XMRV and Culturing, HERV's and more

[starryeyes]

Bravo Gerwyn!! That post explains the science in a much more understandable way. I am very pleased that you did that for us and I really like the colors. It sure helps delineate the points you are making and helps a foggy brain keep them straight plus they're uplifting.

Thank you!! :victory::sofa:

 

[Lily]

GREAT JOB Gerwyn, THANK YOU MUCH

I appreciate all the time and effort that must have taken.

 

[Countrygirl]

:Sign Good one: GERWYN That you for the time and effort you put into this. It certaily deserves front page status. :Retro smile::Retro smile:

 

[Marco]

Thank you Gerwyn.

Now THAT I can follow.

 

[Gerwyn]

Here's the section about MMTV as a superantigen. I bring this up because I want to remind people to keep their minds open. Just because bacteria are the most common examples of superantigens, it doesn't mean that viruses (and even fungi) can't be direct superantigens. No one knows yet what the mechanism is for this illness and I am not ruling out that CFS could be a retroviral illness that induces superantigens as a mechanism, among other ideas. I believe those researcher and docs who are willing to keep an open mind will not only solve this condition(s) but will change fundamental ideas we now hold or discover new paradigms, e.g. the discovery that HHV-6 can be transmitted via integrated chromosomes in families when it was previously thought herpes viruses did not intergrate into chromosomes.

Mouse mammary tumour virus (MMTV)

MMTV, a milk-transmitted B-type retrovirus, causes murine mammary
carcinomas. The MMTV SAgs were discovered first by Felstenstein
in 1974 and were referred to as minor lymphocyte
stimulating (Mls) antigens. The T cell response to Mls antigens is
similar to the response to bacterial SAgs with expansion of unique
TcR Vb subsets [53]. The SAg gene was identified later within the
3 long-terminal repeat (LTR) of the MMTV genome and did not
show any homology to the bacterial SAg genes [54]. The gene
product is a 45-kDa type II transmembrane protein with a 10–14
amino acid polymorphic region at the C-terminus, which is responsible
for the TcR Vb specificity. Infectious MMTV is present in
mammary tissue and breast milk of only a few mouse strains. The
SAg molecule is an essential component of the virus life cycle, providing
efficient viral replication in newly infected gut B cells by
recruiting Vb mediated T cell ‘help’ and promoting B cell proliferation.
The endogenous SAg is inherited in Mendelian fashion
and causes T cell deletion as a result of self-tolerance induction in
the thymus. As a result, the transmission of an infectious virus carrying
the identical SAg will be hampered by the lack of responder
T cells, thereby protecting the mouse from MMTV infection.
E

(BTW, MMTV is being studies in human breast cancers and primary biliary cirrhosis.)

Yes viruses have the genomic equipment to manufacture SAg EBV is a common example .if SAg was causative in ME then anyone who had an ebv would develop the illness.it is not a question of just keeping an open mind but attempting to explain observations

MMTV is a betaretrovirus which has specific genes thet code for a Sag .A herv does not-fact

MMRV only infects mice Fact

 

[Gerwyn]

Thank you Gerwyn.

Now THAT I can follow.

Thankyou but you are right.After reviewing the paper and trying to "translate" it iI realise that I slip into science"shorthand" too often.

When I was talking about cell cell transfer I should have explained it far better

Basically cells are connected by "molecular corridors" In B cells(where XMRV lives)there are molecules(the restrictive factors in the post you referenced) which could damage or destroy replicating XMRV if it spent too long in the cytoplasm..

One technique to avoid this is to travel between cells using these corridors actually using the energy of the intrinsic molecules to power the process of movement.These thing are so clever!

 

[valia]

What everyone else said goes for me too.


I would also like to thank you Gerwyn for breaking up and spacing text the way you do,

you are one of the few who appreciates how difficult it is for some of us to read large blocks of text.

 

[Cort]

I appreciate the time you took to do that. (The colors were really tough for me by the way). I appreciate your pointing out the differences between the Science study and the UK studies.

It appears that you've answered my question: the WPI did not activate the cells prior to doing the PCR.

