Nature: Spoonful of Medicine: There's no tiring of controversy in the XMRV-CFS link

[barbc56]

Interestingly, Dr Singh found XMRV in about 5% of the normal population in her tissue study, but was unable to detect any XMRV in her blood study. She has stated that she is unable to explain the differences. Although there are apparent discrepancies between the results of her different studies, Singh still felt able to declare, categorically, that XMRV does not exist in ME patients even though she does not understand her own results. This, apparently, is OK for everyone, and is good science at work.

Although Dr Singh's work confirmed the WPI's work, with respect to the normal population, I don't see anyone accusing her of fraud or of sloppiness or whatever.

Bob, this was from her earlierl work. She later determined that she was picking up contamination. She revised her conclusions based on the data from her experiment
From PubMed:

Absence of XMRV retrovirus and other murine leukemia virus-related viruses in patients with chronic fatigue syndrome.
Shin CH, Bateman L, Schlaberg R, Bunker AM, Leonard CJ, Hughen RW, Light AR, Light KC, Singh IR.
SourceDepartment of Pathology, University of Utah, 15 North Medical Drive East, Suite 2100, Salt Lake City, UT 84112, USA.

Abstract
Chronic fatigue syndrome (CFS) is a multisystem disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently, CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blind manner using molecular, serological, and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS patients. We did not find XMRV or related MLVs either as viral sequences or infectious viruses, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses, including XMRV, and the off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.

http://www.ncbi.nlm.nih.gov/pubmed/21543496

Bold is mine.

 

[ukxmrv]

Barb,

If your argument is correct I would expect Dr Singh to have concluded that the results on her prostate cancer samples were due to contamination as well?

It would be illogical for Dr Singh to argue that she would find XMRV in tissue samples in prostate cancer but that blood testing only was definitive and the final answer for CFS when she hadn't even tried a method that supposedly worked (i.e. tissue).

 

[Firestormm]

I think this is a valid point UKXMRV. Those Prostate Cancer studies....hmm.... Hopefully Singh and others will review them again. Well, I hope so as it would tick another box.

Interestingly, (due to my reading lately) Patrick Moore I think made the comment that something like 'How can Prostate Cancer relate to CFS?'. Was always a concern I guess and another question that perhaps has now been answered? Well I think it has personally.

Prostate Cancer will be contamination along with CFS.

 

[Bob]

Bob, this was from her earlierl work. She later determined that she was picking up contamination. She revised her conclusions based on the data from her experiment
From PubMed:

Absence of XMRV retrovirus and other murine leukemia virus-related viruses in patients with chronic fatigue syndrome.
Shin CH, Bateman L, Schlaberg R, Bunker AM, Leonard CJ, Hughen RW, Light AR, Light KC, Singh IR.
SourceDepartment of Pathology, University of Utah, 15 North Medical Drive East, Suite 2100, Salt Lake City, UT 84112, USA.

Abstract
Chronic fatigue syndrome (CFS) is a multisystem disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently, CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blind manner using molecular, serological, and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS patients. We did not find XMRV or related MLVs either as viral sequences or infectious viruses, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses, including XMRV, and the off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.

http://www.ncbi.nlm.nih.gov/pubmed/21543496

Bold is mine.

Thank you for the info barb, but it doesn't address the points that I raised.
The discrepancy, that I referred to, is between the positive results in the prostate cancer study, and the negative results in the CFS study.
Whereas this only addresses the discrepancies between this CFS study, and other CFS studies.

 

[currer]

http://aboutmecfs.org/forums/conten...ncer-Results-Validated-Progress-on-CFS&page=3

This is a link to the PR blog by Cort on Dr Singh.

It mentions that her antibody findings mirrored her immunohistochemistry positives at @ 30%

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739868/?tool=pmcentrez

XMRV is present in malignant prostate epithelium....Schalberg et al.

