Endogenous RNA viruses and Methylation

[redo]

I'd really appreciate to get your opinion on this Richvank. It's from mid December 2011.

Silencing of endogenous retroviruses: when and why do histone marks predominate?
Retrotransposons, such as endogenous retroviruses (ERVs), have colonized the genomes of all metazoans. As retrotransposition can be deleterious, numerous pathways have evolved to repress the expression of these parasitic elements. For example, methylation of the fifth carbon of the cytosine base in DNA (5-methylcytosine, 5mC) is required for transcriptional silencing of ERVs in differentiated cells. However, this epigenetic mark is generally dispensable for ERV silencing during early stages of mouse embryogenesis and in mouse embryonic stem cells (mESCs). In this Opinion, we evaluate recent findings on the exceptional role of covalent modifications of histones in ERV silencing in these cell types. In addition, we discuss the potential role of TET proteins, which catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), in perturbing transcriptional silencing, and propose that histone modification-based pathways may be used to silence ERVs during those developmental stages when DNA methylation-mediated silencing is compromised.

http://www.ncbi.nlm.nih.gov/pubmed/22178137

I apologize if ERVs have been covered previously in your work on methylation. The first thing I am going to do when my health allows for it, is to read myself up on it.

 

[richvank]

Hi, redo.

I'm pretty sure that there are others here who know a lot more than I do about this, and maybe one or more will pop in and give us a better explanation, but here are some comments.

First, they are discussing endogenous retroviruses. As I understand it, these are retroviruses that were incorporated into the genome of the host a long time previous. They mention that the expression of this retroviral DNA is usually silenced by methylation, and I think this has been known for some time. Then they go on to comment that in early stages of the development of the mouse embryo and in embryonic stem cells, these endogenous retroviruses are not blocked from expression by methylation, but instead, histones play this role. Histones are proteins that are associated with DNA, and are involved in winding it up, as on spools. They also mention that TET proteins (I'm not familiar with them) can cause the methylated cytosine to be oxidized, thus overcoming the gene silencing, and apparently they suggest that the histones can do the job when that happens, too. I haven't seen the full paper, but that seems to be what the abstract is saying.

As you know, it still isn't clear what if any role retroviruses play in ME/CFS. If they are important, it will probably not be endogenous ones that are. If it turns out that retroviruses are important, then methylation would seem to be a big deal in relationship to them. If retroviruses are present and are being subdued by methylation, then if something perturbs the methylation, such as glutathione becoming depleted by stressors of various types, leading to a functional B12 deficiency, in turn leading to a partial block in methionine synthase and a methylation deficit, then it would seem that this might allow the retroviruses to be expressed. Judy Mikovits is very familiar with this. When I first met her at the IACFS/ME conference in Fort Lauderdale in January 2007, we had a great conversation about methylation. She was one of very few people at the conference who knew anything about it or thought it might be important in ME/CFS. She has mentioned this in various talks she has given, including one in Santa Rosa, CA.

I hope that the Lipkin study will give us a fairly clear yes or no about retrovirus involvement in ME/CFS. If it turns out that they are involved, the GD-MCB hypothesis will be able to embrace them readily. If they aren't, this hypothesis will still stand up, in my opinion. I do look forward to us finding this out, though!

Best regards,

Rich

 

[redo]

Thank you for a thorough reply Richvank! I think retroviruses will turn out to be a component in ME, so I am glad to see how the methylation theory fits into this. I actually got a metylation protocol from my CFS doc some years ago (very likely that it's inspired from your work), it was immunothione and b vitamins. I added SAM-e myself, but I only used it for some days, as I got blue taking it. I have heard getting more utbeat, or no change in the mood is more commom (correct me if I am wrong).

Could you say anything about how long it normally takes before people improve on a methylation protocol? Once my symptoms stabilize I plan to start up again.

 

[richvank]

Thank you for a thorough reply Richvank! I think retroviruses will turn out to be a component in ME, so I am glad to see how the methylation theory fits into this. I actually got a metylation protocol from my CFS doc some years ago (very likely that it's inspired from your work), it was immunothione and b vitamins. I added SAM-e myself, but I only used it for some days, as I got blue taking it. I have heard getting more utbeat, or no change in the mood is more commom (correct me if I am wrong).

Could you say anything about how long it normally takes before people improve on a methylation protocol? Once my symptoms stabilize I plan to start up again.

Hi, redo.

