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Why does 5AZA matter?

Discussion in 'XMRV Research and Replication Studies' started by Lee, Oct 6, 2011.

  1. Lee

    Lee

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    Bob,

    JM claimed to have found virus using simple techniques. Of course those many labs that dropped some part of what they were already doing, followed up the Science paper, tried to confirm and perhaps extend JM's (alleged) work - and found it didn't work. Now, it seems, we know why.

    JM claimed "XMLV is expressed in the activated PBMCs of CFS patients." That is the claim that people were confirming and trying to follow up. You're asking them to have umped to look for something else that she didn't claim?

    5AZa and demethylating agents find lots and lots of things. Even the experiment I describe isn't definitive - If one finds some kind of virus-like expression, one still hasn't ruled out that CFS makes PBMCs respond differently to demethylation. That also is a reasonable hypotheses - remember, this is a p neuro-immune disease, and it makes the immune system act differently. That would be a preliminary experiment, start of a whole ton of work, and no lab is going to commit several people to that work, instead of something currently productive, without evidence that there actually is a virus. Right now - there is no evidence that there actually is a virus. What there is, is evidence the JM et al made claims that were not true and that they cant support.

    That's part of the tragedy here - no one knows what might be real and what is made up, and it is going to be a rare lab that, given a choice of productive work they have going that is leading toward helping people, and pursuing this work on CFS which they have no way to know actually ever really happened - is going to choose this path.

    I hope that part of the investigation goes through JM et al's notes and results and tries to identify if some of what JM did is actually real.

    I hope more work toward trying to identify causes of CFS goes forward. Virsu screening - broad-based, not just following up this s*storm - should be pursued. It looks like the funding and some labs are going there. There are interesting hypotheses about persistent activation of ERV expression, which I think is ongoing and worth pursuing.

    But following up the JM stuff? I wouldn't do that, fi I were running a lab and choosing research directions and wanting my lab to be funded and active and successful n 5 years. Not now, not after this.
     
    Sam Carter likes this.
  2. Lee

    Lee

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    Bob:

    "You can say that as many times as you like, but it is still just speculation based on a partial knowledge of events."

    Mp it is NOT speculation. They have admitted to changing the labels on the gel, and presenting a gel as one thing when it was actually something different. They have admitted to using a demethylating agent, in an experiment sensitive to demethylating agents, and not telling us they did it.

    That's enough. That is discrediting. That means they can't be trusted - they mislead us. They told us they did it.

    I'm off soon for Kol Nidre, and I've pretty much said my piece here anyway - I'll check back in after the Holy Day is over. But I'll add just this. Every scientist I know who is following this does not trust a single piece of this work now. None of it. That's what happens when a scientist finds out another scientist intentionally substituted data and misled readers about their experiment.
     
    Sam Carter likes this.
  3. leela

    leela Slow But Hopeful

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    My recollection is that she said it was *not* easy to detect using simple methods. My recollection is that she has had to
    explain this repeatedly.
     
  4. Bob

    Bob

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    Well, it's a shame that you are not prepared to engage in a conversation and read other people's posts, but prefer to continuously repeat your opinions without considering all of the facts and all of the issues that have been raised on this thread.
     
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  5. Bob

    Bob

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    Well, if other researchers are not prepared to put the work in to look for a possible novel retrovirus and do exploratory work, but they just want it handed to them on a plate by another researcher, then I'm glad that Judy Mikovits came along and did what no one else was prepared to do.

    I'm beginning to have more respect for Judy Mikovits than I've ever had before, now that I realise just how much work she has done compared with some other retrovirologists. It's suddenly dawned on me why these other researchers had no chance of ever detecting a novel human retrovirus.

    I was beginning to think that XMRV might be not be a human virus after all, but now I realise how stupid I was to assume that the authors of the negative studies had actually devoted time, expertise and resources to do independent investigative research.

    Thank you for opening my eyes to reality.
     
