CROI: positive Spanish study - B cells XMRV reservoir

[kurt]

True Kurt but some of the contamination studys might also be using non fresh samples ( i might be wrong ) apolgies if i am. but i havent heard that complaint from you, about those studys if true ?

The freshness vs banked issue is more relevant if we are discussing positive findings with known risk of contamination. Banked samples would seem to have more contamination risk. However, with negative findings, what is the risk of contamination? A contaminant can not cause a negative, to my knowledge anyway.

Besides that point, the real challenge to this report is that these were cell lines, which generally means they have been maintained for a long time, perhaps incubated, fed, worked on, etc. That increases risk of contamination and MuLV viruses are common contaminants, and a lab using MuLV species (such as for positive controls in an XMRV study) is more at risk. Even if they have never used MuLV before, there is contamination risk as MuLV is sometimes found to contaminate commercial reagents.

However, if you want my complaint, here it is. False positive/negative risks do vary by type of PCR test, I looked into that a year ago, if memory serves, real-time PCR (most of the 0/0 studies) has higher risk of false negative, and traditional PCR (WPI) has higher risk of false positive, in some documented cases. So there is a possibility that any lab on both sides could be wrong. This is early stage research and we are waiting for a preponderance of evidence that will be accepted by the scientific community.

My other complaint is that the CFS world has latched on to XMRV as if it is the answer and that is most definitely not proven at this point. I think we need a more balanced view, look at strengths and weakness on both sides.

 

[richvank]

Hi, all.

Note that when EBV transforms B lymphocytes, it immortalizes them. These "memory B cells" can continue to replicate, such as cancer cells do. They thus become places where the EBV virus has a safe haven for the life of the individual, and they account for the fact that about 90% of the world population harbors latent EBV in memory B cells. In order for viruses to remain in the body in the latent state, they need to reside in cells that live a long time or can replicate in an immortal manner. The memory B cells are a cell of this type. Neurons are, also, by having a long individual lifetime, and that's where herpes simplex 1 (cold sores) and herpes zoster (varicella zoster) (chicken pox and shingles) viruses hang out. How clever of XMRV to take advantage of the home that EBV has made for itself, and become its roommate!

Best regards,

Rich

 

[SOC]

My other complaint is that the CFS world has latched on to XMRV as if it is the answer and that is most definitely not proven at this point. I think we need a more balanced view, look at strengths and weakness on both sides.

Get a grip. Like most of us aren't aware this isn't a done deal? Most of us understand that XMRV is not absolutley proved. That will take a long time, and correctly so. It has, however, met the criteria of multiple validations, which is plenty good enough to move forward with.

We also understand that XMRV existence is not disproved. That could be done relatively quickly if anyone chose to do it properly. They haven't.

The preponderance of evidence, at present, supports the discoveries that XMRV exists and that it exists in the ME/CFS community at a higher rate than in healthy controls. The tide could still turn; that's the way science works. It could turn out that XMRV doesn't exist, or if it does, that it is not at the root of our illness. Lots of things could turn out to be true as the science develops.

Most of us have a much more balanced view than you seem to, so how's about you have a nice calm sitdown in your glass house and quit throwing stones? Just because people have a different opinion than you, doesn't mean they are behaving irrationally. Is it necessary that everyone else share your opinion?

I've tried to be very clear that I believe everyone is entitled to their own opinion about what the state of the research tells them at any point in time. Your opinion is a perfectly fine opinion. So is everyone else's. I'm getting pretty fed up with your "complaints" about other people's opinions. State your own opinions. Fine. Provide evidence for why you believe what you believe. Great. But would you please stop with the complaints about other people's decisions?

Another relevant grandmotherism: You tend your garden and let others tend theirs.

 

[SOC]

Hi, all.

Note that when EBV transforms B lymphocytes, it immortalizes them. These "memory B cells" can continue to replicate, such as cancer cells do. They thus become places where the EBV virus has a safe haven for the life of the individual, and they account for the fact that about 90% of the world population harbors latent EBV in memory B cells. In order for viruses to remain in the body in the latent state, they need to reside in cells that live a long time or can replicate in an immortal manner. The memory B cells are a cell of this type. Neurons are, also, by having a long individual lifetime, and that's where herpes simplex 1 (cold sores) and herpes zoster (varicella zoster) (chicken pox and shingles) viruses hang out. How clever of XMRV to take advantage of the home that EBV has made for itself, and become its roommate!

