CROI (Retrovirology and Opportunistic Infections, Boston) on XMRV and CFS

Bob

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...I thought that there was now a wide range of MLVs being found in CFS patients, including Mink focus forming virus - another mouse virus. (If I've remembered it correctly)
Specifically attacking XMRV will not be enough as other murine viruses are now associated with CFS/ME and it will be necessary to assess the significance of all of them.
Hi currer,
Do you have any further info, or references, about these other MLV's please?
Thanks,
Bob
 

kurt

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I was not looking foward to this conference, from what you guys are mentioning it doesnt seem to be depressing as much as i thought it would, though i havent heard Kurt give hes take on it yet, wonder if kurt will notice the under hand attempts to manipulate information from comments by both stoye and switzer. i never trusted j stoye, add switzer to that list plz
free, do you have a specific comment in mind? Can you give a position in the video that was posted early in this thread?

I had time to watch part of the conference video that was posted. Seems they believe they have found the origin of XMRV. What I saw in that video was realization that XMRV has the potential to to become a nosocomial infection, some lab workers may be at risk. Patient risk would seem minimal at the moment unless they work in labs. What also seemed clear was that they like XMRV as a topic of study because it is interesting, but they don't think the connection with disease will hold up (other than risk to lab workers which they now must manage).

None of these researchers were saying we are not sick, they are just saying XMRV is a lab artifact and not a likely cause of our illness, based on their studies. I think any moderation of the comments was probably just to keep discussion on that topic. I do think their retrovirus findings are credible and should not be dismissed.
 

kurt

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William M. Switzer of the CDC has a bachelor's degree in biology with a concentration on microbiology. His graduate degree is a Masters in Public Health. If he is hit with any serious technical questions he can always claim ignorance. The fact that he continues to present CDC results, instead of more qualified people, should speak volumes.
Actually, based on a mutual friend, Switzer is very competent. Just look at his publications record, the studies he has been involved with. Pubmed lists him as primary or coauthor in 77 retroviral related studies, going back to the 1980s (Pubmed search: 'switzer wm'). He is primary author of 14 of the studies, I think he is qualified to present results.

Mikovits is also well published, but not as much as Switzer. Pubmed lists her as primary or coauthor in 29 studies, also going back to the 1980s (Pubmed search: 'mikovits jm'). She is primary author of 8 of the listed studies.
 

currer

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Hi Bob,
If my memory serves me correctly I think it was in Judy Mikovits Santa Rosa lecture on jan 17. In the section where she gives her talk. She mentions that they have found XMRV, MLVs and one case of mink focus forming virus. (I hope I have remembered the virus correctly, there is so much info on these threads that its hard to remember exactly as I search through for infomation quickly.) I ought to take notes.
 

*GG*

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Actually, based on a mutual friend, Switzer is very competent. Just look at his publications record, the studies he has been involved with. Pubmed lists him as primary or coauthor in 77 retroviral related studies, going back to the 1980s (Pubmed search: 'switzer wm'). He is primary author of 14 of the studies, I think he is qualified to present results.

Mikovits is also well published, but not as much as Switzer. Pubmed lists her as primary or coauthor in 29 studies, also going back to the 1980s (Pubmed search: 'mikovits jm'). She is primary author of 8 of the listed studies.
Do the number of publications have a positive relationship with a quality researcher? Not sure what it takes to get a MPH, but it seems to me that his educational background is not that impressive.

Wouldn't it make more of a difference in what publications (some have very high standards(Science?) and my impression is that some are rags) a person has their research published?

GG

PS I think it depends upon where you work (Gov't vs Private) and how far up the ladder you are in your workplace in order to be included as a co-author on papers. I believe that it is better if you work in Public Institutions and if your research gets published, isn't that supposed to help you get tenure?
 

kurt

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Do the number of publications have a positive relationship with a quality researcher? Not sure what it takes to get a MPH, but it seems to me that his educational background is not that impressive.
I agree, most researchers this well published have more credentials. But there are exceptions, and Switzer appears to be one prolific and successful example. Maybe he has more training than appears, we don't know details of what coursework he took at what level. Anyway, there are plenty of PhDs out there that would be envious of his publication record.

Wouldn't it make more of a difference in what publications (some have very high standards(Science?) and my impression is that some are rags) a person has their research published?
He has published in the major journals in his field, particularly 'Virology,' 'J of Infectious Diseases,' and 'Retrovirology', similar to most other credible retrovirologists. He also was first author for a study published in Nature in 2005.