Here is a quotation about that from the "Results" section of the paper Dr. Nathan and I wrote in 2009 about our clinical study of the simplified treatment protocol:


"Various patients reported some early exacerbation of symptoms, which in most cases was followed by a greater improvement in symptoms. Three of the patients found it necessary to decrease their dosage frequency to every second or third day for several days, until they could tolerate the full daily dosage schedule.

"Sixteen of 30 patients (53%) reported an initial worsening of symptoms, beginning in most of these cases within 3 or 4 days, but in some cases beginning at up to 2 weeks. Most of the symptoms were mild, and none of the patients discontinued usage of the supplements during the first 3 months. The most common side effects were gastrointestinal (pain, cramps, constipation, or diarrhea), reported by 6 out of 30 patients or 20%; increase in pain, reported by 4 out of 30 or 13%; and increase in fatigue, reported by 3 out of 30 or 10%. Other symptoms, reported by one patient each, were a decrease in appetite, poor sleep, weak legs, flu-like symptoms, and an increase in anxiety and depression.

"For those who experienced improvement, the time to self-reported improvement on the protocol was an average of 5.6 weeks, with a range from immediate improvement (which was rare) to as long as 8 weeks before improvement was experienced."

I hope this helps. Here is the most recent version of the simplified protocol:


March 30. 2011

SIMPLIFIED TREATMENT APPROACH
FOR LIFTING THE PARTIAL METHYLATION CYCLE BLOCK
IN CHRONIC FATIGUE SYNDROMEMarch 30, 2011 Revision
Rich Van Konynenburg. Ph.D.
(Based on the full treatment program
developed by Amy Yasko, Ph.D., N.D.
which is used primarily in treating autism [1])

SUPPLEMENTS

1. General Vitamin Neurological Health Formula [2]: Start with tablet and increase dosage as tolerated to 2 tablets daily
2. Hydroxy B12 Mega Drops [3]: 2 drops under the tongue daily
3. MethylMate B [4]: 3 drops under the tongue daily
4. Folinic acid [5]: capsule daily
5. Phosphatidyl Serine Complex [6]: 1 softgel capsule daily (or lecithin, see below)

All these supplements can be obtained from http://www.holisticheal.com.
The fourth supplement comes in capsules that contain 800 mcg. It will be necessary to open the capsules, dump the powder onto a flat surface, and separate it into quarters using a knife to obtain the daily dose. The powder can be taken orally with water, with or without food.
These supplements can make some patients sleepy, so in those cases they take them at bedtime. In general, they can be taken at any time of day, with or without food.
Phosphatidyl serine can lower cortisol levels. Patients who already have low evening cortisol levels may wish to substitute lecithin [7] (at one softgel daily) for supplement number 5 above. Lecithin is also available from http://www.holisticheal.com.
For those allergic to soy, lecithin from other sources is available.
GO SLOWLY. As the methylation cycle block is lifted, toxins are mobilized and processed by the body, and this can lead to an exacerbation of symptoms. IF THIS HAPPENS, try smaller doses, every other day. SLOWLY work up to the full dosages.
Although this treatment approach consists only of nonprescription nutritional supplements, a few patients have reported adverse effects while on it. Therefore, it is necessary that patients be supervised by physicians while receiving this treatment.

[1] Yasko, Amy, Autism, Pathways to Recovery, Neurological Research Institute, 2009, available from http://www.holisticheal.com or Amazon.
[2] General Vitamin Neurological Health Formula is formulated and supplied by Holistic Health Consultants LLC.
[3] Hydroxy B12 Mega Drops is a liquid form of hydroxocobalamin (B12), supplied by Holistic Health Consultants. 2 drops is a dosage of 2,000 mcg.
[4] MethylMate B is a liquid form of (6s)-methyltetrahydrofolate supplied by Holistic Health Consultants, based on Extrafolate S, a trademark of Gnosis S.P.A. 3 drops is a dosage of 210 mcg.
[5] Folinic acid is 5-formyltetrahydrofolate. capsule is a dosage of 200 mcg.
[5] Phosphatidyl Serine Complex is a product of Vitamin Discount Center. 1 softgel is a dosage of 500 mg.
[7] Lecithin is a combination of phospholipids without phosphatidylserine. One softgel is a dosage of 1,200 mg.


Best regards,

Rich

 

[redo]

Yes, that helped a lot. Thank you. I am hopeful about giving this treatment a try again, I think it makes good sense.

When this is described as the simplified treatment approach, are there are wider range of supplements for another treatment approach? I've got some B12 syringes from last time (1mg/1ml cyanocobalamin) and Sterop, which will expire later this year, but are fine for now. And I've also got almost a hundred 400gram SAM-e tablets (Doctor's best brand) which expires later this year.

