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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. Gerwyn

    Gerwyn Guest

    notice nothing was speculation all latent viruses etc is a fact you are quite welcome to look them up.

    just for the record i'm not asking kurt to do anything at all
     
  2. jspotila

    jspotila Senior Member

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    So say we all.*

    (a Battlestar Galactica reference, if you are less of a geek than me, which is probably a plus for you in any case!) :D
     
  3. julius

    julius Watchoo lookin' at?

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    Yeah, I know gerwyn. I said that I'm not questioning your statements. I agree people can look them up, just like they can look up statements made by other posters. I think it's silly to ask someone to footnote every sentence in a post. This isn't wikipostia.

    I hope you understand that gerwyn. Not an attack on you at all.
     
  4. Gerwyn

    Gerwyn Guest

    i know that Julius thank you my point is really that you can look up statements and verify or refute them.

    you cant do that with speculation however often such speculation is stated. it is difficult for people without a science background to tell the difference

    as you know there is a world of a difference between offering a hypothesis and speculating

    the former is based on explaining observations the latter is not

    no reflection on you either i take your point
     
  5. Hope123

    Hope123 Senior Member

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    Since the question of HERVs and superantigens was brought up, I came across this review article on superantigens while actually looking for something entirely different. (I was looking up something on shock.) I haven't read it all but there is a small section on viral superantigens. Interestingly, the mouse mammary tumor virus, which is a retrovirus, is discussed here. It also talks about EBV and HERVs. The plot thickens.........
     

    Attached Files:

  6. Cort

    Cort Phoenix Rising Founder

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    I can assure you that Gerwyn has not patiently explained everything (and honestly patience is not one of his virtues). Quite frankly I don't appreciate anyone inferring that I or anyone else simply 'didn't like the inferences'. Kurt has gone to a great deal of trouble to try and understand this stuff and I am digging into PCR methodology (that's alot of fun!). Unless you're willing to go to same degree of trouble then please don't infer about his or my or anyone else's motives. Everybody has their points here.

    I am trying very hard to figure this out. I have several times posted large sections from the methods section for Gerwyn and others to look out. I have outlined the sections I considered problematic. I waited for a day before I published a recent blog for someone to find something wrong with my assertions so that I wouldn't make an idiot out of myself. I've specifically asked them to comment and I've received nothing substantial. In fact I assumed that their silence spoke volumes so I went ahead and published.

    My rant is over; there is some really positive news on the XMRV front for a change - check out the Lightning in the Darkness blog
     
  7. julius

    julius Watchoo lookin' at?

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    @gerwyn

    The quotes I chose were the ones that seemed mostly like verifiable facts. That was my point, which I think is the point you are making too, which is that one doesn't have to footnote every fact. People can look it up if they wish.

    However, several of them could be considerd opinion, such as;

    'She knows that Mulv lives in lymphoid cells'
    and
    'No one but a complete idiot would try to figure out if a test worked'

    And there are several points of pure opinion and speculation that I didn't include.

    But again, my intention was not to question your statements (which I know you are aware of), but to show that EVERYBODY DOES IT, and that IT'S OK.

    We are (or should be) all friends here. Let's throw some thoughts and ideas around. Let's grind up a few sausages....hell, make some soup even. But every thread ends up in insults and people getting PO'd about nothing.

    I decided a while back to leave these fora because everything seems to turn into a fight. Seriously, EVERYTHING. But I decided to stay because good things can come of these type of discussions, you just have to get there before it gets stupid.

    So lately I have been trying to put out fires if I can. It's probably a little presumptuous of me, but somebody has to take the middle path.....Koan? you gotta be able to back me up on the middle path thing at least.
     
  8. julius

    julius Watchoo lookin' at?

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    That's really uplifting about the Dr Light study. I agree, that can't be interpreted many different ways.