PS I think it depends upon where you work (Gov't vs Private) and how far up the ladder you are in your workplace in order to be included as a co-author on papers. I believe that it is better if you work in Public Institutions and if your research gets published, isn't that supposed to help you get tenure?
To be a co-author you generally must participate in the study. To be principle author you are usually the PI (principal investigator and author of the research design). But yes, this varies, I don't know the pattern in retrovirology, in my former research field the boss did not get included in the list of authors unless she or he participated directly in the research.
 

Ecoclimber

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Miller's rebuttal of Gerwyn's comment on Retrovirology

Miller's rebuttal of 'psych graduate' Gerwyn comment ( I wonder what PA institute means, hmm?) on
Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection in Retovirology



[
B]Gerwyn Morris[/B] (26 February 2011) PA institute

The authors are clearly not familiar with MLV viruses

MLV viruses have a preference for integrating within certain genes involved in the promotion or repression of tumours. A classic example would be PIM-1(1) or P53 (2) n-myc(3)is also a common integration site.
Thus mulv virus' display the propery of integrating into the same sites repeatedly.It is surprising that the authors have not researched into the subject. Hence the following section of the paper is completely incorrect

"With the exception of a single early publication on avian
sarcoma-leukosis virus, which was refuted by later work [10], sequencing studies of
thousands of retroviral integration sites have to our knowledge never identified exactly the
same site twice."

The following table demonstrates just how inaccurate the author's comments are. They illustrate the integration sites within genes of the class of virus to which xmrv belongs

BMC

The reader will note that the activity of these genes are all related to the development of cancer in humans

The authors are stating beliefs in the guise of fact.
They state that the sequences were unlikely to have been transfered into the DU125 cells from the patients examined without nothing but their biases as evidence.

Likewise they voice their belief that the other integration sites are probably caused by contamination despite failing to provide any evidence despite a rigorous effort to discover said evidence.

The integration of a mulv class virus into the PIM-1 gene leads to the overexpression of PIM-1(1).The degree of overexpression of PIM-1 correlates with severity of prostate cancer in humans(1)

I submit that patients with prostate cancer are entitled to anyone ,purporting to be scientists, investigating the link of XMRV to their condition limit themselves to publishing scientific evidence and not speculation in the guise of scientific evidence as is,sadly,the case here

references

(1)Wong KS, Li YJ, Howard J, Ben-David Y. Loss of p53 in F-MuLV 14induced-erythroleukemias accelerates the acquisition of mutational events that confers immortality and growth factor independence. Oncogene. 1999 Sep 30;18(40):5525-34.

2)Cuypers HT, Selten G, Quint W, Zijlstra M, Maandag ER, Boelens W, van Wezenbeek P, Melief C & Berns A. (1984) Cell 37: 141–150.

(3)van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H & Berns A. (1991) Cell 65: 737–752
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Response to the comment by Gerwyn Morris posted 26 February 2011

A Dusty Miller (02 March 2011) Fred Hutchinson Cancer Research Center, Seattle

Morris' criticism of the article by Garson et al. is unwarranted, and stems from a misunderstanding of the difference between retrovirus integration near a gene versus integration at exactly the same site in the whole human genome. It is true that murine leukemia viruses (MLV) can often be found within or near oncogenes involved in the progression of tumors in MLV-infected mice. However, integration within 1,000 or 10,000 nucleotides of a gene is quite different from integration at exactly the same nucleotide position in a whole mammalian genome. None of the references provided by Morris show integration of a retrovirus at the same nucleotide position.

I do agree that it would be nice to have formal support for the statement that "With the exception of a single early publication on avian sarcoma-leukosis virus, which was refuted by later work [10], sequencing studies of thousands of retroviral integration sites have to our knowledge never identified exactly the same site twice." However, given our current understanding that retrovirus integration is semi random and can target most of the 3 billion base-pair-long human genome, the authors' contention seems quite reasonable.

Lastly, derogatory comments like "The authors are clearly not familiar with MLV viruses" have no place in these posts.
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response to Miller

Gerwyn Morris (02 March 2011) PA institute
in response to the letter by Miller

Would he argue with the points below?

The authors state that the sequences were unlikely to have been transfered into the DU125 cells from the patients examined without nothing but their biases as evidence.

Likewise they voice their belief that the other integration sites are probably caused by contamination after failing to provide any evidence despite a rigorous effort to discover said evidence.