 

[richvank]

Yes, that helped a lot. Thank you. I am hopeful about giving this treatment a try again, I think it makes good sense.

When this is described as the simplified treatment approach, are there are wider range of supplements for another treatment approach? I've got some B12 syringes from last time (1mg/1ml cyanocobalamin) and Sterop, which will expire later this year, but are fine for now. And I've also got almost a hundred 400gram SAM-e tablets (Doctor's best brand) which expires later this year.

Hi, redo.

The reason I called it the "simplified" approach is that it was derived from the full treatment program of Dr. Amy Yasko, used primarily for cases of autism. This full program is much more complex and expensive, and is based on genomic testing and tailoring to the individual person. I don't favor using high dosages of cyano B12 because of the cyanide, though there is a little in the Yasko multi that I have used. I don't know what Sterop is. Some people do well with SAMe, while others do not. I had it in the first version of the simplified protocol in 2007, but took it out because several people couldn't tolerate it. The rest of the protocol will bring SAMe up, so it isn't really necessary, and I think it causes too much sulfite for some people, especially if they are low in molybdenum, which supports the sulfite oxidase reaction. High sulfite can produce headaches, breathing problems, rashes, and further lowering of glutathione.

Best regards,

Rich

 

[redo]

Ohh, now I see. Beginning on the simplified would seem like the logical thing to do than. Sterop is some high dosed Belgian hydroxocobalamine shots. They come in ampullas of 10 mg per 2 ml. It's used by KDM on CFS patients, but I don't know what the other uses for it is.

 

[richvank]

Ohh, now I see. Beginning on the simplified would seem like the logical thing to do than. Sterop is some high dosed Belgian hydroxocobalamine shots. They come in ampullas of 10 mg per 2 ml. It's used by KDM on CFS patients, but I don't know what the other uses for it is.

Hi, redo.

O.K. I knew that KDM gives high-dose injections of hydroxocobalamin. Dr. Cheney does this also, and I think that Dr. Cindy Bateman does as well, since she mentioned it in her talk at the "State of the Knowledge" NIH workshop. I have personally tried to encourage all of them to add active forms of folate to their protocol, because the combination of active folate and high-dose B12 is what is needed to get the methylation cycle working better B12 alone will not do it. But for whatever reasons, I have not been successful, as far as I know. I think there will have to be a statistically-significant, randomized, double-blind, placebo-controlled trial published in a peer-reviewed journal before some of the physicians will try it. This gives them protection from licensing boards and malpractice suits. If I were them, I might have to do the same.

Best regards,

Rich

 

[redo]

Thanks!

This is really OT, but about the discussion of endogenous/exogenous (post 2), there's a reason (or several reasons) why I think that if there indeed is a virus, it's endogenous. The most important reason is the various ways ME is triggered (I wrote some about that here). I actually emailed Lipkin, and elaborated about this, and although he didn't mail me back, in the message he released some weeks later he specified they will "assess results obtained in individual laboratories for consistency and evidence for or against an association between retroviral signal and disease. I use the term signal because any finding related to a retrovirus, whether infectious or noninfectious, genetic material, protein, or antibody, may provide insights (...)".

Also, there's the issue of if it is a exogenous RV which is spreading, it's strange that it's only rampant in the West. If it's spread with normal behavior, ME should be more prevalent also in third world countries than it is now.

The most fascinating research on endogenous retroviruses which is done to date, is by Perron et al., on multiple sclerosis. I would really recommend this article in Discover Magazine on the topic. The link between human endogenous retrovirus (HERV, a gammaretrovirus) and MS is strong, but one thing is seeing it's prevalent, another thing is causation. He has taken the virus from MS patients and injected it to mice, the mice became clumsy and died (of course, no proof of it being pathogenic - but an indication), he than injected it to mice, but gave them a monoclonal antibody specifically designed to stop HERV, and they lived. He is now beginning phase I studies of giving this monoclonal antibody to MS patients, you can read about that here.

But, what I am very glad to see, is that the methylation problems theory and the human endogenous retrovirus theory seems to be very compatible. I think the whole methylation thing really makes more and more sense the more I study it. I have not come long on that yet, but I plan to dig deep, and hopefully get more answers from you. All the time and effort you have put in for the benefit of all, means a lot, to a lot of people.

 

[aquariusgirl]

Sorry if this is off topic..for this thread, but I just saw Rich's post & I wanted to say it seems like hydroxy B12 with Nexavir has some good effects for me..This is the combo that Kenny de Meirleir suggests. Early days but I will keep you posted. It seems to have a good effect on the brain.

