Xenotropic Murine Leukemia Virus-related Virus in Healthy Blood Donors in Japan'.

    Furuta RA, Miyazawa T, Sugiyama T, Kimura T, Hirayama F, et al.
    Cold Spring Harbor Retrovirus Symposium 2009



    WPI XMRV/CFS study

    68/101(67%) CFS patients and 8/218(3.75%) of healthy controls XMRV, 'Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome'.

    Lombardi V, Ruscetti FW, Gupta JD, Pfost MA, Hagen KS, et al. Science. 2009 Oct 23;326(5952):585-9. http://www.ncbi.nlm.nih.gov/pubmed/19815723



    German prostate cancer study #1

    1/105 non-familial prostate cancer patients and 1/70 tissue samples from men without prostate cancer XMRV, 'Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer'.

    Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, Aepfelbacher M,
    Schlomm T.J Clin Virol. 2008 Nov;43(3):277-83. http://www.ncbi.nlm.nih.gov/pubmed/18823818
  9. shrewsbury

    shrewsbury member

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    Rita - thank you for doing this. I found it so helpful. What a splashy entrance to the forums! Welcome.

    There's also the US study below that seems to get forgotten:

    2008 10 US XMRV and Prostate Cancer #2
    Integration Site Preference of Xenotropic Murine Leukemia Virus-Related Virus, a New Human Retrovirus Associated with Prostate Cancer
    Sanggu Kim,1 Namshin Kim,3, Beihua Dong,5 David Boren,2 Serena A. Lee,2 Jaydip Das Gupta,5 Christina Gaughan,5 Eric A. Klein,6 Christopher Lee,3 Robert H. Silverman,5 and Samson A. Chow1,2,3,4*
    Biomedical Engineering Interdepartmental Program,1 Department of Molecular and Medical Pharmacology,2 Molecular Biology Institute,3 UCLA AIDS Institute, UCLA School of Medicine, Los Angeles, California 90095,4 Department of Cancer Biology, Lerner Research Institute,5 Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio 441956
    Received 22 June 2008/ Accepted 3 August 2008
    Journal of Virology, October 2008, p. 9964-9977, Vol. 82, No. 20
    0022-538X/08/$08.00+0 doi:10.1128/JVI.01299-08
    Copyright 2008, American Society for Microbiology. All Rights Reserved.
    http://jvi.asm.org/cgi/content/full/82/20/9964?view=long&pmid=18684813

    and the article from 2008 that summarizes a 2008 UK study into XMRV and ALS and previous research

    1. 2008 03 06 XMRV and ALS (2008 British research and references to earlier work)
    Article: Evidence of Retrovirus Identified in Serum of ALS Patients
    Talan, Jamie
    Neurology Today: 6 March 2008 - Volume 8 - Issue 5 - pp 1,14doi: 10.1097/01.NT.0000314454.74483.a9
    http://journals.lww.com/neurotodayo..._Retrovirus_Identified_in_Serum_of_ALS.1.aspx
    “A retroviral marker has been quantified in serum from patients with amyotrophic lateral sclerosis (ALS) but scientists say that it is still not clear what the virus is or whether it is damaging motor neurons.”
  10. Cort

    Cort Phoenix Rising Founder

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    Very nice Rita and IslandFinn
    Thanks for explaining why its so time consuming and expensive. Lombardi did say low viral loads.
  11. natasa778

    natasa778 Senior Member

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    XMRV detection in blood versus tissue

    Is it hypothetically possible that XMRV could be widespread in/within certain tissue cells (I'm especially interested in glia/CNS and endothelial/vascular tissue as autism my focus) while at the same time almost absent from blood, or present at levels that are hard to detect through PCR methods? Could this be the reason for some of the false negative results and would an antibody test surpass this problem? Or would it open floodgates for false-positives?

    I remember reading something similar to be the case with Herpesvirus6 - it being so hard to detect through blood tests as it spreads from cell to cell in a very direct manner. References on this on HHV-6 foundation site.

    If this is a distinct possibility with XMRV would it be a wise approach to carry out immunochemistry or similar (FISH?) on biopsy or postmortem tissue?
  12. natasa778

    natasa778 Senior Member

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    P.S .from HHV-6 Foundation site on tissue infection versus blood levels

    ... just to illustrate the point:

  13. Ok, so I looked for a bit to see if there was any evidence as I remembered there was, years ago.......So I found this so far.
    Is this of interest perhaps? I don't know if you guys are talking about XMRV here, or have diverged somewhat to common CFS viruses such as HHV-6, but here's something:

    ''Over the last decade a wide variety of infectious agents has been associated with the chronic fatigue syndrome (CFS) as potential etiologies for this disorder by researchers from all over the world. Many of these agents are neurotrophic and have been linked previously to other diseases involving the central nervous system (CNS). Because patients with CFS manifest a wide range of symptoms involving the CNS as shown by abnormalities on brain MRIs, SPECT scans of the brain and results of tilt-table testing, we sought to determine the prevalence of HHV-6, HHV-8, EBV, CMV, Mycoplasma species, Chlamydia species and Coxsackie virus in the spinal fluid of a group of patients with CFS. We found evidence of HHV-8, Chlamydia species, CMV and Coxsackie virus. Attempts were made to correlate the clinical presentations of each of these patients, especially the neurological examinations and the results of objective testing of the CNS, with the particular infectious agent isolated. It was also surprising to obtain such a relatively high yield of infectious agents on cell free specimens of spinal fluid that had not been centrifuged”.


    The above may tie in with the below........

