XMRV in ME/CFS:Video Presentation by Mikovits in Goteborg on 02/12/2010

[shannah]

Apparently contains some new unpublished data so there's a chance it could be taken down.

XMRV Global Action

Videos of Dr Mikovits' presentation in Goteborg, Norway (from Dec 2, 2010). We've posted a summary of the Goteborg presentations (Dec 14th). Here are the 4-part videos, with thanks to thx1138mindlock on YouTube.

Part 1:
[video=youtube;pAD2_YfgNV4]http://www.youtube.com/watch?v=pAD2_YfgNV4[/video]
Part 2:
[video=youtube;n3kdU5FhH2U]http://www.youtube.com/watch?v=n3kdU5FhH2U[/video]
Part 3:
[video=youtube;fNJe5ltD2K0]http://www.youtube.com/watch?v=fNJe5ltD2K0[/video]
Part 4:
[video=youtube;C-4BSPFuNHU]http://www.youtube.com/watch?v=C-4BSPFuNHU[/video]

 

[LJS]

The first two videos are set to private so we are unable too watch them. The third video address is copy and pasted wrong and the forth video address is invalid. Thanks for the info though, hopefully they will be released to the public again.

 

[LJS]

Here is a link to the discussion on these videos from the XMRV global action facebook page: http://www.facebook.com/permalink.php?story_fbid=117674931635469&id=216740433250

Here are some posts in the discussion of these videos

XMRV Global Action
FYI: At ~5:30 on the second video, Dr Mikovits discusses a slide entitled, Assays to Detect Anti-XMRV Antibodies. She talks about Dr Rachel Bagnis work in developing an ELISA on plasma. Preliminary results for this test:
34/39 CFS p...atients positive
10/77 donors positive (a whopping 12.99%!)
NB: Rachel is doing a confirmatory Western Blot to prove that these are indeed positives, and not a nonspecific response. (Cant wait for that research)
M.S.


XMRV Global Action
At approx 8:30; Video 2
Slide: Possible Reasons for Disparity in XMRV Detection
- False positives due to PCR or contamination with mouse cells

Dr Mikovits (quoted on Dec 2, 2010): And of course if PCR is the only technique that youre study...ing to isolate the virus, you could have contamination from mouse cells

Retrovirology - tell us something new!
M.S.


XMRV Global Action
At approx 12:00; Video 2
Slide: Detection of gag XMRV RNA in Plasma of CFS Patients in UK Cohort
- PCR with standard primers not most sensitive assay to detect XMRV

And yet Hue et al in Retrovirology 2010 addressed Taqman PCR primers, while ...Sato et al reported mouse contamination in standard PCR kits from Invitrogen.


XMRV Global Action
Aak Karin - thank you! (mental head slap . I stand corrected!
Here's another goodie: At approx 14:00, 2nd video

"...(Shyh-Ching Lo) and I ARE THE ONLY TWO PEOPLE THAT IM AWARE OF IN ALL OF THE STUDIES WHO PROCESS THEIR OWN SAMPLES. And... thats really important because IF YOURE LOOKING FOR A RETROVIRUS IN A SAMPLE THAT HAS BEEN THAWED AND REFROZEN, YOULL BREAK UP THE NUCLEIC ACID AND YOU WONT BE ABLE TO ISOLATE IT OR DETECT IT BY PCR. So its important to understand sample processing as part of virus detection.

Its not part of the publication but Frank Ruscetti Harvey (Alter) asked Frank if he would isolate the virus from these patients. He did so. He detected XMRV, suggesting that our cell line preferentially replicates XMRV. Importantly Rachel Bagni also showed an immune response in these 9 samples (from Dr Komaroff, Harvard) 15 years later."
M.S.

.....
.....

Xand Xmrv Andrea was asking me to find out wether Judy has given permission to publish this, because She thinks it is unpublished material... could you please double check? thanks in advance...

 

[shannah]

Thanks for the discussion notes LJS.

The videos were all working earlier so I guess they disabled them due to the unpublished material.

