Why does 5AZA matter?

[Bob]

Mark, you get the 'best post of the month' award, from me. Thank you so much for spelling out all of that with such clarity, while keeping a cool head (something i've not been able to do this week). I really appreciate the amount of thought, patience and effort that you put into it. You make a whole load of crucial points, and I agree with everything that you've said.

I'll just pick out one point to comment on for now... The following had crossed my mind as well... (my underlining)...

- Indeed, since Ruscetti says that the failure to mention the 5AZA treatment of patient samples in Figure 2c was a trivial omission not necessary to the paper, and since Mikovits has asserted that the bulk of samples were treated exactly the same for patients and for controls, the most reasonable way to reconcile all these statements is that Figure 2c was included in the paper as the most clearly-defined image from their various experiments, because it best illustrated the contrast. That may seem a little misleading, but in the wider context of all the materials the researchers had at their disposal, and in the context of the review process re-editing the original paper down to a fraction of its original length, it seems to me most likely that they just presented the case in the strongest possible way by selecting the strongest image. If other slides which did not depend on the use of 5AZA, or where 5AZA was applied to both patient and control samples, showed similar contrasts, then this choice may not have been particularly misleading.

I didn't know that the paper had been edited down so much by the editors of Science (or i'd forgotten!)

 

[Bob]

My point is that we have no reason now to trust what they've told us - we don't know what they did - because where we've been able to verify, we know that what they told us is either wrong (the PCR results) or not what they did (the western blot). We therefore have no reason to believe that any of the rest of that paper is valid.

My personal suspicion is that everything remaining in that paper is an artifact of 5AZA - it would certainly explain all the negative followup results. But I don't and cant know that yet. What I do know is that nothing they said in that paper can be trusted - right or wrong, it is now worthless - because they have admitted to telling us things that just ain't so.

Lee, your technical explanations have merit, and have been helpful, thank you.

But the information available to you is incomplete, and therefore you have made your conclusions based on your own assumptions.

Therefore your opinions and conclusions are speculative.

I refer you to Mark's post to explain why:
http://forums.phoenixrising.me/show...es-5AZA-matter&p=210834&viewfull=1#post210834

You do not know all of the facts, and so you have, out of necessity, made omissions in the data that you have presented in order to make your case.

Therefore, by your own logic, nothing you say can ever be trusted. Ever again.

I don't actually believe that of course, but I'm just highlighting the weakness of your arguements.

 

[Bob]

Nah - that's a poor example. It was an error, but not an important one for supporting their argument, and one that was admitted as soon as it was pointed out. It looks as if originally all of the data was correct, then the scoring method was changed, so some figures on a graph became inaccurate (ugh at the paper though. These 'send out the questionnaires' CFS papers, and they way they end up being cited to make really broad and tenuous points, are a bit of a bug bear. People who do a lot feel more tired? There's a moderate correlation between psychological distress and feeling of fatigue? These are findings that should guide the way we treat CFS for the next two decades!)

I think Mark's point is that we don't yet know the significance of the 5AZA omission.
So if we are going to denigrate the reputations of Lombardi et al, based on a single omission for which we don't yet have full info about (and therefore don't know it's significance), then it is right to tear apart any other paper based on a single omission or error.

 

[Bob]

That is also what Dr. Mikovits said in the "naturenews" article. They said it was activated, they just didn't think back then that it is worth mentioning by what substance it was activated. Anyway, they wrote that BOTH PBMCS FROM HEALTHY CONTROLS AND FROM PATIENTS were activated. So, unless someone has something that tells us that only the PBMCs from the patients were activated, I don't see how in any way this could be a fraud or something.
What it is doing, and that is why Dr. Mikovits started talking about 5-AZA, is that it might be causing researchers not to find evidence for HGRVs when they are in fact there. They didn't think about it back then, they do think about it right now, and that is why back than they just wrote "activated" and right now they are also mentioning what it was activated with.

Anyway, if someone would explain to me why he thinks this is a fraud, it would be nice. Because I don't see why someone would think so.

Well, you'd think that a good investigative scientist might try testing all their samples with and without 5AZA anyway.
Why on earth wouldn't experts such Singh, Coffin, McClure carry out the study using 5AZA on some of their samples by way of exploration?
It seems like it might be quite an obvious thing to do if you are a retrovirologist, and you know about these things, especially before declaring that XMRV doesn't exist.