Note that they said the cells for the DNA and RNA extraction were stored as unactivated cells in Trizol. As I noted earlier there is no mention of activation or culturing anywhere in the PCR section. WPI used PBMC's for PCR; they did not break the cells up into their differents - which is where the culturing came in. The WPI later said activation was necessary but there's no evidence I can find that they did that. I want XMRV to work - I'm not trying to bash it - but logic is logic; if they didn't do it they didn't it and nothing that I've yet read convinces me that they did it. I want to be fair to every side - not just the WPI.

From Gerwyn “PBMC were isolated by layering the diluted blood onto Ficoll-Paque PLUS (GE Healthcare, Waukesha, WI), centrifuging for 22 min at 800 g, aspirating the PBMC layer and washing it once in PBS. The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 C for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 C for DNA and RNA extraction and analysis.”

Note the unactivated PMBCs were frozen for culture and further experiments

OR

resuspended in Trizol for nucleic acid extraction OR further experimentation

“PCR methodology on extracted DNA

PCR. To avoid potential problems with laboratory DNA contamination, nested PCR was performed with separate reagents in a separate laboratory room designated to be free of high copy amplicon or plasmid DNA. Negative controls in the absence of added DNA were included in every experiment.

Identification of XMRV gag and env genes was performed by PCR in separate reactions.

PCR amplification for sequencing full-length XMRV genomes was
performed on DNA amplified by nested or semi-nested PCR

PCR analysis performed on 20 of the identical patient PBMC DNA
specimens stored at the NCI (Frederick, MD) since 2007 confirmed nearly identical gag sequences, thereby diminishing the possibility of laboratory contamination as a source of XMRV”

This dealt with the issue of contamination and replicated the findings of this part of the experiment.

It is important to note that PCR was the approach taken in the Imperial college and Groom studies. The claim that the PCR methodology needed validation is objectively false. It had already been achieved by an independent laboratory.

“Nested RT-PCR for gag sequences was done as described”

Neither the Imperial college study nor the Groom study even attempted to do this despite looking for a RNA virus. RT –PCR looks for viral RNA. You don’t need replicating virus for this test but you do for PCR

Hence both studies based their approach on the assumption that the virus if present would be replicating.

The significance of the RT approach will be apparent in the conclusion.

 

[MEKoan]

Many thanks, my liege, Wizard of Wales!

Your grateful subject,
Koan

 

[Cort]

Here's the section where Gerwyn points out that the culturing took place: its titled NEXT Experiment: that's the experiment AFTER the PCR took place. It doesn't appear to me to have anything to do with the PCR test. Note that with regards to the cultured cells they're talking about leukocytes, T-cells and B-cells. They simply used 'PBMC's ' for the PCR. PBMC's also contain monocytes and macrophages and NK cells.

Here's the definition of PBMC 10- A Peripheral Blood Mononuclear Cell (PBMC) is any blood cell having a round nucleus[1]. For example: a lymphocyte, a monocyte or a macrophage. These blood cells are a critical component in the immune system to fight infection and adapt to intruders. The lymphocyte population consists of T cells (CD4 and CD8 positive ~75%), B cells and NK cells (~25% combined).

NEXT EXPERIMENT. All sections also contained controls using the same parameters isolated from healthy donors

Leucocytes (a kind of white blood cell) were isolated from the FROZEN PMBCs of patients and fresh PMBCs of healthy donors.

These were then activated (PHA).

They were then cultured for 42 days with sub-culturing taking place every 3 to 5 days.

That was the first amplification procedure.

CD4+ T and CD3+ T cells were isolated and further cultured.

That was the second amplification procedure

The CD4+T cells were further amplified and activated by culturing with IL2.

CD19+B cells were also isolated from the activated leucocytes and the concentration amplified by further culture.

Ok now we have hugely amplified concentrations of activated T and B cells to work with.

 

[MEKoan]

Hey Cort,

I wish I understood this better. I keep thinking it's been answered but I'm not at all science savvy! Not at all!

Anyway, have you thought about asking WPI directly?

I worry that we will wear out our dear Wizard. Even wizards must have limits - especially wizards with ME!

Peace out,
koan

 

[MEKoan]

That's what I thought I understood, Gerwyn!

I may be learning!

 

[Robin]

Cort, the second experiment with culturing you're referring to was prep-work to a nested PCR, and the demonstration of infectiousness.

I think I understand what Gerwyn is saying. Let me try to reformat the concluding section without the rainbow colors so maybe it's more clear. (Apologies to Gerwyn if I did this wrong.)