 

[shannah]

Recommended viewing!

These videos ARE WORKING TONIGHT.

THEY CONTAIN SOME UNPUBLISHED AND INTERESTING DATA.

Links in first post.

 

[alex3619]

Hi Shannah, thank you for these links. Unfortunately the third and the first weren't working when I tried them just now.

From the first and the second I got these snippets (not direct quotes, my take home message):

AY: The first is a comment from me. When Judy was discussing reverse transcriptase PCR, it occurred to me that this was far far better than PCR for this virus because it has less risk with mouse dna contamination.

JM: Cell culturing uses a prostate cell line.

AY: It was not mentioned how often this was cultured by itself to find XMRV, but if controls are cultured and the culture was infected with live XMRV initially, all the controls would have been positive too.

JM: Virus proteins are identified from the culture using gel electrophoresis. Culturing depends on reverse transcriptase, and cannot amplify dna directly, it requires an active virus.

JM/AY??? Did I mishear: they can reduce culture time to two weeks by adding reverse transcriptase. This would of course increase cost so is not commercially a good idea unless you are more worried about time than cost.

JM: the antibodies used to isolate XMRV were mouse XMLV antibodies.

JM: the UK study used bedridden or homebound patients, with blood drawn in their home. This made the criteria even stricter than the original study, and they still found 65% XMRV (antibodies and virion), which is almost identical (67%) to the Lombardi study.

AY: It occurs to me that if Lo found up to 8% of PMLVs in 94 or prior, that using fresh blood draws we will have a good idea of how these viruses are spreading. I personally think its now over 10%, but there is no substitute for doing the science.

Bye
Alex

correction: the third and fourth weren't working

 

[free at last]

Hi shanna, The links seem to be working here now thanks for posting, hard not to belive that judy has really found the major antagonist on the immune systems of a large group of cfs patients, even after 16 years of good recovery, i still get the fluey attacks, milder for sure, but still enough to affect dreams in a ill way, with moaning and tossing and turning. such times making the legs ach similair to normal flu. and more mucle pain. likely cytokin toxicity i reckon, had it last night and today. snakes and ladders is what this illness is.

I think HTLV1 will show how many do not get sick with the virus, but some do, just like xmrv, some of our partners ( maybe most infact ) do not get sick, and it seems obviouse htlv1 shows us this is not as unlikely as it might first seem

Thanks Alex for your assesment, was hoping those who understand these things more would comment. cheers for that

 

[Frank]

edited/ updated your post shannah

 

[Cort]

An Overview by me

Dr. Mikovits noted that Dr. Sandra Ruscetti has worked on mouse viruses for almost 40 years. After work on HIV halted work on MLV’s and cancer she saved her reagents….. and these were the reagents the WPI used. Dr. Ruscetti came out of retirement to help out with the current research and she’s still there, and, in fact Ruscetti has committed to five more years of work. Dr. Mikovits noted that the mouse retrovirus program at the NCI was undergoing review as she spoke. It was an amazing stroke of luck that Dr. Mikovits just happened to capture a retrovirus type her mentor’s wife had worked on for so many years.

Basics - Dr. Mikovits provided some basics; whatever XMRV is doing that is bad is probably going to be caused by one part of it - the env gene that has been shown to cause neurological problems and cancers in mice. She noted, that, yes, XMRV is very similar to endogenous retroviruses (ie retroviruses embedded in mouse DNA) but it is nonetheless different something that retrovirologist Dusty Miller recently pointed out when he talked about the Hue Retrovirology paper

The main problem with Hue et al. is they propose that XMRV is a mouse virus, but I have looked at all of the mouse sequencing data available, and can find no copy of XMRV in the genome of any mouse sequenced to date. That includes the 129x1 strain mentioned in their paper. Sure one can find snippets of DNA that look similar to regions of XMRV, but nothing very extensive.

