me.cfs.forums: Congratulations to Dr Enlander on his new collaborative endeavour

We wish to congratulate Dr Enlander on his new collaborative endeavour.  The team put together by Dr Enlander looks impressive and we now have an opportunity to answer questions definitively. If the researches were willing to establish baseline immune profiles very much in the same manner as demonstrated by Dr Judy Mikovits we could establish unequivocally that ME is a neuroimmune disease.


Rituximab doesa lot more than deplete mature B cells. To begin, there is a molecule called IL-2 which is elevated in people examined by Dr. Mikovits who were ZMRVpositive. This molecule is a signal molecule and is pivotal in activating the immune system, and more importantly from our perspective, keeping it active. It  also controls the level of certain kinds of T regulator cellcalled FoxP3.   Without sufficient FoxP3 the T cells start to attack the immune system, and an over-high ratio of T Helper 17 (TH17) cells is created, as is found to be the case in auto-immune and chronic inflammatory diseases.  If IL-2 (an interleukin, a type of cytokine) remains high, then the T regs response is suppressed, and an autoimmune situation will arise.   Rituximab also acts as an antibody against IL-2 molecules and hence switches off or at least dampens down the activity ofthe immune system.


Rituximab could also be helping by removing TH17, a major source of autoimmune symptoms. It also reduces the level of a molecule called NF-kappaB. This is always high in people with an activated immune system and/or inpeople with activated microglia in the brain.  The activated microglia are the ultimate source of neurotoxicity and mitochondrial damage. Importantly cytokine levels are maintained by NF-kappaB as is the continued activation of microglia, yet another way in which Rituximab administration could aleviate the symptoms of someone with neuroimmune activation.

Much of the molecular biology surrounding HIV has been revealed using a mouse model of the effects of a gammaretroviral infection. The virus used is a gammaretrovirus like XMRV and ZMRV and thecondition has been dubbed Murine Aids.  This condition is caused by a replication defective retrovirus coding a protein, which causes B cells to expand once the retrovirus has infected the cell.  This is called clonal expansion.  The disease created is multisystemic likeME.  Obviously if the ME retroviruses are found in B cells, as theyare in Murine Aids, then reducing the number of these B cells will aleviate symptoms. Finally, the B cells first meet the antigens (bugproteins) produced by the Murine Aids virus in the spleen, lymph nodes and the payers patches in the gut.  Now, B cells tend to spend considerably more time in the compartments of the body where they first encountered anantigen than they do in the blood.  In fact only about 4% of preactivated CD20 B cells are present in the blood at any given time.  So looking for a ZMRV which inhabits CD20 B cells in the blood is not a very clever idea. 


If base line Th17  Nf-kappa B  and Tregs were established we could monitor the effects of rituximab directly and that would give the ammo for a specific licence application.


If then they are willing to use Dr Mikovits's/Dr Ruscetti's reverse transcriptase assay in its entirety, and Dr Lo's assay, we can establish the presence of ZMRVs once and for all.


Finally, there is good theoretical evidence that ZMRVs are not likely to be found in the blood at detectable copy numbers, hence a variation specifically looking for ZMRV sin gut tissue would blow the negative studies out of the water.


4 experts + onestudy = definitive answer.



Finally, for this blog we must comment on the increasing censorship endured by supporters of Dr Judy Mikovits and her research on patient forums. Wethink that hidden agendas should have no place on forums which are suppposed toserve the interests of patients rather than the intersts of individual administrators and moderators. We will endeavour to make this a safe place from which to receive unbiased information and a blog which unashamedly supports thecause of scientific research into our neurological disease and advocates for oppressed sufferers. The following is a post from Angela Kennedy a long term andhighly respected advocate.

I wanted to let people know that I have been prevented from posting on threads because I REPORTED OTHERS for blatantly breaking this forum's rules, and for them attacking other forum members, and using a campaign of intimidation and humilating tactics to silence, and called said others on what they were doing.

The moderator then said that what the person who started doing this on a particular thread, was 'fine', and has allowed it to stand.

This forum has been subject to abuse of moderator power for a long time, where moderators punish victims of abuse of forum rules, and use their power to advance their own partial positions, allowing others free rein to attack and intimidate others into silence and implied acquisance.

Free speech has been banished, and agent provocateurs of both the ideological and employed kind proliferate, spreading demoralisation and powerlessness.

