New Paper - Gammaretroviruses - Maureen Hanson, David Bell

[Mark]

Thanks Sam. The date given in that link is actually
Volume 43, Issue 3, September 2008, Page 245
and it was posted in December. I don't see where "Monday, October 13, 2008" comes from?

Anyway, it just goes further towards confirming what Bob and I have been saying and fleshes out a little more detail. This is an even earlier version of the results, it looks like about 11 patients and 2 controls were not included in the later presentation, and 50 of these weren't included in the XMRV cytokine paper. That's one explanation, but of course this could be another entirely different cohort, or another experimental run on roughly the same cohort - if they can run the high-throughput tests in 6 hours, then maybe they've run this study several times.

If they did just do one study, and removed a few samples before later publication, they haven't stated why they decided to exclude some of the samples from their results, and if the point is that they should have explained this, then I would have to agree that if this is all the same study there seems to be detail missing here, but it looks to me like it's either a process of progressive refinement of the cohort as they did further testing, or they ran this study a few times. Would be interesting to ask them if these were all results from the same round of testing, or multiple studies.

 

[Mark]

Not quite done, it would seem. ;-)

I think I will have to be done with it now because I really don't have time for this and there's still just nothing in this at all. I'll keep an eye on the thread, but I've pretty much given up hope that any decent evidence or substance will be presented to back up what's been suggested.

 

[Sam Carter]

Thanks Sam. The date given in that link is actually
Volume 43, Issue 3, September 2008, Page 245
and it was posted in December. I don't see where "Monday, October 13, 2008" comes from?

Anyway, it just goes further towards confirming what Bob and I have been saying and fleshes out a little more detail. This is an even earlier version of the results, it looks like about 11 patients and 2 controls were not included in the later presentation, and 50 of these weren't included in the XMRV cytokine paper. That's one explanation, but of course this could be another entirely different cohort, or another experimental run on roughly the same cohort - if they can run the high-throughput tests in 6 hours, then maybe they've run this study several times.

If they did just do one study, and removed a few samples before later publication, they haven't stated why they decided to exclude some of the samples from their results, and if the point is that they should have explained this, then I would have to agree that if this is all the same study there seems to be detail missing here, but it looks to me like it's either a process of progressive refinement of the cohort as they did further testing, or they ran this study a few times. Would be interesting to ask them if these were all results from the same round of testing, or multiple studies.

Sorry, Mark, I got the date from here (note this is quite a large pdf file) and added it to the top of the co-cure post.

I'm sure you're right about the multiple data runs.

Now it's deffo time for me bed....

 

[RustyJ]

Mark, I totally understand your frustration.
It's so hard to work out all the facts re MLV research, without the added confusion of people posting innaccurate and misleading information.
So it's especially annoying when people post information as 'facts', when on closer inspection, it turns out to be either baseless or plain wrong.
This has happened twice recently: on this thread, and on another thread re Singh's study.
Presenting misinformation as 'fact' causes other people to do so much work to find out the real facts, that it's simply unreasonable.
I'm particularly pleased that you've managed to establish that RRM's assertions, re the cytokine study, are baseless.
I was quite surprised that RRM would make such baseless allegations, and I'm very annoyed by it.
I'm annoyed that I was misled, and that it's taken so much work of other forum members to work out the truth.

But I really appreciate the time and effort you've taken to explore these issues.
It's been really interesting to read all of the recent discussions, and I've learnt a few things that I didn't know previously.

I agree with Mark and Bob's assessment of RRM et al's contribution to the XMRV debate. Inevitably these contributions have misdirected forum members - not because the quality of information was deficient (it is dependent on members to ultimately judge the veracity of such information), but because it was portrayed as "the inside story", from those who should know. It is a shame it was allowed to happen, given the repercussions for individual patients and for the damage to the wider research community.