I submit that scientists who publish unfounded opinion and not fact is the main issue here

Miller talks of unacceptable phraseology.I am therefore somewhat surprised that he has not highlighted the extract below

"we suspect that some or all
of them may also be the result of contamination with DNA from experimentally infected DU145 cells. It is striking that there have been no independent reports of patient-derived XMRV integration sites nor have there been any descriptions of polytropic or modified polytropic MLV integration sites in human samples despite the apparent detection of these viruses in CFS patients [5]."

The authors are showing bias and are the use of the word "apparent" are denigrating the work of others

I would submit that this is an example of wording which has no place in any journal

On the other hand the authors appear to have no issues with the fact that their findings have not been supported or that they could not demonstate any evidence that the 12 integration sites were not genuine

Considering the evidence as a whole the results can easily be explained by the transfer of proviral DNA from patients into the cell lines.

Would Miller dispute that and why?

How would Miller explain the integration of XMRV into CREB5 and NFATc3?

The essence of the scientific method is hypothesis testing

Hypotheses are tested via challenge or an active attempt to disprove the proposed explanatory model

The authors have clearly not engaged with the scientific method in this study

I am surprised that Miller has not raised this fact

When the authors describe integration sites they appear to be discussing integration into the same genome or region of a genome rather than a single nucleotide as raised by Miller.In which case the statements made by the authors are erroneous as per my original references

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Citation request by Dr. Miller--

Abigail Smith (02 March 2011) OUHSC
If you do not want a list of every integration site ever (heh), Mitchell et al might be suitable citation: "Retroviral DNA Integration: ASLV, HIV, and MLV Show Distinct Target Site Preferences"

They identified 3127 insertion sites of HIV-1, ASLV, and MLV and they did not mention observing any duplicates. As far as MLV goes, their analysis of Chromosome 11 showed, at max, 3 MLV insertion sites within the same 2 Mb region.

I do not know of any published duplicated insertion sites myself. It certainly strains credulity that a lab only sequenced a handful of integrations, and found not just one duplication, but two. And the duplications just happen to have identical LTRs as well.
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the study cannot demonstrate direction of transfer

Gerwyn Morris (03 March 2011) PA institute


The authors did not follow the scientific method and demonstrate that the DU125 cells did not contain integrated DNA before the start of the experiment. If the cell line was "contaminated" with human XMRV then the results would have been exactly the same

The other problem they have is that none of the studies that identified and isolated XMRV used the the DU145 cell line in any way whatsoever

The experiment that isolated integrated XMRV directly from the prostste tissue of infected patients also did not invole the use of DU145

The question of identical insertion sites is a probabalistic argument and purely determined by sample size and the affinity of a retrovirus for GPG islands

XMRV has been demonstrated a greater affinity for these sites than any known retrovirus.

DU145 cell lines began in 1978 yet Towers asserts that the origin of the XMRV detected in prostae cancer is the "XMRV" in the 22RV1 cell line which began in 1993

Schalberg et al(2009) reported that RT PCR only detected xmrv in samples taken from 6% of prostste cancer suffere while IHC showed 22% of paients to be positive

This is an interesting observation if XMRV is a Mouse contaminant!

I note Miller has not replied to any of the other points I raised

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Response to the comment by Abigail Smith posted 02 March 2011

A Dusty Miller (04 March 2011) Fred Hutchinson Cancer Research Center

Thanks for the citation (Mitchell et al., PLoS Biology 2:E234, 2004). While these authors did not mention observing any duplicate integrations, they do state in Materials and Methods that only novel integration site sequences were deposited at the NCBI, implying that some sites were not novel and thus were duplicates. Duplicates would not be unexpected in a single experiment, because PCR amplification results in many copies of what might have initially been a unique site, and if one clones and sequences enough sites, duplicates are bound to be found. For example, if 1,000 cells were infected with 10 of their replication-defective HIV vector particles, resulting in 10 integrated proviruses, cloning of 100 integration sites from DNA isolated from these cells would result in detection of multiple identical (duplicate) integration sites. On the other hand, infection of a million cells with 100,000 vector particles followed by cloning of 100 integration sites would be very unlikely to identify identical sites.

Thus, to get at the question of whether retroviruses integrate at identical sites at much higher rates than would be expected by chance, and thus might explain the identical XMRV integration sites identified by Garson et al., one needs to sequence integration sites from at least two independent experiments, and compare integration sites between experiments for duplicate integrations. The data to make this comparison is obviously available, but I’m not aware of anyone having performed this analysis. If someone has done so, I’d appreciate being informed.

