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

Cort

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I'm confused. What is that statement in Post #32, where does it come from, and why is it posted in advance of the meeting where the evidence is to be presented and the subject discussed?
that was posted by Khaly Castle in her blog. She apparently received it from someone in the meeting. I don't think it was posted in advance. (?)

The twitterer's are not tweeting very much.......
 

Jemal

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Thanks for the updates Cort, much appreciated.

4 samples... I shudder to think what will happen when the world is faced with a bigger health crisis. Oh wait, it already happened with HIV and millions died and are still dying :(
 

Cort

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This is from Lo?

3 main contamination concerns:

Contam by PCR amplicons
Contam by MuLVs or vial vectors studied in the labs
Contam by mouse DNA -- Mouse DNA genome contains endogenously many closely rleated proviruses of MLS or mERVs

Built in 300-400 negative controls!

On Mouse DNA: highly sensitive nucleaic acid PCR assaying mouse mitochondria DNA sequences. Semi-nested PCR assay to detect mouse DNA was 1000 times more sensitive than gag gene specific amplicon PCR.

Bottom line - no mouse DNA in any human samples.

Reviewing testing of new blood from 8 of the sample pts. Viral gene copy numbers appeared to decrease.
They seem simply to be going over the Lo study - yes they did mtDNA - no contamination but did not do some test that would have clinched it, I guess....which I don't know....

Interesting that viral #'s decreased over time in the patients they retested -years later

Here's the big question - Coffin wants to know if they have isolated the virus. If they can do that they're in darn good shape - but they haven't been able to do that yet.

Coffin: No virus to go with these sequences as yet? Lo - no.
I suggested mitochondrial DNA as contaminant. Wide variation in cells that could, many sites where could arise. Extraction procedures might differentially extract mitoch vs gene. MY lab has developed an assay that's much better. 2 papers, will show results where CFS pts and non-contemporaneous controls show same sequences, but CFS pts were the negatives, not the healthy. In other study, found mouse DNA. Contam w/some lab DNA.
Coffin is clearly worried about contamination. He can now tell whether a sequence came from a mouse or XMRV........he has found mouse DNA in CFS patients.
 

Cort

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Mitochondrial DNA may not be sufficiently sensitive.
Have you gone back to compare sensitivity of assay? Lo-thinks mitochondria DNA is much more sentitive. Hard to believe we have a selective isolation of mito CNA. (I'm not following this effectively). Of coruse, contam is a concern. But if we amplify and sequence a million of those mouse DNA, we rarely see intact product. Always see many diff interruption. Can't completely rule out selectivity, but unlikely.
Lo - didn't use muscle cells with high mito DNA, used spleen cells cuz concentration much lower.
Coffin continues to challenge/question.
Lo-most measurement coming from nuclear DNA
This is what the retrovirologist told George and I: the mtDNA genome is too small - its good but you can miss things - best to do the other form. Lo used spleen cells with lower mtDNA - which I would guess doesn't help.

Lo is saying that mouse contaminants don't show up like our gene sequences did - they are broken up - degraded - ours were too clean to suggest mouse DNA (I think that's what he's saying....)

Coffin - cloned/sub-clones from products? We found amplification gave rise to mixtures of products, needed further dilution. Lo-when subcloned, major if sequence exactly the same. 10-15% have a mutation, replacement here and there. (Coffin - asking if monomorphic) Lo very confident.
There's clearly a debate about this.....but Lo is very confident - it's amazing they can't just get this down. I guess this IS rocket science.....
 

Cort

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Coffin - samples from pts and donors from diff times and places. Nightmare: a warehouse somewhere w/sodium phosphate sitting in it with mice crawling around, ends up in your reagent.
Not saying Lo is wrong, saying standard of proof is very high. Lo-totally agree. We did pay attention to it. We put in many neatives. have never seen in any results from lab. Understand that contam can come in at any time (e.g., while drawing blood).


New questionare: Did you attempt to isolate virus? Lo-tried very hard. Haven't successfully isolated virus. Have seen changes in infections, but not yet isolated.
Our retro contact says this is a big deal. You can't create a virus out of a string of mouse DNA that slipped in. (the virus could still slip in but at least you've completely the removed the threat of some mouse DNA floating into your sample at some point) The ability of Ruscetti to do this is what really got his attention. Too bad they are unable to do it thus far.

