Ponderings and speculations about purinergic signaling, in pursuit of a unified ME/CFS theory

dreampop

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Genuinely wondering, what actually is the concrete evidence of CDR in CFS/ME? In Naviaux's paper he calls CFS the opposite of CDR ("chronic CDR"), so we probably shouldn't even call it that. However, the specifics of that were not all replicated - for example, the shingolipids, which were a major abnormality in that study. There are many aspects of cellular danger signaling, but I've not seen any evidence that are elevated in CFS (DAMPS, NLRP3). I'm sure some are intracellular and some are local to the just outside the cell. But if you look on his original paper of metabolic features of CDR, the metabolic features of CFS are not the opposite of summer metabolism. Perhaps most importantly, it shows ATP being more intracellular.

And even more strangely, he recommends suramin for CDR, but also CFS, when he says they are opposites. I think his suggestion is that, "a postexposure adaptation or mitocellular hormesis" (whatever that is) gears the cells to the opposite of CDR response through the same CDR pathways, esp purinergic. Buy you can't just make every pathway do the opposite.Well, I really assume you can't. How could an entire series of very different pathways be downregulated when they are encoded by different genes.

@Murph You could read Naviaux original paper on CDR, it talks about mTOR being the summer fuel sensor and AMPK being the winter fuel sensor. I believe per his talk at the OMF summit, he believes we were in winter metabolism.

Maybe I've hopelessly misunderstood everything.
 
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nandixon

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@necessary8 gave an explanation on the first page in his replies.
Are you meaning the concentration gradient idea of applying enough extracellular ATP to have it flow into the cell? I'm afraid that's not workable because before you reach a high enough extracellular concentration of ATP to do that you reach cytotoxic signaling levels of ATP that destroy the cell.

This is actually described in the first reference (2015) that I cited above, which is titled:

ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor

(It would be somewhat interesting if they did inadvertently use such a large amount of ATP because that would mean the normalization that occurred with the nano-needle would be due to the apoptosis of some number or subset of cells.)
 

anni66

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Thank you Murph, it's amazing, the female control is so low, almost as low as ME Males!!!
Could it be an explanation about the higher prevalence of the disease in women??
Why would you want lots of AMP - would this not only emphasis a dysbiosis in energy metabolism. The preferred route for recycling is ATP-ADP-ATP ( ie not AMP) - lots of AMP would suggest that there is a lack of balance ( too much ADP- ie ATP is not being recycled quickly enough) AMP is not easily converted ( it's suggested most is lost in urine- which in essence reduces ATP)- I don't know what the energetics would be for this process
 

dreampop

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Are you meaning the concentration gradient idea of applying enough extracellular ATP to have it flow into the cell? I'm afraid that's not workable because before you reach a high enough extracellular concentration of ATP to do that you reach cytotoxic signaling levels of ATP that destroy the cell.

This is actually described in the first reference (2015) that I cited above, which is titled:

ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor

(It would be somewhat interesting if they did inadvertently use such a large amount of ATP because that would mean the normalization that occurred with the nano-needle would be due to the apoptosis of some number or subset of cells.)
That would be interesting and probably good for Ron to know for future experiments. @Janet Dafoe (Rose49)

If it didn't destroy the cell and normalized impedance and suramin also normalized impedance, that's a real enigma. Perhaps an unknown ligand interacts with P2X receptors? It seems unlikely.
 
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A really long time ago, back in 2004, a patient with lymphatic cancer got better from mecfs after being given methotrexate for -bcell depletion by two Norwegian oncologists. That inspired them to look into Rituximab as a better treatment.

Interestingly, in another autoimmune condition, rheumatoid arthritis, response to Methotrexate can be determined by expression of cd-39 on t reg cells.


Red dots (R-MTX) are the responders to methotrexate, blue dots (U-MTX) is non responders


Screen Shot 2017-11-09 at 7.11.12 PM.png

The more I think about the shortage of adenosine relative to ATP the more I think @necessary8's theory of a problem with cd39 is a good one.

I wonder if expression of cd39 might help predict response to Rituximab? Hopefully it is something Fluge and Mella measured in their patients before administering Rituximab in Phase III.
 

nandixon

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The more I think about the shortage of adenosine relative to ATP the more I think @necessary8's theory of a problem with cd39 is a good one.

I wonder if expression of cd39 might help predict response to Rituximab?...
It's interesting because I think that Fluge & Mella have noted that severe ME/CFS patients may be less likely to respond to rituximab.(?) If so, then just off the top of my head, looking at the CD39 (ENTPD1) SNP rs10748643, which I previously mentioned here, might be useful to determine who rituximab may be more likely to work for. (Short story is that the GG genotype makes more CD39 and so might respond to rituximab better, versus the AA genotype which might respond less well and which might be more common in severe patients - under a CD39 deficit theory.)