Even if RRM's theory (theories) about how contamination could have occurred were plausible (which hasn't been proven), it is far more feasible to consider that Mikovit/Lombardi's lab assay might actually work. So far that hasn't been tested. It's like saying "Well, we're not actually going to test your way of doing things, we just going to theorize why it won't work", or, "We can't make it work with our assays (which we wanted to patent), therefore we refuse to believe your assay might actually work."

The BWG was just a gigantic exercise to prove which assays didn't work (something they already knew).

 

[currer]

I have a feeling that the activity seen recently in this thread in response to items of minor interest and sometimes items of NO interest is a measure of the furore that will probably break out once there is a major retroviral report.

It has been quiet recently.......

 

[barbc56]

What are"the real facts" to someone is misleading to another. It's a matter of perspective.

Debate is fine but I find it incredibly sad and unproductive that people are making personal remarks, either outright or implied. Are we really lowering ourselves to the point that we don't want to har other's opinions? I think we are better than that.

IMHO, I think we all need to lighten up and step back a bit. So here's a funny yet a rather apt quote.

"Get your facts first, then you can distort them as you please."

Mark Twain



Barb C.:>)

 

[barbc56]

I have a feeling that the activity seen recently in this thread in response to items of minor interest and sometimes items of NO interest is a measure of the furore that will probably break out once there is a major retroviral report.

It has been quiet recently.......

I think you are spot on about this. Unfortunately.

Barb C.:>)

 

[Bob]

Debate is fine but I find it incredibly sad and unproductive that people are making personal remarks, either outright or implied. Are we really lowering ourselves to the point that we don't want to hear other's opinions? I think we are better than that.

Well, I've thought about what I said earlier (which I said in frustration.)
And maybe I was unfair.
Clearly we all get passionate about this subject, and we often want to promote our own points of view, and to highlight information that supports our opinions.
And I'm sure that I have presented some information in the past, as 'fact', which turned out either to be inaccurate, misinformed or unproven.
So I've probably been unfair.

But having said that, when people repeatedly post misinformation or speculation as 'fact', and put other people to a load of work in finding out the actual details, then it is highly frustrating. And it's frustrating to find out that you've been misled.

But, yes, I don't think I've been shy in promoting my own point of view in the XMRV discussions, including saying some things that turned out to be inaccurate, so I should more tolerant.

RRM's posts often challenge my own opinions and that is useful, and it stimulates discussions.

 

[SilverbladeTE]

Just wait folks

Such contenous issues rarely ever get resolved quick, and like it or not, it is contenous because it would throw up a HUGE HUGE mess.
And it isn't gonna get resolved fast anyway as it's cutting edge but not massively funded or seen as "Important".
Time will tell, either way.

 

[beaker]

Would like to ask a big favor.
My eyes are crossing. Have not been well enough to tackle the paper and this thread is overwhelming. I am sure I am not the only one !

Is anyone that pulled this paper apart able to do a simple summary write up all in one paragraph kind of thing ?
I look at the sections in the thread that mark pulled apart and discussed but it was too choppy for my eyes these days.
( that is my eye issue not anything against mark : ) it's great he was able to dive in )

Thanks if someone can. and if you can't........ no worries. I know why.

 

[Bob]

Would like to ask a big favor.
My eyes are crossing. Have not been well enough to tackle the paper and this thread is overwhelming. I am sure I am not the only one !

Is anyone that pulled this paper apart able to do a simple summary write up all in one paragraph kind of thing ?
I look at the sections in the thread that mark pulled apart and discussed but it was too choppy for my eyes these days.
( that is my eye issue not anything against mark : ) it's great he was able to dive in )

Thanks if someone can. and if you can't........ no worries. I know why.

OK Beaker, i'll give it a go...

They detected sequences that were closer to Lo's P-MRVs than to XMRV.

But they couldn't repeat their positive test results reliably.

"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."

The authors say that it was a negative study because they couldn't reliably repeat most of their test results.

In total, I think they found roughly between 16 and 31 patient samples that tested positive (out of a total of 40 patient samples) for sequences, at least once, but most of these results could not be reliably repeated. So that was between 40% and 78% of the patient samples that they tested positive at least once. (They don't seem to provide enough info to know exactly how many tested positive.)