    Giddyup! :horse:
     
  9. Gerwyn

    Gerwyn Guest

    I think i have been very patient Cort when repeatedly presented with the same speculative arguments time and time again .Kurt keeps recycling the herv and cross infection arguments without any scientific evidence to back them up. I repeat scientific hypotheses are used to explain factual observations . There was a specific reaction with XMRV env which rules out cross reaction as a problem.That is scientific fact and not speculation . Do you really think that many months of peer review would not have considered that. Three days of peer review on the other hand is not enough time to consider anything in that sort of depth.Anyone is entitled to their opinion but an opinion is not a hypothesis merely a subjective viewpoint. I have answered your posts several times today so please dont say you have not recieved anything substantial .I specifically told you i would explain the issues you raised in a post so that everyone could understand what was going on in the study which was attempting an isolation process XMRV in blood.Something never attempted before.

    I have repeatedly told him that the evidence he quoted on Hervs is in vitro or in glass and not applicable to real living cells.i have also told him that the cross reaction theory would not apply in the WPI methodology.it is clear enough in the paper. i understand that facts may be inconvenient but that does not make them less true.

    I know you have outlined sections you consider problematic but the Science peer reviewers did not.They are professional retrovirologists

    i am quite happy to discuss facts with anyone.Any scientist will always challenge speculation
     
  10. Cort

    Cort Phoenix Rising Founder

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    Honestly I was talking about my question - not Kurt's.

    Ok - looking forward to that.

    We are all getting a little touchy - me included.

    In any case we do have some very good, if anecdotal news on the XMRV front. Maybe these questions about the Science or other studies will be moot soon.
     
  11. kurt

    kurt Senior Member

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    That is older but still a nice find, Hope! Here is the section on EBV for those who do not want to wade through the article:

    Then later in the article more discussion of pathogenic effects

     
  12. bel canto

    bel canto Senior Member

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    Kurt, this is not meant to be a personal attack - you may be quite well-meaning. But I think it's important for you to understand how your posts come across to some of us, and, whether it's true or not, why it feels like you could be a mouthpiece for someone determined to undermine WPI. Maybe it's your references to "outside experts", "outside researchers", "inputs I receive regularly from other labs", "I receive inputs from other labs (and also individuals)", many "ifs", ":it seems to me's", "maybes", "hope". "I have been told", "I think", etc.

    You present no new facts and seem to ignore those presented by others -you just reiterate your (or others) theories - you seem determined to convince us that WPI's research is very iffy.

    Cort, I don't think that the criticism re: speculation, etc. is aimed at you. As you noted, your comments, questions, etc. are based on the science that has been published or spoken about by the researchers, and you seem to truly want to understand. That feels quite different. You have been caught in the crossfire.

    And thank you a million times over for your post re: possible good news. Not a rumor, not an opinion, just an eyewitness report of one person's experience in an ongoing study.
     
  13. Gerwyn

    Gerwyn Guest

    the most common cause of Sag manufacture are bacterial infection This work once again involves IN VITRO experimentation

    In 1996, Sutkowski et al. observed that
    Epstein–Barr virus (EBV) infected human B cell lines note human B cell lines

    Although there is no direct evidence of SAg
    involvement, it has been suggested that S note yet again no evidence just a suggestion

    So i,m afraid the plot does not thicken at all
     
  14. Gerwyn

    Gerwyn Guest

     
  15. Adam

    Adam *****

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    Originally Posted by oerganix
    Yes, it seems to me that Gerwyn or someone else has patiently explained or countered every item, then someone has doggedly circled round the block and brought it up again and again. I don't know if that means they didn't understand the explanation, or they just didn't like the inference.

    And FWIW, I don't need anyone to keep me from getting "too hopeful" (whatever that is) or to keep my feet on the ground. No need to save me from myself. I feel insulted and talked down to if anyone is posting negative speculation on XMRV just to keep me from getting too excited about it. Like Adam, the rumors and speculations I've seen here have been predominately negative on XMRV. Of course, if there is some real science that disproves WPIs preliminary study, bring it on. None has appeared so far.