  6. Snow Leopard

    Snow Leopard Senior Member

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    It is a non-sequitur to assume the money spent on XMRV research after the publication of the Lombardi would have been less than if it that study had not been published. The same debate was going on with the prostate cancer results, it is quite likely that a similar amount of money would have ultimately been spent, only it would have occurred over a longer period of time. It would have been different, but not necessarily better or worse. Instead, due to the controversy, there has been much more XMRV research and we now know that all of the results (in prostate cancer too) are highly suspect - we now have a reasonable answer. Given the risk, it is hard to say that the overall money was a waste as the virus can demonstratively infect human and macaque cells, we are simply fortunate that no one has yet been infected (as far as we know).

    In terms of the whole 5AZA issue, is there any evidence that the Science referees did not actually know about this aspect of the methodology? Because otherwise you are out on a limb.
    Regardless, I would put any consequence of this detail down to a failure of the peer review process. (peer review is rarely ideal)

    But mistakes and methodological grey areas happen more often than you think. I know of more than a few stories where researchers have omitted potentially relevant details (such as only publishing the results of modified protocols as the standard protocols did not work, neglecting to mention the failed experiments). This is more common than you might think, it is accepted so long as the full details of the actual method used are provided to the referees. It is actually a major oversight of scientific publishing these days - the results of many failed experiments are not published.

    Still, at the end of the day, scientists need to have the freedom to make mistakes.
     
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  7. ukxmrv

    ukxmrv Senior Member

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    KFG, you may find it hard but some patients who have a knack, or a high IQ, or training in similar areas or access to researchers and information find it is a hard slog to keep up with the science but apply themselves and are pretty good with their understanding.

    It's not for everyone and if you personally cannot do this just respect the patients who are able to.

    CFS has been filled with scientists, researchers, doctors and govt agencies with vested interests.

    I'm been reading the research in journal form for over 20 years. It can be hard to keep up but so many parts of what we hear are just opinion.

    Scientists have got it wrong before. It doesn't need to be a conspiracy. Some people are just wrong and it takes time for the truth to come out.

    We've been hearing opinions. I'm in touch with scientsists who don't agree with the HGRV detractors. They don't feel the need to join the patient groups though and think it unprofessional.

    Just because you have heard only only type of opinion doesn't mean that it is right.
     
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  8. Bob

    Bob

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    KFG, Lee is correct in his analysis of how the widespread use of 5AZA would affect the outcome of the Science paper.
    However, he cannot know the significance of the single 5AZA slide, because he is not in possession of all of the facts.
    He has made assumptions in order to come to his premature and possibly misplaced conclusions.
    Please read Mark's post to find out why he is making assumptions and why he cannot legitimately make the conclusions that he has made.
    None of us, including Lee, can possibly know the significance the 5AZA slide based on our current knowledge about it.
    One other thing... Lee does not represent Scientists... He is just expressing his own opinion.
    He may or may not know a few researchers (we don't know if he does), but even if he does, that doesn't mean that he knows the opinions of the majority of retrovirologists.
    Like you say, we will have to wait until Science complete their investigation.

    There are a number of very high profile names attached to the original Science paper, and I find it very hard to believe that they were all involved in some sort of mass fraud.

    KFG, you say that the comments directed at Lee are unwarranted, but if he insists on making unsubstantiated extreme accusations about the conduct and integrity of such a large number of ME/CFS researchers, then he is bound to attract controversy. Lee has a rigid point of view, and he has clearly joined the forum to promote that point of view in no uncertain terms. However, his opinions do not stand up to scrutiny.

    His opinions and conclusions may yet prove to be right, but at this stage he does not have the evidence to justify his statements.

    Again, please see Mark's post which explains all of the reasons why Lee's opinions cannot be justified at this point in time.