I'm not sure exactly how many controls they found to be positive, but it looks like there were very few.

They don't know what the origin of the sequences are, or how they entered the samples.

But they say they have ruled out mouse contamination, because they tested for it.

The 40 patient samples included 30 patients, some of whom were severely affected, and 10 'recovered' patients, provided by Dr Bell and Susan Levine.

See my earlier posts for a break-down of the results:
http://forums.phoenixrising.me/inde...en-hanson-david-bell.17574/page-2#post-268299

Well, that's my quick summary, based on one quick reading of the paper.
I've not studied the details in depth, and so I've probably not got it exactly accurate, but anyone can correct me if they spot any errors.


Results:

Single round and Nested PCR results:
5 samples from severe patients
2 from recovered CFS patients
3 from control subjects resulted in detection of gag PCR products.

LNCaP cultures:
Between 16 and 24, from patient samples (?)

 

[Bob]

Contamination of the LNCaP master cell line?

- their LNCap cell-line probably became contaminated
- they tested the uninoculated LNCaP master cell line by single-round PCR and found:
--- 8/84 replicates +ve for gag sequences (this should have been 0/84 if there was no contamination - and assuming I've understood this section correctly...)
--- 0/10 replicates negative for IAP (ie. the IAP test failed)
--- their reagents, HotStart-IT FideliTaq master mix (USB) and NEB master mixes, were also negative for IAP

Because of this they looked at an earlier sample of the same LNCaP cell-line and found:
- 1/21 replicates +ve for gag sequences with the USB master mix
- 4/41 replicates +ve for gag sequences with the NEB master mix

ie. this was also contaminated and USB/NEB master mixes gave different results.

Eventually new LNCap cells were bought:
- 0/21 replicates showed gag sequences with the USB master mix
- 0/41 replicates showed gag sequences with the NEB master mix

ie. the new batch was uncontaminated.

Thanks for that Sam. Like you, I wasn't too sure if this meant that there may have been contamination in the LNCaP cell lines, or not. (If it does, then I wonder at what stage could the contamination have entered?) Have you thought any further about this, or has anyone else got any insight into this?

Also, do you know if they did further patient sample culturing/testing with the new LNCaP cells? And if so, were there any positive results?

 

[beaker]

OK Beaker, i'll give it a go...

They detected sequences that were closer to Lo's P-MRVs than to XMRV.

But they couldn't repeat their positive test results reliably.

"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."

The authors say that it was a negative study because they couldn't reliably repeat most of their test results.

In total, I think they found roughly between 16 and 31 patient samples that tested positive (out of a total of 40 patient samples) for sequences, at least once, but most of these results could not be reliably repeated. So that was between 40% and 78% of the patient samples that they tested positive at least once. (They don't seem to provide enough info to know exactly how many tested positive.)

I'm not sure exactly how many controls they found to be positive, but it looks like there were very few.

They don't know what the origin of the sequences are, or how they entered the samples.

But they say they have ruled out mouse contamination, because they tested for it.

The 40 patient samples included 30 patients, some of whom were severely affected, and 10 'recovered' patients, provided by Dr Bell and Susan Levine.

See my earlier posts for a break-down of the results:
http://forums.phoenixrising.me/inde...en-hanson-david-bell.17574/page-2#post-268299

Well, that's my quick summary, based on one quick reading of the paper.
I've not studied the details in depth, and so I've probably not got it exactly accurate, but anyone can correct me if they spot any errors.


Results:

Single round and Nested PCR results:
5 samples from severe patients
2 from recovered CFS patients
3 from control subjects resulted in detection of gag PCR products.

LNCaP cultures:
Between 16 and 24, from patient samples (?)

Thank you very much Bob. That helps.
What confuses me is that the authors are saying it is a negative studying where o'keefe and posts I have seen here say positive study.
I guess it is both depending on how you look at it. : )

I hope they continue to follow up on these findings.