Best regards,

Rich

Thanks, Rich! I think I'm starting to get a handle on this now.

 

[alex3619]

So alex you are saying they are finding different strains, because i believe that is main concern for scientists.

Hi Grape Funk, one of the problems is that much of the research is not being published. I think some of the older research from, for example, the WPI should be published in lower prestige journals just to combat the misinformation that is out there. There do appear to be multiple strains of XMRV - which means considerable genetic divergence. However, some of this may be due to there being similar MLVs in people which are being confused. We just don't know enough yet. Bye, Alex

 

[Otis]

They seem to have a technigue for finding XMRV. I don't have the knowledge to understand their reason for looking at EBV transformed B-cells, but I feel good that they are considering what co-infections could have on XMRV.

Their mention of the 24nt deletion rings a bell, but thats about it? Someone else??

I blabbed about this some time ago and it's a key characteristic of XMRV as we know it. The sequenced copies of XMRV all have a significant deletion in the glyco-gag region, which is 24nts in length, as compared to the most similar MLVs. The polytropic sequences that Lo/Alter found had a 9nt deletion in the same region. I was curious so I used some online tools and Excel to compare these sequences and to see these deletions for myself.

 

[FunkOdyssey]

Curcumin again

J Surg Res. 1999 Nov;87(1):1-5.
Enhanced apoptosis mediates inhibition of EBV-transformed lymphoblastoid cell line proliferation by curcumin.

Ranjan D, Johnston TD, Reddy KS, Wu G, Bondada S, Chen C.

Department of Surgery, University of Kentucky, Lexington, Kentucky 40536, USA.
Abstract

BACKGROUND: Epstein-Barr virus (EBV)-associated B-cell lymphomas occur more frequently in immunodeficient states such as organ transplantation and HIV infection. We have previously reported that B cell immortalization with EBV was promoted by cyclosporin A (CyA) and that curcumin (Cur), a natural phenol with known antioxidant and antitumor properties, blocked EBV-induced B cell immortalization. In the following experiments we show that Cur inhibits the proliferation of EBV-transformed lymphoblastoid cell lines (LCL) via enhanced apoptosis.

METHODS: LCL were generated by infecting freshly isolated human B cells with EBV (B95-8) for 12 h and coculturing with predetermined optimal concentrations of CyA (500 ng/ml) for 4 weeks. LCL were then either frozen for future use or propagated for immediate experiments. These cells were then plated in 96-well plates with 20 microM Cur or 0.1% DMSO (vehicle control). The number of immortalized colonies/well, cell count, and (3)H uptake were used as an index of immortalization. To assess apoptosis rate LCL were cultured with 0.1% DMSO or Cur (20 microM) for 0, 18, and 42 h in culture flasks and then stained with MC540 and H33342, as markers for apoptosis, and analyzed by FACS.

RESULTS: A profound inhibition of proliferation was seen in the LCL with 20 microM curcumin compared to 0.1% DMSO control. The colony count reduced from 34.5 +/- 3.4 to 0/well (P = 0.005), cell number reduced from 101,250 +/- 12,093 to 3750 +/- 1500/well (P = 0.002), and (3)H uptake reduced from 40,889 +/- 3669 to 70 +/- 5.2/well (P = 0.001). The apoptosis rate of LCL in the DMSO control at 24.07 and 16.87% increased significantly with 20 microM Cur to 76.4 and 95.1% at 18 and 42 h, respectively (P = 0.02).

CONCLUSION: Cur is a potent inhibitor of EBV-transformed LCL. This effect appears to be mediated through enhanced apoptosis. A further investigation of this effect may be useful in prevention and therapy of B-cell lymphoma in immunodeficient patients.
Copyright 1999 Academic Press.

PMID: 10527697

Am Surg. 1998 Jan;64(1):47-51; discussion 51-2.
The effect of curcumin on human B-cell immortalization by Epstein-Barr virus.

Ranjan D, Siquijor A, Johnston TD, Wu G, Nagabhuskahn M.