    You say that we should respect the professionals. Should that respect extend to Alter, Lipkin, Silverman and Ruscetti?
    Should it also extend to Singh who has found XMRV in 5% of the normal population?
    Should it also extend to Switzer who has found XMRV in prostate cancer patients?
    And should it extend to Coffin who has found XMRV in human cell lines?

    We just don't know enough about XMRV yet to make any conclusions.
    Sure, there have been many negative XMRV studies but, as Lee explained, not many of these studies were exploratory in nature.

    Even if XMRV turns out just to be some sort of contamination in the ME studies, there is still a lot to find out about this novel human retrovirus.
    And if it turns out to be a retrovirus contaminant, then that doesn't make Judy Mikovits ( or Silverman, or Ruscetti, or Lombardi, or Peterson) bad scientists or mean that they are involved in fraud.
    Whatever the outcome, they will have furthered the field of retrovirology, they have futhered science, and they have deepened our understanding of human gammaretroviruses and retrovirology and they have attracted a lot of interest and futher research in ME.
    Thay may also have discovered a biomarker and a further avenue of research in relation to the antibody results.

    However, I do agree that we do not know how the XMRV story will end.
     
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  9. Bob

    Bob

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    Hi KFG,

    Thank you for your friendly message.

    It's really refreshing to have a friendly and supportive conversation with someone who doesn't necessarily have the same points of view as I do.

    Yes, I'm afraid that I have been feeling frustrated with the forum over the past week or two. The forum has been a bit tense, and maybe I haven't reacted very well to it all.

    I'm sorry that you've had a rough time of it as well. I think most of us are feeling the same at the moment, whatever our points of view about the XMRV situation.


    I do try to understand other people's points of view, and I like to engage in discussion and I try to stay open minded.

    But when non-patients join the forum just to impose their pre-formed, rigid, but unsubstantiated and controversial opinions onto ME patients, and state their opinions as fact, then I'm afraid it does lead to a certain amount of frustration. Certain people clearly don't have an interest in ME patients (nor do they understand ME, the history of ME or how research affects the lives of ME patients), but only seem interested in promoting a certain point of view based on their own narrow interests. I probably haven't dealt with this frustration as well as I'd like to.

    Even if you and I don't share opinions about XMRV and the WPI, at least we both understand the perspective and lives of ME patients, so it's helpful and interesting to hear your point of view.

    And we can both understand each others frustration with the current situation.

    Thanks again,

    Bob
     
  10. Guido den Broeder

    Guido den Broeder *****

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    Coming from another field of research, I want to stress the following.

    When I finish a research project, I publish a research report. The report contains ALL the information, including all the details of the applied methods. Only then will I consider writing an article for a journal - which obviously contains much less material and detail due to lack of space.

    I find it quite disheartening that in a field where details can be so crucial, it is apparently considered normal to jump to the publication of an article right away, thereby skipping a layer of documentation. Something will always be missed. If not 5AZA, then it would have been something else. This mess is therefore the WPI's responsibility, not that of any individual scientist.


    Now, about 5AZA. I can understand very well why it was one of the many details that did not make it into the Science article. The reason must be that they all believed that Silverman had already identified XMRV and that, therefore, the image was merely an illustration.

    Only now that we know that Silverman's result was a contamination, the data must be re-examined. And it could well be that the results for these two patients now form the most important piece of evidence that was produced. Because, while it is possible that it merely shows the presence of an innocent ERV, that is not likely.
     
  11. kurt

    kurt Senior Member

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    The other researchers did not need to use 5AZA as they were running PCR testing for the most part, and more advanced real-time PCR tests with higher resolution than the ones used by WPI. This type of PCR can find a sequence regardless whether it has been 'activated' or stripped out by a demethylating agent. Modern PCR also finds embedded sequences just fine.. As was explained to me by one researcher, 5AZA and this whole issue is irrelevant to the fact that PCR testing by dozens of labs failed to find any evidence of ANY member of the MLV family in ME/CFS patients. This is because unlike WPI, they tested the conserved portion of the genome (the pol gene, which must be preserved in all viral family mutations as it controls reproduction of the virus).

