XMRV Buzz - Take a Deep Breath/the Big XMRV Meeting, Singh On XMRV, Sample prep?

[Cort]

Twiving XMRV II

XMRV Buzz from the 29th

Dr. Rein and Dr. Racaniello on Twiv cont.- Dr. Rein emphasized an important point on sequence variation. He noted that if the sequence you’re looking for is a little different from the published sequence then you can miss it. He said that was an inherent ‘risk’ in PCR and can lead to false negatives. (That risk does not apply to the immunohistochemistry results - which are just coming on CFS now). From that we can conclude that sequence variation is a real question given the newness of XMRV and how little researchers understand it. How antibodies constructed to match up XMRV and other similar viruses should be able to pick it up.

The Big "C" again (and again). They went into a scary scenario - which does not seem to playing out at all - of the water controls researchers use possibly being contaminated from mouse DNA from mouse droppings coming from reservoirs. (No water controls have tested positive….this is not an issue with the XMRV studies). They then went on for a while about contamination possibilities…a millionth of a microliter of mouse DNA (much less than a cell) would be detectable da da da….no need to spell out all the gory details.

The 22Rv1 cell line - Not a Smoking Gun - The 22RV1 cell line has become an area of controversy. Dr. Rein reported that he found that the cell line produces XMRV; ie has become infected with XMRV probably when it was passed through nude mice a couple of decades ago. One of the papers found that XMRV was less diverse than the XMRV in the cell line and therefore concluded that XMRV was probably derived from it. This may have been THE major finding of the four papers - and it seemed very convincing at first but doubts have definitely crept in….Dr. Rein did not agree it was possible to conclude that XMRV was derived from that cell line, and after being initially rocked by the finding, Dr. Racaniello came to the same conclusion. In fact 3 of the 4 commentators - after cheerily espousing on all the dangers of contamination - all agreed that this central finding had been overstated and one of them actually put up a blog post about that. The papers do put a stronger focus on the possibilities of contamination - and the 22RV1 theory could be correct but it has certainly not been proven to be correct or to apply to the WPI’s or other studies. They all agreed that what the field needs is what it is doing; creating strict protocols and sharing samples between labs.

A Definitive Test - XMRV Integration into human DNA. What clinched XMRV for prostate cancer was showing that it was integrated into human DNA. Dr. Rein wanted that finding replicated by other labs and it to be extended to CFS. He noted that Illa Singh is trying very hard to do that; the problem is that it takes a lot of DNA and prostate tumor biopsies do not provide much of that....

What about those Antibodies? They finally got to the big question the patients have been asking - what about those antibodies? Doesn't THAT prove a real infection has occurred? Dr. Rein simply said… another lab needs to replicate those findings. (Dr. Mikovits has reported that Dr. Bagni’s antibody test at the NCI confirms the WPI’s findings; her study has not been published yet.) Of course many labs are developing their own antibody tests and some are finding it and some are not.....

Doesn’t the ability to transfer virus from blood to cells in culture mean virus is there? - This has been a big argument from the WPI. Dr. Rein stated that if XMRV is floating around in the lab then it could get into the samples that way.

How could ME/CFS samples consistently show higher rates of XMRV infection than the controls? Dr. Rein suggested if the ME/CFS samples were handled more it might….but otherwise he didn’t have an answer to that and said he didn’t think ‘we’ have an answer to that.

What is next? Using validated assays…exchanging samples…..using different labs. Dr. Rein does not believe that large-scale case-control human studies are called for - which makes sense; what assay, after all ,are you going to use to test all those people? Instead he called for developing a consensus assay and then moving on - which is essentially what is occurring. He was clear that he believes it’s possible that XMRV was a ‘mistake’ (I think he leans this way given the way his prostate studies have turned out) or that it is is a virus infecting people.

Conclusion - discussing the four Retrovirology papers was never destined to be a fun experience. However, all the researchers agreed the XMRV's options are still very much open and that we still have much more to learn and that several of the ongoing should tell us a lot.

Next Up - an interesting seminar from Dr. Mikovits in Sweden (thanks to Ann!).

 

[Sam Carter]

....(No water controls have tested positive….this is not an issue with the XMRV studies).....

I think the Japanese Red Cross team found that water could be contaminated:

"""""""""""""""""""""""""""""""""""
A positive band was frequently detected at the expected product size in the negative control (water) using primer sets to detect a partial gag region of XMRV.
""""""""""""""""""""""""""""
http://www.retrovirology.com/content/pdf/1742-4690-7-110.pdf

ETA: on rereading the paper I see they're not suggesting the water itself contained mouse DNA; it was the PCR test kit itself that contaminated the water.

