Myra McClure and Retrovirus Contamination

Marco

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Myra McClure seems to be convinced that contamination was an issue in the positive studies.

Digging around a little for her background it appears she has published on this problem before in relation to MLVs.

ABSTRACT

Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to the Gammaretrovirus genus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01/F7 cells to produce gibbon ape leukemia virus. These findings highlight the importance of routine testing of cell lines for retrovirus contamination to prevent potential experimental artifacts and allow correct biohazard assessment.

FULL PAPER

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593302/


The science is more than a little beyond me but I can't help feeling that this is significant in either casting doubt on the XMRV story or conversely possibly even supporting it?

Sorry to be obtuse. You'll see what I mean when you read it.
 

pictureofhealth

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I see what you mean Marco. Whether the findings are interpreted as 'contamination' or 'additional infection in an a patient already infected with HIV' is apparently still very open to debate. You can see where the doubt is coming from. Let's hope WPI, the Ruscetti's and Lo/Alter think of even more ingenious ways to prove it is a genuine infection, which hopefully it is! What about this testing to see if the XMRV and MLV's are included in the ME/CFS patients' genomes, or whatever the clever testing was that the PNAS reviewer suggested in July that Lo/Alter have yet to do?
 

lansbergen

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What about this testing to see if the XMRV and MLV's are included in the ME/CFS patients' genomes, or whatever the clever testing was that the PNAS reviewer suggested in July that Lo/Alter have yet to do?
That was a tricky one. When it is in the human germline it is endogen. Then is should be called a HERV

The question should have been: is it in the genome of infected cells.

If it is not in the genome of infected cells it can not produce virions. No virions means it can not spread.
 
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But how could that be that contamination effects CFS samples in a total different percentage compared to results from test persons in the healthy arm? Also a second test in Alters study gave the same positive number. This rules out contamination to my understanding.
 

Marco

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Well my simplistic interpretation is this.

Myra McClure found evidence of retrovirus contamination (MLV particularly) in human cell lines commonly used in labs and concludes that there is a danger that this contamination could skew research results. This contamination presumably results from either murine materials being used in the preparation of the cell lines or murine materials used in the same lab that produces human cell lines and contamination results. Labs using these materials could find that they are either picking up MLV traces from the use of the cell lines themselves in experiments or again that their use in the labs results in the lab being contaminated.

She goes on to test, in her own case, whether it is her lab that is contaminated, or the contamination is already in the muman cell line products. She concludes the latter.

With respect to the positive XMRV studies, she concludes that their experiments are picking up this contamination either through the direct use of human cell lines in their tests (I don't know if this is the case) or they have used these products in other work and the lab itself is contaminated. Perhaps from this perspective the patient percentages were higher because more PCR rounds were used? It also explains why, if only the negative studies have carefully excluded contamination, the negative studies found nothing at all.


A very worrying scenario.


The other interpretation of course of her finding MLV in human cell lines is that they were already there and do not result from murine contamination.

This would suggest to me that XMRV/MLV might just be pretty ubiquitous in humans but does not replicate in high numbers in the majority of people. As Coffin? suggested, the disease may 'cause' the virus. I'd also be surprised if the 4-7% of healthy controls found with XMRV weren't harbouring some form of disease, either of a different type or in a latent stage.

Certainly, the findings of antibodies in patients would suggest that lab contamination wasn't responsible.
 

Mark

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Begs the questions...

1. Is there any connection between the sort of cell lines that are potentially contaminated and the processes involved in the positive studies?

2. Is it plausible that such systematic contamination could explain false positives in all 4 of the WPI tests, in 3 labs, plus also Alter's findings, plus other studies that have detected XMRV?

3. I've heard it said that the antibodies could no way be evidence of such contamination, lots of people I respect all seem agreed on that point.

4. Even if it were true that the "contaminants" come from the primers it would be extremely valuable to understand why they are expressed more often in CFS patients at 85% vs 4% in controls.

5. Even if they are ubiquitous and are simply expressed more in CFS patients (Coffin mentioned the possibility) it still seems that would suggest they may well have a role in the pathophysiology of CFS.

6. If they are widespread contaminants in human cell lines and similar results have often been observed in the past, why have those results not been explained and does the "contamination" not suggest that there is something going on here that needs explaning properly anyway?

7. Contamination or no, if this test were fully validated it is a test, maybe a candidate biomarker?

8. If it's known that this gammaretrovirus can infect human cell lines, doesn't that constitute evidence in itself that XMRV can infect human cells?

9. Where exactly did this "contamination" come from, how did it become so widespread in human cell lines so as to be showing up repeatedly all over the world, does that not mean that it is still happening systematically, and why does this sort of thing keep showing up in association with human diseases?

10. Is it not the case that these XMRV sequences come from 2 strains of laboratory mice and have infected human cell lines, is there any explanation of how and why, and is this not a cause for serious concern?
 