Sadly forums have become one of the ways in which people who wish to advocate for this community to protect sufferers of ME/CFS against medical abuse. It is where people learn to mobilise, to discuss the issues, gather information. This forum is stopping that, and various other forums are on the way to this behaviour too.

I am speaking out because the level of abuse is blatant - I eventually recognised it for what it was. It took me a while because I actually do try and moderate my own behaviour to be reasonable. I'm no shrinking violet, and I don't have ME, so I can only imagine how dreadful this situation must be for people who are so ill - and find themselves powerless in the grip of such abuse of power and advancing of positions not in the interests of this community.

I empathise with those people on this forum - and I know there are enough of you because of discussions away from this forum- who have been intimidated and moderated into silence while others blatantly flout forum rules and intimidate others in order to advance points of view that are potentially and/or actually damaging to the interests of the ME community.

All I can hope is that this post is read before it is removed, and people will find some comfort in understanding the problem, and seek to get things changed. Whether that means leaving this forum and starting elsewhere, or having the courage to tell Cort and friends where they are going wrong has to be up to individuals.

This community has been under siege for too long. Friendly fire has ALWAYS been a problem, and the level of friendly fire has intensified with the advent of Mikovits's research. Sadly this is far more serious an issue than silly spats on a forum.

No evidence that a strain of XMRV was created in a cell line in the 1990s

Update on the paper Paprotka et al.

The argument in Paprotka et al. (a study led by Pathak and Coffin) is that the sequence isolated by Silverman, which he claimed came from patient VP-62, is a result of a unique recombination event. This unique recombination event occurred between a virus, found in many species of mice, and a hitherto undiscovered replication defective erv, when the xenograft which was involved in the creation of the 22Rv1 line was passed through mice.

They claim, but cannot prove that these mice were NU/NU and or Hsd mice, as they contain the viruses or at least the virus fragments they deem to be necessary to further their hypothesis.

They argue that their assays did not amplify XMRV in the early xenografts, but after several passages of the cells through the proposed mice their assays were able to amplify XMRV. Thus XMRV must have been absent from the early xenografts and created by this unique recombination event.

So we have to judge between two explanations. One is that the VP-62 sequence was generated by a unique recombination event, or that the PCR assays used lacked the clinical sensitivity needed to detect low copy VP-62 sequences in the early xenografts.

Given that a unique event is so very unlikely they must provide very strong evidence that VP-62 equences were indeed absent from the early xenografts and that their apparent absence was not caused by the failure of their PCR assays. Absence of evidence is not evidence of absence.

If one focuses on the initial quantitative PCR assay using one envelope primer it becomes clear that there is no evidence provided regarding the copy number of VP-62 sequences per 100 cells in the NU/NU or Hsd mice, later xenografts or early xenografts. This is because the PCR amplified what were known to be VP-62 sequences from the DNA taken from 22Rv1 cell line and the CWR-R1 cell line. The problem here is that the copy number of VP-62 in these cells is huge, being 2000 copies per 100 cells and 3000 copies per 100 cells respectively. We do know that this assay could detect ERV sequences in the mice and the early xenografts, but we don’t know what the copy number of ERV sequences is compared to the copy numbers of VP-62. Thus it is quite possible for VP-62 sequences to be present but undetectable, due to a lower copy number for example. There are other more complex reasons. In any event absence of evidence is not evidence of absence. We also have the problem that this assay with the env primer was not used to screen other species of lab mice or wild derived mice. Had this assay being able to detect either XMRV or ERV sequences in these mice the paper would have collapsed. Certain vested interests have argued that it would be silly to use this assay with this primer to screen the other mice because the primer could amplify both VP-62 and ERV sequences. In that case why use that primer to screen NU/NU and Hsd mice, and indeed why use that primer at all. I wonder what the defense is for not using this primer and assay to any screen wild mice. Wild derived mice are not wild mice.

There is no evidence that this assay can amplify XMRV specific env sequences other than in the DNA taken from 22Rv1 and CWR-R1 cells. There is no evidence that this assay with this primer could detect VP-62 sequences in the DNA extracted from lab mice, the early xenografts or the later xenografts. The later xenografts were not screened using this assay. In fact figure S3 in the SOM (Supporting Online Material) shows that provided this primer was used at all to screen the later xenografts using the second single round PCR, then that PCR assay could not amplify any product. As this primer was used to screen the early xenografts with the second PCR assay (Figure S3), not to use it to screen the later xenografts would be a serious departure from the scientific method.