 

[redo]

Thanks for the list. I am thinking about buying supplies for three months. There are two I am not sure about. Hydroxy B12 Mega Drops and MethylMate B Drops, both in 15mL bottles. Do you know how many of those would I need for them to last three months?

 

[richvank]

Sorry if this is off topic..for this thread, but I just saw Rich's post & I wanted to say it seems like hydroxy B12 with Nexavir has some good effects for me..This is the combo that Kenny de Meirleir suggests. Early days but I will keep you posted. It seems to have a good effect on the brain.

Hi, AQG.

That's good to hear. Thanks for the information.

Rich

 

[richvank]

Thanks for the list. I am thinking about buying supplies for three months. There are two I am not sure about. Hydroxy B12 Mega Drops and MethylMate B Drops, both in 15mL bottles. Do you know how many of those would I need for them to last three months?

Hi, redo.

Sorry, I don't. Maybe someone who has experience using them will comment on this.

Best regards,

Rich

 

[Freddd]

Thanks for the list. I am thinking about buying supplies for three months. There are two I am not sure about. Hydroxy B12 Mega Drops and MethylMate B Drops, both in 15mL bottles. Do you know how many of those would I need for them to last three months?

Hi Redo,

As far as b12 goes, nobody has so far been able to document extent of absorbtion. The trials I ran with sublingual tablets, which get wet and release b12 in seconds, is that 45-120 minutes produce typically 15-25% absorbtion with the amount absorbed proportional time in tissue contact. Methylmate B could be a disaster for some people containing glutathione. Part of the problem is that we have no idea what percentage or identfying characteristyics of those who crash from glutathione except that all the people who crashed worst were also all sucessful, 100%, on the active b12 protocol. Others who started on the active protocol had zero sucess until stopping the glutathione.

 

[redo]

Hi Redo,

As far as b12 goes, nobody has so far been able to document extent of absorbtion. The trials I ran with sublingual tablets, which get wet and release b12 in seconds, is that 45-120 minutes produce typically 15-25% absorbtion with the amount absorbed proportional time in tissue contact. Methylmate B could be a disaster for some people containing glutathione. Part of the problem is that we have no idea what percentage or identfying characteristyics of those who crash from glutathione except that all the people who crashed worst were also all sucessful, 100%, on the active b12 protocol. Others who started on the active protocol had zero sucess until stopping the glutathione.

Hi Freddd,

Thank you for your input. I am not sure if I am following you. Are you saying that Methyl mate B contains glutathione? And that some people crash when they are given glutathione? What's the active B12 protocol?

 

[Freddd]

Hi Freddd,

Thank you for your input. I am not sure if I am following you. Are you saying that Methyl mate B contains glutathione? And that some people crash when they are given glutathione? What's the active B12 protocol?

Hi Redo,

I looked at the ingrediants listed for it and it contains glutathione. When one take glutathione, where directly or as precursors, it has the same effects. One of most immediate effects that is purely chemistry is that glutathione combines with various forms of b12 in the body and makes them into glutathionylcobalamin and almost instantly flushing them from the body. When I took it as a trial my urine tuyrned red, Normally it is slightkly tinted by the amount of mb12/adb12 I take. WIthin hours it turned as red as if I were taking 180mg/day instead of 30mg. That as followed by an incredibly severe folate deficiency that worse day by day causing what is most commonly called "glutathione detox reaction" or "NAC detox reaction". NAC is a precursor of gltutathione. After a couple of weeks b12 defieincy symptoms were building up to. Every one of the other 9 people who did the trail, all successful on the active b12 protocol, had a similar response.


What's the active B12 protocol?

http://forums.phoenixrising.me/showthread.php?11522-Active-B12-Protocol-Basics

 

[redo]

Hi Redo,

I looked at the ingrediants listed for it and it contains glutathione. When one take glutathione, where directly or as precursors, it has the same effects. One of most immediate effects that is purely chemistry is that glutathione combines with various forms of b12 in the body and makes them into glutathionylcobalamin and almost instantly flushing them from the body.

I've heard several stories about people taking glutathione IV, and whom got great and swift effects which have lasted for some days, what's your thoughts on that? If glutathione binds to B12, why not just take more B12?

[...] That as followed by an incredibly severe folate deficiency that worse day by day causing what is most commonly called "glutathione detox reaction" or "NAC detox reaction". NAC is a precursor of gltutathione. After a couple of weeks b12 defieincy symptoms were building up to. Every one of the other 9 people who did the trail, all successful on the active b12 protocol, had a similar response.