They did three different experiments with PCR. The most successful techniques involved frozen blood that was amplified and activated. The least successful is on fresh blood with no prep.

Hit rates on different types of PCR technique

1. Nested PCR on fresh blood PMBC's yields a 20% hit rate.

This means that at this concentration of PMBC's derived from 8 ml of fresh blood you would need to run a PCR assay 4 times to get a hit on viral RNA. If you realise that McClure used 2 ml of frozen whole blood, then even if she had concentrated her sample she would have had to run her PCR 20 times to get one hit. Of course as the sample was much more dilute she would have had to run her assay many more times than that.

2. RT-PCR on fresh blood PMBC isolates yields a 50% hit rate.

This is the one the English did not bother with at all. You need to run it twice to get a hit.

3. Nested PCR on amplified and activated PMB cells from FROZEN blood yields a 90% rate.

You virtually only have to run one PCR assay.

Conclusion: This is the point: if you want to try to isolate viral DNA from frozen blood then your chances are hugely improved if you isolate the target cells, activate them and expose them to multiple amplification procedures BEFORE PCR.

 

[Hope123]

Yes viruses have the genomic equipment to manufacture SAg EBV is a common example .if SAg was causative in ME then anyone who had an ebv would develop the illness.it is not a question of just keeping an open mind but attempting to explain observations

MMTV is a betaretrovirus which has specific genes thet code for a Sag .A herv does not-fact

MMRV only infects mice Fact

I'm not saying that EBV is THE cause or only cause of CFS but rather that the idea of superantigens is interesting.

Here's the article on MMTV in Wiki. There is ongoing research into this because of a similarities found in human breast cancers (called H (human) MTV).

http://en.wikipedia.org/wiki/Mouse_mammary_tumor_virus

Also, for some people, EBV is the major factor as there are people I know who have recovered (yes, 100% by their own reckoning, back to work full-time, and exercising/socializing) after treatment for EBV over several months. These folks were diagnosed with CFS, were sick for years, and had EBV IgM (not just IgG) but were not taken seriously. This may be a very small group within CFS but it's there.

I'm going to stop commenting on this as it is off the topic of the thread.

 

[Gerwyn]

I appreciate the time you took to do that. (The colors were really tough for me by the way). I appreciate your pointing out the differences between the Science study and the UK studies.

It appears that you've answered my question: the WPI did not activate the cells prior to doing the PCR.

Note that they said the cells for the DNA and RNA extraction were stored as unactivated cells in Trizol. As I noted earlier there is no mention of activation or culturing anywhere in the PCR section. WPI used PBMC's for PCR; they did not break the cells up into their differents - which is where the culturing came in. The WPI later said activation was necessary but there's no evidence I can find that they did that. I want XMRV to work - I'm not trying to bash it - but logic is logic; if they didn't do it they didn't it and nothing that I've yet read convinces me that they did it. I want to be fair to every side - not just the WPI.

i decided my first reply to your post was unfair.You obviously had a great deal of difficulty inreading it because of the colour scheme. While most people have apparently found it to be beneficial I can see that not everyone would

I will try and keep this post as clear and simple as possible so that it makes it less likely that you form erroneous conclusions because you find the text difficult to understand

The Paper is devided into THREE DIFFERENT EXPERIMENTS

Initially 8 ml of FRESH BLOOD was taken from each patient That is FRESH blood.

The PMBCs were seperated from the blood. to be simple from now one i will call them white blood cells.

The reason they did this was TO CONCENTRATE the sample of white blood cells

these cells were devided into two equal amounts

One lot was frozen for later use and the other lot was broken up and the DNA AND the RNA extracted

They Ran nested PCR on the DNA sample and found that they had a 99.9% match to XMRV as first identified and genotyped by its original discoverers.They also found that there was not any DNA belonging to any other MuLV gamma retrovirus present. this is important because XMRV is a member of that family of viruses so any future tests on the sample would be difficult if there was DNA belonging to another member of the family present

The exact same experiment was carried out at the cleveland clinic with exactly the same results ruling out contamination

The RNA was also investigated by RT-PCR.Now this is a different kind of PCR .The reason that virologists do this when trying to isolate a RNA virus is that this technique can detect an RNA virus when it is not replicating at all.Normal PCR cannot do this

Now just to make it clear PCR cant isolate viral DNA unless the virus is replicating. RT-PCR can

The results of the RT-PCR confirmed the presence of XMRV .NOW THAT IS THE END OF THE FIRST EXPERIMENT THERE IS IS GOING TO BE SOME MORE PCR LATER SO KEEP READING

How does this compare with the English studies.