 

[Sam Carter]

He [Dr Rein] was clear that he believes it’s possible that XMRV was a ‘mistake’ (I think he leans this way given the way his prostate studies have turned out)

He also said that even if XMRV was a genuine infection it was present in so few prostatic cells it was not compatible with know mechanisms of tumorigenesis and seemed to believe it may be a passenger virus.

 

[Cort]

Yes, this is yet another argument...at least for the prostate tissues. If it is so rare - how could it be causing the cancers; particularly when viral caused cancers tend to be full of the virus....

 

[Cort]

I think the Japanese Red Cross team found that water could be contaminated:

"""""""""""""""""""""""""""""""""""
A positive band was frequently detected at the expected product size in the negative control (water) using primer sets to detect a partial gag region of XMRV.
""""""""""""""""""""""""""""
http://www.retrovirology.com/content/pdf/1742-4690-7-110.pdf

ETA: on rereading the paper I see they're not suggesting the water itself contained mouse DNA; it was the PCR test kit itself that contaminated the water.

Thanks Sam, I lucked out

 

[illsince1977]

Yes, this is yet another argument...at least for the prostate tissues. If it is so rare - how could it be causing the cancers; particularly when viral caused cancers tend to be full of the virus....

If it is so rare can it be causing our illness? I guess better minds than ours (Singh, Ruscetti, Mikovits) are working on this, but I've wondered more than once how so few copies of a virus have such profound effects, or don't they? Please enlighten me, anyone who understands this.

 

[alex3619]

If it is so rare can it be causing our illness? I guess better minds than ours (Singh, Ruscetti, Mikovits) are working on this, but I've wondered more than once how so few copies of a virus have such profound effects, or don't they? Please enlighten me, anyone who understands this.

Hi illsince1977, the short answer to your question is that nobody knows how XMRV could be causing our illness, or cancer, at least not right now.

This does not mean that there aren't plausible hypotheses, only that they aren't proven (unless some researchers are awaiting publication of course).

The first is that ME/CFS might be a reaction to XMRV. Immunological and biochemical changes to the local tissues might promote cancer formation or growth. Systemic changes (hormonal, immunological, neurological) might induce ME/CFS symptoms. The body tries to fight the virus, but can't win because it is a retrovirus.

The second is that the neurotoxic envelope of XMRV might be attacking the nervous system. While we find that XMRV is rare, we don't know how much envelope is being produced. It might be a lot. Indeed, this envelope could induce the problems I mentioned in the first hypothesis.

I don't doubt there are more explanations: we just have to find and test them.

Bye
Alex

 

[Cort]

If it is so rare can it be causing our illness? I guess better minds than ours (Singh, Ruscetti, Mikovits) are working on this, but I've wondered more than once how so few copies of a virus have such profound effects, or don't they? Please enlighten me, anyone who understands this.

it could that there they are looking at cells it cam't replicate in - which is true. Dusty Miller has said it should be able to infect nerve cells - so you could have a nice infection up there and very little i nthe blood

 

[Sean]

Dusty Miller has said it should be able to infect nerve cells - so you could have a nice infection up there and very little i nthe blood

And hence -myelitis?

 

[Cort]

Yes! He thinks XMRV fits a viable profile for causing CFS - if they can prove its there....XMRV is so tantalizing really - it can infect infect nerve cells.....just gotta get over the hump!

 

[Cort]

XMRV Buzz Jan 4th

Exhausted by Illness and Doubts - the NYTimes plopped a largely sympathetic and even-handed article on XMRV and CFS into its popular Tues Health section. Highlighting what a volatile issue XMRV has been over the past year, the article documented the ups and downs of the past year; the FDA panel recommending that blood donations be banned, the four Retrovirology papers that shortly followed, the emphatic statements by Greg Tower that it was over, Dr. Racaniello's agreement, then his backtrack then his outright rebuttal and Dr. Mikovits refutation. Mary Schweitzer got in there and Michael Allen got the last word in saying essentially 'walk in my shoes for awhile and then tell me not to try anti-retrovirals"

The Blog! - Even better, truly are the comments underneath a NYT's blog pointing to the article. The NYT probably guessed they would get a boatful of comments and they did - with 112 comments already - helped out in part by a "Dr. Santana" who said all people with ME/CFS need to do is, yes, exercise more.....to get better... and 'Skeptic' clearly hailing from the UK, who believes we will all being see psychs in the future.....