This discussion gets at the heart of the contamination issue raised by Garson et al. In the experiments they discuss, 14 XMRV integration sites in prostate cancer tissue are compared to ~500 XMRV integration sites found in acutely-infected DU145 human prostate cancer cells, and two identical (duplicate) integration sites are observed in these theoretically independent experiments. Given the number of possible integration sites in the human genome (~3 billion), cross contamination almost certainly explains the result. That is, unless there are some unusually hot spots for retrovirus integration, which likely would have been identified by now.
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Response to the comments by Gerwyn Morris posted 02 March and 03 March 2011

A Dusty Miller (04 March 2011) Fred Hutchinson Cancer Research Center

You note that I have not replied to your previous comments. This is because of their voluminous nature, your inability to correctly interpret what is basically a sound article, and because I have other things to do!

In your post of 02 March, the main substantive point questions the proposed direction of contaminant transfer, from the XMRV-infected DU145 cells to the prostate cancer tissue. In the Kim et al. 2008 paper describing these studies, they infected DU145 cells with XMRV at a multiplicity of 0.1 and the cells were grown for 3 days. By this time, it is likely that all of the cells carried at least one integrated copy of XMRV, because XMRV is replication-competent and can readily spread in cultured DU145 prostate cancer cells. In contrast, the number of integrated copies of XMRV in fresh prostate cancer tissue is estimated to be low, on the order of 1 copy per 100 cells, if it is there at all. Therefore, contamination of the prostate tissue DNA with heavily-infected DU145 cell DNA is more likely to result in false positive results than the reverse. Furthermore, the real question here is whether XMRV can be found integrated into prostate cancer cells from a human, not whether the DU145 cells are infected. The finding of identical integration sites in these two samples suggests contamination of the prostate cancer samples with XMRV-infected DU145 DNA or PCR products amplified from this DNA, and threatens the conclusion that XMRV is found integrated into the DNA of prostate cancer tissue from humans.

In your post of 03 March, the main substantive point appears to question how DU145 cells could have been involved in the studies of XMRV integration sites in prostate cancer tissue. The answer is that the analysis of XMRV integration sites in XMRV-infected DU145 cells was performed at the same time and in the same lab as was the analysis of XMRV integration into DNA from human prostate cancer tissue, thus the possibility of cross contamination must be considered. Many of your other questions make no sense to me, and I don’t have the time to delve into your underlying meanings. I suggest you seek further clarification and answers to your questions from knowledgeable colleagues.
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Cort

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Honestly Ana this does nothing for Gerwyn's stance for me....I think Miller believes Towers knows how to do that experiment correctly and did not introduce XMRV into the cells prior to doing the study. For me - its hard to read, let alone trust, someone who is continually taunting the other.

Look at this

By this time, it is likely that all of the cells carried at least one integrated copy of XMRV,
Gerwyn:

Now he enters speculative mode. Note: this is not even the paper I raised.


Well, yes he is speculating. Why? Because past results of experiments in the past undoubtedly suggest that this is true. Since they didn't actually measure this he had to assess that based on his knowledge...Gerwyn chooses to call informed reasoning - mere speculation...

Then as Miller brings the findings of another paper to bear on the subject - as researchers often due - Gerwyn simply dismisses this as 'not even the paper I raised"....?

because XMRV is replication-competent and can readily spread in cultured DU145 prostate cancer cells. In contrast, the number of integrated copies of XMRV in fresh prostate cancer tissue is estimated to be low, on the order of 1 copy per 100 cells, if it is there at all.
Gerwyn: Note "if at all." and so what?

???? - yes, given the recent findings - it is entirely appropriate to say, if at all regarding XMRV's presence in prostate tissue. In fact, any researcher should say that - but Gerwyn doesn't like to have his favorite finding bashed.....Notice that Miller leaves open the possibility of XMRV being there as well

The finding of identical integration sites in these two samples suggests contamination of the prostate cancer samples with XMRV-infected DU145 DNA or PCR products amplified from this DNA, and threatens the conclusion that XMRV is found integrated into the DNA of prostate cancer tissue from humans.
Gerwyn: The point, of course, is that if the sequences were introduced during the experiment the conclusions of the study are false!

I think I do understand Gerwyn's point - he's frustrated because Miller had not responded to his main question - which is understandable.

The answer is that the analysis of XMRV integration sites in XMRV-infected DU145 cells was performed at the same time and in the same lab as was the analysis of XMRV integration into DNA from human prostate cancer tissue,
Gerwyn: No evidence is given to support that comment of course.