Have seen changes in infections, but not yet isolated
Not sure what this means but 'changes' in general are good because real viruses change subtly over time while 'sameness' is a red flag because contaminants do not. I suppose they can be degraded by processes in the body...?
 

pictureofhealth

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Is that Dr. Lombardi with Dr. Mikovits??


This is from http://plixi.com/p/62953856
Could be Dr Shyh-Ching Lo (FDA - From the Alter/Lo study,) and Maureen Hanson?

ie. They are giving the Group C. study talks -

C. "Recent Studies of Epidemiology of MLV-related Human Retroviruses":

i. U.S. Study, Shyh-Ching Lo, M.D., OCTGT, FDA (15’)
ii. U.S. Study. Maureen Hanson, Ph.D., Cornell University (15’)
iii. UK Study, Judy Mikovits, Ph.D. Whitmore Peterson Institute (15’)

Makes sense they might all be seated together.
 

Cort

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Alter fights back

Alter - Was very impt to get 8 samples from 15 yrs ago. Drawn in clinic, sent immed to Dr. Lo. If contam from a reagent, doesn't make sense that sequences differed, original blood draws from different times, third sample tested immed so less opportunity for contam.

Lo-We checked the IPA. See mitoch DNA much more sensitive than Coffin method, also used it for comparison.
Score Big Time - :thumbsup::thumbsup::thumbsup:

How do you get the same contaminant twice 15 years apart? What are the chances of that? They are using different reagants, different sampling techniques, maybe different tubes, they are processing the sample immediately - yet the same contaminant shows up........Very strong point by Alter

It sounds like Lo says their method for checking contamination is the most sensitive...They sound quite confident.

I think that's it for them...why only 15 minutes for a presentation??

Surprised they haven't fully sequenced what they found but they do seem confident...
 

Esther12

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I'm surprised there's such disagreement. I thought the big researchers there would know the BWG results and that would have created some consensus over the big picture.
 

Cort

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Hansen is up - she found pMLV's in Bells original pediatric cohort

Hanson, Cornell, unpublished 20/20 study, still in progress. Samples from Dr.s David Bell (father and son).

40 Ss selected by Dr. Bell. 10 "severe CFS", 10 "recovered CFS," 20 healthy controls.

Bells administered various surveys.

Cornell blinded to samples status, altho Hanson had to be unblinded for Sept 10 meeting. Lab members are still blinded.

Very severe:
Less than 3 hrs daily upright. Fukuda criteria.

Recovered:
15.5 avg upright daily. Lindenville group.

Healty - all live in W NY. Screened to have never lived with CFS, FM or PC. Some have close CFS friends. Appeared to be completely healthy.

Severe pts had sig diff scores on surveys (much worse) than other 2 groups. Recovered better, but not as well as healthy. Feel well enough to donate blood.

Have tried several diff assays under develop. PBMCs from EDTA tubes. Nucleic acid made from PBMCs immed frozen or culture 5-10 days.

Recently PCR assay of WB.

Assay for proovial and cDNA in plasma, incubated LNCap Cells. Make RNA, convert, make genomic DNA, do PCR.

Contam controls - mitochrondial. Considering switching to Lo methods, cuz so sentive/validate.

Explanation of precautions against contam. Hoods, no mice ever, etc.

Have not had good luck w/a single round of PCR in any assay. Gag sequences after min of 4 transfers, good after 6 transfers.

W NY sequences more like Lo/Alter sequences than to Genbank XMRV. More like PMLVs. Don't have a deletion in glycogag region of XMRV PC samples.

7/10 severe
7/10 recovered
4/20 healthies

So far.

High controls - suspect becuz samples were taken from an outbreak area.

Why so many reports of failures? PCR primers that will not detect viruses lacking the deletion in the glycogg region.

All other neg labs optimize PCR conditions of VP62 XMRV, not for any MLV-like viruses possibly present in their samples.
She found some contamination in her controls (CHECK - wrong interpretation!) - which in a weird way is good because it means she can find it (at least in part) if its there. She's considering using mtDNA techniques that Alter uses.