 

[Bob]

Thank you very much Bob. That helps.
What confuses me is that the authors are saying it is a negative studying where o'keefe and posts I have seen here say positive study.
I guess it is both depending on how you look at it. : )

I hope they continue to follow up on these findings.

Yes Beaker, like you say, I think it's a case of interpretation...

I think that the authors suspect contamination, but were unable to prove it or locate the source:

"It seems likely that our LNCaP cell line, which was maintained for many months, became contaminated at very low levels, or there was environmental contamination at the time of DNA preparation."

Whereas O'Keefe seems to suspect that the sequences might be more significant.

 

[beaker]

Yes Beaker, like you say, I think it's a case of interpretation...

I think that the authors suspect contamination, but were unable to prove it or locate the source:

"It seems likely that our LNCaP cell line, which was maintained for many months, became contaminated at very low levels, or there was environmental contamination at the time of DNA preparation."

Whereas O'Keefe seems to suspect that the sequences might be more significant.

or maybe the authors are just being super super cautious considering what has happened in the past.
For some reason doing rv research on this plague seems to be a career breaker.
more work is needed (understatement of the year! ) lol

 

[barbc56]

Well, I've thought about what I said earlier (which I said in frustration.)
And maybe I was unfair.
Clearly we all get passionate about this subject, and we often want to promote our own points of view, and to highlight information that supports our opinions.
And I'm sure that I have presented some information in the past, as 'fact', which turned out either to be inaccurate, misinformed or unproven.
So I've probably been unfair.

But having said that, when people repeatedly post misinformation or speculation as 'fact', and put other people to a load of work in finding out the actual details, then it is highly frustrating. And it's frustrating to find out that you've been misled.

But, yes, I don't think I've been shy in promoting my own point of view in the XMRV discussions, including saying some things that turned out to be inaccurate, so I should more tolerant.

RRM's posts often challenge my own opinions and that is useful, and it stimulates discussions.

Bob, I am not specifically referring to your posts. Again I have to ask why people perceive statements as inaccurate when they may just be someone elses opinion.

Little mistakes, sure and not surprising as it's a lot of information to process. We've all done that

My opinion is that many people still thinking that XMRV is not a contaminant are misleading, inaccurate and not helping our community. But this is my opinion and interpretation of studies helped by scientest and posters I feel are accurate. I am also passionate about this.

If people want to debate then you have to put in a lot of work and research and this may require extra time. If someone doesn't want to put in this extra work then maybe they should think twice about posting. I hope you continue to post as this is how we learn.

Barb C.:>)

 

[Bob]

Bob, I am not specifically referring to your posts. Again I have to ask why people perceive statements as inaccurate when they may just be someone elses opinion.

Little mistakes, sure and not surprising as it's a lot of information to process. We've all done that.

Yes, well, I did agree that we all tend to promote our own opinions, and that we all post inaccuracies at times.

But RRM's stuff about 100% of patients testing positive for XMRV in the WPI's cytokine study, was not just a 'little mistake'. It painted the WPI's cytokine study as almost fraudulent. On close inspection, it turned out to be baseless speculation. But forum members had to do a load of work to get to the truth. Even if it was an honest mistake, surely you must be able to see that this might cause a bit of annoyance and frustration?

But, yes, I know we can all get carried away on these threads sometimes. But not usually to that extent!

My opinion is that many people still thinking that XMRV is not a contaminant are misleading, inaccurate and not helping our community. But this is my opinion and interpretation of studies which I will find and post.I could say the same that these statements cause a lot of frustration and extra work.

Well, all our different opinions create interesting discussions, so although we can get frustrated with opposing views, it all leads to interesting discussions in the end.

I'm open minded to XMRV being a contaminant, but I'm also interested in finding out exactly what the source is for all these MLV-like sequences that have been found in various studies, including non-ME studies.