Mark

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If it is not in the genome of infected cells it can not produce virions. No virions means it can not spread.
Is that definitely the case? Lo's earlier work concerned gammaretroviruses "packaged up" inside other larger cells to bypass immune system detection; can we really be confident that if a gammaretrovirus is not actually integrated into an infected cell's genome there is no (currently unknown) mechanism by which it can go on to produce virions?
 

Mark

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Perhaps from this perspective the patient percentages were higher because more PCR rounds were used?
Surely it can't be true that they all systematically treated the patient samples differently from controls? That would be crazy!

It also explains why, if only the negative studies have carefully excluded contamination, the negative studies found nothing at all.
Alter and Mikovits have both stated that they have taken extraordinary steps to rule out contamination. Alter in particular should surely be considered highly credible when he says this.

The other interpretation of course of her finding MLV in human cell lines is that they were already there and do not result from murine contamination.
Quite: if it's accepted they can be present in human cell lines, why assume that's contamination? Where's the evidence that it got into those cell lines from murine contamination in the laboratory? Could the widespread use of these cell lines even have been a potential route for the original spread of XMRV?

This would suggest to me that XMRV/MLV might just be pretty ubiquitous in humans but does not replicate in high numbers in the majority of people. As Coffin? suggested, the disease may 'cause' the virus. I'd also be surprised if the 4-7% of healthy controls found with XMRV weren't harbouring some form of disease, either of a different type or in a latent stage.
This has always been a plausible "worst case" scenario and if this is the case then the finding is still a major breakthrough for us: one can go on to try to understand why they replicate in higher numbers, rather tha just rubbishing the findings and not following up on this lead.

Certainly, the findings of antibodies in patients would suggest that lab contamination wasn't responsible.
Probably the most crucial point.
 

lansbergen

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Is that definitely the case? Lo's earlier work concerned gammaretroviruses "packaged up" inside other larger cells to bypass immune system detection; can we really be confident that if a gammaretrovirus is not actually integrated into an infected cell's genome there is no (currently unknown) mechanism by which it can go on to produce virions?
Retroviruses have a specific livecycle. Virions enter the hostcell. Virion RNA is converted to DNA and as provirus intergrated in the cell genome. Activated provirus produces the genectic materiaal by which the cell can make virions. Virions spread to other cells.

I asked a virologist I trust whether something new was discovered. His answer was: Not that I know off. This virologist obtained his doctorate with a dissertation on a retrovirus.

Absence of evidence is not evidence of absence and miracles happen so yet unkown cannot be excluded. But ........another livecycle would cause a revolution. As I have not seen any mention of another reproduction route by the critics I have no reason to doubt the classic livecycle.
 

alex3619

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Hi Mark,

I think they are all good questions, but I have reservations about this one: antibodies as evidence. Antibodies are nonspecific. An antibody reacts to a protein sequence, usually at least eight amino acids long (but there are exceptions). Anything that has the same sequence, and is shaped the same way (its not just about sequence, but about 3D structure) will have the same antibody response. Think of an antibody as a piece of a 3D jigsaw puzzle. Sure, it might have the same shape, and so appear to fit in this puzzle, but it could really be meant for an entirely different puzzle. An antibody to xmrv/pmlv/hmrv is indicative, but hardly conclusive evidence. We have no idea what target the antibody is really after.

Bye
Alex

3. I've heard it said that the antibodies could no way be evidence of such contamination, lots of people I respect all seem agreed on that point.
 

julius

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This has always been a plausible "worst case" scenario and if this is the case then the finding is still a major breakthrough for us: one can go on to try to understand why they replicate in higher numbers, rather tha just rubbishing the findings and not following up on this lead.
Problem there is that a Wesselys and Reeveses can easily say, "Oh, their immune systems were sluggish because of depression/anxiety, thus allowing the RV to replicate."
 

Hope123

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Hi Mark,

I think they are all good questions, but I have reservations about this one: antibodies as evidence. Antibodies are nonspecific. An antibody reacts to a protein sequence, usually at least eight amino acids long (but there are exceptions). Anything that has the same sequence, and is shaped the same way (its not just about sequence, but about 3D structure) will have the same antibody response. Think of an antibody as a piece of a 3D jigsaw puzzle. Sure, it might have the same shape, and so appear to fit in this puzzle, but it could really be meant for an entirely different puzzle. An antibody to xmrv/pmlv/hmrv is indicative, but hardly conclusive evidence. We have no idea what target the antibody is really after.

Bye
Alex
I agree with Alex. There are diagnostic antibody tests out there (not for CFS) that are good but still have a false-positive rate -- i.e. when you get a positive, you have to make sure other clinical conditions that might cause the false-positive are ruled out to correctly interpret the result.

I wished someone would move on to tissue sampling -- i.e. lymph nodes, etc. -- as this would provide another view of the situation than just the various blood tests.

For those interested in reading about cell lines, the HeLa cell line used for growing viruses were originally derived from the cells of one woman, who never gave official consent for their use before her early death. Her family didn't know about it until recently. A vial of HeLa sells for $10,000. Fascinating story: http://www.virology.ws/2009/02/09/the-amazing-hela-cells-of-henrietta-lacks/
THE IMMORTAL LIFE OF HENRIETTA LACKS. - by Rebecca Skloot came out last year.
 