 

[Sam Carter]

...

Well, you'd think that a good investigative scientist might try testing all their samples with and without 5AZA anyway.

Why on earth wouldn't experts such Singh, Coffin, McClure carry out the study using 5AZA on some of their samples by way of exploration?

It seems like it might be quite an obvious thing to do if you are a retrovirologist, and you know about these things, especially before declaring that XMRV doesn't exist.

(my bolding)

I think you make an important point, Bob - anyone trying to replicate the experiment that produced the contested image would be unable to do so because the methods were not correctly described, even in the supporting material where space was not constrained.

 

[Bob]

I think you make an important point, Bob - anyone trying to replicate the experiment that produced the contested image would be unable to do so because the methods were not correctly described, even in the supporting material where space was not constrained.

Yes Sam, we have established that 5AZA was not mentioned in the Science paper, and that we don't know the facts surrounding this.

I was asking a wider question. I was asking why these other expert retrovirologists wouldn't have used 5AZA on at least some of their samples, before declaring that XMRV does not exist?

I don't know much about 5AZA, but they are leading expert retrovirologists (or at least some of them are supposed to be), and so 5AZA must surely be very familiar to them?

They must know that a retrovirus can be latent and that, to make it more easily detectable, 5AZA can be used.
I would have thought that they might use their initiative, and use all techniques available to them to tease out the latent virus.
I would have thought that they would do this without needing to refer to any other study, because they are experts in retroviruses and would surely try to use all techniques available to them?

But I'm just asking a question here. I don't know the answer.

 

[Esther12]

I don't really know what would be normal.

I just had another look at the later paper they published, going in to more detail about methods, and why they thought others may not be successfully replicating: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073172/

This bit makes me think that, at the time, they did not think activation was necessary, and there was no specific mention of 5AZA (my bold):

In our experience from performing the multiple methods on the same 57 blood samples, the most sensitive blood-based assays for detection of XMRV in decreasing order (Table 3) are: (1) performing nested PCR for gag sequences from LNCaP cells that have been co-cultured with subject's plasma or activated PBMCs, (2) the presence of antibodies to XMRV Env in subject's plasma, (3) presence of gag products by nested PCR on stimulated PBMCs or detection of viral proteins expressed by activated PBMCs with appropriate antisera, (4) nested RT-PCR of plasma nucleic acid or PCR from cDNA from unactivated PBMCs and (5) PCR of DNA from unactivated PBMC prepared from subject's blood.

I'm not going to pretend I understand the details of that paper (and I'm feeling a bit worn day by too much on-line chatting), but to me it seems that someone trying to confirm their results would be unlikely to think they should try using 5AZA.

Anyway - if 5AZA was all that was needed to validate their science results, they should have got better results from the BWG. Maybe some replication attempts did try 5AZA, and it didn't help?

 

[duckndive]

Hi Lee,
Can I ask another question of you please? There was some discussion that the term 'activated' is rather ambiguous in the context of whether 5AZA was or wasn't used. There is a description of the by now infamous slide in a patent application here:

http://www.freepatentsonline.com/y2011/0117056.html

Lysates from normal PBMCs (lanes 1, 2, 4, 5 and 7) or from subjects WPI-1235 (lane 3) and WPI-1236 (lane 6) were activated for 7 days with PHA and IL-2 and then analyzed by Western blot using rat anti-SFFV env monoclonal antibody (top panel) or goat anti-R-MuLV p30 (bottom panel). Lane 8: SFFV-infected mouse HCD-57 cells. Molecular weight markers are shown on the left.

That seems to my inexpert eye quite clear that activated doesn't include use of 5AZA?

 

[Advocate]

What Mark said.

 

[duckndive]

Thanks Bob! Trying to put the jigsaw pieces together in my non-science head!

 

[Bob]

Thanks Bob! Trying to put the jigsaw pieces together in my non-science head!

Actually, you might have a point duckndive... I've just deleted my previous post...
Figure 2D in the patent looks like it's the same as Figure 2C in the Science paper...
I'm having a look at it now...

EDIT:

Yes, sorry, I was looking at the wrong image.

Figure 2D in the patent is the same as Figure 2C in the paper.

