NIH SoK - Alter's wrap of Mikovits and Coffin's presentations

[video=youtube;hWN3rkbXCm4]http://www.youtube.com/watch?v=hWN3rkbXCm4[/video]

When two women claim to be the mother of the same child, King Soloman made a judgement to cut the baby in half and resolve the dilemma, and it was resolved when the real mother gave up her share to save the baby's life. Well, I'm no Soloman, but I'm stuck here between the diametrically opposed viewpoints of Judy Mikovits and John Coffin, both of whose science I greatly respect, and I don't truthfully know who to cut or what to cut, except that I'd like to cut out of here. (laughter)
So I think I'll just try to review the two presentations and then we'll see see where we go from there.

So Dr Mikovits told us that she had detected gag and env sequences of XMRV and more recently of PMLV's in a high proportion of patients with this syndrome, and a dramatically lower number in healthy controls. In the original publication 67% versus 3.7%. She has also detected antibodies in the majority of patients who have these viral sequences. She has detected XMRV in serial samples from the same patient over time. She has cultured XMRV from PBMC's and even better from plasma, from patients that are PCR positive from either gag and/or envelope using these LNCaP prostate cancer cell lines. She has found no evidence for contamination of the LNCaP cell line, or of the reagents that she's utilised in her lab, she's tested innumerable healthy controls, and assay controls, they've always tested negative, she's tested negative controls to be negative in coded panels.

She claims never to have had the 22rv1 cell line in her laboratory, the one that Dr Coffin found to be contaminated. So Dr. Mikovits concludes that mouse leukemia-related gamma retroviruses can infect humans and are strongly associated with Chronic Fatigue Syndrome, and it sounds quite plausible.

This is just one data slide from her to show the link, the coexistance of the virus and the antibodies in most of the patients tested, although some have one, or the other.





So, if you just saw that, you'd be relatively content, but Dr Coffin presents his data from Oakes where wherever XMRV gag sequences were found they also found evidence for mouse mitochondrial DNA by those assays, or he prefers the IAP assay.
Not only that, but many of the samples that tested negative also had mouse genomic sequences.



They concluded that the positives are due to sporadic contamination with mouse DNA.

Dr Coffin went on to show that exogenous murine leukemia viruses endogenised in mice, but at some point the receptors changed, and these endogenous viruses became unable to infect mice but now were xenotropic and then could infect other species, including man. But the key in his analysis here is that in the original prostate cancer studies they took prostate cancer tissue and they took it from the tumor and passaged it in nude mice, grafted it in nude mice, and then again and again, and it's possible that as it was passaged in the nude mice that xenotropic viruses in the mouse contaminated the prostate cancer cell line, and this happened over time, so that early on in these passes there was very little evidence for XMRV but over time it became stronger and stronger, and that this particular 22rv1 that has been widely used is contaminated with a virus that was actually generated in these mice and that this came after the recognition of Chronic Fatigue Syndrome. He further shows that XMRV might be a a hybrid, a recombinant of two pre-XMRV viruses, one of them which is replication competent, the other of which is not, and if you put these two together it almost perfectly matches XMRV.

So Dr Coffin's conclusions is that mouse mitochondrial DNA and/or these intracisternal A particles (IAP) are found in many samples that test XMRV positive, as well as in those that test negative.

That XMRV evolved when XMRV-negative prostate cancer xenografts that were passaged in these nude mice and then became infected over time.

That XMRV is a recombinant of two pre-XMRV strains, only one of which is replication competent,

That XMRV was created in a lab long after ME/CFS was recognized as a clinical entity

And that XMRV is a contaminant that does not warrant further study. That is I think sort of a quote.

So, where do we go, with these two very very divergent pieces of data? I do want to give you this cautionary note, the fact that contamination can occur, and nobody doubts that, does not mean that it has occurred in any given laboratory. There is as yet no direct evidence for contamination in either the Mikovits lab or the Lo laboratory.

So, what are we going to do? I think, rather that cutting the baby in half, the resolution may come - first of all we have to resolve this issue, is it contaminant or is it real, and I think that it will be addressed by two coded panels that are currently in preparation. There's a National Heart Lung and Blood panel that employed samples that have already been found to be XMRV positive, and will distribute these under code with multiple controls to five laboratories including WPI which are actually supplying these samples, but won't know when their own samples coming back.

The second panel is done by NIAID, it's getting the name as the Lipkin panel. Dr Lipkin's in Colombia, and he's coordinating this whole very massive process. Here they're going to collect samples from five or six different centres of CFS excellence, picking out the most classic cases of Chronic Fatigue so that people can't argue at the end that this was poor selection. They will draw large volumes of blood. These patients have never previously been tested for XMRV so there's no selection bias. They will then be sent to Colombia, separated and coded in triplicate, with matched controls, and then sent to the WPI, the FDA lab of Dr. Lo, and to the CDC. If the controls test positive, then contamination will have been proved and the case will be closed. Coffin will have won this argument. If the controls are negative, and the majority of patient samples test positive, for XMRV or PMLV then the association with CFS will have been confirmed in a blinded way. But even if the association is confirmed, causality will not have been proved.