Next we examine the second PCR single round assay with a gag primer, which is only capable of detecting VP-62 gag sequences. Figure 2D and 2E suggest that this assay, with this primer, was used to screen the early xenografts. This is however in direct conflict with the text, which states that all the primers used with the single round assay were capable of amplifying both VP-62 and ERV sequences. This is prima facie evidence of misfeasance and at the very least should be investigated. In any event, it seriously weakens if not invalidated their claim that gag sequences were not present in the early xenografts. Indeed we once again have the scenario that there is no evidence that this single round assay can detect VP-62 gag sequences in anything other than the DNA taken from the 22Rv1 or CWR-R1 cell line either. So they cannot provide evidence that their assay could detect VP-62 GAG sequences in mice or later xenografts let alone the early xenografts.

Thus I will leave it to those without vested interests to consider whether these authors have provided convincing evidence that VP-62 sequences are absent from the early xenografts, bearing in mind that absence of evidence is not evidence of absence and that the alternative explanation is a unique recombination event, which cannot have happened in the past and its future occurrence is a virtual impossibility.

Finally the paper does not fulfill the minimum requirements of surviving a peer review process. There is not enough evidence to enable the scientific merit of the study to be judged or enough data in the methodology section to enable replication. The claim that it is custom and practice among a certain group of retrovirologists not to include such details unless they deem it necessary merely means that none of their studies should have passed the Science peer review process or indeed any rigorous peer review process. Contrary to statements made by certain vested interests the Science magazine is not published merely for the perusal of certain retrovirologists but also other scientists from different disciplines. I would remind everyone what the requirements of science are:

"Data and materials availability
All data necessary to understand, assess, and extend the conclusions of the manuscript must be available to any reader of Science."

Paprotka et al., ‘Recombinant Origin of the Retrovirus XMRV’ does not meet the requirements as specified by Science. 

Permission to repost 

Thank you to Pumpkin for the post.

The serology assay used in Lombardi et al. could never have detected an erv.

There are 16 human endogenous retroviruses (erv's) capable of producing a full length env protein.

Three are naturally expressed in PBMCs.  They are HERV-K  HERV-R and HERV-FRD. None are latent or methylated.

Only HERV-FRD is related to gammaretroviruses. HERV-FRD is better known as syncytin 2.

Hence only HERV-FRD would have been a candidate for reacting with the monoclonal antibody to SFFV env in figure C in Lombardi et al.

HERV-FRD would have produced an antibody response whether 5-AZA had been added or not.

The molecular weight of the FRD env is known and does not correspond to the molecular weight of the env protein detected in the patients in lombardi et al.

Thus the speculation that the culture of PBMCs activated by AZA could have activated a latent erv is not justified and is not supported by any scientific observations or other scientific evidence.

ME, retroviruses and prions

A family of gammaretroviruses (HGRVs) has been discovered in the blood of people with ME.  These viruses in mice (MuLVs) are notorious for causing neuro-immune disease and cancer.  Is it possible that in humans it is the relationship of these viruses to PrP, a cellular prion protein found in humans and other animals, which creates the multi-systemic disease Myalgic Encephalomyelitis (ME)?

Cellular prion proteins are essential for normal neurological and immune function, but in their misfolded form they can lead to a number of diseases, such as Creutzfeld-Jakob disease (CJD).

As can be seen from the following excerpt from Gabus et al. (2001), MuLVs in particular are also believed to interact with misfolded prions.  This interaction between the retroviruses and prions is called “nucleotide chaperoning”.

“Although PrP is a highly conserved protein in vertebrates, its role remains to be identified. Interestingly, PrP null mice develop normally and appear to be healthy (2). Nonetheless, a number of functions have been proposed for PrP such as super- oxide dismutase activity, involvement in copper metabolism (reviewed in Ref. 10), and, very recently, participation in signal transduction during neuronal differentiation (11). In addition, PrP was shown to interact with sulfated glycans (12), RNA aptamers (13), and large nucleic acids (14, 15), causing the formation of nucleoprotein complexes similar to HIV-1 nucleo- capsid-RNA complexes formed in vitro (16). A recent report shows that MuLV replication accelerates the scrapie infectious process (17), suggesting possible in vivo interactions between retroviruses and PrP.” 

http://www.jbc.org/content/276/22/19301.full.pdf

If a retrovirus infection caused cellular prion protein misfolding, via nucleotide chaperoning and vice versa, we have a mechanism that would result in a multisystemic disease.