    And to describe any of Dr Mikovits's statements, at least those I'm aware of, as "lamblasting" anyone is hyperbole itself. Dr Vernon's speculation, if what Cort says of her is true, that purported flaws in WPIs study might somehow lower the interest of the rest of the scientific community in XMRV just isn't supportable by the facts. There are studies on XMRV going on all over the world. Governments, private researchers and BigPharma are all interested, no matter whether the criticisms of WPI are born out or proven to be more smoke.

    What I am seeing is that whenever someone expresses a hopeful sentiment, there are some who then express a negative speculation, even when the hopeful sentiment was qualified with the acknowledgement that we don't know anything for sure. That feels like an attempt to squelch hope and I don't think that's helpful or healthy.

    Ditto. I am big boy now. All grown up. I'd still give Judy M a big fat kiss even if XMRV bombs.

    I don't get negativity. If I had been negative about my illness I would be a bunch of pills by now.
     
  16. Gerwyn

    Gerwyn Guest

    The following is a guided tour through the original XMRV study conducted by the following personnel.

    I have added some contextual information and punctuation where I think appropriate to facilitate understanding


    Vincent C. Lombardi, 1* Francis W. Ruscetti, 2* Jay dip Das Gupta, 3 Max A. Pfost, 1
    Kathryn S. Hagen, 1 Daniel L. Peterson, 1 Sandra K. Ruscetti, 4 Rachel K. Bagni, 5
    Cari Petrow-Sadowski, 6 Bert Gold, 2 Michael Dean, 2 Robert H. Silverman, 3 Judy A. Mikovits1†


    This is the cohort information that seems to cause so much confusion:


    Banked samples were selected for this study from patients fulfilling the 1994 C.D.C. Fukuda Criteria for Chronic Fatigue Syndrome (S1) AND
    The 2003 Canadian Consensus Criteria for Chronic Fatigue Syndrome/myalgic encephalomyelitis (CFS/ME) and presenting with severe disability.

    These are patients that have been seen in private medical practices, and their diagnosis of CFS is based upon prolonged disabling fatigue and the presence of cognitive deficits and reproducible immunological abnormalities. These included but were not limited to perturbations of the 2-5A synthetase / RNase L antiviral pathway, low natural killer cell cytotoxicity (as measured by standard diagnostic assays), and elevated cytokines particularly interleukin-6 and interleukin-8. In addition to these immunological abnormalities, the patients characteristically demonstrated impaired exercise performance with extremely low VO2 max measured on stress testing. The patients had been seen over a prolonged period of time and multiple longitudinal observations of the clinical and laboratory abnormalities had been documented.”

    Note the objective criteria for diagnosis and the MEASURED impairment of Vo2. I struggle to see why experienced people can’t see that this is a different patient cohort to the English studies. If I am reading the above correctly then ALL patients satisfied the Canadian and the FUKUDA criteria.

    I have written seeking confirmation

    It is important to remember that the paper consists of quite separate experiments.

    Here is the first one:


    DNA and RNA isolation.

    Whole blood was drawn from subjects. The blood was fresh not frozen.


    PBMC were isolated by layering the diluted blood onto Ficoll-Paque PLUS (GE Healthcare, Waukesha, WI), centrifuging for 22 min at 800 g, aspirating the PBMC layer and washing it once in PBS. The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 C for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 C for DNA and RNA extraction and analysis.”

    Note the unactivated PMBCs were frozen for culture and further experiments

    OR

    resuspended in Trizol for nucleic acid extraction OR further experimentation

    “PCR methodology on extracted
    DNA

    PCR. To avoid potential problems with laboratory DNA contamination, nested PCR was performed with separate reagents in a separate laboratory room designated to be free of high copy amplicon or plasmid DNA. Negative controls in the absence of added DNA were included in every experiment.

    Identification of XMRV gag and env genes was performed by PCR in separate reactions.