THIS EXPERIMENT DOES NOT COMPARE AT ALL because they Used FROZEN WHITE CELLS in whole blood

So lets look at the experiment that was carried out in the science study which looked at extracting XMRV from FROZEN WHITE BLOOD CELLS

THE first difference is the concentration of white blood cells at the start.The Science researchers used white blood cells which had been isolated from blood.

The English studies did not.The English sudies began with a much lower concentration of white blood cells to begin with

The science researchers activated the white blood cells.The English researchers did not.Activation stimulates and induces replication now remember that PCR only works if a virus is replicating

The science researchers massively increased the number of activated white blood cells by repeated culturing.The English researchers did not

The science researchers ran their PCR on massively amplified numbers of white blood cells which had been activated.The English researchers did not

The science researchers detected XMRV.The english researchers did not.

In summary the English researchers used frozen diluted white blood cells. In one experiment the Science researchers also used frozen white blood cells which were concentrated

The English researchers performed their PCR without any attempt at concentrating their sample .They did not activate the white blood cells or attempt to amplify their numbers.The Science researchers did

The English researchers could not detect xmrv and did not The science researchers could and did

I hope that this is clear now

 

[Gerwyn]

Here's the section where Gerwyn points out that the culturing took place: its titled NEXT Experiment: that's the experiment AFTER the PCR took place. It doesn't appear to me to have anything to do with the PCR test. Note that with regards to the cultured cells they're talking about leukocytes, T-cells and B-cells. They simply used 'PBMC's ' for the PCR. PBMC's also contain monocytes and macrophages and NK cells.

if you read it properly it may" appear" otherwise

 

[Gerwyn]

There seems to be some confusion about the PCR run by the Science researchers on the pmbcs that were frozen after extraction thawed activated and amplyfied by culture. here it is

Genotyping. The rs486907 R462Q SNP was genotyped using Applied
Biosystems’ Taqman 5' nucleotidase assays, Taqman Universal PCR Master
Mix: No AmpErase UNG, and 5 ng of genomic DNA. The thermal cycling
conditions consisted of an initial hold at 95o C for 10 minutes followed by 50
cycles of a 15 second 95o C denaturation step and a one minute 60o C annealing
and extension step. A 7900HT instrument was used to detect fluorescent
probes, and Sequence Detection Software (SDS) v2.2 was used to discriminate
alleles and call genotypes (Applied Biosystems, Foster City, CA

It is sometimes called allelic PCR and confirmed the presence of whole genome XMRV in the activated highly concentrated white blood cells originating from the blood of patients properly diagnosed with ME/cfs

 

[Gerwyn]

I'm not saying that EBV is THE cause or only cause of CFS but rather that the idea of superantigens is interesting.

Here's the article on MMTV in Wiki. There is ongoing research into this because of a similarities found in human breast cancers (called H (human) MTV).

http://en.wikipedia.org/wiki/Mouse_mammary_tumor_virus

hervs do not carry the genetic equipment to manufacture Sag full stop

 

[Cort]

The RNA was also investigated by RT-PCR.Now this is a different kind of PCR .The reason that virologists do this when trying to isolate a RNA virus is that this technique can detect an RNA virus when it is not replicating at all.Normal PCR cannot do this

Now just to make it clear PCR cant isolate viral DNA unless the virus is replicating. RT-PCR can

The results of the RT-PCR confirmed the presence of XMRV - Gerwyn

OK they used RT-PCR without activating the cell to look for XMRV. They then did PCR on activated cells even tho they never explicitly said they did.

What they should have said here - simply they apparently spelled everything else out elsewhere - was

The PBMC (approximately 2 x 10 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 C for further culture and analysis or resuspended in TRIzol (Invitrogen, Carlsbad, CA) and stored at -80 C for DNA and RNA extraction and analysis.

