Ruscetti Acknowledges Resuse of WB Image 2

Wow! Ruscetti just confirmed ERV's information that the same slide was used twice, to show two different things!
Here is the story: http://news.sciencemag.org/scienceinsider/2011/10/xmrv-researcher-fired.html
And here is the quote:

Mikovits's collaborator, Francis Ruscetti of the National Cancer Institute (NCI) in Frederick, Maryland, who ran all of the Western blots, confirms that the Ottawa slide uses the same image that appears in Lombardi et al.

Even worse, Ruscetti says that the Conference image was closer to the original than the Science image. But Ruscetti and Mikovits say that it wasn't fraud, because they witheld some information on their protocol from the Science paper, so they were not lying when the labeled the conference image. Quote is below:

As far as the use of 5-Azacytidine, Ruscetti and Mikovits stressed in their e-mail that "there was no attempt in the original paper to hide anything." They say for the purposes of Lombardi et al., the use of 5-Azacytidine was not germane: They were simply trying to demonstrate that CFS patients had viral proteins not seen in controls. By the time of the Ottawa meeting, they say they realized that this experiment did not in fact show XMRV but proteins from a broader family of gammaretroviruses.

So what they are saying now is:

1. We were really sloppy with the image.
2. We didn't report properly on the use of 5-Azacytidine in the science paper, even though it was an important part of our protocol.
3. But it's not fraud.

They might be right on that last point (if they are telling the truth), but in terms of their credibility, it's completely destructive.

V99 and Angela, are you going to apologies to ERV now? In the end, she was right about that image.

Joshua (not Jay!) Levy

Reinterpreting evidence in light of further information is common, it's how science is supposed to work, using the same data as was used to justify an initial hypothesis to backup a more eleborate form of the same (or even a different) hypothesis doesnt mean the first hypothesis was fraudulent, it simply means it was superceded by a, hopefully better, one. If you cant understand that then......

You appear to be jumping up and down in glee, thinking that Ruscetti (initally someone you seem to trust statements from) has said something that supports your position that JM and FR are incompitent at best and probably frauds, although why you would trust ANY staement from someone you seem to doubt the integrety of is beyond me. If you trust him then you should trust his assertion that everything is above board, if you dont how do his statements backup your position?

I also dont see what the big deal over 5-Azacytidine is, if the people who designed the protcol decided it wasnt a relevant factor to the hypothesis they were advancing in the Science paper who are you to decide otherwise. If based on subsequent work they then decide that it be was relevant, as they modified their hypothesis, how does that invalidate the original work? Or to be more blunt, how does it make the orginal work fraudulent?

The key point surely, SOC, is that patients and controls were handled differently and this wasn't disclosed in the paper.

If 5-Azacytidine makes it easier to detect XMRV proteins then treating patient but not control samples with it will bias the experiment in favour of the hypothesis that XMRV is associated with ME.

But we know that did not happen in this case, because they changed the labels on the slides. If they were just reinterpreting, then they would have used the same slides (same information), but had different textual description (different interpretation of those slides) to explain what was going on. By changing the labels, they were trying to change the data, not the interpretation. (Obviously, the patient numbers are not part of the interpretation of anything, so why change those?)

For that particular action, I think the term "scientific misconduct" would be better than "fraudulent". Remember that the whole point of a scientific paper, is so that others can run the experiment themselves and get the same results. So if you don't describe the experiment you really ran, then it could be considered "scientific misconduct". If you hide part of your procedure or lie about it, for example. So if the original experimenters used 5-aza (or anything else) and did not report it, that's a serious problem. Also, I'm pretty sure they claimed that the experiment was done double blind; that the researchers didn't know which samples came from CFS/ME and which from controls. But if one group got 5-aza and other group did not, well then: the experiment was not double blinded (at least not for some of the researchers). So that would be another serious problem, that goes directly to their credibility. Especially since, one of their most important points, repeated over and over, was that they saw XMRV in patients but not controls. But if the researchers were not double-blind, or if the two groups were treated with different chemicals (like 5-aza), that's an obvious explanation.

