New Paper - Gammaretroviruses - Maureen Hanson, David Bell

Thanks for the analysis from those that have read the full paper, esp Mark.

This looked interesting to me:

IIRC, Judy Mikovits said they scored inconsistently reproducible results as a positive. I wonder how much this different approach to scoring could account for the difference between the Science results and these latest ones? [excluding the retracted Silverman results from the Science ones].

Good point Mark. Scientists should be curious about things that "dont fit" They are clues to the unknown.
That is why I have been so dismayed by the energy with which Dr Mikovits findings have been ridiculed and dismissed.
I know that not all researchers have responded in this way, but many of the most prominent seem to have been guilty of it.

There is quite a history of new retroviruses having been initially associated with a disease, only to experience the same problem of reproducibility with PCR testing we have seen with "XMRV"
All these early disease associations from the 1970s and 80s have been discarded.

I seem to remember that there were viral associations made in leukaemia which could never be reliably reproduced once researchers came to rely exclusively on PCR to validate their initial findings. Could be that PCR is misleading in some unpredictable and unsuspected way and these old viral associations, now discarded, could be correct?

I remember an early talk in which Mikovits remarked that the present generation of retrovirologists have been trained to rely too exclusively on PCR findings, and contrasted that with earlier approaches taken by "classically trained" virologists - before they could just rely on the technology.

Mikovits used to score the inconsistent positives as positive because she said it depended on the life cycle of the retrovirus,which had to be caught at a point when it was not locked up in the DNA.

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Hi Mark

I'm not sure that JM repeatedly testing her samples to get a few positives is fully consistent with the O'Keefe finding: it sounds like JM had negatives on the first test and positives on later tests too - rather than lots of early positives that couldn't be replicated (possbily because of inhibiting contamination). Maybe someone has more information on this. And does anyone know if JM explicitly confirmed that controls were tested as many times as patients? That was exactly what concerned me when she first made the comment but never saw any clarification. This isn't a question of insulting this or that scientist but trying to fully understand the science. Maybe JM did clarify this but if so I missed it (it wasn't easy to keep up with the XMRV maelstrom). Thanks

Just thinking about the O'Keefe paper. I don't think it should apply to successive testing if changing to a different type of PCR test. I think it only applies to successive testing using the same PCR slides, or to any testing kits that have been previously contaminated. In other words, it's the slides that get contaminated, and not the blood samples. A fresh slide is not automatically exempt from contamination, because the contaminating product can be used before the experiment for calibration. Therefore successive tests using the same PCR test might be affected, but so might the first test. But when switching to a new type of test, the chances for contamination start afresh.

That's going from memory and a bit of guess-work, but without re-reading the O'Keefe paper, I can't remember the exact details.

Confused? I am!

Novel MLV-GAG sequences detected in blood samples of ME/CFS patients

http://okeefe-lab.blogspot.com/2012/05/novel-mlv-gag-sequences-detected-in.html

Alex, we can just do the other studies with no spinal injections???
I think the one they are really chasing first is testing nk function and comparing cfs to rheumatoid arthritis and ms. Its sounds like they have a plan for alot more in the future.

cheers!!!

Thanks for that Eco. Very interesting.

Denise O'Keefe has clearly got her 'open-minded thinking-hat' place firmly on her head!

Its interesting what she says about other researchers having similar unexplained results.

It doesn't go anyway towards explaining why the results have been so inconsistent though.

I've managed to run my eye over it now, but I've not read it carefully yet, and so I may have made some mistakes, but here are some extracted titbits:


Study Subjects:

David Bell provided 10 severely ill patient samples (Fukuda), 10 'recovered'* patients, and 20 control samples.
Susan Levine provided samples from 20 CFS patients and 4 healthy controls.
12 controls who have never been diagnosed with CFS were recruited from Ithaca, New York.

So that's 30 patients, 10 'recovered'* patients and 36 controls, if i've added those up correctly.

* The 'recovered' patients, once fitted the Fukuda criteria but now consider themselves recovered.


A round-up of the positive results:

MLV-like gag sequences were obtained following PCR of gDNA and cDNA from PBMCs from subjects in Western New York:

"Taken together, 5 samples from severe patients, 2 from recovered CFS patients, and 3 from control subjects resulted in detection of gag PCR products. All of these samples were negative in mouse mtDNA assays. All PCR results were confirmed by sequencing."

So that's 7 positives from patients, and 3 positives from controls.


MLV-like gag sequences detected in LNCaP cultures:

"Two samples gave gagO/gagI PCR products: 11F2-P4 and 14F2-P4. With gagL PCR, 7 samples were positive: 2F1-P6, 4F1-P6, 9F1-P6, 10F3-P6, 11F2-P4, 12F2-P6, and 13F3-P6. In addition, 5 additional samples irreproducibly gave gagO/gagI PCR products and 11 sometimes were PCR positive for gagL; due to the inconsistency, we decided to score these as negative."

So that's at least 18 that were positive for gagL, and 7 positive for gagO, out of 40 subjects for this test. (I assume that the '40 subjects' breaks down as: the 30 patients, and the 10 'recovered' patients.)
It doesn't give numbers for the controls as far as i can see, and it doesn't give a break-down for the 30 patient samples and the 10 'recovered' patient samples.
And it doesn't give a total for how many were positive for any sequences, as far as I can see.


gag PCR of DNA from PBMCs of [12] control subjects in Ithaca, New York:

"We observed a gagL PCR fragment only one time in an Ithaca control sample"


Phylogenetic Trees:

The phylogenetic trees show that the sequences were closer to Lo's PMRVs than to XMRVs, and were distinct from 22Rv1. (I'm not yet certain if this applies to all of the sequences.)

http://www.plosone.org/article/slid...RI=info:doi/10.1371/journal.pone.0037482.g008

http://www.plosone.org/article/slid...RI=info:doi/10.1371/journal.pone.0037482.g009

"The trees illustrate that the sequences amplified from the samples of this study were related to polytropic MLV-like sequences, and that all gagL sequences were distinct from 22Rv1 XMRV, which contains deletions in the gag leader region."

