Blood Products Advisory Committee Meeting Announcement (BPAC) December 14-15, 2010

Cort

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Judy's up


Judy - working on more sentitive methods for biological and molecular amplification. HMRV in blood and plasma cell.

Methods used to determine incidence of HMRV in UK cohort of ME/CFS using CCC criteria.
She's saying HMRV's. Subtly thumbing her nose at the medical establishment. Not sure that's such a smart thing to do.

What a cohort, though. 50% homebound. Flu-like onset. CCC criteria - this is THE group that XMRV should show up...

ME post-viral fatigue dx
CCC criteria
duraing 9-26 yrs, >50% homebound
onse childhood/puberty
Symptoms include
severe cog dysfunction
multi joint pain, migraines
vertigo
lymphadenopathy
mitchon dysf
GI disturbances/dysbiosis
chronic infections, flu-like onset

Current age of Ss 19-70.
50 total - equal numbers of male/female
They shipped samples to the National Cancer Institute - impeccable credentials. (Our retro contact says Ruscetti's rep is really solid). Very smart :) A new lab there as well - never tested for XMRV - they are covering the bases! And they used two independent labs....everything was blinded...what more could you want?

Drawn by PSI, code and ship samples to NCI, lb w/no previous XMRV murine research.
2 days after collection - Plasma and PBMC isolated.
Tested in 2 indep labs, blinded.
50 health controls from London run concurrently (didn't have fresh draws from them)
Samples tested using 4 methods:
plasma HMRV RNA
Cell free transmission fo HMRV to LNCap
plant atnibodies to HMRV viral protens
Western blot.

Sequencing of isolates.

Detection of gag HMRV RNA after delayed processing.
 

Cort

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Culture for 21-42 days - a long time but it makes sense if the virus is at the limits of detection as Hansen suggested and she notes other studies have only cultured for 1-2 weeks. Is that the difference there? They didn't let the samples sit for another couple of weeks (aaarghhhhhh!). Using DERSE (?) XMRV does not show up in the controls and it shows up in spades in the patients - 78%!.

They did find PCR negative patients from whom they could culture the virus.....(limits of detection again? Has the WPI just worked harder to find the virus than anyone else?)

2 pts were positive using Dr. Lo's PCR that weren't orig identified by 2nd round PCR.

compared to original WPI Science study. Carry this sample through to new sample.

Control samples, very few HMRV RNA. 2/50 from healthy controls were pos for gag.

WPI isn't a PCR lab, so used assays to detect and isolte SMRV using PC Cell Line LNCaP), know there are hormone-responsive elements to make virus replicate more. Culture for 21-42 days. Alot of culturing. Follow w/Western blot for monoclonal antibodies, spleen-focus forming virus antibody from 1982. This anti-body could detect all of the viruses. PCR neg pts from whom we could culture and sequence whole virus. Other studies culture maybe 1-2 weeks.

Develop assay with inactivated green protein in it. 4-18 days. Needs reverse transcriptase and something...darn. Flow cytometry to count green cells. Used in UK samples. DERSE Assay. 78% of CFS pos using this assay (hehehe)
Now for the questions from Coffin: this will be interesting :)
 

Cort

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what is the significance of saying HMRV ?
Dr. Mikovits decided to rename the virus to illustrate that it is a human virus...Dr. Singh thought the idea of Dr. Mikovits renaming it on her own was rather wild - since she doesn't have any control over what the virus is called......Its a minor point but her calling it HMRV when everyone else is calling it XMRV will undoubtedly tweak some people off a bit.....It doesn't hurt XMRV but I don't think it helps Dr. Mikovits much...
 

CBS

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Interesting. As many patients with whatever she found (not classic XMRV - poly MLV's) recovered as remained ill - so if what she found is what the WPI found or related to it - you don't necessarily need to take anti-retrovirals to recover....if all this holds up.
"Recovery" is a pretty poorly defined term here. From what has been posted, the "recovered" group was not as well as the healthy control group. Just as likely is that something is still going on (not exactly recovered) that can vary overtime in terms of the symptomology.
 

Cort

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And here we have it - they confirm their original findings - and they are different from the Hansen/Lo findings. I would have thought they would have started to pick up those pMLV's but maybe they are searching differently.

gag and env proteins in Western blot confirming transfer of virus from plasma to LNCaP.

SU Sequences of virus transmitted from plasma of UK Me/CFS pts to LNCaP are more similar to XMRV than to PMLVs
 

bertiedog

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And here we have it - they confirm their original findings - and they are different from the Hansen/Lo findings. I would have thought they would have started to pick up those pMLV's but maybe they are searching differently.
Could this possibly be anything to do with the fact the cohort are from the UK and not the US?
 