WHERE DOES THE NUCLOTIDE CHAPERONING TAKE PLACE?

Prions and retroviruses both make use of exosomes and endosomes, which act like tiny cellular submarines, ferrying proteins between different islands in a cell and between cells.  Retroviruses specifically use exosomes and endosomes like submarines to disguise themselves and avoid the immune response, whilst prions travel between the surface of the cell membranes and the inside chambers of the cellular islands (organelles).

The shared use of these submarines allows for the retroviruses and prions to meet and interfere with each other, potentially changing the shape of each other’s proteins.  Once started each will increase the number of misfolded proteins of the other, over and over again.

SUPPORTING EVIDENCE

• Gammaretroviruses are known to increase the number of mishapen prions  and gammaretroviruses are known to cause spongiform encephalopathies.  Mad cow disease is an example.

• Change a prions shape and you get neuroimmune toxicity.

• Change shape of retrovirus proteins and you get neuroimmune toxicity.

• MuLVs cause neurotoxicity in mice as a result of a misfolded env protein (region of the virus), which is not incorporated into the virion.

• In 40% of HIV patients on HAART neurotoxicity still occurs despite the HAART treatment. 

 So could ME be a prion disease? 

The Recombinant Origin of XMRV paper - why the Paprotka et al. paper failed to support the authors' conclusions

Paprotka, Coffin, Pathak et al. wrote the paper 'Recombinant Origin of the Retrovirus XMRV' (Paprotka, 2011, DOI: 10.1126/science.1205292).

This paper claimed that the XMRV variant of Human Gamma Retro Viruses (HGRV) was accidentally created in a lab when human prostate cancer cells were passaged through mice some time between 1993 to 1996. That XMRV did not exist before that time (1993) and that the original patient was not infected.

They surmise that the laboratory cell line thus created (22Rv1) then carried the XMRV virus to other labs, where it then contaminated human samples of blood or tissue, resulting in the findings in all the positive HGRV papers.

This is problematic, firstly because the WPI, NCI/Ruscetti, FDA/Lo and NIH/Alter labs have never had this 22Rv1 cell line in their labs. Secondly, just because a cell line is infected it does not mean patients cannot also be infected. Thirdly, it cannot account for other variants of HGRVs. And more importantly, this paper failed to include details on a third assay used in the study (RT-PCR) and failed to properly screen the cells to determine whether they contained the XMRV variant.

What follows is a description of how the authors used the three assays. To help the reader understand this section, an analogy has also been included at the bottom of the page.

ASSAYS USED IN PAPROTKA ET AL.

As you can see from the diagram below, cells were initially taken from a patient with prostate cancer in 1992. It has been hypothesised that this person must have been infected with the XMRV variant, because the 22Rv1 cells, represented by the 3rd jar from the left, are known to contain the virus.

The authors of Paprotka et al. would have you believe that the patient was not infected and that XMRV was created from two mouse viruses when tumour cells taken from that patient were transplanted into laboratory mice. This is called xenografting and was done repeatedly to the cell line between 1993-1996. After which the cells continued to be developed for research purposes (no xenografting involved) and eventually two sub-lines were created, 22Rv1 and CWR-R1. The latter is represented by the jar on the far right.

To support this argument, the authors of Paprotka et al. screened several generations of the entire cell line derived from the patient, including early cells that had been xenografted into mice and later cells that had been xenografted into mice. These are represented by the first two jars on the left. They state that the early xenografts do no contain XMRV, but the later ones do contain XMRV and that therefore XMRV was created during this transplantation into mice.

"We conclude that XMRV was generated as a result of a unique recombination event between two endogenous MLVs that took place around 1993–1996 in a nude mouse carrying the CWR22 PC xenograft." (Paprotka, 2011)

Click on image to enlarge

It is now know however that a third assay was used in the paper. Vinay Pathak in his presentation of this paper at CORI 2011, a conference on retroviruses, specifically stated that the later xenografts (2nd jar on the left) were screened with an RT-PCR assay. This assay was not included in the paper, was not used on any of the other cells, and we have no knowledge of its sensitivity (minimum amount of virus the assay can detect). Consequently, it is impossible to know the amount of XMRV that was found in the cells of the later xenografts.

It is also known from the paper, that these cells were not determined to be from the same patient as the other cells. This can be seen in the small grey box, labelled B, at the top of the image. The later xenograft's cells were given the following designations, 2152, 2524, 2272 and 2274, none of which were included in that experiment.