    Guido, I also was a published researcher in another field prior to ME/CFS, and agree that this was sloppy but common when pubs are rushed to print. However, from my own research experience, my complaints with WPI are based on the non-scientific handling of the whole situation the past two years. They have behaved more like marketing company than a research lab. I guess given the background of the principals at WPI that is not a surprise (The Whittemores own an energy drink company and Mikovits is an executive level vitamin salesperson, in a MLM company, they both knew how to sell). Anyway, were the gel issue a critical detail I think there would be more reason to investigate. However, in light of the many fully competent PCR tests that produced 0/0 results, this whole gel flap is a non-issue. Much ado about nothing. Going after JM or WPI at this point will not help us much, that game is finished. XMRV can not be found by well-designed PCR, nor can any of its MLV varients, for me that is sufficient to conclude the hypothesis can not be validated, and move on to the next research topic. The many arguments about whether the outside lab tests were competent have been based for the most part on a poor understanding of lab testing methods, anyway that is what I have been told by experts. But people only hear what they want to hear.

    The question I have had all along that has yet to be answered is this: if ME/CFS were caused by a blood-borne retrovirus, then how did we all contract this illness? And how exactly did the outbreaks happen? I don't think there is much probability ME/CFS is caused by a single blood-borne infection such as a HGRV. That is a big-picture perspective, I think we need to make certain in the future that when specialists study ME/CFS they first learn about our illness, and prove that they understand enough to avoid making obvious errors like looking for a blood-borne pathogen in an illness that has a completely different epidemiology, is not an STD, etc.
     
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  12. Bob

    Bob

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    Interesting points you make Kurt.

    If there is an interplay between genes and a pathogen(s), then some people could be carriers without being ill, and maybe that could explain why it does not look like the behaviour of an infectious pathogen. Only those people who are genetically vulnerable would become ill. This would suggest that ME/CFS might run in families, and I don't think that we have had large scale epidemiological studies to tell us whether it does or not.

    So I certainly agree that we need a better understanding of the epidemiology, and it would be helpful if a quality large scale epidemiological study was carried out.
     
  13. alex3619

    alex3619 Senior Member

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    Hi Kurt, you may well be right about XMRV and ME although we wont know for sure either way until after Lipkin finishes the NIH study.

    What is wrong about your argument is something I have explained many times on PR, based on a model I was working on several years before XMRV was ever heard of by patients.

    This is the two hit hypothesis (although it could just as easily be three hits or twenty hits, so maybe multiple hits hypothesis is better, but thats not what I called it at the time). It is widely regarded that developing ME requires multiple factors. Some think genetic, toxic and viral factors are nvolved for example.

    Taken in context with a slow replicating retrovirus, first you get infected. Then it slowly grows. The immune system is a little weaker. Then there is an epidemic of something else - EBV being a common one, but coxsackie and other viruses will do it too. Indeed, it does not have to be a virus, just anything that stresses the immune system. The key factor is a severe immune challenge. Toxins may well fulfill this role.

    The retrovirus can multiply by normal viral replication, and spread rapidly in the body, during such events. In addition, rapidly replicating immune cells can cause viral replication via clonal expansion. Now you have a massive retroviral presence and the immune system takes a sudden nosedive. Now an unnoticeable infection becomes very noticeable.

    The incubation period for ME clusters thus appears to be the incubation period of the new infection - but the underlying cause may have been contracted many years prior.

    This explanation makes a lot of sense, but just because it can be so does not make it right.

    There is also the possibility that a retrovirus is present that is not even a gammaretrovirus. I keep wondering why nobody is trying to do sensitive reverse transcriptase testing. Is it because a slow retrivirus that uses mainly clonal expansion is a poor candidate so they never bothered? Did they look and not find it enough to justify publication? Hasn't it been shown before? I vaguely recall that it has, perhaps with deFreitas.