She is not finding XMRV.......She can tell because there is a specific deletion in the gag protein in XMRV that is not found in pMLV's and she's looking right at that area and its not showing up....

Her percentages are very close to the WPI - but she really is finding a different virus....Yet she is right in step with the WPI on how to test for; do not use the VP62 clone as a base - you'll miss it. The really weird thing is that the WPI used the VP62 clone as a base for their first study I believe - they had to - they had no other base to work off of I don't think. Later they said - no don't use it....Dr. Hansen agrees.

I think some of the translation is off; Dr. Hansen is finding pMLV's which do not have the deletion - yet she is saying she found them....I am a bit confused...

Have not had good luck w/a single round of PCR in any assay. Gag sequences after min of 4 transfers, good after 6 transfers.
This is different from the WPI as well - they found XMRV in the first pass - something that McClure questioned. Hansen is saying it took at least four passes?????
 

Jemal

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I'm surprised there's such disagreement. I thought the big researchers there would know the BWG results and that would have created some consensus over the big picture.
I am glad though that a renowned researcher like Alter has entered our arena. His reputation is now at stake as well and it looks like he is defending it well.
 

anciendaze

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I am glad though that a renowned researcher like Alter has entered our arena. His reputation is now at stake as well and it looks like he is defending it well.
This virus violates some assumptions that are very convenient for researchers. The alternative looks so difficult it is frightening. Nature does not have to play by rules set by researchers. We are seeing the beginning of a paradigm shift.
 

pictureofhealth

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Re Hanson findings - Cort's post #54 above:

That's:

14/20 for Severe/Recovered CFS (in total),
4/20 only, for Healthy controls.

Big difference.
 

Deatheye

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This virus violates some assumptions that are very convenient for researchers. The alternative looks so difficult it is frightening. Nature does not have to play by rules set by researchers. We are seeing the beginning of a paradigm shift.
Details? which assumptions are you Talking about?
 

Cort

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have seen diffs from diffs brands of PCR machines in their lab.
That's wild....different brands of PCR machines in the same lab give different results...that will probably drive people nuts...

Work not done at Cornell:
Healties NIH, 6 reacted w/ at least 1 antigen, 3 with both
WPI, detected 7th and reacted w/both NIH antigens

7 recovered, NIH 4 reacted w/ at least 1 antigent, 1 w/both
WPI, detected other 3 and also detected 1 that reacted

DANG!!! Missed it. Control samples serology consistent with PCR results.
Hard to tell but it looks like some outside confirmation (maybe?). Good news that serology matches with PCR in controls.....since some controls were positive....
 

Cort

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Coffin - You do not have a virus that goes with these sequences yet? (no)
Bulk sequencing or cloning? Based on bulk sequencing. But don't just take sequence, get actual traces, too. Can see if there 15% of diff seq will see in bulk sequences, can see some single nucleotide polymorphisms. If had 15% or more of single nuc poly's, would be able to see them. Don't have resources to clone every one. Onlysee occasionally.
Coffin wants to see that virus isolated. (So do we!!). She might be saying that she can see variations in the viruses, again, which is good. Actually I don't think the answer to that question is reported....too b ad...

New Questioner: Tested only once? If more than once, concordance? Cornell - done it twice w/ at least 2 methods -- what I'm reporting. We are at limits of detection. PCR is really tricky. Inconsistent results were sometimes related to which PCR machine.
At the limits of detection! Yes...Some PCR's can pick it up and some can't. Could it be all about newer vs older machines? Or better vs not quite as good machines? Could it all come down to something like that? It starts to make a bit of sense given the inability to explain the disparate results, though....At the limits of detection.....Clever of her to try two different PCR machines :)

Nancy - congrats on the nice study. Interesting to compare severity levels. Interesting that recovered pts pos as frequently as severe. Hanson - we've never seen "classic" XMRV. Only polytropic sequences from LNCap cells so far. Not all indivs were ppl from outbreak. Some more recent folks mixed in, scattered in both pts and control grps, but samples too sample to test for sig diffs.
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.