In this context, what's successful?

 

[richvank]

Hi, redo.

Normally, glutathione is beneficial in the B12 metabolism, because it assists the CblC complementation group to remove the beta ligand (methyl-, adenosyl-, or cyano-) from forms of B12 that come into the cells, and it also protects B12 from reacting with toxins by forming glutathionylcobalamin as a buffer form of B12. Then the cells are able to reform methyl- and adenosyl-B12 in the amounts they need. In Freddd's case, and apparently in several other cases, this does not work properly, apparently because of a polymorphism in the MMACHC gene, which codes for the CblC complementation group. In his case, glutathione traps the B12, and his cells are not able to use it. We don't yet know what fraction of the ME/CFS population has this genetic issue. I am hopeful that we will be able to identify the polymorphism or polymorphisms involved, and then perhaps using the 23andme.com panel, we will be able to predict which people will have this problem.

Best regards,

Rich

 

[Freddd]

Hi, redo.

Normally, glutathione is beneficial in the B12 metabolism, because it assists the CblC complementation group to remove the beta ligand (methyl-, adenosyl-, or cyano-) from forms of B12 that come into the cells, and it also protects B12 from reacting with toxins by forming glutathionylcobalamin as a buffer form of B12. Then the cells are able to reform methyl- and adenosyl-B12 in the amounts they need. In Freddd's case, and apparently in several other cases, this does not work properly, apparently because of a polymorphism in the MMACHC gene, which codes for the CblC complementation group. In his case, glutathione traps the B12, and his cells are not able to use it. We don't yet know what fraction of the ME/CFS population has this genetic issue. I am hopeful that we will be able to identify the polymorphism or polymorphisms involved, and then perhaps using the 23andme.com panel, we will be able to predict which people will have this problem.

Best regards,

Rich

Hi Rich,

I think you place far too much weight on my possible genetic processing of cyanocbl and hydroxcbl. Glutathione causes the immediate flushing form the body of massive amounts of b12 and that has noting to do with genetics. Maybe it leaves 1% of the b12 in the body and that is more useless to me than to others, but the immenzse flushing of b12 is simple chemistry. That can be deonstrated by anybody who wants to do so very easily.

 

[redo]

More on methylation and endogenous retroviruses.

CpG Methylation Directly Regulates Transcriptional Activity of the Human Endogenous Retrovirus Family HERV-K(HML-2)
A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) transcription remains poorly understood. We investigated in detail the role of CpG methylation on the transcriptional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-K(HML-2) proviral 5? LTRs are methylated at different levels in the cell line Tera-1. Methylation levels correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5? LTR reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5? LTR activity, as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in evolutionary old HERV-K(HML-2) 5? LTRs suggests that CpG methylation had already silenced HERV-K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG methylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC538560/

DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas.
Since DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome, hypomethylation in human tumours may lead to their reactivation. The methylation status of LINE-1 retroposons was determined in 73 samples of urinary bladder cancers, 34 specimens of renal cell carcinoma and in the corresponding normal tissues by Southern blot analysis. LINE-1 sequences were strongly methylated in normal tissues and were significantly hypomethylated in 69 (95%) urothelial carcinomas, but in none of the renal carcinomas. Hypomethylation in bladder cancers was independent of stage and tended to increase with grade. The methylation status of HERV-K proviral DNA in normal and transformed urothelial cells paralleled that of LINE-1 sequences (r2 = 0.87). It was shown by ligation-mediated polymerase chain reaction that hypomethylation also extended to the LINE-1 promoter sequence located at the 5'-ends of full-length elements which is repressed by methylation in somatic tissues. Accordingly, full-length LINE-1 transcripts were detected by Northern blot analysis in two urothelial carcinoma cell lines. In contrast, transcripts from HERV-K proviruses were restricted to teratocarcinoma cell lines. Our data indicate that genome-wide DNA hypomethylation is an early change in urothelial carcinoma, but is absent from renal cell carcinoma. The coordinate changes of LINE-1 and HERV-K DNA methylation suggest that hypomethylation in urothelial cancer affects a variety of different retroelements to similar extents. We speculate that decreased methylation of LINE-1 retroelements, in particular, may contribute to genomic instability in specific human tumours such as urothelial carcinoma by rendering these normally repressed sequences competent for transcription and recombination.

http://www.ncbi.nlm.nih.gov/pubmed/10424731