Viral Integration into the Human Gene....1/2 Way There - Dr. Racaniello gave us a science lesson on viral integration in his Virology Blog. We keep hearing that viral integration into the human genome will go a long way to lay to rest some researchers fears about XMRV. It turns out that viral integration is just like it sounds. Once a virus gets into a cell it uses an enzyme called reverse transcriptase to turn its single-stranded RNA genome into the double-stranded DNA used by our cells. Then it uses an enzyme called integrase to integrate the viral DNA into our DNA. How to confirm this has happened? 'Simply' amplify the viral DNA sequences and determine if they are bordered by human DNA. ('Simply' may considerably understate the complexity of this task since it has not occurred with ME/CFS yet and only partially prostate cancer). If viral DNA is bordered by human DNA you have evidence that the virus has entered a human cell and burrowed into human DNA. At that point it doesn't matter where the virus came from; it's now infected a human cell and is now an infectious human virus.

Why is XMRV only halfway there? Because the Silverman team proved that one side of the virus was bordered by human DNA but not the other. Although they were able to show human DNA was present in every sample, and if one side is integrated in human DNA, it would seem strange that the other side wouldn't be, Dr. Raccaniello wants more.

The isolation of the entire proviral DNA, including both flanking integration sites, from patients with prostate cancer or chronic fatigue syndrome would be additional evidence that XMRV is a virus that infects humans.

Finding XMRV was integrated into human DNA would still apparently not clinch it for Dr. Racaniello - note that he'll only go so far as to say 'it would be additional evidence'...One wonders what would clinch XMRV being a human infectious virus for him (and I will ask in his blog).

Down the Rabbithole - Dr. Alan Dove, in a comment posted to the blog, wants to dot even more i's and cross some more t's. He stated that

Besides looking at the other end to check for duplication, I'd also want to see some blinded samples from healthy controls. That's especially important in this case, as they're doing a preliminary linear amplification on the patient samples before doing the junction PCR. With that much amplification, and with the first step open-ended, I think there's some room for PCR artifacts.

 

[Cort]

XMRV Buzz Jan 4th

Exhausted by Illness and Doubts - the NYTimes plopped a largely sympathetic and even-handed article on XMRV and CFS into its popular Tues Health section. Highlighting what a volatile issue XMRV has been over the past year, the article documented the ups and downs of the past year; the FDA panel recommending that blood donations be banned, the four Retrovirology papers that shortly followed, the emphatic statements by Greg Tower that it was over, Dr. Racaniello's agreement, then his backtrack then his outright rebuttal and Dr. Mikovits refutation. Mary Schweitzer got in there and Michael Allen got the last word in saying essentially 'walk in my shoes for awhile and then tell me not to try anti-retrovirals"

The Blog! - Even better, truly are the comments underneath a NYT's blog pointing to the article. The NYT probably guessed they would get a boatful of comments and they did - with 112 comments already - helped out in part by a "Dr. Santana" who said all people with ME/CFS need to do is, yes, exercise more.....to get better... and 'Skeptic' clearly hailing from the UK, who believes we will all being see psychs in the future.....

Viral Integration into the Human Gene....1/2 Way There - Dr. Racaniello gave us a science lesson on viral integration in his Virology Blog. We keep hearing that viral integration into the human genome will go a long way to lay to rest some researchers fears about XMRV. It turns out that viral integration is just like it sounds. Once a virus gets into a cell it uses an enzyme called reverse transcriptase to turn its single-stranded RNA genome into the double-stranded DNA used by our cells. Then it uses an enzyme called integrase to integrate the viral DNA into our DNA. How to confirm this has happened? 'Simply' amplify the viral DNA sequences and determine if they are bordered by human DNA. ('Simply' may considerably understate the complexity of this task since it has not occurred with ME/CFS yet and only partially prostate cancer). If viral DNA is bordered by human DNA you have evidence that the virus has entered a human cell and burrowed into human DNA. At that point it doesn't matter where the virus came from; it's now infected a human cell and is now an infectious human virus.

Why is XMRV only halfway there? Because the Silverman team proved that one side of the virus was bordered by human DNA but not the other. Although they were able to show human DNA was present in every sample, and if one side is integrated in human DNA, it would seem strange that the other side wouldn't be, Dr. Raccaniello wants more.

The isolation of the entire proviral DNA, including both flanking integration sites, from patients with prostate cancer or chronic fatigue syndrome would be additional evidence that XMRV is a virus that infects humans.

Finding XMRV was integrated into human DNA would still apparently not clinch it for Dr. Racaniello - note that he'll only go so far as to say 'it would be additional evidence'...One wonders what would clinch XMRV being a human infectious virus for him (and I will ask in his blog).