Department of Surgery, University of Kentucky, Lexington, USA.
Abstract

Cyclosporine is a commonly used immunosuppressant in solid-organ transplantation. It is, however, associated with an increased incidence of Epstein-Barr virus (EBV)induced post-transplant lymphoproliferative disorder (PTLD). In this study, human B lymphocytes isolated from healthy volunteers were immortalized in vitro with EBV. The effect of oxidative stress mediated by cyclosporine A or hydrogen peroxide on in vitro B cell immortalization was studied by coculturing immortalized B cells with cyclosporine A and hydrogen peroxide. Curcumin, a phenolic extract of the spice turmeric, was then used to observe its effect on this process. We found that in vitro B-cell immortalization with EBV was promoted by the oxidative stress induced by cyclosporine A and hydrogen peroxide, with the maximum effect seen at concentrations of 500 ng/ml and 100 microM, respectively. Curcumin blocked the B-cell immortalization in a dose-dependent fashion with nearly complete inhibition at 20 microM. We conclude that, because both hydrogen peroxide and cyclosporine A strongly promote in vitro B-cell immortalization with EBV (the putative process responsible for PTLD) and curcumin, an extract of a common spice is an effective inhibitor of this process; curcumin may be an effective adjunct in the prevention of PTLD in the patients undergoing therapy with cyclosporine A.

PMID: 9457037

20 microM is a huge amount of curcumin though, higher than you could reach with any reasonable dose of curcumin currently available (best you could do is maybe 4-5 microM with several grams of BCM-95). Also at these micromolar doses it becomes cytotoxic and may have some negative effects. Still I'm going to check out the full text because if it had at least *some* impact at lower doses it should still be worthwhile.

 

[FunkOdyssey]

Looks like lower doses were useful too, although all still in the micromolar range (1000mg of BCM-95 produced serum levels of about 1 microM in humans for reference). I doubt anyone with CFS has taken that much or more than that for extended periods, so the real world effects are presently unknown.

 

[heapsreal]

Hi, all.

Note that when EBV transforms B lymphocytes, it immortalizes them. These "memory B cells" can continue to replicate, such as cancer cells do. They thus become places where the EBV virus has a safe haven for the life of the individual, and they account for the fact that about 90% of the world population harbors latent EBV in memory B cells. In order for viruses to remain in the body in the latent state, they need to reside in cells that live a long time or can replicate in an immortal manner. The memory B cells are a cell of this type. Neurons are, also, by having a long individual lifetime, and that's where herpes simplex 1 (cold sores) and herpes zoster (varicella zoster) (chicken pox and shingles) viruses hang out. How clever of XMRV to take advantage of the home that EBV has made for itself, and become its roommate!

Best regards,

Rich

Does anyone else not produce antibodies to other infections even though they have had the infection in the past?? I have got blood test results of positive EBV igg around the same time of my onset of cfs(within 6 months) and had 2 blood test since, one approx 2 years after initial test and one 12 months ago, both came back negative. Also hep B vaccine i had a number of years ago and retested 10 years later and had no antibodies from the vaccine which my doc at the time said was strange as normally u have some antibodies. Although still producing antibodies to cmv. I wonder if not producing antibodies is a type of immune defiency. I know i have poor NK function and wonder if immune defiencies is why some dont have antibodies to xmrv or they are no longer in the blood but sitting in tissue or the nervous system doing their damage??

cheers!!!

 

[dannybex]

Most of us have a much more balanced view than you seem to, so how's about you have a nice calm sitdown in your glass house and quit throwing stones? Just because people have a different opinion than you, doesn't mean they are behaving irrationally. Is it necessary that everyone else share your opinion?

Sickofcfs -- I'm kind of stunned. Where is Kurt throwing stones and saying others need to share his opinion, or that they're behaving 'irrationally'?

I've tried to be very clear that I believe everyone is entitled to their own opinion about what the state of the research tells them at any point in time. Your opinion is a perfectly fine opinion. So is everyone else's. I'm getting pretty fed up with your "complaints" about other people's opinions. State your own opinions. Fine. Provide evidence for why you believe what you believe. Great. But would you please stop with the complaints about other people's decisions?

First of all, I think his complaint was with peoples opinions, but rather with aspects of certain studies protocols. But with all due respect, isn't that kind of what you're doing here -- complaining about Kurt's opinions and decisions?

And isn't he providing evidence for why he believes as he does?

Seriously. I don't get it.

???

 

[August59]

Hi Grape Funk, one of the problems is that much of the research is not being published. I think some of the older research from, for example, the WPI should be published in lower prestige journals just to combat the misinformation that is out there. There do appear to be multiple strains of XMRV - which means considerable genetic divergence. However, some of this may be due to there being similar MLVs in people which are being confused. We just don't know enough yet. Bye, Alex

I would hope that some of these completed studies would get published as well. A lot of this information could be crucial to perhaps another study being done by another researcher. If anyone could explain how the process goes for getting a paper or study published, I sure would appreciate it? If there is some good science sitting on a desk and the information is not conveyed due to lack of publishing, then something about this process is flawed or in any case needs to be changed.