While I'm thinking about it, one thing needs to be clarified:

Basically, extracellular ATP is an important co-factor in almost every step of immune activation, having pro-inflammatory effects. Except for NK cells. For NK cells, the presence of extracellular ATP actually reduces their cytotoxic activity.[source] (Sound familiar?)
That source is actually saying that the reduction in natural killer cell activity seen with extracellular ATP is due to a reduction in proliferation of the NK cells and not a reduction in their function. The latter is what is seen in ME/CFS (likely due to elevated TGF-beta, I believe). The source says:

Miller et al. equally confirmed that ATP and related adenine nucleotides play a role in decreased NK cell activity [187] but in contrast to the previous studies, the authors reported that this was in fact due to an inhibition of proliferation because despite a reduced thymidine incorporation induced by extracellular adenine nucleotides, NK cells maintained their capacity to lyse K562 cells in their experiments [187].
(Emphasis added)

And indeed, it is found that CD39 expression (which reduces ATP levels) actually inhibits NK cell function:

We show functional CD39 expression on CD4(+)Foxp3(+) Tregs suppressed antitumor immunity mediated by natural killer (NK) cells in vitro and in vivo… Conclusion: CD39 expression on Treg inhibits NK activity…[Reference]
 
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ljimbo423

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just speculation, but this is connected to AMPK--someone claiming that trans-reservatrol helps activate AMPK
Chris Armstrong (CFS researcher) thinks that AMPK is already over-active in CFS. You might want to look at this 13 minute video. At 8:25 he talks about how AMPK gets turned on and creates a positive feedback loop.

 
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Why would you want lots of AMP - would this not only emphasis a dysbiosis in energy metabolism. The preferred route for recycling is ATP-ADP-ATP ( ie not AMP) - lots of AMP would suggest that there is a lack of balance ( too much ADP- ie ATP is not being recycled quickly enough) AMP is not easily converted ( it's suggested most is lost in urine- which in essence reduces ATP)- I don't know what the energetics would be for this process
Outside the cell, you probably want a normal amount of AMP relative to ATP, because it would be a sign that any extracellular ATP is being broken down. If eATP is not being broken down then it might perpetuate its danger signal.

This is the main finding in the Naviaux data that can support his idea of purinergic signalling imo. The eATP seems to be stuck as ATP, not being dephosphorylated to AMP and the immuno-suppressor adenosine.

Naviaux did not find higher levels of eATP in patients. But if it's ratios of ATP: adenosine outside the cell that matter*, that might cause more ATP to be pumped out of cells. (The ratios might be sensed if the adenosine sensing p1 receptors are being activated less than the p2y receptors)

This would be the vicious purinergic signalling cycle Naviaux proposes and would also cause a likely intracellular shortfall of ATP causing metabolic disturbances including nonstop AMPK activation...

*I haven't seen evidence that the ratios do matter rather than the absolute levels - altho it seems plausble.


I feel obliged to say again that I know I'm not an expert in this! I've been doing a lot of reading and trying to get a grip - and I've found the process very satisfying - but I know I'm still miles from catching up. Please anybody correct me where I'm going wrong. :)
 

nandixon

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To see how consistent @necessary8 ‘s CD39 deficit hypothesis might be for ME/CFS, it's interesting to look at some of the research where mice have been studied that do not express CD39 at all, i.e., CD39/ENTPD1-null nice. One such study is:

Deletion of Cd39/Entpd1 Results in Hepatic Insulin Resistance

Outwardly, these mice appear perfectly healthy, which I thought was sadly funny given most of our experiences with the perception of this disease.

Metabolically, though, the CD39-null mice have some changes. One of the more interesting is that they produce more leptin than normal mice. This might be consistent with Jarred Younger’s work which showed that fatigue severity in ME/CFS was significantly correlated with leptin levels.

Another change is that they produce more pro-inflammatory cytokines. This might be consistent with Jose Montoya’s recent study which showed that the level of such cytokines correlated with severity of illness in ME/CFS.

These null mice also have perfectly normal blood sugar levels but yet have higher insulin levels than normal. I'd not looked at insulin before but just happened to come across a 1997 study that showed higher insulin in a group of ME/CFS patients. I mention it only because our friend Simon Wesley was a co-author. I can just hear him telling the mice that it's all in their heads...

On a more serious note, these mice are highly predisposed to liver cancer, which isn't seen at any higher rate in ME/CFS, but then these mice aren't producing any CD39 at all. (Reference)
 
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Hip

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In his theory, @necessary8 points out that in order to prevent a runaway activation of the immune response from ATP activating P2X, which pumps out even more ATP, there is a counter-balancing negative feedback loop which mops up the extracellular ATP.