    Arguments exist as to the cause of CFS. Some think that it is an example of symptom amplification indicative of functional or psychogenic illness, while our group thinks that some CFS patients may have brain dysfunction. To further pursue our encephalopathy hypothesis, we did spinal taps on 44 patients (31 women and 13 men) fulfilling the 1994 CDC criteria for CFS and on 8 women and 5 men serving as healthy controls. Our outcome measures were white blood cell count, protein concentration in spinal fluid, and cytokines detectable in spinal fluid.

    We found that significantly more CFS patients had elevations in either protein levels or number of cells than healthy controls (30% versus 0%), and 13 CFS patients had protein levels and cell numbers that were higher than laboratory norms; patients with abnormal fluid had a lower rate of comorbid major depression than those with normal fluid (0 vs 27%, p<.04). In addition, of the 11 cytokines detectable in spinal fluid, (i) levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) were lower in patients than controls (p<.03), (ii) levels of interleukin-8 (IL-8) were higher in patients with sudden, influenza-like onset than in patients with gradual onset or in controls (p<.007 for both comparisons), and (iii) IL-10 levels were higher in the patients with abnormal spinal fluids than in those with normal fluid or controls (p<.025).

    The presence or absence of FM did not alter the outcome.

    The results support two hypotheses: that some CFS patients have a neurological abnormality that may contribute to the clinical picture of the illness and that immune dysregulation within the central nervous system may be involved in this process. Lower levels of GM-CSF (a growth factor) in the presence of IL-10 and IL-8 can lead to a downregulation of the immune response to certain bacterial infections. Alternatively, IL-10 may have downregulated the GM-CSF in these patients.''
  14. natasa778

    natasa778 Senior Member

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    yes, but my point being that not that much is known about XMRV anyway, so in terms of testing and detection it could be useful apply knowledge we have on other "common" viruses that have been implicated in chronic conditions...
  15. Dr. Yes

    Dr. Yes Shame on You

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    natasa778 -
    I think that's a very good question. I wonder if the WPI or other groups have plans to test biopsied tissue for XMRV (brain tissue would be especially interesting, but that of course is rarely biopsied and would have to be taken postmortem).
  16. usedtobeperkytina

    usedtobeperkytina Senior Member

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    I think this is entirely possible, if not evidently probable.

    We know XMRV can be in tumor cells. We know it can be in immune system cells. And somewhere on here and I think also postulated by Klimas, XMRV can be in neurological system.

    Seems I remember something about blood / brain barrier and that some viruses can go into the brain or neurological system to hide from the immune system.

    Now, I remember reading in Osler's Web that Peterson and Cheney noted that the fatigue symptoms seemed to be first, but even if the fatigue lessened in year two and three of the illness, the neurological symptoms often worsened in that time.

    I have noticed that for myself. Although I it is not conclusive. In October I got the flu. (I took that as a sign that maybe I was gettingbetter, that my immune system may be back to normal since this was the first cold or flu I had gotten in five years.) A week after the flu was gone, I had a CFS relapse. It took three months for me to get back to the level I was at during the summer. But as I was recovering from the relapse, I noticed the cognitive problems get much worse. I was leaving the stove on. Did that three times in two days. Making big mistakes in driving. Loosing things, y'all know the story. I wondered if this was part of the pattern of CFS, likely due to the virus moving to different systems. But at the same time, I thought it could be that the cognitive problems are just as bad when the fatigue is bad, but I can't tell because the fatigue is so bad I don't do hardly anything. If I am on the recliner for days, then I'm not doing anything that would show my cognitive problems.

    But I do remember Mikovitz saying that any immune system activation can turn on the XMRV. So I think the flu I got caused the relapse a week later. The relapse was a week and a half in the bed or dragging in a hungover type haze.

    Tina
  17. ANGELONDUTY

    ANGELONDUTY

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    Greeting's cort...& all,

    i have just posted a fewshort posts, .....bad day. .......

    New here....................u guys are rocking !

    Just wanted to report, that as of a few months ago - i tested pos w/ xmrv - dna ---neg, rha (?)
    of couse, like all..been sick for many yrs....but have become a advade researcher.........& slow w/ emails

    just thanks, for being here !

    Put me down for the ' pos--group ' 4 / xmrv ( is this the right thread 4 this ? )

    live in nc,usa
    grew up in tx.
    Age 58

    blessing's........to all,
    angelonduty
  18. I looked up some of the molecular biology re retroviruses.The reverse transcriptase of a retovius has no proofreading ability leading to considerable variation in the dna sequences of the inserted genome.In short the viruses drift all over the place and produce antigenic shifts similar to the flu virus this with a slow replication rate ,fast degredation in sera make xmrv one difficult bug to detect unless you target the specific deletion sequence in the env gene -this is the only way fto know for sure(if such a thing really exists) that its there and you dont have an endogenous virus this bug is a doozy -on the face of it relatively simple but with real twits in its replication process-----if you dont really understand this virus--designing a study to find it is really tricky-It doesn,t behave like Viruses of this type are supposed to----how it got detected in the first place amazes me----If anything can be tagged as a stealth virus this is it!.
  19. I,ve always thought that ME should be called mitochondrial encephalopathy-----the symptoms of MLAS, a known mitochondrial disorde,r and ME/CFS are virtually the same, virus gets in --immune system activated hpa axis down mitochondrial damage immune system permanently swiched on hpa axis stays down complete autonomic deregulation encephalopathy-------it certainly explains the whole range of symptoms
  20. Advocate

    Advocate Senior Member

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    I looked it up. If some people are peeled off by XAND, maybe others will be peeled off by MELAS.

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