?? Again this kind of childish retort - the evidence is apparently in the papers cited and is easy enough to assess by looking at them.

Gerwyn may have a point - I don't know. His posts have been described to me by researchers as a mishmash of sometimes valid and other times invalid points....Its hard for us to assess because Gerwyn has delved deeper into the field than most of us.
 

Sean

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This should be a scientific issue. I don't give a damn if XMRV pans out or not. I DO CARE about finding out what has wrecked my life. This may be XMRV or it may not. This is a scientific issue. It's vitally important that XMRV is not trashed before it's properly checked out. On the other hand, we don't want researchers spending time and resources on something which is not relevant. That's why we are in a very difficult position at present.

What won't help is anyone being obnoxious or beligerent or intolerant. I don't read the other forums because the people who stopped posting here and went there had a different standard of acceptable forum behaviour. Let's debate 'til the cows come home but keep it civil. If you don't keep it civil you scare people off.
Couldn't have put it better myself.
 

Mark

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Thanks for the abstracts Rita.

Paper # 237
A Sensitive Real-time PCR Assay for the Detection and Quantification of XMRV
Laura Li*, M Raines, and T Robins
Quest Diagnostics Clin Trials, Valencia, CA, US

...we have developed a sensitive real-time PCR assay that reliably detects XMRV from CFS patients...

...The performance of our quantitative real-time PCR assay was determined, and then it was compared with the current XMRV detection method...only the quantitative real-time PCR assay could detect XMRV in non-co-cultured PBMC.
The WPI have been working with Quest, I think? So I'm not sure how much that queries their status as 'independent' confirmation...but how far away is this Quest Diagnostics team's claim from confirming WPI's findings? One randomised control trial away?...They are saying they can "reliably detect XMRV from CFS patients" !!!
 

urbantravels

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What won't help is anyone being obnoxious or beligerent or intolerant. I don't read the other forums because the people who stopped posting here and went there had a different standard of acceptable forum behaviour. Let's debate 'til the cows come home but keep it civil. If you don't keep it civil you scare people off.
I agree with all of the above 100%, except that I would add: If you don't keep it civil, you damage your own credibility and the credibility of the argument you are making.

The best debaters are those who can keep their statements civil even in the face of provocations by others to drop below that standard.
 

SOC

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This should be a scientific issue. I don't give a damn if XMRV pans out or not. I DO CARE about finding out what has wrecked my life. This may be XMRV or it may not. This is a scientific issue. It's vitally important that XMRV is not trashed before it's properly checked out. On the other hand, we don't want researchers spending time and resources on something which is not relevant. That's why we are in a very difficult position at present.

What won't help is anyone being obnoxious or beligerent or intolerant. I don't read the other forums because the people who stopped posting here and went there had a different standard of acceptable forum behaviour. Let's debate 'til the cows come home but keep it civil. If you don't keep it civil you scare people off.
[snip]
Let's all be on the side of the CFS patient. Life's too short and exhausting for all that other stuff.
I agree 100%. Let's work for the best science possible, wherever it leads. We want to know what's making us ill and how to treat it.
 

jace

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Do bear in mind, while analyzing personalities, that Greg Towers (author of the paper under discussion) is the author of the misleading press release that stated that it had been proved that XMRV is a contaminant, over-blowing the results of the December 20th Retrovirology papers.

Bear in mind also that Gerwyn does not stand to gain money or status, he is working to shine light on the research purely because, like us, he wants to be well.

ETA
Gerwyn may have a point - I don't know. His posts have been described to me by researchers as a mishmash of sometimes valid and other times invalid points....Its hard for us to assess because Gerwyn has delved deeper into the field than most of us
Cort, this unsubstantiated allegation is worth precisely nothing. While Gerwyn tackles Miller point by point, you throw out a generalisation, appealing to authority (described to me by researchers) without giving any facts, like the identity of these "researchers". Gerwyn is putting his reputation on the line for us. Vague criticisms of his work, like yours above, are unhelpful, and bad for your reputation.
 

biophile

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Honestly, I don't know which side is correct, but as I understand it, Gerwyn appears to be holding his own despite lacking formal qualifications (correct?).

When it comes to ME/CFS, experts having their ass handed to them by mere lay patients isn't all that surprising for the soft psych sciences, but when it comes to a field such as retrovirology I would expect this to be much more unlikely. I can only imagine the embrassment a "renouned expert" like Miller would feel if XMRV/MLV turned out to be significant and Gerwyn had been right all along.