 

[barbc56]

From the National Cancer Institute:

2011:

In contrast to previous findings, and in keeping with reports that subsequently emerged in the literature, two independent NCI studies failed to detect XMRV in humans. In other studies, it was observed that XMRV replication is severely inhibited in human white blood cells, making it doubtful that XMRV replicated efficiently in the blood cells of CFS patients as previously reported. Additional evidence was obtained showing that previous data was potentially confounded by the presence of contaminating mouse DNA, and/or by contamination with XMRV virus in the laboratory.

http://www.cancer.gov/newscenter/qa/2011/xmrv_qa

As far as I know, Singh has not said anything recently one way or another about prostate cancer and XMRV.

I would think (opinion) she would go with the scientific current thinking based on recent studies. If she didn't it would seem more likely that she would have said something.

We are talking about two different experiments by Singh. Each with a different disease. It does not mean she was contradicting herself. She was simply reporting her conclusions from the results of the data. This is how science should work. It evolves as new information is gathered.

 

[Bob]

We are talking about two different experiments by Singh. Each with a different disease. It does not mean she was contradicting herself.

Indeed, they are two different experiments with two different methodologies - one investigating blood and the other investigating tissues - so different results can be expected and both sets of results can be correct. So, strictly speaking, the results are not necessarily inconsistent

But because of these different results, specifically in relation to the normal population, I question why Dr Singh is so certain about her results with blood in the CFS study, when the tissue study gave different results within the normal population.

The differences have not been explained by anyone including Dr Singh so I would like to see further investigation carried out to explain them.

She was simply reporting her conclusions from the results of the data.

If she was just reporting her data, then she would not have concluded that there is no association between ME and XMRV.
She would just have said that she was unable to detect XMRV.
She did not test the prostate tissue of ME patients, when prostate tissue had previously shown positive results in non-prostate-cancer-patients, so I question her certainty with regards to her blood study results.

This is how science should work. It evolves as new information is gathered.

Yes, I totally agree, but I don't hear that sort of charity and room-for-evolving-knowledge being equally directed towards the WPI.
A bit of consistency would be welcome.
When there are discrepancies or questions with the WPI's results, or the science develops, Judy Mikovits is accused of all sorts.
Whereas where Dr Singh, Switzer and others are concerned, there doesn't seem to be any sort of negative focus (which is how I think it should be).

As barb says; this is how science should work - Studies are published, and the science evolves as new information is gathered.

 

[currer]

The Schlaberg et al paper is an impressive piece of work and as I havent heard any calls for its retraction, I will assume its authors stand by their findings.

Singh analysed 233 prostate cancers and 101 controls.
She detected XMRV DNA by PCR in 6.2% of prostate cancer and 2% of controls.

However her antibody results showed XMRV proteins in 23% prostate cancer samples and only 4% of the controls.
She observed XMRV specific staining predominantly in the malignant prostatic epithelial cells.

She found that the presence of XMRV correlated with higher tumor grades as correlated by the gleason score.

Her conclusions were that the presence of XMRV correlated with more aggressive cancers, and that XMRV proteins are expressed almost exclusively in cancerous epithelial cells.

She suggests that antisera specific to XMRV offer a more sensitive means of viral detection than the use of the monoclonal antibody to SFFV gag.

Interestingly, if my interpretation of her paper is correct, she found it easier to detect the presence of XMRV using antisera, than by PCR.(23% positive, as opposed to 6%)

Positivity in her controls ranges from 2% to 4%.
This is why her findings in ME were questioned. From her own past research, she should at least have found 2-4% positives if her assays worked in her ME paper.

Does anyone know if this paper is about to be retracted?
I have nothing against Singh, but it does seem unfair that Dr Mikovits should be singled out if other researchers are also guilty of finding viruses that are not there.

So what do you think Firestormm? Does this look like contamination to you?

 

[Wonko]

It doesn't matter what the study type, or the tissue type, or who did it. If XMRV is a manufactured RV, or contamination, and doesnt exist outside a lab/cell lines (i.e. it doesnt exist in humans), then ANY study finding it anywhere in human tissue must logically be wrong - even if said study isnt associated with ME.