This moping up of extracellular ATP is performed by CD39, a molecule which along with CD73 is found in especially high amounts on T-regs.

The hypothesis that @necessary8 posits is that CD39 function may be impaired in ME/CFS, leading to unregulated immune activation and inflammation from extracellular ATP. Three possible ways that CD39 may become impaired are suggested:

(1) Not enough T-regs that express CD39 and CD73 are being produced.

(2) The CD39 T-regs are not being activated (in mice, CD39 is only switched on when the T-reg cell is activated).

(3) Inhibition of CD39 activity, which might be caused by an anti-CD39 autoantibody.



Thus the theory would suggest that stimulating the production and activity of T-regs might benefit ME/CFS. Here are some supplements and drugs that do just that:

Selenium enhances the activity of T-regs. 1 (I got major improvements from high dose selenium)

Andrographolide, the active compound from Andrographis paniculata, can induce active T-regs. 1

Grape seed extract increases the T-regs to Th17 cell ratio. 1

Cordyceps sinensis increases T-regs to Th17 cell ratio. 1

Fucoxanthin (found in brown seaweed) induces T-regs. 1

IGF-1 stimulates T-regs. 1

Vitamin D boosts T-regs. 1

The cytokine IL-2 stimulates the development and function of T-regs. 1
> Inosine (or at least Imunovir) increases IL-2. 1
> Oxymatrine increases IL-2. 1

Rituximab seems to boost T-regs. 1

High TGF beta-1 destroys T-regs, according to Dr Ritchie Shoemaker. 1 Losartan lowers TGF beta-1 (a drug used by Shoemaker).
 
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dreampop

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In his theory, @necessary8 points out that in order to prevent a runaway activation of the immune response from ATP activating P2X, which pumps out even more ATP, there is a counter-balancing negative feedback loop which mops up the extracellular ATP.

This moping up of extracellular ATP is performed by CD39, a molecule which along with CD73 is found in especially high amounts on T-regs.
@Hip, there are a few more which may be interesting to share, although, at @necessary8 's request I was trying to avoid taking about ways to increase CD39 or Tregs.

However, one is interesting in that it is also related to the theory and what @Murph has been pulling out of his bag of tricks - it's extracellular adenosine.

  1. First, Tregs express, highly, CD39 & CD73 and are capable of producing extracellular adenosine local to the Treg
  2. Inhibiting CD39 or CD73 inhibits Treg function suggesting extracellular adenosine is critical to Treg functioning
  3. Tregs are less efficient knocked out for A2A (adenosine receptor) or with an A2A antagonist.
  4. These results indicate that adenosine produced from Tregs executes immunosuppression by triggering A2AR-dependent inhibition of effector cell activation. This mechanism is functional in both mice and human.
  5. Furthermore, adenosine may suppress antigen-specific activation of T cells by interfering with the migration of T cell and APCs in the draining lymph node (110).
"Thus, Tregs not only utilize adenosine as one of their immunosuppressive mechanisms, but also receive positive regulation from adenosine to enhance the number and immunosuppressive activity of Tregs"

So, now imagine this in the context of one of OP's theory, a CD39 antibody. It's been mentioned before the concept of an antibody that begets it's own production. A CD39 antibody (or block, or deficit, or genetic defect) would reduce the function of one of the body's main ways of curtailing auto-immunity. NK cells can also be activated by the A2A receptor.

That paper has a really interesting discussion of TGF-B, IL-10 (basically the only cytokines found high in CFS)* and Il-6, and t-cell differentiation that I want to read more about later because I'm gassed at the moment.

*Correct me if I'm wrong on this.
 
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If this illness in some part purinergic mediated, would they inflammasone be actived? It is in Fibromyalgia (1) but I could find no studies of it in ME/CFS. Many of the mediators are intracellular, but il-1b and il-18 would be detectable maybe.
In my theory, no. The thing about ATP-mediated IL-1b and IL-18 secretion, is that it's dependent on macrophage polarization, which is modulated by TLR activation and cytokines. If you activate a macrophage with eATP only, without actual pathogens present, it can secrete IL-1b and IL-18 with just that, but that secretion will stop shortly, as the macrophage shifts towards M2 polarization.[source]

That source is actually saying that the reduction in natural killer cell activity seen with extracellular ATP is due to a reduction in proliferation of the NK cells and not a reduction in their function. The latter is what is seen in ME/CFS (likely due to elevated TGF-beta, I believe). The source says:
No. The review says that there is solid evidence for eATP decreasing NK cell activity, but the data is not conclusive whether this is due to their reduced cytotoxycity, or reduced proliferation. They cite 3 sources for the former, and 1 source for the latter. The fragment you quoted is out of context, and kinda misleading.