Cells where activated with X,Y, Z and then

DNA was isolated from TRIzol preps according the to manufacturer’s protocol and also isolated from frozen PBMC pellets using the QIAamp DNA Mini purification kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol, and the final DNA was resuspended in RNase/DNasefree water and quantified using the Quant-iT Pico Green dsDNA Kit (Invitrogen, Carlsbad, CA). RNA was isolated from TRIzol preps according to the manufacturer’s protocol to make it more explicit

In the culturing section

For isolation of CD4+T cells, CD8, CD11b, CD14, CD19, CD33 and CD56 positive cells were removed using magnetic activated cell sorting (MACs) methods according to the manufacturer’s instructions (Miltenyi Biotec, Inc., Auburn, CA). After isolation, the CD3+, CD4+ T cells (>95% pure) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate and antibiotics. CD4 T cells were activated by culturing with 20 units/mL of IL-2 and 1 g/mL PHA.

They should have noted the cells were refrozen again (ie. Cells were refrozen for PCR analysis) because PCR section states they used frozen cells . I assume that freezing/refreezing cycles can have an effect on viability.

Its messy to me. To me you have to infer that the cells were activated prior to either PCR. I can see how people would wonder. Its very detailed account of the process. Its designed to demonstrate exactly how to replicate it -yet it doesn't explicitly spell out some key steps.

Later they explicitly say the T-cell cultures were used in the viral transmission experiment:

Frozen cell-free plasma and 0.22 m filtered cell free supernatants from PBMC and T cell cultures were diluted 1:1 with tissue culture media and 600 L aliquots were added to a six-well culture plate with the LNCaP
ocell line (50% confluent) or a million primary activated CD4+ T cells isolated from healthy donors.

Its odd they would note the culturing and activation in the viral transmission section but not in the PCR section.

Maybe its time to move on- we'll have new studies soon I hear to dig into. What they did or didn't do in the first study will eventually be moot.

One of the reasons I've stuck with those is not to get at the WPI but point out how people like Dr. Vernon, might, based on these finding legitimately wonder if the patient cohort was very different from those in England. I know that you noted that section of the paper and they appear to be very different but then WPI personnel have repeated stated that they were 'typical patients'.

In any case, based on our 'data' from the Light/Bateman study my sense is that the three CFS studies (really two CFS studies) made a critical error somewhere - as you have surmised throughout - and we're going to get very different results as better studies come out.

 

[Gerwyn]

OK they used RT-PCR without activating the cell to look for XMRV. They then did PCR on activated cells even tho they never explicitly said they did.




Cells where activated with X,Y, Z and then



In the culturing section



They should have noted the cells were refrozen again (ie. Cells were refrozen for PCR analysis) because PCR section states they used frozen cells . I assume that freezing/refreezing cycles can have an effect on viability.

Its messy to me. To me you have to infer that the cells were activated prior to either PCR. I can see how people would wonder. Its very detailed account of the process. Its designed to demonstrate exactly how to replicate it -yet it doesn't explicitly spell out some key steps.

Later they explicitly say the T-cell cultures were used in the viral transmission experiment:



Its odd they would note the culturing and activation in the viral transmission section but not in the PCR section.

Maybe its time to move on- we'll have new studies soon I hear to dig into. What they did or didn't do in the first study will eventually be moot.

One of the reasons I've stuck with those is not to get at the WPI but point out how people like Dr. Vernon, might, based on these finding legitimately wonder if the patient cohort was very different from those in England. I know that you noted that section of the paper and they appear to be very different but then WPI personnel have repeated stated that they were 'typical patients'.

In any case, based on our 'data' from the Light/Bateman study my sense is that the three CFS studies (really two CFS studies) made a critical error somewhere - as you have surmised throughout - and we're going to get very different results as better studies come out.

cort a competent virologist could easily follow those instructions they were not constructed for laymen but proffessional retrovirologists

it may well "seem" messy to a layman but to the science peer reviewers it was clear enough as indeed it is to me.Now Mclure and Groom have many o rders of magnitude more knowledge in this area than me.You tell me why the methods and proceedures were not clear to them.

the WPi said that their patients were typical of patients with ME/cfs using their diagnostic criterea NOT typical of the patients labelled with CFS by the Oxford mythodology.The two patient cohorts are clearly different and Dr Vernon should be repeatedly stating that