A Timely Find - The WPI found the gene sequences belonging to XMRV but still needed to grow the virus. Finding a cell line that will grow a virus is often not easy but they got ‘lucky’when it turned out that Dr. Mikovits hunch about a T-cell Lymphoma cell line defective in producing the interferons and that was susceptible to androgens which effect prostate tissues). Luckily she guessed right - it’s not always easy to find the right cell line - and the WPI was off and growing the virus. This is still the only cell line known that XMRV proliferates in.

Progress in Testing #1 - if I got this right it appeared to me that Dr. Mikovits stated they have made a big step forward in their ability to speed up their tests - an important issue. Not only does this test cut down the assay time now by almost a month but it can also quantify how much virus there is; ie it seems they finally have a way to measure viral load. Not being able to measure viral load has been a sticking point for those who call for treatment trials; most retrovirologists want to be able to measure how effectively anti-retro viral drugs are knocking down the virus - this test appears to give them the ability to do that.

Progress in Testing #2 - Of course, the most important thing to do right now is to actually prove that XMRV is there and this is where the second step forward may have come. Dr. Mikovits stated that Dr. Bagni at the National Cancer Institute has created an XMRV specific antibody test that is able to validate the WPI’s results. Independent lab verification of the WPI’s right now is something of a holy grail. The WPI, by itself, however, cannot convince the research world that XMRV is present - they need other labs to do that. Dr. Mikovits reported that Dr. Bagni was able to correctly identify 34/39 patients from the WPI. This has not been published but if my notes are right it sounds like the trial was blinded . This would be an important finding for the WPI because it could help wash away the contamination question. Of course it would then need to be replicated by other independent labs to be fully verified.

The Disparate Study Results - Dr. Mikovits ran through all the possible reasons for the differing study results; different methods, genetic variability, contamination…and then spent most of her time on two of them; one ‘old’ one which has been mentioned almost from day one - the patient cohort - and a newer one - sample storage.

The HTLV Saga - But first she talked about HTLV - which may be the closest thing in human retroviruses to XMRV. HTLV presents an interesting case of how wacky retrovirus distribution can be. It’s found across the world in about 20 million people but is found in distinct clusters; it’s endemic in southern Japan the Caribbean and parts of South America and Africa but not in the US or Europe. In fact even in individual countries HTLV1 can show distinct clustering that is not well understood. For instance, it’s found in high rates in southern Jap but not in northern Japan and not in Japan’s neighbors such as Korea.

ME/CFS Trivia question - who discovered HTLV1? It was none other than Frank Ruscetti and his coworkers in Dr. Gallo's lab.

The UK Study - The Patient Cohort AGAIN - - A similar clustering effect could potentially explain some of the negative XMRV study results but the WPI’s UK study (assisted by Invest in ME) suggested this was not the case. The fact that the WPI found high rates of XMRV using nested PCR indicated to Dr. Mikovits that it wasn't about geography but instead was ‘ all about the patient cohort” - and this was a very ill patient cohort ’ ; Dr. Mikovits reported they were bedridden or housebound necessitating the use of phlebotomists who traveled to the participants homes to collect their blood. Once the collected the blood XMRV was apparently readily picked up by nested PCR (if I understood it correctly) suggesting that in the right patients the pathogen is easily found. This suggested to Dr. Mikovits that finding the right type of patient is very important. Interestingly they did not find the pMLV’s Dr’s Alter/Lo found in their study.

New Storage Issue - Dr. Mikovits brought up a new sample storage issue . She stated that her and Dr. Lo were the only ones in all the studies that processed their own samples - something she stated was ‘really important’. The factor she keyed in on was the number of times a sample has been frozen and thawed and then refrozen. She said “if you're looking for a retrovirus and a sample has been thawed and refrozen that will break up the nucleic acids and you won't be held to find the retrovirus again”. It appears that freezing and thawing and then testing is okay but freezing and thawing and then freezing and thawing and retesting again is not okay.

Ruscetti Isolates Virus - Dr. Mikovits reported that Dr. Ruscetti was able to isolate XMRV from Dr. Lo’s samples which suggests that the LNCap cells they are using preferentially grow XMRV and not pMLV’s and that they would need another cell line to grow pMLV’s in.​