And your quote was correct.

"(FIG. 2D) Lysates from normal PBMCs (lanes 1, 2, 4, 5 and 7) or from subjects WPI-1235 (lane 3) and WPI-1236 (lane 6) were activated for 7 days with PHA and IL-2 and then analyzed by Western blot using rat anti-SFFV env monoclonal antibody (top panel) or goat anti-R-MuLV p30 (bottom panel). Lane 8: SFFV-infected mouse HCD-57 cells."

I agree that this (alongside the labeling of Figure 2 in the Science paper) probably clears up any ambiguity about what the word 'activated' was refering to in the Science paper.
And anyway, Mikovits and Ruscetti have already said that they didn't refer to 5AZA in the Science paper, so I think I was wrong when I said that the word 'activated' was ambiguous.

 

[asleep]

Fantastic post Mark!

And THERE it is, the elephant in the room. Lee has done a great job spouting out techinical details (not a crime!). It's the unsafe prejudicial value judgements that get tacked onto the end of these long technical explanations that people have to watch for, which show Lee is here to push an agenda: and that is to claim Mikovits and her colleagues as disreputable.

This is a very concise description of exactly what's going on here.

 

[Sam Carter]

Yes Sam, we have established that 5AZA was not mentioned in the Science paper, and that we don't know the facts surrounding this.

I was asking a wider question. I was asking why these other expert retrovirologists wouldn't have used 5AZA on at least some of their samples, before declaring that XMRV does not exist?

I don't know much about 5AZA, but they are leading expert retrovirologists (or at least some of them are supposed to be), and so 5AZA must surely be very familiar to them?

They must know that a retrovirus can be latent and that, to make it more easily detectable, 5AZA can be used.
I would have thought that they might use their initiative, and use all techniques available to them to tease out the latent virus.
I would have thought that they would do this without needing to refer to any other study, because they are experts in retroviruses and would surely try to use all techniques available to them?

But I'm just asking a question here. I don't know the answer.

Hi Bob,

Sorry, after looking at your post again I see my reading comprehension has failed me - again ...

I searched on pubmed for 5-azacytidine and got 4562 hits. (All of which I've read of course ...

I found one paper [*] which said "latent proviruses are responsive to TNF alpha treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2." which does support your case that probing cells with 5-Aza would be useful for teasing out viruses.

It's also interesting to see that they used the same agents, PMA and IL2, as the Lombardi group to activate cells.


[*] Retrovirology. 2008 Apr 25;5:37.
HIV-1 latency in actively dividing human T cell lines.
Jeeninga RE, Westerhout EM, van Gerven ML, Berkhout B.

 

[Bob]

Hi Bob,

Sorry, after looking at your post again I see my reading comprehension has failed me - again ...

I searched on pubmed for 5-azacytidine and got 4562 hits. (All of which I've read of course ...

I found one paper [*] which said "latent proviruses are responsive to TNF alpha treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2." which does support your case that probing cells with 5-Aza would be useful for teasing out viruses.

It's also interesting to see that they used the same agents, PMA and IL2, as the Lombardi group to activate cells.


[*] Retrovirology. 2008 Apr 25;5:37.
HIV-1 latency in actively dividing human T cell lines.
Jeeninga RE, Westerhout EM, van Gerven ML, Berkhout B.

Thanks Sam. That's very interesting.

 

[Lee]

Bob:

"I think Mark's point is that we don't yet know the significance of the 5AZA omission."

The significance of the 5AZA omission is that it was an a omission, and that it isn't just a minor technical detail. Using 5AZa means it was a completely different experiment. They did a different experiment from the one they claimed they did.

That gel is meaningless, worthless, because they didn't tell us what is on it. We still don't know - that's why people are arguing about it here. They presented it as if it meant something different from anything it actually could mean. It doesn't matter if they used it because it 'looked better' than the actual experiment gel. In science, you don't get to substitute one result for another. If they could not get a clean good-looking, clear gel for the experiment they described - that is relevant, and they have to show that.

As it is, they have not presented ANY data for the claim they made about gag expression in patients. None. But they told us they did. That fact means that they can not be trusted. period. They are untrustworthy, by their own admission. They told us they did something different from what they claimed.

They have admitted that they told us things in the Science paper, and in the Ottowa presentation, which are not true.