Either that or the assertion that XMRV doesnt exist in humans is wrong.

One or the other - they cant use both arguements depending on convienence.

 

[barbc56]

Yes, I totally agree, but I don't hear that sort of charity and room-for-evolving-knowledge being equally directed towards the WPI.
A bit of consistency would be welcome.
When there are discrepancies or questions with the WPI's results, or the science develops, Judy Mikovits is accused of all sorts.
Whereas where Dr Singh, Switzer and others are concerned, there doesn't seem to be any sort of negative focus (which is how I think it should be).

But it is consistent. I would use the same standards for any scientiest. I haven't seen any inconsistencies with Dr. Singh, therefore she doesn't get the focus. Not ony are there serious questions about the original Science paper, Judy has been the most vocal of the lot and thus gets attention, good or bad. It falls where it falls.

 

[justinreilly]

But it is consistent. I would use the same standards for any scientiest. I haven't seen any inconsistencies with Dr. Singh, therefore she doesn't get the focus. Not ony are there serious questions about the original Science paper, Judy has been the most vocal of the lot and thus gets attention, good or bad. It falls where it falls.

Dr. Singh has been inconsistent as Bob, currer and wonko explained. I don't fault her science at all, but i do fault her inconsistent interpretations of the data that she put in the paper and spread in the media. Her coming to conclusions like ME and XMRV were unrelated wasn't supported by her own published data.

 

[barbc56]

Dr. Singh has been inconsistent as Bob, currer and wonko explained. I don't fault her science at all, but i do fault her inconsistent interpretations of the data that she put in the paper and spread in the media. Her coming to conclusions like ME and XMRV were unrelated wasn't supported by her own published data.

I guess I am not understanding yours as well as others you mention, take on this. I see the second Singh study as a very well designed study as well as extremely through. She eventually found the contamination. That says a lot.

It appears you and others are saying because her first study did not have the same conclusions as the second study, she isn't consistent.

As far as the discrepant results in the prostate study and the me/cfs, I still don't see in inconsistency.

But why are we talking about Singh here when the bottom line is that XMRV as well as HGRVs didn't pan out in either me/cfs nor prostate cancer.

Are you trying to say that since you believe Singh is inconsistent we can't find fault with Judy Mikovits?

I would ask you to elaborate but I actually answered as well as others on the forum, this exact same question on another thread and we are now going around in circles.

We have different takes on things and I have moved on to other related topics.

Science will win out in the end. Time will tell.

 

[currer]

Hi Barb,

This is just what I am trying to establish.

Has Silverman or Singh retracted their prostate cancer findings connecting XMRV to prostate cancer?

Do you have a link or reference for that statement please?

It seems to me that many of these discussions are going ahead in a very confused way because the scientific findings are all up in the air and no-one knows which findings are valid now.

I am trying to establish exactly where we stand.

So please do you have a link to prove that prostate cancer is not connected to XMRV?

 

[Bob]

I guess I am not understanding yours as well as others you mention, take on this. I see the second Singh study as a very well designed study as well as extremely through. She eventually found the contamination. That says a lot.

It appears you and others are saying because her first study did not have the same conclusions as the second study, she isn't consistent.

As far as the discrepant results in the prostate study and the me/cfs, I still don't see in inconsistency.

But why are we talking about Singh here when the bottom line is that XMRV as well as HGRVs didn't pan out in either me/cfs nor prostate cancer.

Are you trying to say that since you believe Singh is inconsistent we can't find fault with Judy Mikovits?

I would ask you to elaborate but I actually answered as well as others on the forum, this exact same question on another thread and we are now going around in circles.

We have different takes on things and I have moved on to other related topics.

Science will win out in the end. Time will tell.

But I think you are making a premature mental leap here barb. You appear to be assuming that because contamination was found in Dr Singh's CFS study, that it applies to her earlier studies, and all other prostate cancer studies.