"There are differences among the sequences we obtained and between our sequences and those of Lo et al. [9]; however, it should be noted that relatively small sequences, approximately 400 nt, are being compared."


Some interesting quotes:

"Whether a retrovirus is involved in inciting or maintaining CFS/ME will require further investigation using other types of assays."

"Like a number of other investigators, we have identified MLV-like gag sequences following PCR of nucleic acid samples from whole blood or PBMCs from human subjects. Nested PCR analysis of our initial batch of 30 samples resulted in a significant difference in frequency of gag PCR products between patients and controls; however, continued analysis failed to maintain this association."

Thanks for all this, very helpful.

The LNCaP results look like 8 consistent positives (2 for GagO, 7 for gagL with 11F2-P4 positive for both), plus 16 more that were inconsistently positive and so scored negative.Total 24 positive at least some of the time ex 40 patients?

If I've got that right (not sure I have) that's an interesting finding both for the number of confirmed positives - and the inconsistent positives that Hansen scored negative, but presumably the original Science paper would have scored positive. 24/40=60% 'positives' which I think tallies broadly with the Science paper.

Hi oceanblue, I added a little bit more text, for extra info, after you quoted me, re the number of 'recovered' patients included in the study. Just letting you know.

Yes, it looks like 8 in total were positive, and then the additional samples were 'irreproducibly' positive: 5 samples gave gagO/gagI PCR products, and 11 gave gagL PCR products. But does this mean there were 16 additional samples, or was there an overlap between gagO and gagL? It isn't clear.

So in total, it could be between 19 (8+11) and 24 (8+16) samples that were positive, out of 40 patient samples, 30 of which had a current diagnosis and 10 of which had 'recovered'.

19 would be 47% positive
24 would be 60% positive

But this doesn't take into account the 7 positives from the other tests (PCR of gDNA and cDNA from PBMCs), which might have been additional positive samples. I don't think they've included enough data for us to work out the total positives in either the patient samples or the control samples. I haven't found the info yet anyway. If those 7 positives were additional, then it could be a total of up to 31 positive patient samples out of 40 (30 current patients, and 10 'recovered'), which would be 77%.

I can't find many figures for the controls, beyond the 3 in the 'PCR of gDNA and cDNA from PBMCs' tests, so I can't work out the total for controls.

Thanks, Bob. You're right, the range is 47%-60% positive, and that excludes positives from the non-LNCap tests. Would have been nice if they'd given tallies for positives/sometimes-positives across all tests, but from the data given it does look like their results broadly tally with the original Science paper if intermittent positives are scored 'positive' rather than 'negative', which makes this a very interesting paper indeed.

Yes, it is very interesting, isn't it. It is actually a positive replication study, or sorts, of both the Mikovits and the Lo papers.
But they have no ideas why they are getting these results. From what I understand, so far, this study completely vindicates Judy Mikovits, and confirms her research. The only difference is the interpretation.

What gives me hope, about the science in general, is that this Hanson paper has been completely open and honest about publishing their exact results, with no obfuscation, and no over-blown conclusions. Nothing is hidden, so the information is out there for others to follow up.
And people like Denise O'Keefe are clearly interested in seeing this research followed up.
Maybe we are at a stage with technology now, where these results may be understood over the next couple of years.

Apart from anything else, they are finding unexplained MLV-like sequences which are not mouse contamination, as far as they can determine, and are not 22RV1. So what are these sequences, and where do they originate? No one seems to have any idea at all. If it isn't mouse contamination, then what is it?

(I haven't scrutinised the sequences yet, and so I don't know how similar they are to mouse ERV sequences - Has anyone else had a look yet?)

Has this already been posted? This is the patent Denise O'Keefe was posting about I think.

http://www.freshpatents.com/-dt20120503ptan20120107338.php

Thanks for posting Jemal.

There's a parrallel thread, about O'Keefe's reaction to the Hanson paper, and the patent details have been posted on there:
http://forums.phoenixrising.me/inde...here-too-scared-to-publish.17596/#post-268293

(Maybe the two threads should be merged?)

Basically, unless I've misunderstood, it's another piece of positive MLV-related research?

That's how I read it. For me it's another indication that XMRV/MLV's might not be so innocent...

Hi Bob

Yes, these results are fascinating and in line with the original Science resutls. However, I don't think they wholly exonerate Judy Mikovits as declaring inconsistent results as positives is a questionable practice, particularly as this wasn't mentioned within the original Science paper.

As you say, the other really interesting aspect is that Hanson found the sequenced MLV's were closer to Lo's than XMRV clones. JM also said she was finding non-XMRV but MLV-related sequences, though I don't think this was actually published.

I don't know how this new paper's results square with the definitive negative papers published before, but it does seem to rule out a simple contamination explanation.

Maybe we still need to wait for the Lipkin study to see how this all pans out.

I thought she had declared that she got inconsistent results. It was widely known that she had to test individual samples multiple times, and if she got one positive, then she would mark that up as a positive sample. But I can't remember how much of that was actually published in the original paper. Is that questionable practise if you are clear and transparent about your methodology?

In any case, it seems to be a difference of interpretation, rather than a difference in the actual results. Would you agree?

Yes, I think Judy said she was finding P-MRVs as well.

Yes, it doesn't rule out contamination, but it does rule out a 'simple' contamination explanation. An important difference, considering everything.

Yes, but if Lipkin doesn't answer any of these questions, then where does the science go from there?