Which means that nothing they said can be trusted.

THAT is the significance.

 

[Lee]

"I was asking why these other expert retrovirologists wouldn't have used 5AZA on at least some of their samples, before declaring that XMRV does not exist? "

As i said in my original post, the argument JM made at Ottowa about proviruses, and use of 5AZA to reveal them, is a reasonable hypothesis adn a reasonable experiment to do.

But even with the labels she used in Ottowas- - and we know that those labels ae STILL not what was actually on the gel - she did not show that experiment.

To do that experiment properly, one needs four conditions:
healthy control - no 5AZA
healthy control - 5AZA
patient - no 5AZA
patient - 5AZA

Each of those needs to be done multiple times, and a presentation gel would need to show all four conditions at least 3 times on the same gel

If JM had done this experiment, she would have the gels. Even if she had done it improperly without all the proper controls, without enough replications she would still have the gels.
But she showed us the gel from years earlier, which does not have the proper controls.

I make my living working with scientists, mostly biologists and chemists. Every scientist I know who is following this, including several sho study retroviruses, has looked at what she actually showed, taken into consideration that we already know they were willing to mislead us about what is on the gels, and immediately concludes - "I don't believe she did it - or she would have showed it."

 

[redo]

Lee. Your insight on this topic is valuable. About the study (and I am asking cause I don't know); when the authors wrote that they had "activated" the samples, does "activate" imply some specific sort of activation? Did the authors only activate the patient samples, or the controls as well? And if they only activated one of the groups, was it specified that only one part was activated?

 

[Lee]

2redo:

'Activate" or 'activation' means something very specific in immunology - roughly, it means to turn on the cells immune functions. There are techniques known to do that, they are standard.

In Science 209, they activated the PBMCs by treating them with PHA and interleukin. They describe this in the legend to Figure 2A, and if yo look at figure 2D from the patent (which is the same as
figure 2C from the paper) they specifically claim they used PHA and IL.

Activation has nothing to do with 5AZA - its is something entirely different.

Ruscetti, in one of the recent commentaries (Science or Nature - for some reason they aren't loading for me right now, and I dont remember which one) Ruscetti was claimed to have said something like '5AZA was an unimportant detail, and anyway, we said we activated the cells' - implying that 'activation' covered the 5AZA use.

My biologist friends and clients who read that - ~5 of them that I've talked with - universally said something close to: 'He said WHAT?!?!?!?"

 

[Bob]

But Lee, that doesn't explain why the other retrovirologists did not use 5AZA themselves in their own work, or any other helpful techniques, stimulated by their own expertise, initiative and knowledge.

No wonder they got negative results if they were only superficially looking for the virus, without investigating more deeply using their own specialist knowledge and expertise.

I'm not very impressed.

I mean, if I was a retrovirologist, and I knew that 5AZA can tease out a retrovirus, so that it can be detected, then I might think to myself to try that out, even if it isn't mentioned in the Science paper. It's not as if they religiously followed the Science paper methodology anyway - many of them used their own techniques.

It seems blindingly obvious to me that they would want to try it before declaring that XMRV does not exist.

 

[Bob]

The significance of the 5AZA omission is that it was an a omission, and that it isn't just a minor technical detail. Using 5AZa means it was a completely different experiment. They did a different experiment from the one they claimed they did.

That gel is meaningless, worthless, because they didn't tell us what is on it. We still don't know - that's why people are arguing about it here. They presented it as if it meant something different from anything it actually could mean. It doesn't matter if they used it because it 'looked better' than the actual experiment gel. In science, you don't get to substitute one result for another. If they could not get a clean good-looking, clear gel for the experiment they described - that is relevant, and they have to show that.

If you read Mark's post, you'll see that he has explained why this is not necesarily the case.

As it is, they have not presented ANY data for the claim they made about gag expression in patients. None. But they told us they did. That fact means that they can not be trusted. period. They are untrustworthy, by their own admission. They told us they did something different from what they claimed.

They have admitted that they told us things in the Science paper, and in the Ottowa presentation, which are not true.

Which means that nothing they said can be trusted.

THAT is the significance.

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

I refer you again to Mark's post.

If the Science editors forcibly retract the paper as a result of misconduct, based on the same scenario that you have presented, then I will accept that.