CBS

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what is the significance of saying HMRV ?
Singh actually felt that the use of multiple terms for something we really don't have nailed down yet was likely to cause confusion in the literature. I got the sense that she understood the political reasons for doing so but she felt that at this point it wasn't worth the potential cost.
 

Cort

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Now for the questions

Coffin - re Derse cells, can you grow out? Could be growing something that resulted from handling, not in samples.

Ruscetti - no diff in env and gag regions.
Coffin - To my knowledge, no-one has grown a virus with polytropic or xenotropic.
What does this mean??? I think we're missing some relevant pieces.

New Questioner: Is this a cause or result? Have pts been studied for other co-infections?

Judy - haven't in UK becuz it's a psychosomatic disease in the UK and ppl can't get those kinds of test there. In US, our studies show all kinds of co-infections. Looks like an AIDS pt with hypothesis that underlying deficiency that allows pathogens to proliferate.
Someone suggests going back and looking in large blood repositories to see when XMRV first appears. Judy says, you can't do that right now (but this is a given if XMRV proves out - they'll jump right on that as soon as XMRV is validated).

Questioner: Positive controls need to be followed to establish temporality. Can find and follow in large repositories to go back and look for disease incidence.

Judy - can't do it with existing assays, very good question. Need high through-put assays to do this type of epid studies.
Coffin suggesting yes, it could be there, somewhere in the body that is hard to detect. (What is PPL?) Is he suggesting that it is present in the population but something about CFS could turn it pathogenic??? That would make sense...In any case - a more positive outlook, at least temporarily, from Dr. Coffin.

Coffin - could be in difficult place to detect, could be in large number of ppl. Could be something in CFS pts that allows it to be pathogenic.

Judy - if were in large number, would be easier to find...
Again... the limits of detection??? A virus you need to culture alot for? One that some PCR machines can find and others cannot not.....These would be good answers to the XMRV dilemma :D:D:D

Stoye is next but before that - a cheer from the crowd :)

(Lo, Hanson, and Mikovits were/are AWESOME!!! Not to mention Alter and Ruscetti chiming in from the audience.)
 

Sasha

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Interesting. As many patients with whatever she found (not classic XMRV - poly MLV's) recovered as remained ill - so if what she found is what the WPI found or related to it - you don't necessarily need to take anti-retrovirals to get better....if all this holds up.
Many of us have had sustained periods of remission (without taking anti-retrovirals, obviously) even if we relapsed later - I suspect those who have "recovered" are basically in a long and maybe indefinite period of symptomless remission. I hope they don't develop serious disease (cancer, heart disease etc.) later.
 
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Singh actually felt that the use of multiple terms for something we really don't have nailed down yet was likely to cause confusion in the literature. I got the sense that she understood the political reasons for doing so but she felt that at this point it wasn't worth the potential cost.
so it may be premature. thanks.
 

Cort

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"Recovery" is a pretty poorly defined term here. From what has been posted, the "recovered" group was not as well as the healthy control group. Just as likely is that something is still going on (not exactly recovered) that can vary overtime in terms of the symptomology.
I agree, recovered did not sound like really recovered - just no longer as severely ill. I changed it to 'get better'. Good point.
 

Cort

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She doesn't like Stoye - I agree that a virology lecture is hardly what's need at this point. Who is he talking to? Isn;t he talking to a bunch of virologists????:confused::confused: Honestly, this always seems to happen at conferences.

Stoye embarrassing himself by giving a basic virology lecture that really should have come first, as planned. It might have set the stage for some of the BWG members.
Causality in PCA: Insertion of XMRV into human DNA has been demonstrated in a few tumors.
(Val - What is IHC? Not sure I've even heard of this one.)
"One has to worry if get a result from one technique, but not another."
Comparing 3 studies, if drop input level, get much lower positives. Have to push your PCRs in order to find XMRV.
If LoD is 1/1000 cells, then must come up with plausible disease mechs to explain diseases.

OK Immunohistochemical (IHC) studies: 0/0 studies, pos studies -- methods seem very similar, why the differences?

Sneering about WPI Science study. "Appeared to be robust." If 4%
There's the 'insertion of XMRV into human DNA' - very important that they can show that its gotten into, ie infected human DNA....This means there is no doubt that it is the 3rd or whatever infectious human retrovirus......

He does agree with the need to "push your PCR" - this is a rare virus in the blood...and he does question how such a rare virus could be causing disease...of course they've only looked in the blood. In monkeys it appeared - spread through the blood - then basically disappeared. So its maybe a one time infection type of thing. It gets in there - goes to where it will go then is disseminated no more! It's not in the blood anymore - its not going anywhere since that is how viruses move around the body - except in the localized areas where it got in during the initial infection.

I don't know if stating the 4% finding appears to be robust constitutes "sneering". It sounds more like validation to me - but I'm not there.