"We verified that the xenograft samples (736, 777, 9216R, 9218R, 8R and 8L) and the 22Rv1 or CWR-R1 cell lines were all derived from the same person by performing short tandem repeat (STR) analysis at 7 loci (Fig. 1B and fig. S1)" (Paprotka, 2011).

Without having assessed the later xenograft cells to see if they were from the same patient, it cannot be assumed that they were. Without this evidence it is impossible to argue that XMRV was create during xenografting between 1993 to 1996. Cross-contamination of cell lines is also a common problem, and again without this data we cannot be sure that the cells were not already contaminated from a separate source. So why were the later xenograft cells not included in this experiment?

Given these omissions, the paper cannot be said to have shown that the XMRV variant was created in this prostate cancer cell line during xenografting.

In addition, of the two assays actually named in the paper (qPCR and PCR), the qPCR was demonstrated to have at best a sensitivity of 1-3 copies per 100 cells and did detect XMRV in the early xenografts, but then the authors used a PCR assay, with no known sensitivity, to re-screen the early xenografts. It is this PCR assay they based their results upon.

This assay (the PCR assay represented by the blue line) was at most shown only capable of detecting as low as 2000 copies per 100 cells, but did not detect XMRV in the early xenografts, even though the qPCR assay had shown the level was at most 1-3 copies per 100 cells. Far below the proven ability of that PCR assay. The paper therefore also failed to provide evidence that the early xenografts were not infected and failed to present evidence that the patient was not infected.

As authors are obligated to provide data on all assays used to produce results published in scientific journals, here the authors failure to do so in this paper cannot be seen to be in the spirit of science and can only harm future progress in our understanding of human gammaretroviruses.

Finally, a number of laboratory mice were screened (no wild mice) to see if they contain viruses that may predate the XMRV variant. However, it is impossible to support any argument of contamination on this evidence as the mice used for creating the 22Rv1 cells are unknown, and no direct link can made to the cell line. The authors also failed to isolate from a single source one of the viruses (found in the authors chosen mice) that they believe was one of the two that combined to create XMRV (PreXMRV-1). Instead independent sections of virus were combined and called a whole virus. Thus, a hypothesis cannot be constructed as one of the two viruses has not been shown to exist.

SUMMARY

• We have four types of cells; Early Xenograft, Later Xenograft, 22rv1 cells and CWR-R1 cells

• A qPCR assay detected XMRV in the 22rv1 and CWR-R1 cells, but not in the Early xenograft cells. It was not used to examine the Later xenograft cells.

• A RT-PCR assay was used to examine these Later cells and found XMRV, but was not used to examine any of the other cells. This type of assay is also more sensitive than PCR alone.

• Details of the RT-PCR assay were not included in the Paprotka paper.

• No one knows where the Later xenograft cells came from or if they were contaminated.

• Paprotka et al. argue that XMRV was created by the recombination of two different gamma retroviruses when the Early xenograft cells were passed through specific mouse cells. They say the recombination event is so rare that it could only ever have happened once.

• There is no evidence that the mice tested in the paper were involved in creating the cell line originally, and the very existence of one of those mouse viruses they claim recombined to create XMRV, is unproven.

ANALOGY

The following is an analogy to help readers understand why the type of PCR used is important.

Imagine you are given two beakers, one red and one blue. Each beaker contains about an inch of what looks like sand. You are asked to find tiny bits of iron filings which might exist in one or both beakers. You are allowed to pour the sand out of the beaker onto a magnetic plate, shake off the sand, and use a magnifying glass to see if there are any tiny pieces of iron filings stuck to the plate. You are given a rack of magnifying glasses to choose from, because the filings are so small that they are right at the limit what you can see.

You pour out the sand from the red beaker and can see no filings at all. You do the same with the blue beaker and are able to see iron filings stuck to the plate. The problem is that you used different magnifying glasses to examine the contents of the red beaker and the blue beaker. You also find out later that the power of the magnetism in each plate was different, but you don't know which was the stronger.

Nevertheless, this paper assumes, to extent the analogy, that the blue beaker contained the iron filings but the red beaker did not.

The sensitivity of PCR, a type of test, is dependant upon the adjustment of a number of variables: magnesium, salt, buffers, annealing temperatures, and so on. By not determining the sensitivity of the PCR assay before its use the authors have rendered the interpretation of the results void.