But we cannot make that assumption. The other studies may or may not have been contaminated. We just don't know. There is no definite evidence that they were contaminated, or that contamination was responsible for all of their results.

Even if VP62 did not actually exist in Silverman's original prostate cancer study, there may have been other MLV or XMRV sequences. They need to do further research to understand the potential for contamination in the positive prostate cancer studies.


My earlier point was that there is no fundamental difference between the Science paper and the other positive XMRV studies. They all detected XMRV or MLV sequences.

So if the Science paper needs to be withdrawn now, based purely on other researchers not finding XMRV, then so do the other positive XMRV studies, if there is to be consistency.

And I still cannot see any reason why Dr Mikovits is attacked any more than Dr Singh, based on their positive XMRV results, and their similarly strong but opposing statements about XMRV in CFS patients. I don't think that anyone should be attacked for carrying out honest science. If there is criticism to make, then it is the strong statements made by scientists on all sides of the debate.

Judy Mikovits has always said that XMRV is a novel human retrovirus, and she has been proved right about that. The only question is whether it infects humans in the wild, or only infects human tissue in the lab. Even Harvey Alter agreed that his research showed that MLV-like retroviruses infect humans, although I don't know what his current opinion is. Switzer of the CDC went so far as to agree that XMRV infects humans in the wild, and demonstrated that infection in his research, along with publishing sequences that confirm a wild infection.

 

[Esther12]

But I think you are making a premature mental leap here barb. You appear to be assuming that because contamination was found in Dr Singh's CFS study, that it applies to her earlier studies, and all other prostate cancer studies.

But we cannot make that assumption. The other studies may or may not have been contaminated. We just don't know. There is no definite evidence that they were contaminated, or that contamination was responsible for all of their results.

From what I remember Singh still sounded positive about the link between XMRV and PC at the time of her CFS study. This was a long time ago though, prior to the recombination type stuff (I can't believe how long we've been paying attention to XMRV).

I'd be very surprised if the association with PC held up. The association with PC is much less politically wrought that the association with CFS, so controversy about these findings has received far less attention.

 

[currer]

It does seem that all the positive XMRV papers are now in the same boat.
Although they do show a clear difference in positives between controls and patient samples.

Silverman states that his initial interest in investigating gammaretroviral infection in prostate cancer was sparked by using a DNA microarray to screen for conserved sequences of viral genomes.
The positive signal for gammaretroviral infection resulted in the eventual cloning of three viral genopmes, VP35,42 and 62.

At the time I do not think it was realised that the 22RV1 cell line was producing the VP62 strain of this MLV.

Mikovits herself also used the virochip DNA microarray to initially screen her samples for viral genomes. This is why she became interested in the possibility that a gammaretrovirus was in her ME samples.
Remember that her serology also suggests a gammaretroviral infection as her samples showed an antibody reaction to gammaretroviral proteins, that has to happen in life, and that could not happen if the VP62 plasmid had contaminated thiose samples at a later date.

I suppose the question still is unanswered as to what gammaretrovirus exists in these samples - if it is a gammaretrovirus causing this antibody reaction.

It seems to me that Silverman could have been cloning the VP62 virus from the 22RV1 cell line without realising it if he had unacknowledged contamination in his lab or in his samples.

There still remain the other two clones, which do not appear to have come form the 22RV1 cell line.
Dr Yes, from the other forum has pointed out that the WPI and NIH could not find Silverman's env genes in their samples which suggests that their retrovirus is distinct from VP62

An interesting further point is that an infectious murine leukemia virus DG75 is produced by a human B-lymphoblastoid cell line.
Given that there is a connection between lymphoma and ME, and that Dr Snydermans CLL retrovirus is about to be sequenced, it will be interesting to see if there is any connection between DG75 and any human cancer outside a laboratory cell line.

The paper by Zhang et al, "Frequent detection of Infectious xenotropic murine leukemia virus in human cultures established from mouse Zenografts" discusses the history of contamination of cell lines by these viruses.