    The main competing theory to the retrovirus idea that explains most of the data is essentially the neuroimmune disturbance hypothesis. I have talked about this at length in the past, but it is interesting to note that Freidman is now saying that most ME and CFS researchers think it is dominant.

    This is a problem which something triggers an immune response. The immune system is weakened by whatever this is - toxin, virus, bacteria, injury, whatever. The degree of the immune damage is critical. One or more viral infections that are latent reactivate if the current infection is not viral. The viruses spread but are incapable of properly replicating - the cause of this is not specified, but it is known to be demonstrated clinically in up to 80+% of patients. The body induces emergency antiviral measures for the duration ... but they don't eliminate the virus. Because of that the emergency never ends, and the body keeps fighting.

    NK cells may play an important part in this. I suspect that initial NK cell dysfunction is key, and this may not just mean usual activity. Something may be stopping them from activating like they should. We need to understand what is happening here.

    My version is slightly different in that I proposed that RNase L was critical here. I proposed that the 37 kDa RNase L was part of an emergency antiviral defense - and that means elastase and possibly other enzymes are the trigger. This model is not limited to RNase L however.

    We now know that elastase is elevated by LPS translocation from the gut (I am still not convinced of bacterial translocation). So increased gut permeability or a failure in gut and liver detox could be a key part (the gut is the first detox step, and the liver eliminates the rest of the LPS). The existence of prior IBS is thought to be a precursor to ME by some but there is not enough research to be sure of this.

    So the pattern is either -

    retrovirus - slow growth - trigger event - rapid retrovirus expansion - immune crash

    or

    immune trigger- severe immune challenge/high tissue viral load - emergency defense - viral load remains - permanent emergency defense. (LPS translocation may belong here somewhere.)

    This latter theory appears to be gaining dominance. Various versions of the activated emergency state also exist, although I tend to think of them as complicating factors for now - like the NO/ONOO theory or methylation cycle failure.

    Another feature of my version was the nature of the infection. Is it just severity? I don't think so. I think there is likely to be part of the body, maybe several locations, where immune insult will trigger the emergency defense. Leading candidates for me are heart, brain and spine. The viruses that are common triggers for ME attack all three.

    Bye
    Alex
     
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  14. kurt

    kurt Senior Member

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    Alex, I don't know enough to critique your theory in depth, but I do see some clear reasoning with what you say. But the point that I think is most important in that type of analysis is to realize there can be multiple hypotheses that can explain the same phenomenon. Unfortunately in any scientific area like ME/CFS research only people with expertise will fully understand what is and is not credible. So what has happened among patients is that people have listened to non-expert theories so much there are patients who actually believe a retrovirus is the only rational explanation for ME/CFS. In fact I believe the epidemiology issue is a hole in the blood-borne retrovirus theory, a hole big enough to drive a truck through. If ME/CFS were caused by blood transfer that would have been obvious right from the start. And Genetic differences and universal (vaccination) exposure ideas don't really work either unless there is a specific ME/CFS gene and the HGRV is only a problem for someone with that gene, and that seems highly unlikely given the variations seen in ME/CFS. That type of scenario is binary, either you have it or you don't and if you have it the biological profile is clear and consistent from patient to patient, there are many diseases like that but ME/CFS is not one of them.

    Retroviruses are NOT the only reason an immune system can malfunction!!!! There are autoimmune conditions, allergy conditions that lower immune levels, even prolonged stress alters immune function. In fact there are ordinary viruses that behave as if they were retroviruses, they multiple rapidly and create similar immune trouble to a retrovirus. Norovirus (or 'cruse ship' virus), for example, is so virulent it is classed as a pseudo-retrovirus, even though it is an ordinary virus. The Herpes viruses can be as virulent as a retrovirus and they can alter immune function. Candida and EBV and some parasite/protozoa infections alter immune function. HPA problems alter immune function, even low ATP from methylation or mitochondria disorders can cause immune problems as ATP is required in immune activation. Then there are genetic immune system problems that can be related to this (RNasL, probably others). There are MANY different scenarios that can work to explain the immune and neurologic wierdness of ME/CFS. There are probably hundreds of possibilities, many to track down.