So I think that's where we stand. These panels are going to come late this spring, early summer. They'll take quite a while to do, because they are so complex, but hopefully we'll have some answers this year. So I just want to leave you - I'm somebody who, in an hour and a quarter may be out of work (laughter) and I just want to tell you something about government workers. We get a bad rap.

We're considered to be quite lazy, and we don't work anyway is kind of the feeling, but I want to tell you that I always give 100% at work.



(applause, laughter) It's just that there's a peculiar distribution. So today is Friday, I gotta get out of here!

Judy do - anybody want to come in here?

JM I would like to say something, I would like to say that what I've seen here this week says that clearly XMRV at best what we originally set out to do five years ago was a systems biology approach, we used those genomic technologies with Vi Sceen (?) and Mary Carrington, we used the microarray from the NCI so we could screen all the pathogens, and what we saw was what we heard from Mary Schweitzer was a lot of active pathogens, things like shingles, things like enteroviruses, things like EBV, CMV, HHV6, they are all totally on in that microarray.

In NCI's infinite wisdom, it did not put XMRV on it, that's how we ended up going back, but we did not look with a hypothesis for retroviruses. We took the systems biology approach, collected samples from well-defined patients who had the infectious characteristics that Tony Kormaroff talked about, that were in the Lo study, that Dan Peterson had done, these patients had data on them for decades.

We took samples over three years, so it wasn't that we found the sample in every sample indicate - like the Macaque study where it quickly went into the tissue, like the mouse study that came out this week that said that the antibody responses were weak and transient. We don't know everything about this virus, but HIV does not cause AIDS. The CDC definition is HIV and one of 25 co-pathogens, so the Lyme, the EBV, the enteroviruses - Martin Lerner's patients who don't get better with Valcyte - this is a reasonable hypothesis because we see the same thing.

We've developed a cytokine signature that is distinct from Nancy's cytokine signature and from Ben Natelson (?) so this is a marker to follow on clinical trial improvement. There is no doubt these people are infected - with HTLV1, if you are seropositive and you are sick, you can get some kind of treatment. I'm not saying antiretrovirals, I'm saying immune modulators - so the patients that are found to be infected now, and there are thousands of them, need something now, not three years from now when Lipkin decides there is an association.

Whatever their disease is, they are infected and sick, and I know John Chia has patients that are coinfected and they don't treat the same way. So we can get together with the physicians who have coinfected patients, even Lyme doctors who were working with across the country, and start doing something now, take it out of CFS. It's not about CFS, it's about a retrovirus we don't understand very well as Frank Ruscetti said in a meeting a month or so ago, if this were HIV, it would be 1983. That's all.

Vernon Judy, I think you need to be careful, you said when Lipkin decides whether or not XMRV

JM I know, I'm just saying - Lipkin's a wonderful man - I'm just saying when the Lipkin study's done - two years from now - they way it's set up there's 1300 samples, and I've got to do what I do and it takes two months on every one of them - oh yeah, great!

John Kusiak Just a word as a neurologist who again believes this ailment is the brain - the brain is the organ being affected, we looked at our spinal fluids using both broad viral panel and a specific look at XMRV - a paper just came out a few days ago, and we found nothing, unfortunately

JM But the damage at a distance, the microglia, Sandy Ruscetti's entire work, the affected cell doesn't have to be the brain, and we don't know anything about the stability in the CNS.

JK Well obviously there are examples of CNS infection that don't go to spinal fluid, but in HIV it does, but certainly it's negative but it doesn't tell us - hopefully there are other explanations. I'm just reporting the data.

? John. John Kusiak, in the neurology.

JM Two samples. It was not a good study. We'll go away. Totally flawed.

JK I'm always doing it wrong

JM There's no validation of looking at CFS in spinal fluid. There's no validation, it's a different sample maker, it's not blood.

 

Well just have to keep working at it - and not in any confrontational way. Of course the pathogen or whatever crosses the BBB into the brain (areas controlling basic function). Just keep looking. As someone who could not recall my own name or recognise anything familiar around I know it affects the brain.

 

Prostate cancer cells were passaged through nude mice back in the 70-80s:
http://cancerres.aacrjournals.org/content/37/11/4049.full.pdf

But somehow the newly 'created' virus at the very least has become a common laboratory contaminant and likely multiple contamination events - eg Coffin was detecting XMRV with mice DNA, Alter was detecting MLV (would require separate event) only and apparently no mice DNA. Mikovits again, no mouse DNA. The 'contamination' findings in Retrovirology also had contradictory findings with regards to pinpointing the likely source (eg requiring separate events).

General discussion about contaminated cell lines and risk of contamination of biological products here:
http://www.ncbi.nlm.nih.gov/pubmed/19003296

If it has infected humans, it is likely though contaminated biological products, as this is the only way the low level of transmission genetic variability can be explained:
http://www.frontiersin.org/virology/10.3389/fmicb.2010.00147/abstract
http://forums.phoenixrising.me/show...cell-lines-used-for-production-of-biologicals

However, this hypothesis only makes sense for isolated clusters - those that had been exposed to the contaminated products in question, rather than CFS sufferers in general (still faintly possible given the mixed results). Either that or the virus is widespread in the population and doesn't cause CFS on its own.