I'd be quite interested to hear why you think that people who still think that XMRV is not a contaminant are not helping our community. In what way do you think this opinion does not help our community? Or were you just making a philosophical response to one of my previous posts?

.

 

[barbc56]

TBH, I will go with RRMs opinion unless he states otherwise.

I could write a whole thesis on why XMRV is a contaminant and shouldn't have to go over this once again. Read my previous posts. Look at what the major retrovirologist/scientest are saying. I am analyzing them critically and not just following blindly and agree with many. Yes, I even think ERV is a credible source despite her way of presenting information. The core of what she says is accurate, IMHO.

I think the people who are still so desperately hanging on to the theory that xmrv is harmful, are hindering other theories. Yes, and I have said this before ,even looking for other RVs or viruses is important but the latter should at this point way down the list of priorities. If this were some other issue it would have been discarded by now. So my opinion goes beyond the philosophical.

I could be wrong but I think looking into our immune systems is a more probable explanation.

How many negative studies is it going to take?

Barb C.:>)

ETA

It painted the WPI's cytokine study as almost fraudulent

How is that different from people claiming there is a conspiracy by certain scientiest?

 

[Bob]

TBH, I will go with RRMs opinion unless he states otherwise.

Why would you blindly follow RRM's opinions, when it has clearly been demonstrated that, on this occasion, his information is pure baseless speculation? Did you not read anybody else's posts?

I think the people who are still so desperately hanging on to the theory that xmrv is harmful, are hindering other theories. Yes, and I have said this before ,even looking for other RVs or viruses is important but the latter should at this point way down the list of priorities. If this were some other issue it would have been discarded by now. So my opinion goes beyond the philosophical.

Well, 'desperate' is rather a pejorative term, and a little insensitive, considering that many of us do actually feel quite desperate with this illness.

There is a lot of other research going on, Barb. I'm only aware of one XMRV study that is currently being carried out. Although, there might be one or two others that I'm not aware of. So I hardly think it's a priority.

I agree that other research is essential, and could potentially be a more fruitful route to better answers.
But being interested in XMRV doesn't rule out encouraging, supporting, or engaging with other research.

The Lights are doing fascinating research with genes and biomarkers.
Nancy Klimas has got a number of really interesting projects that she's involved with.
Bond University, Australia, is carrying out some amazing research at the moment.
Lenny Jason continues to do amazing stuff with definitions and epidemiology.
There's the new ICC which is in its infancy.
Then there's the Rituximab research.
And the CDC seem to be finally taking CFS/ME seriously, or semi-seriously, and rumour has it that they are working on a revised diagnostic criteria.
And the MRC in the UK have just funded a few decent looking biomedical projects for the first time ever.

As far as I understand, there is a bigger pool of research being carried out now than at any time in the past. Definitely in my own experience anyway.

So I don't quite see how XMRV is either taking up valuable time, or resources, when no one is carrying out any XMRV research, apart from a few retrovirologists who wouldn't be doing any CFS research anyway.

I could be wrong but I think looking into our immune systems is a more probable explanation.

I probably agree with that.

.

 

[Bob]

The reason that people on 'this side' (for want of a better description) of the XMRV debate get frustrated, is that we have watched various people dismiss XMRV from day one, for apparently spurious reasons. And yet it's been demonstrated that the research was worth persisting with, for very unexpected reasons. As per the Hanson study, we still don't have definitive evidence that these new sequences are a result of contamination, let alone what the source of any potential contamination might be, so we want to see the MLV retrovirology research continue in order to find the source of these sequences, whether they are ME-related or not. I expect it's being worked on. And if there is even the slightest, smallest, tiniest, chance that any MLVs could be associated with ME, then for obvious reasons, I want to see it fully explored. And I wouldn't be surprised if the research led us to some serendipitous unexpected answers - related to, or unrelated to, MLVs. Already, XMRV has unexpectedly drawn Ian Lipkin into ME/CFS research, to carry out a full pathogen study, which is a total bonus as far as I'm concerned.