    PCR amplification for sequencing full-length XMRV genomes was
    performed on DNA amplified by nested or semi-nested PCR

    PCR analysis performed on 20 of the identical patient PBMC DNA
    specimens stored at the NCI (Frederick, MD) since 2007 confirmed nearly identical gag sequences, thereby diminishing the possibility of laboratory contamination as a source of XMRV”

    This dealt with the issue of contamination and replicated the findings of this part of the experimen
    t.

    It is important to note that PCR was the approach taken in the Imperial college and Groom studies. The claim that the PCR methodology needed validation is objectively false. It had already been achieved by an independent laboratory.


    “Nested RT-PCR for gag sequences was done as described”

    Neither the Imperial college study nor the Groom study even attempted to do this despite looking for a RNA virus. RT –PCR looks for viral RNA. You don’t need replicating virus for this test but you do for PCR

    Hence both studies based their approach on the assumption that the virus if present would be replicating.

    The significance of the RT approach will be apparent in the conclusion.

    The DNA extracted above was used to from the whole viral genome which was then compared to all other known Mulv viruses both exogenous and endogenous this was also done at the National cancer institute laboratory.The presence of all MuLV viruses were excluded during this analysis.

    Both sets of researchers confirmed that the viral genome corresponded almost exactly with the VP^2 genome as produced by the original discoverers of the virus. The match was greater than 99.9%

    Ok, good point for a summary


    The FIRST experiment showed that there was a presence of XMRV gag and XMRV env in the sample of concentrated Peripheral mononuclear cells (PMBCs) taken from FRESH blood. The Viral DNA was sequenced and compared to all known MuLVs and was different to any. It was then compared to the DNA sequence of the original XMRV isolated and genotyped by the discoverers of XMRV and it was an almost perfect match. The PMBCs of patients diagnosed according to the criteria in the study contained XMRV DNA.

    NEXT EXPERIMENT. All sections also contained controls using the same parameters isolated from healthy donors

    Leucocytes (a kind of white blood cell) were isolated from the FROZEN PMBCs of patients and fresh PMBCs of healthy donors.

    These were then activated (PHA).

    They were then cultured for 42 days with sub-culturing taking place every 3 to 5 days.

    That was the first amplification procedure.

    CD4+ T and CD3+ T cells were isolated and further cultured.

    That was the second amplification procedure

    The CD4+T cells were further amplified and activated by culturing with IL2.

    CD19+B cells were also isolated from the activated leucocytes and the concentration amplified by further culture.

    Ok now we have hugely amplified concentrations of activated T and B cells to work with.

    Bear in mind that the English studies not did not isolate PMBC cells from FROZEN blood and amplify them in any way. The concentrations of virus in this experiment were huge in comparison to the levels to be found directly from whole blood.

    The next part of the experiment is divided into two parts:
    [/e cultCOLOR]
    The cultured T and B cells were separated from the medium they grew in (the supernatant).

    The cells were broken up (lysed). The protein content was isolated (Flow cytometry and Western Blotting).

    Protein products of gp55 env, gp70 env, p30gag and p10 gag. were identified.


    Next step was to see if there was infectious virus.


    Was the virus able to leave T and B cells and get into the supernatant

    LnCap cells were cultured with the supernatant using an accelerated culture/ transfect ion investigating cellular infection and cell transfer.

    The protein content of the LNCap cells was analysed as above and produced an exact match.

    Nested PCR revealed the presence of XMRV gag.

    Allele specific PCR revealed the presence of the XMRV genome VP62 which was the same sequence identified in experiment one.

    Conclusion
    There was infective XMRV present in the original PMBC’s from the CFS patients diagnosed by the criteria used in this study.

    Experiment 3.

    Plasma from 9 out of 18 cfs patients provided a specific response with SSFV-env.

    Plasma also blocked the reaction between SSFV env and SSFV env Mab

    Conclusions

    XMRV mounts an immune response in plasma from patients with ME/CFS.

    The possibility of the presence of other Mulvs had been excluded in experiment one. Hence no possibility of a cross reaction.