In a sense, they are claiming that they did not properly describe their procedure in order to make people believe that they did not misrepresent their results. It's the scientific version of pleading to a lessor crime to avoid a more serious one. Not a good position to be in.

In another sense, it doesn't matter exactly what words are used. Their credibility is gone.

Joshua (not Jay!) Levy

On that only, we agree

Thanks for posting the link. I'm glad to hear Ruscetti and Mikovits' explanation. To me, the explanation of the different labels on the Ottawa slide is plausible, but not mentioning the use of 5-Azacytidine in the original paper is troubling. Especially after its publication, when there were questions about the methodology.

If it was just a couple of positive samples that they applied 5-Azacytidine to, for purposes of illustration, I still think it's odd to say it was "not germane" to mention it. But afterwards, when the accusations of contamination started coming in, Dr. Mikovits said that if it was contamination, it should have appeared in controls in equal rates as patients, because she said they had handled the samples the same way. That made sense to me, and made the contamination charges less believeable. But this sounds as though they applied 5-Azacytidine only to patient samples. Even if they were doing it just for illustration, shouldn't it have been done to patient and control samples equally? Otherwise, it doesn't show that gammaretroviruses occur in patients MORE than in controls; without that, you have no disease association.

Am I missing something here?

It isn't credible, SOC. If the viral proteins are not seen in controls you need to demonstrate this by treating patients and controls identically.

Just wanted to say; Thank you guys for this productive and intelligent discussion; very good arguments on both sides.

Hi SOC,

Did your post grow a little bit, or are my faculties failing me (it's quite late here in Enger-land)?

Anyway, you say "I thought it was already established that the association of XMRV with ME was not proved by the Science paper", but in fact, this was precisely what the Science paper was arguing. That is, it did show a clear association between XMRV infection and ME. It didn't argue causality, but did demonstrate a very strong association.

Sam

It is late here -- I hope I haven't misrepresented your argument
ETA: I hope I haven't misrepresented my own argument either.....

You've interpretted what Dr Ruscetti said differently from the way I read it. I thought he said that the Ottawa slide was an earlier version than that published in Lombardi et al. That they changed the patient numbers prior to publication to protect patient ID, and that they failed to do that when they used the same picture in the Ottawa slide. I don't see that changing the other labels on the slide, when it was meant to illustrate a different point, is deceptive. Not if they're saying, in essense, we thought it was XMRV but it's not, it's some other gammaretrovirus.

Hi, Some seem worried about the different treatment of controls from patients. Let me draw attention to the ScienceInsider quote:

"Mikovits described how she had treated cells from two CFS patients with a chemical, 5-Azacytidine, that takes methyl groups off DNA."

The bolding is mine. The facts are only coming out slowly and too many are leaping to unsubstantiated conclusions, either for or against various points of view.

Two patients tested differently? My guess is this was a test of an idea that they weren't sure about but ran anyway. Since it was only two patients it was way too small to include as any kind of evidence in the Science paper and was quite rightly omitted in my view. If scientists had to explain everything in minute detail then they might as well be submitting video logs of everything they do. This was not a significant factor at the time of publication of the Lombardi paper.

Later on they realized this could be important, which is why I think it was mentioned in Ottawa. Since this is minor evidence it is more proper to discuss this at a conference than in a major paper.

This whole episode is a non-event blown up to something major by media coverage. If there is a problem then the investigation by Science will uncover it, and I trust them more than I do unsubstantiated accusations. Wait for their report.

The real news is that Dr. Judy Mikovits is no longer with the WPI. This has real ramifications. This includes changes made to the research capacity at the WPI and issues over research rights.

Bye
Alex

Alex,

I've followed all of this unfolding but have had difficulty understanding this whole situation and piecing it all together. Your explanation makes sense.

Thanks for your take on this.

This is bad

Ruscetti confirms that the version of the slide - the LABELING of the slide - at Ottowa is original and correct, and that the Science figure came after.

Although he tries to excuse it, if the Ottowa version is correct, this mean that the version - the labeling - in the Science article is wrong.
Not the patient numbers - that isn't a problem, it seems, and that part of what he say makes sense. But the figure is wrong in OTHER major, substantive and misleading ways. Plural.