@dreampop, you're correct that the production of adenosine by CD39 also contributes to its immunosuppresive capabilities. I actually mentioned it in my initial post:

It was shown in many studies to downregulate purinergic signaling, first by reducing eATP concentrations, and second, along with 5'-NT (CD73), it produces adenosine, activating some of the P1 receptors, which have anti-inflammatory functions in many immune cells.[source]
A2A receptors are actually a type of P1 receptor (yes, this naming convention is kinda confusing)

The paper you've found looks interesting, I will read it properly when I have more time and energy.

*Correct me if I'm wrong on this.
TGF-b was high, IL-10 wasn't. At least in the big Montoya/Mark Davis study.

Thus the theory would suggest that stimulating the production and activity of T-regs might benefit ME/CFS.
Thats only in the case of the first two possibilities. In the case of the third one, it's kinda fifty-fifty, whether it would help, or disregulate the immune system even more. This all depends on a loooot of factors. All of the possible treatments you mentioned also do a bunch of other stuff in the body, and while I don't necessairly discourage trying them, their successes or failures wouldn't realistically serve to validate or invalidate my theories. In this regard, direct antagonism of purinoreceptors seems like the best approach.

And regarding the whole nanoneedle ATP experiments, and also this:

Are there any questions / simple experiments we can collate from this discussion to submit to Ron Davis and Robert Naviaux for consideration?
...I want to hopefully discuss all that with Ron myself, and I'll message Janet about it shortly, it's just that before I do I want to post Part 2, because it's almost finished, and because I think it's even better and more relevant to ME/CFS than Part 1. Obviously I'm also reading this whole thread, and I'm open to suggestions about possible experiments that I maybe haven't thought of. Part 2 will also list my own ideas for those in detail, so you can all read them and comment what you think. But yeah, I would really appreciate if you guys just wait a few days and let me contact Ron about all this myself, once I have the Part 2 ready.
 
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nandixon

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No. The review says that there is solid evidence for eATP decreasing NK cell activity, but the data is not conclusive whether this is due to their reduced cytotoxycity, or reduced proliferation. They cite 3 sources for the former, and 1 source for the latter. The fragment you quoted is out of context, and kinda misleading.
@necessary8, That's not correct. Note that those 3 references are older references (1984, 1995 & 1992, respectively). Note that I also cited an even newer (2010) reference in addition to the Miller (1999) one that the review missed that clearly shows that CD39 expression (i.e., reduced extracellular ATP) inhibits NK cell function. (It's at the very end of my post so you might have missed it.)

This seeming anomaly isn't problematic for your hypothesis, because elevated TGF-beta (perhaps arising from a T cell activation process not kept in check by CD39) would likely be the cause of the reduced NK cell function in ME/CFS.
 

nandixon

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The other day I checked Naviaux' data for ATP and adenosine. ATP was normal but adenosine was low. Today I went back to have a look at AMP, which is the intermediate state between ATP and adenosine. My thinking was that maybe looking at it could tell us a bit more about exactly where any failure to breakdown eATP is occurring.
@Murph, Naviaux says in one of his mouse model autism studies that:

Testing the hypothesis that purinergic signaling is chronically increased in the MIA model of ASD cannot be achieved by measuring tissue or plasma concentrations of nucleotides like ATP and ADP. The relevant concentration of nucleotides is confined to a thin shell, or pericellular halo, that defines the unstirred water layer (UWL) around the effector cells where receptors and their ligands meet. Concentrations of metabolites in the UWL can be 1000-fold higher than in plasma or interstitial fluid [45]. Hence, we selected purinergic receptor downregulation as a surrogate for chronic hyperpurinergia. [Reference]

This same localized concentration effect problem is going to apply in trying to use the plasma data from Naviaux’ Metabolic features of chronic fatigue syndrome paper as well.
 
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@necessary8, That's not correct. Note that those 3 references are older references (1984, 1995 & 1992, respectively). Note that I also cited an even newer (2010) reference in addition to the Miller (1999) one that the review missed that clearly shows that CD39 expression (i.e., reduced extracellular ATP) inhibits NK cell function. (It's at the very end of my post so you might have missed it.)

This seeming anomaly isn't problematic for your hypothesis, because elevated TGF-beta (perhaps arising from a T cell activation process not kept in check by CD39) would likely be the cause of the reduced NK cell function in ME/CFS.
A study being old does increase the probability of it being incorrect, but does not guarantee it. To be honest, to settle this matter, we'd have to carefully compare the methodologies of all those studies and look for errors. I don't think it really matters that much, so I'm not gonna bother, and I'm just gonna stick to data not being conclusive, as that review did.

@Murph, Naviaux says in one of his mouse model autism studies that: [...]
This same localized concentration effect problem is going to apply in trying to use the plasma data from Naviaux’ Metabolic features of chronic fatigue syndrome paper as well.
Yes. This. Thanks for finding the exact quote.