Reports of XMLV strains being present in human xenograft cultures appeared in the 1970s, yet this information was forgotten, and the authors state that most present day scientists appeared unaware of the problem.

This paper concludes that 26% of xenograft cultures tested were positive for XMLVs, sometimes with multiple strains.
Supernatent fluids release large numbers of infectious viral particles, with a potential for horizontal spread to ther cell lines or lab personnel.

Indeed horizontal spread can occur rapidly.
The XMRV virus has been shown to infect another cell line, the RKO colorectal cancer cell line, after only being kept in the same facility for a few days.

It seems to me that this unacknowledged problem with contaminated cell lines, cell lines infected with murine leukemia viruses, suggests that it will be very difficult to determine whether any patient samples are free from contamination.

Equally should patients themselves be genuinely infected with these lab-created retroviruses, it could be tricky to find a lab that is sufficiently uncontaminated, has uncontaminated reagents and uncontaminated personnel, for a clear and unquestioned result.

In this case perhaps the serology, the evidence from antibody reactions, might be a preferable way forward.

 

[Bob]

Very interesting post thanks currer.

The research in a very confusing place right now.

I can't see them sorting out these issues for ages.

If there are MLVs/HGRV's in humans and they are present as contamination in the research at the same time, then it's going to be especially difficult to disentangle them both.

It seems to me that further serology research might be the best way forwards for anyone continuing with retrovirus research.

 

[barbc56]

When a sample is activated, it is often picking up all sorts of background noise which includes bits and pieces of HGRVs. At this time this background noise does not act like a contagious HGRV. They act like contmination. People assume that all HGRVs are dangerous.

 

[currer]

Hi barb,

The Zhang paper,
http://www.landesbioscience.com/journals/5/article/15955/

is specifically discussing infectious MLVs and their spread in cell culture. The authors excluded any cell line from their research which tested positive for mouse DNA.

They obtained 26 xenograft cultures from seven independent laboratories.
Nine samples proved positive for XMLVs, however three of those also contained evidence of mouse DNA and were excluded.

Six of the remaining 23 xenograft cultures (26%) were positive for one, or in one case, two strains of XMLV virus.

Supernatant fluid was available from five of the six positive lines. "Four of these released large amounts of potentially infectious viral particles, indicating possibilities of horizontal spread to other human cultures as well as posing a biohazard of unknown potential to lab. personnel."

"We also found evidence of widespread contamination of non zenografted cultures maintained in the same tissue culture facilities with zenografted cultures....to rigorously prove that the virions released.... were infectious for human cells, we collected supenatant fluid from 3 XMLV positive xenografted cultures and used them to infect five other human cell lines...."

 

[barbc56]

Hi barb,

The Zhang paper,
http://www.landesbioscience.com/journals/5/article/15955/

is specifically discussing infectious MLVs and their spread in cell culture. The authors excluded any cell line from their research which tested positive for mouse DNA.

They obtained 26 xenograft cultures from seven independent laboratories.
Nine samples proved positive for XMLVs, however three of those also contained evidence of mouse DNA and were excluded.

Six of the remaining 23 xenograft cultures (26%) were positive for one, or in one case, two strains of XMLV virus.

Supernatant fluid was available from five of the six positive lines. "Four of these released large amounts of potentially infectious viral particles, indicating possibilities of horizontal spread to other human cultures as well as posing a biohazard of unknown potential to lab. personnel."

"We also found evidence of widespread contamination of non zenografted cultures maintained in the same tissue culture facilities with zenografted cultures....to rigorously prove that the virions released.... were infectious for human cells, we collected supenatant fluid from 3 XMLV positive xenografted cultures and used them to infect five other human cell lines...."

If you could cite your source it would be easier for me to read. I'm not sure what the copyright policy is here but on some forums if you quote something you need to cite the source. If this has been asked before and I missed it, someone let me know. Thanks!!