First, at Ottowa , lanes 2 and 5 are labelled as CFS patients without 5AZA treatment. In Science, they are labelled as Normal controls. In other words, thos are lanes showing MECFS patients who DO NOT express viral proteins. The science figure is making the argument that people with CFS express viral proteins. Changing a patient non-expressing lane to say they were controls, is fundamentally misleading.

Think about that. The Science paper is making the argument that people with ME/CFS are infected with XMRV. That figure is making the point that people with ME/CFS express viral proteins, while people healthy people do not. They have two lanes showing people with ME/CFS and NO VIRAL PROTEINS - but they change the label to say those are normal controls.It is a misrepresentation of the experiment and of the data. At best, it is unforgivable sloppy, mislabeling a key piece of data in a way that causes it to say something other than what the experiment actually said. This - to put it in terms that scientists will be using to evaluate all of this - is really, really bad.

Second. 5AZA treatemnt of cells can cause derepression of endogenous retroviruses. That is jargon. What it means is that our own human DNA contains a lot of old crippled viral sequences. They cant make functional virus - but they can make viral proteins. They are turned off by methylation - they don't make protein. Treatment with a demethylating agent - 5AZA - can allow them to turn on and make viral proteins. This is we known I'm not a virologist - I'm an out-of-dae evolutionary geneticist - but it is well known, I knew it, and I've confirmed it with virologists, verbally and by going into the literature.

There is no way, based on the Ottawa labeling, to tell if the proteins they are detecting on that blot are XMRV viral, or human. They don't do the proper control - which would be to treat control with 5AZA to compare.

But worse - in the Science paper they never reveal that they used 5AZA. This is NOT a trivial omission. It fundamentally changes the interpretation of that figure. If they had revealed that their patient cells were treated with 5AZA, but the controls were not, that figure would probably not - should not - have gotten through review. The figure does not say what they claim it says.

This is all bad enough, and Ruscetti's statement either ignores them (the 'control label) or does not adequately address them (the 5AZA issues).

Third - and if it is true, worst of all - the original image of the gel seems to have been uncovered by uncropping and unmasking one of those figures. That image is now out on the web, and the original lab labeling does not match EITHER of these two versions.

Changing labels so that patients are represented as controls in a way that hides data contrary to the hypothesis that ME/CFS patients are infected with XMRV - at the very least, this is very, very bad.

Lee,

Just checking to see if you have followed all the relevant news because you are new to the forum. Apologies if this is all old stuff to you. We don't get many "well" people here and you are very welcome.

How do feel about Dr Silverman and his contamination problem? How do you feel it contributed to the wasted money on XMRV testing by all the scientists who did negative attempts.

Do all the negative XMRV papers need to be followed up and looked at because of that?

What's your feeling on Alter/Lo? How should we be following that up?

What would you like to see happen about the MLV's/HGRV's that Lombardi et al found.

Obviously having seen your friend suffer like that you would want them to get treatment if they did have a HGRV.

I suspect that even if money turns out to have been "wasted" on XMRV, it was not money that would have been spent on anything else that is ME or CFS related. Indeed, that spending has drawn attention to the issues and led a lot of researchers to have another look at ME and CFS. About the only negative, presuming the argument that XMRV research was a waste is correct (something I do not concede), is that some research funds that could have been spent on something else wasn't. Given the size of redirection of funds allocated to CFS research in the past into other endeavours, I doubt this could have even come close to redressing the balance. In terms of the massive underfunding on ME and CFS it is not even a blip on the radar toward parity.

MS is a worthy disease that I think is still underfunded. While more lethal than ME it is less disabling on average according to three morbidity rankings I have read over the years. It is also less common. Yet in one year MS gets more research money than ME has ever had, by which I mean the total funding since the 1930s epidemics. If MS research is underfunded, what does that make ME research?

The real issue is a lack of interest and funding, period. The huge sums wasted on the PACE study for example could have been far more beneficial if they investigated immunological issues instead.

Bye, Alex

@ ukxmrv:

Silverman? I think he showed definitively that the positive 'XMLV' PCR results were due to plasmid contamination, and confirmed that the early criticisms that the sequences were too similar to the plasmid, and didn't show enough sequence diversity, were correct.