 

[Grape Funk]

Anyone know if once a studied is submitted to a journal, and the journal accepts it, and publishes it, can it be used once again? Especially if the paper is for a bigger journal the second time around, can it be printed again, or does the first journal automatically have exclusive rights?

 

[Grape Funk]

Does anyone else not produce antibodies to other infections even though they have had the infection in the past?? I have got blood test results of positive EBV igg around the same time of my onset of cfs(within 6 months) and had 2 blood test since, one approx 2 years after initial test and one 12 months ago, both came back negative. Also hep B vaccine i had a number of years ago and retested 10 years later and had no antibodies from the vaccine which my doc at the time said was strange as normally u have some antibodies. Although still producing antibodies to cmv. I wonder if not producing antibodies is a type of immune defiency. I know i have poor NK function and wonder if immune defiencies is why some dont have antibodies to xmrv or they are no longer in the blood but sitting in tissue or the nervous system doing their damage??

cheers!!!

I have heard that heaps, people not producing antibodies to certain infections. But, the only people were PWC, i don't know about healthy persons.

 

[free at last]

The freshness vs banked issue is more relevant if we are discussing positive findings with known risk of contamination. Banked samples would seem to have more contamination risk. However, with negative findings, what is the risk of contamination? A contaminant can not cause a negative, to my knowledge anyway.

Besides that point, the real challenge to this report is that these were cell lines, which generally means they have been maintained for a long time, perhaps incubated, fed, worked on, etc. That increases risk of contamination and MuLV viruses are common contaminants, and a lab using MuLV species (such as for positive controls in an XMRV study) is more at risk. Even if they have never used MuLV before, there is contamination risk as MuLV is sometimes found to contaminate commercial reagents.

However, if you want my complaint, here it is. False positive/negative risks do vary by type of PCR test, I looked into that a year ago, if memory serves, real-time PCR (most of the 0/0 studies) has higher risk of false negative, and traditional PCR (WPI) has higher risk of false positive, in some documented cases. So there is a possibility that any lab on both sides could be wrong. This is early stage research and we are waiting for a preponderance of evidence that will be accepted by the scientific community.

My other complaint is that the CFS world has latched on to XMRV as if it is the answer and that is most definitely not proven at this point. I think we need a more balanced view, look at strengths and weakness on both sides.

Ok fair enough i get your point, But the Huber contamination findings used banked samples for the CFS samples, so it doesnt appear intirely true that the banked rather than fresh sample observation, does only apply to positive studys because the positive detections by Huber were deemed as contamination using banked samples ? I think it depends on your definition of positive studys, if you would like to include the defiintion of a positive study being more of a problem with banked samples, when such said positive detection study of the likes of Huber, concludes contamination, as the source of the positive signals. Then yes in full agreement, But your definition wasnt clear on this. At least to me. Not sure what others thought ? So indirectly it can be a important question to negative studys, in as much as the Huber detections are really deemed Negative, in relation to the contamination observation findings using IAP How can this be a positive study when its concluded contamination was the cause of the detections ? Its not its regarded as a negative study in relation to the contamination findings ?

 

[free at last]

Free at last, i have to agree along the lines when you said it is somehow contamination yet a real virus is able to infect monkey tissue and human tonsil when infected with xmrv. It is very contradictory. Now the other report came out that just said it is the cell line contaminate. So maybe people were already supposedly affected by the "endogenous RV" found in the prostate. But why are cdc studies not showing any xmrv then? How about substantial percentage differences?

So alex you are saying they are finding different strains, because i believe that is main concern for scientists. The blog i was looking at was straight up trashing xmrv, giving it no shot at all, though a sizable blog with resonable comments. But not skeptics just doubters.

Its really beyond me Grape, i just dont understand so many contradictions, its there but its contamination, maybe its there, but so is contamination, maybe thats what we are seeing. as for the negative studies, some people think pcr optimization is more important than many scientists are giving credit for in this particular virus, and from what i hear, they dont like that idea, they belive it means the whole feild is suspect, because of this optimization question, replication if you will. Whos right ?

 

[kurt]

Anyone know if once a studied is submitted to a journal, and the journal accepts it, and publishes it, can it be used once again? Especially if the paper is for a bigger journal the second time around, can it be printed again, or does the first journal automatically have exclusive rights?