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Well my simplistic interpretation is this.

Myra McClure found evidence of retrovirus contamination (MLV particularly) in human cell lines commonly used in labs and concludes that there is a danger that this contamination could skew research results. This contamination presumably results from either murine materials being used in the preparation of the cell lines or murine materials used in the same lab that produces human cell lines and contamination results. Labs using these materials could find that they are either picking up MLV traces from the use of the cell lines themselves in experiments or again that their use in the labs results in the lab being contaminated.

She goes on to test, in her own case, whether it is her lab that is contaminated, or the contamination is already in the muman cell line products. She concludes the latter.

With respect to the positive XMRV studies, she concludes that their experiments are picking up this contamination either through the direct use of human cell lines in their tests (I don't know if this is the case) or they have used these products in other work and the lab itself is contaminated. Perhaps from this perspective the patient percentages were higher because more PCR rounds were used? It also explains why, if only the negative studies have carefully excluded contamination, the negative studies found nothing at all.


A very worrying scenario.
.
Thansk for the input . Its perhaps possible but seems very unlikely to me.
 

Mark

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Problem there is that a Wesselys and Reeveses can easily say, "Oh, their immune systems were sluggish because of depression/anxiety, thus allowing the RV to replicate."
Such a theory would of course require evidence that, amongst ME/CFS patients, depression and anxiety (symptoms experienced by - what is it, 25% of ME/CFS patients?) - are correlated with retroviral infection.

Considering the nature of the cohorts so far it would seem they are actually negatively correlated: the cohort considered to be far more largely, perhaps almost wholly, composed of people with depression and anxiety, is precisely the one in which no XMRV was found.
 

acer2000

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The other interpretation of course of her finding MLV in human cell lines is that they were already there and do not result from murine contamination.

This would suggest to me that XMRV/MLV might just be pretty ubiquitous in humans but does not replicate in high numbers in the majority of people. As Coffin? suggested, the disease may 'cause' the virus. I'd also be surprised if the 4-7% of healthy controls found with XMRV weren't harbouring some form of disease, either of a different type or in a latent stage.
I have always wondered about this. They are using prostate cancer cell lines to do the culture for the virus. From what we know, the prostate cancer cells are associated with this virus infection anyways. So they'd have to make damn sure that the cell line they are using to do the culture isn't already infected with XMRV "from the factory" and that what they are observing is actually infectious virus being transmitted from the patient blood samples to the prostate cells upon combination in culture, and not just the virus replicating in the prostate cancer lines because it was already there.

It seems that they way they rule this out is they test the prostate cancer cell lines for mouse mitochondrial DNA before they attempt a culture. I have no idea what the pitfalls of that test are and how accurate/not accurate it is.

Certainly antibodies to XMRV in patient blood that has never been near a prostate cancer cell line would be further evidence against contamination, as long as they are sure those antibodies are specific to XMRV and not a cross reaction.

Finally, if the positive results were due to some sort of contaminant, why would the patients with CFS have a much higher rate of contamination? The studies are blinded, so all of the samples are tested the same way in the same labwhether they are CFS or controls. You'd think the controls would test positive at the same or similar rates if the test kits/cell lines/etc.. were the source of some sort of contamination.
 

Sean

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Finally, if the positive results were due to some sort of contaminant, why would the patients with CFS have a much higher rate of contamination? The studies are blinded, so all of the samples are tested the same way in the same lab–whether they are CFS or controls. You'd think the controls would test positive at the same or similar rates if the test kits/cell lines/etc.. were the source of some sort of contamination.
Agree. You can't get that degree of reproducible difference in a properly double blinded study, and claim it is all just an artifact of poor methodology. Nobody is buying that.

Also, the -ve studies didn't find any at all, in any group. They should have found at least a few % of random +ves.

It is pointing more & more towards limitations or problems in the methods used in the -ve studies.
 

Mithriel

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The large discrepancy between the number of patients against the number of controls is proof in itself that contamination is not an issue. When Huber said that she found her 2 positive CFS samples were contamination, it was not a surprise as her controls had come out positive at much the same rate.

My OH is in a diagnostic laboratory which routinely uses PCR on samples. He found the whole debate about contamination laughable. All equipment is carefully set up to not cross contaminate, and it doesn't happen from anything floating about the lab.

If reagents or cell lines are contaminated it is very apparent. You would expect every sample to be positive.

False positives are better called equivocal, they are usually borderline and retesting gives the correct result. Sequencing is the usual way it is done in research.

The positive studies have done more than enough to show that they are not getting their results from contamination. It is only an issue because it is the only possible way that the negative studies could be correct so these researchers are desperate for it to be true, or to at least muddy the waters.

Mithriel
 

Marco

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I'm not doubting what you are saying Mithriel. In fact I hope and believe thats true.

On the other hand I believe Myra McClure is a more than competent researcher and her 2008 study predated any involvement with Wessely and ME/CFS.

Which brings me back to my second proposition? That the human cell lines showed MLVcontamination because it was already there.