I also think that his reported protections against contamination were stunningly thorough.

I also note that the contaminating plasmids were found in ME/CFS samples and not in healthy controls. This was the result that made me personally sit up and go 'hmmmm.' This is NOT a pattern that one sees with PCR contamination - this is a sample handling issue - at best, patient samples were handled differently from control samples, in a way that caused contamination of patient samples.

I think n evaluating the Silverman retraction, one must also look at JM's claimed ability to reliably find XMRV in patients and only at low levels in controls - and the fact that they were unable to do so when samples were blinded.

Put all this together, and the only conclusion I can find is that - AT BEST - JM's lab were guilty of handling patient samples differently from control samples, in ways that caused contamination of patient samples but not controls.

And given this - I don't believe a thing she has reported. She is - at best - too sloppy to be trusted.

[This post is not directed at any on person. It is something I feel like saying.]

I'd like to point out that failed trials or studies are not automatically bad science.
They are part of the scientific process to study something objectively (we hope) and determine whether it pans out or not.

In the case of XMRV it was not necessarily wasted time and money. It has brought to light the very srong likelihood that HGRVs or
some human retrovirus is a major part of the immune disturbance in ME.

I am really starting to get tired of the people taking XMRV as the axis of their argument against RVs or anything else, when a year ago it was determined that it was a family of retroviruses, not XMRV that was implicated. Further study to really nail down that theory was not ill-spent.

Good science should always be willing to investigate, thoroughly assess, and unabashedly admit to a theory proving out or not.
Societally we have looked upon these outcomes as failures or successes, but the only failure is when we fail to look at a potential problem or solution out of *fear* of failing, or *fear* of social and economic shunning. That is bad science: the unwillingness to look at an inconvenient, or perplexing, or uncertain data set.

Science is absolutely no longer pure. So putting your faith 100% in science, and ignoring the political, financial, and corporate
influences that are very real, is almost like believing in the Wizard of Oz.

I have no answer for it all except to be willing to read between some lines, and pull back curtains wherever possible.

Good points Leela.

If a scientist honestly follows a lead, and carries out their work diligently, then even if their work ends up being proved incorrect, based on further knowledge, then it is not 'bad science'. It is just science in progress. Science learns from past mistakes as well as past successes.

@Bob:
"On the question of hope and being 'misled'" I was referring specifically to my friend, who is spitting-nails angry right now.

@leela:
"In the case of XMRV it was not necessarily wasted time and money. It has brought to light the very srong likelihood that HGRVs or
some human retrovirus is a major part of the immune disturbance in ME."

Actually, given that we now know that they were using 5AZA without reporting it (until the Ottowa conference, in a slide, apparently) we don't know, and cant know, if any of the alleged HGRV results are real.

That's why this is bad science. Not because it didn't work out - lots of good science doesn't work out. It's bad science because it was badly done and badly reported. If they were using 5AZA without telling anyone, and not doing proper controls, then it is possible, maybe likely, that much of the non-PCR results they have reported are simply an artifact of the 5AZA treatment. And they have admitted that they didn't think it was important to let us know about the 5AZA treatment, so we don't know, and we can't know.

JM has shown with the 'blind' results that she cant repeat her own PCR results unless she knows what the answer 'should' be. We know that she has been unwilling to let anyone else have access to her experimental materials to make independent tests for contamination. We know that she failed to reveal a key experimental detail, the 5AZA treatment, this we do know can cause exactly the kind of artifacts that she is reporting as results, and that in the one place she revealed that usage, she did not do the key control, for those artifacts.

Nothing she has done, it is now clear, can be trusted.

At this point, given these issues, nothing that JM has done or reported is credible evidence for any virus, XMLV or HGRV or otherwise.

BTW, I'm not commenting more on the PACE trial because I am not a clinician, and do not have the expertise to easily evaluate that work - and because it is completely irrelevant to the results of the alleged XMLV work. I am an (out of practice) molecular biologist and geneticist, and I do have enough expertise to easily and quickly understand these issues. What I see, each new emerging contaminant and changed label and changed interpretation and previously-unreported experimental detail, makes me really angry.