Down the Rabbithole - Dr. Alan Dove, in a comment posted to the blog, wants to dot even more i's and cross some more t's. He stated that

Besides looking at the other end to check for duplication, I'd also want to see some blinded samples from healthy controls. That's especially important in this case, as they're doing a preliminary linear amplification on the patient samples before doing the junction PCR. With that much amplification, and with the first step open-ended, I think there's some room for PCR artifacts.

 

[Cort]

An addition

Dusty Miller Study Saga Continues - Ecoclimber posted a prospective XMRV pilot study from* Dusty Miller, a noted retrovirologist at the Fred Hutchinson Cancer Institute on the both the Phoenix Rising and ME/CFS Forums to begin to round up people who had tested positive for XMRV. The posting was meant* to be followed up with a fuller explanation of the study and an interview with Dusty Miller explaining* the study and his research but the posting turned out to be premature as IRB approval had not been granted. The post was quickly removed from the PR Forums but unfortunately stayed up on the ME/CFS Forums where it received a reception not unlike a CBT trial might receive.

Soon posts and blogs went up warning ME/CFS patients not to take part in the study..Both Dusty Miller and an assistant showed up at various times to try to answer questions. Miller was accused of not having any experience in MLV's (some fact-checking was definitely missed on that one) and Andy, his assistant - was accused of lying Many members, of course, understandably simply wanted more information on what could be a very important study on XMRV..... Finally it was confirmed that Dusty Miller was Dusty Miller and that Andy was Andy...and a set of questions has been deposited with him that will hopefully clear up some fears.

The fate of the study to my knowledge is unclear. It still needs to receive IRB approval; if and when that happens and the study resumes the scope and methods of the study will be fully revealed and then the discussion will begin anew Check out an evenhanded blog on the happenings.

 

[Esther12]

Ouch... a Dusty Miller thread just came up in one of my random XMRV searches. It seems like there are a few people who really like tearing in to the guy. I may have missed the context, but it looked pretty unfair to me.

 

[Cort]

It's astonishing he's ...a good friend of Dr. Silverman....and Sandra Ruscetti and the folks on the ME/CFS Forums are tearing him a new one....he and a grad asst who tried to defend their pilot study on XMRV went on the Forum to explain and they are off now and will not be coming back...it an amazing turn of events to me and to them and to ecoclimber who is helping fund the study and just wanted to get an experienced MLV retrovirologist involved....That study has not been officially announced and when it is we'll take it from there.

 

[urbantravels]

There is a certain contingent of the ME/CFS community that is so angry and paranoid that you basically can't do anything right - unless you're the WPI, in which case you can't do anything wrong. Maybe someday we'll learn not to cut off our noses to spite our faces.

 

[Esther12]

so angry and paranoid

I'm angry and paranoid... given the way CFS has been treated it's hard not to be, but this Dusty Miller seems fine to me. He was making some nice understated posts in the thread I read, which just made the aggression of the replies even more absurd. Oh well.

 

[Sean]

There is a certain contingent of the ME/CFS community that is so angry and paranoid that you basically can't do anything right - unless you're the WPI, in which case you can't do anything wrong. Maybe someday we'll learn not to cut off our noses to spite our faces.

Sad but very true, and it has to be said. Being so partisan and ideologically pure, and incapable of reasonable productive compromise, is a sure path to impotence and irrelevance.

Sometimes I think we patients are our own worst enemy. Being the victim does not give us a free pass to behave like irrational arseholes. I understand why patients might behave like that at times, we are only human and have been pushed way past our reasonable limits. But such behaviour does not advance our cause one jot. It just plays straight into the hands of hardcore psychs and pseudo-sceptics, further entrenches the view that we are delusional and unpleasant to deal with, and worst of all it drives away competent decent people who could help us.

 

[August59]

Sad but very true, and it has to be said. Being so partisan and ideologically pure, and incapable of reasonable productive compromise, is a sure path to impotence and irrelevance.

Sometimes I think we patients are our own worst enemy. Being the victim does not give us a free pass to behave like irrational arseholes. I understand why patients might behave like that at times, we are only human and have been pushed way past our reasonable limits. But such behaviour does not advance our cause one jot. It just plays straight into the hands of hardcore psychs and pseudo-sceptics, further entrenches the view that we are delusional and unpleasant to deal with, and worst of all it drives away competent decent people who could help us.

I'm guilty, but you are exactly right. Very well said, Sean! Thanks

 

[leaves]

I dunno, I'm sure Miller means well, but I don't think I'm going to give him blood. We need a replication study. stakes are too high, we can't afford the risk.