    But I would agree that XMRV and then HGRV were an elegant idea, easy to understand. A single virus, a single disease, it would have been nice to have an easy answer, a pill to take, and a way to automatic medical and social credibility. XMRV was a very marketable idea, it just did not work out. And as I said before, that means the gel issue is a pointless pursuit. I don't care whether it was caused by carelessness or a pattern of exaggeration or whatever, pursuing the gel issue will not advance our cause, going on a witch hunt will not advance our cause. Only staying the course and continuing to support research and advocacy will advance our cause. And it would be nice if the ME/CFS community could find something to agree on...
     
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  15. Firestormm

    Firestormm Guest

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    I have never believed personally that this was 'fraud' Bob. It was a mistake an oversight, but in light of everything else (especially the partial retraction by Silverman) and the simple fact that the same experiment was being presented differently - it is nevertheless important. In terms of the Lombardi et al paper if the original headings had been shown in full that might have helped - but for some the fact that this 5AZA does not appear to have been applied to the controls appears to be another question mark. And then you throw the BWG conclusions into the mix and well all of the resulting concern is hardly a surprise really.

    To what extent this Western Blot experiment with its' unrevealed labelling will matter in the Science review of the paper - that began before Mikovits and the others were asked to voluntarily retract - I really could not say, as since then of course Silverman has retracted based on contamination and those concerns were (if I recall correctly) the overriding concern expressed by Science. In terms of 'da media' (and patient et al. forums) this experiment and Mikovits' firing together with the seeming 'implosion' over at WPI are matters that are of concern but also something people need/want to talk about. Speculation has happened ever since Science published Lombardi et al. - these latest events were hardly going to go quietly, were they?
     
  16. Guido den Broeder

    Guido den Broeder *****

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    Perhaps. Could that be why Peterson quit?


    That I can't agree to. The PCR studies that produced no results were at best aiming for Silverman's contaminator, which we now know did not produce Mikovits' gel. Some of these studies did not properly select patients, and none (AFAIK) used 5AZA. This game is not finished, rather it is just starting for real.

    As with AIDS, the retrovirus does not cause the disease. It merely compromises the immune system. To cause ME two more factors are needed: the opening of the blood-brain-barrier, which a latent herpes virus can do, and a trigger that leads to inflammation, which is most likely an enterovirus, explaining the possibility of an outbreak.
     
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  17. free at last

    free at last Senior Member

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    Moderation Team: Text removed due to inflammatory content and personal attacks. Please refer to the PR forum rules, section 5 for further clarification.
     
  18. leela

    leela Slow But Hopeful

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    Where Did My Post Go?

    I was sure I posted in reply to Alex's post #77.
    I told Alex I very much liked his post and was looking fwd to rereading it, I asked Kurt what area of research he used to be in.
    Did it get moderated for being overly polite, or did it just fail to post?
     
  19. Dainty

    Dainty Senior Member

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    I can assure you that no such post has been removed by moderation. There's nothing here against being polite! :Retro tongue: Sorry the computer ate it. :(
     
  20. jace

    jace Off the fence

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    Kurt says, in his post #75
    Well knock me down with a feather! I've never heard this before! Is it true Kurt? Is there any substantiation available? I've heard that Mikovits worked in a yacht club bar before, but a vitamin salesperson is a new one on me.

    Actually, I am doubly surprised, in that her career so far with Ruscetti in the NCI labs and gaining her PhD hardly seems to leave time for such commercial endeavours. Mind you, many students do a bit of bar work to fund their studies...

    Leela, doesn't Kurt work on computer based training or some such?
     

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