    PCR Hit rates

    Nested PCR on fresh blood PMBC’s 20% hit rate

    This means that at this concentration of PMBC’s derived from 8 ml of fresh blood you would need to run a PCR assay 4 times to get a hit on viral RNA.


    If you realise that McClure used 2 ml of frozen whole blood, then even if she had concentrated her sample she would have had to run her PCR 20 times to get one hit. Of course as the sample was much more dilute she would have had to run her assay many more times than that. There is a specific issue with the Groom samples which I will post at a later time.

    RT-PCR on fresh blood pmbc isolates rate 50%. This is the one the English did not bother with at all. You need to run it twice to get a hit.

    Nested PCR on amplified and activated PMB cells from FROZEN blood c90%

    You virtually only have to run one PCR assay.

    This is the point –if you want to try to isolate viral DNA from frozen blood then your chances are hugely improved if you isolate the target cells, activate them and expose them to multiple amplification procedures BEFORE PCR.
     
  17. Adam

    Adam *****

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    Many many thanks Gerwyn for a full explanation of what WPI undertook and pointing out the variances witht the other studies in a way that us plebs can follow if not necessarily fully understand.

    I gotta feeling though...maybe...perhaps...you never know...well...I'm just saying...you might not have satisfied everybody.

    I have dubbed you the Welsh Wizard. Please post or PM me if this term of endearment annoys or embarasses you.

    all the best

    Adam
     
  18. Hope123

    Hope123 Senior Member

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    Here's the section about MMTV as a superantigen. I bring this up because I want to remind people to keep their minds open. Just because bacteria are the most common examples of superantigens, it doesn't mean that viruses (and even fungi) can't be direct superantigens. No one knows yet what the mechanism is for this illness and I am not ruling out that CFS could be a retroviral illness that induces superantigens as a mechanism, among other ideas. I believe those researcher and docs who are willing to keep an open mind will not only solve this condition(s) but will change fundamental ideas we now hold or discover new paradigms, e.g. the discovery that HHV-6 can be transmitted via integrated chromosomes in families when it was previously thought herpes viruses did not intergrate into chromosomes.

    Mouse mammary tumour virus (MMTV)

    MMTV, a milk-transmitted B-type retrovirus, causes murine mammary
    carcinomas. The MMTV SAgs were discovered first by Felstenstein
    in 1974 and were referred to as minor lymphocyte
    stimulating (Mls) antigens. The T cell response to Mls antigens is
    similar to the response to bacterial SAgs with expansion of unique
    TcR Vb subsets [53]. The SAg gene was identified later within the
    3 long-terminal repeat (LTR) of the MMTV genome and did not
    show any homology to the bacterial SAg genes [54]. The gene
    product is a 45-kDa type II transmembrane protein with a 10–14
    amino acid polymorphic region at the C-terminus, which is responsible
    for the TcR Vb specificity. Infectious MMTV is present in
    mammary tissue and breast milk of only a few mouse strains. The
    SAg molecule is an essential component of the virus life cycle, providing
    efficient viral replication in newly infected gut B cells by
    recruiting Vb mediated T cell ‘help’ and promoting B cell proliferation.
    The endogenous SAg is inherited in Mendelian fashion
    and causes T cell deletion as a result of self-tolerance induction in
    the thymus. As a result, the transmission of an infectious virus carrying
    the identical SAg will be hampered by the lack of responder
    T cells, thereby protecting the mouse from MMTV infection.
    E

    (BTW, MMTV is being studies in human breast cancers and primary biliary cirrhosis.)
     
  19. dannybex

    dannybex Senior Member

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    Yes, thank you Gerwyn for taking the time and 'energy'(!) to write that all out in laymen's terms. It makes it so much easier to at least try to understand.

    It must've taken you hours and hours to do that -- I really appreciate it!

    Dan
     
  20. Dr. Yes

    Dr. Yes Shame on You

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    Kudos Gerwyn... Hope you can work with Cort in turning this into an article... it deserves front page placement!
     

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