For enough money, a small journal might allow their article to be licensed. But no major journal would reprint an article already published elsewhere, they are very focused on exclusive news/articles. They all want 'the big scoop'. That's how they get their readers. And also that is why journals like Science and Nature try to publish ONLY new discoveries, new scientific findings that will have a major impact on a scientific field. I believe they call this something like 'impact factor' and it is a really big deal to the major journals. However, the major publications do sometimes print letters from scientists that are commentary on hot topics in one of their specialty journals, so that might be a way for them to address something printed elsewhere, if it has become a hot topic.

 

[Cort]

Grape Funk - Free at last, i have to agree along the lines when you said it is somehow contamination yet a real virus is able to infect monkey tissue and human tonsil when infected with xmrv. It is very contradictory. Now the other report came out that just said it is the cell line contaminate. So maybe people were already supposedly affected by the "endogenous RV" found in the prostate. But why are cdc studies not showing any xmrv then? How about substantial percentage differences?

It is incredibly complex...It is a real virus and it can infect numerous human cells. The big question is where did the XMRV in the 22RV1 cell line come from? That cell line was produced from prostate cancer tissue that was grafted onto nude mice. They're saying the prostate cancer tissue was clean but at some point after it was grafted onto the mice XMRV all of sudden showed up. This makes them think that XMRV never infected that person but was created in the lab.

Even so it is still a retrovirus that can infect human cells! Now the big question, I think, is did it leap from the lab into the human population? The fact that XMRV appears, at this point, to be so similar to 22RV1, suggests that it never got into the human body but somehow found its way out of the lab and into reagants and other lab products and then showed up in the WPI's samples.

But why it would show up in their samples and not others? Note that almost no one else has been able to find XMRV. Why would it show up in the WPI's labs and not others? They are not, after all, a mouse lab. You would think that if it was widespread in labs across the world it should be showing up in lots of studies but it's not.

Three labs have thought they found XMRV and after looking at it decided it was contamination but that is an entirely different issue because the WPI has proven, at least in some of their samples, that they have the entire virus.

So how did this 'lab creation' only end up in the WPI's samples?

That is a huge puzzle, I think.

 

[Cort]

One connection, I guess is Dr. Silverman since he discovered the virus. Could materials have been passed from the Silverman lab to the WPI that contained the virus....that, I suppose is a possibility.

 

[Grape Funk]

One connection, I guess is Dr. Silverman since he discovered the virus. Could materials have been passed from the Silverman lab to the WPI that contained the virus....that, I suppose is a possibility.

Did silverman state recently it was contamination with the cell lines? Or just coffin and others. I see Silverman was on the original Study as well.

www.ncbi.nlm.nih.gov/pubmed/16609730

 

[Otis]

One connection, I guess is Dr. Silverman since he discovered the virus. Could materials have been passed from the Silverman lab to the WPI that contained the virus....that, I suppose is a possibility.

And on to every lab finding a positive? That's where this starts stretching a bit thin.

I'm not sold in the idea but it makes for some interesting speculation. If we're dealing with a lab creation, then we need to be thinking in terms of this happening repeatedly creating and thereby different viable infectious retroviruses. They started with the "answer" the the form of the XMRV sequence and went backwards and found two endogenous strains that appear to have combined to "make" XMRV, apparently without too much trouble. C=A+B. Well there's a helluva of a lot of possible substitutions for 'A' and 'B' and even more for 'C' as a result of all the possible combinations of 'A' and 'B', recognizing that we're talking about roughly 8100nt sequences for each.

And although we don't have a complete sequence from Lo/Alter if they're seeing a recombination artifact it's different in significant ways - e.g. 9nt deletion vs. 24 seeming to add some weight to the diversity of these viruses - made by nature or man.

So if we accept the idea that recombination may have have created an infectious virus in the one case where we started with the result and backtracked and happened to find the answer, how many times has nature found a way to take advantage of these xenografts (and other means of such recombination) and created something viable. And how many times have lab workers come in contact with them over the last 3-4 decades? It seems a little light bulb went on here. As CBS mentioned the concern seems to be for their own, not the public at large.

It's ironic how caviler people can be until it's their own asses on the line, which in the case of Stoye is one large ass even discounting his charming personality. If Stoye thinks this is contamination or perhaps now a non-viable recombinate, I find it quite amusing he's testing himself for XMRV.