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

Jesse2233

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Fascinating @necessary8! Really enjoying reading your thought process, it’s great how many connections between existing studies and symptoms you’re making, and from my laymen’s standpoint it all sounds good. I also appreciate your ambivalence, that’s how good hypotheses should be framed. Thanks for compiling this and hopefully your recovery doesn’t take too long

This is another thing I really wish Dr Light has tested in his study - different ratios those metabolites
Would be great to get Light’s thoughts on all this (in addition to Davis and Naviaux). Maybe he has unpublished data he could share, or could quickly test some of the metabolite combos you’re interested in. I know he’s open to correspondence as he’s answered questions on mtDNA I’ve asked him in the past.

So my model of PEM works like this - when an ME/CFS patient overexerts themselves, this causes a large accumulation of eATP in their muscles, inducing the feeling of fatigue as well as activating dendritic cells.
How does this play into PEM caused by cognitive or emotional exertion? Those aren’t exactly micro-injuries although they do involve stress and presumably alterations in physiology

Antibodies don’t cross the blood-brain barrier.
What about neuronal antibodies? I’m thinking of the anti-D1/D2 Abs seen in PANS and the anti-NMDR Ab seen in Autoimmune Encephalitis

Also there’s the possibility of a leaky blood brain barrier (perhaps damaged by infection or chemical exposure) as you alluded to with endothelial dysfunction

higher dissolved oxygen is the trigger for the metabolic changes, and work from there.
Going to embarrass myself by bringing up HBOT and the benefits reported by CFS patients receiving it anecdotally and in two studies (one by Meirlier, one by Turkish researchers). HBOT drastically increases the amount of dissolved oxygen in plasma, and under Naviaux’s model wouldn’t that make the dauer state much worse? FWIW I emailed Naviaux on HBOT but did not hear back

No rush in responding, and again great work!
 

debored13

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Back when Naviaux did his paper on the metabolic features of chronic fatigue syndrome (Dauer, worms, hypometabolism, etc) he released data on all the metabolites he found, at the metabolomics workbench.

At the time I dived in and tried to make sense of it all. If I know little about cellular biology now I knew even less then, but I know how to drive Microsoft Excel so I made a few tables and a few charts.

I decided to have a look back at my graphs and to see where ATP levels were at. It is, sadly, not found to be wildly out of line between patients and controls: levels in female patients were 97% that of female controls. (For some reason he measured it in women but not men).

View attachment 24899

(Patients at left, numbered; Controls at right labelled fctrl_0XX.) The variance is low and there's no clear pattern.

However, another purine was one of the clearest and most consistent results: adenosine.

View attachment 24895


(nicotinamide and continine were further out but showed *extremely* high variance and are related to nicotine, and I suspect smoking may explain them.) As you can see here adenosine levels were within a more narrow range and quite different on average between patients and controls. I haven't done p values on this but I guess I should.

View attachment 24897

Adenosine certainly has a range of interesting roles.

View attachment 24896

I'm personally always interested in vasodilation/vasocontraction problems (because of POTS, widespread problems with alcohol, the Fluge Mella NO patent, etc.). I think endothelial cells, being bloodflow facing immune signalling cells, are a perfect suspect that might decide to turn a local problem (lack of appropriate vascular tone) into a global one: an acute systemic immune response. Which could be a great explanation for PEM.

Adenosine is also tied up in metabolism, of course.

View attachment 24898

I'm certainly interested to hear if anyone can find any strong links from Adenosine to the puringeric signalling theory!

EDIT: this paper may be interesting:
Extracellular Adenosine-Mediated Modulation of Regulatory T Cells
the niacinamide result is particularly interesting, tho do you think that would be explained by smoking as well? I wonder how much one can megadose niacinamide
 

debored13

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Ponderings and speculations about purinergic signaling, in pursuit of a unified ME/CFS theory, Part 1

Introduction

ATP, while inside the cell serving for energy transport, when outside of the cells has signaling functions. Those functions differ between cell types, but a lot of it is pro-inflammatory, where extracellular ATP serves as a danger signal, so-called Damage Associated Molecular Pattern, DAMP. This is because ATP should normally be mostly contained inside the cells, and spills outside when a cell is ruptured, either by mechanical means or by an invading pathogen, therefore making a good danger signal to start the immune response we know as inflammation.

Dr. Naviaux proposed that overactive purinergic signaling sustains many chronic diseases, including ME/CFS and autism. In the case of autism he's also proven that this seems to be the case, by causing symptom remissions with IV administration the anti-purinergic drug suramin. He thinks the same might be happening in ME/CFS, and Alan Light's research which indicates that extracellular ATP is a key ingredient in facilitating the feeling of fatigue itself, supports this hypothesis.

For me, Naviaux's theories seemed fascinating, and made a lot of sense, but many questions arose, like how do we get different diseases from a unified danger response, how does it tie in with other observations in ME/CFS, how exactly does this cause our symptoms, etcetera. Naviaux never explained his theories in detail, only gave an incredibly simplified version at the Stanford symposium. Some details can be understood reading his papers about CDR and oxidative shielding, but those prompted even more questions in me. So I did the crazy thing and decided to dive into Google Scholar trying to understand this stuff myself by reading related papers. As it currently stands, I am very far from any form of full understanding of this subject, but nevertheless, I wanted to share my findings with you all, first to save some trouble other people interested in it, second because there are people here who understand biochemistry better than me (Im gonna tag a few - @nandixon, @Hip, @JaimeS), and might help me in refining this. And third, because our amazing Ron Davis apparently likes to get ideas from the community, which is why I'm tagging @Janet Dafoe (Rose49) to pass this post to him. I imagine he heard a lot of this from Naviaux and other scientists, but maybe a thing or two will spark a novel thought. I'm sorry it's so long, but I assure you I put a lot of thought in which of my findings include here and which not.

Biochemical background for the uninitiated

Purinergic receptors, which detect the presence of ATP and other purines outside the cell, consist of P1, P2X and P2Y families. P2X receptors are the ones most specific to ATP and seem to be most well studied.

ATP release from the cells can also occur without the cell rupturing, but rather to send the danger signal on purpose. ATP can be released vesicularly, but the more prominent and well studied method seem to be pannexin and connexin hemichannels, particularly Panx1. It's basically a protein on the surface of the cell, which when activated creates a large pore, permeable to molecules up to 900Da in size, including ATP. Those have been shown to open in various cell types in response to stressful stimuli - mechanical stretching[source], hypoxia[source]. This is in line with the model of eATP serving as a danger signal.

Interestingly, in some cells, Panx1 channels can be directly opened by activation of the P2X7 receptor.[source] They kinda bundle together and activation of the P2X7 activates Panx1 as well. By this mechanism, when a cell senses large amounts of extracellular ATP, it also releases ATP to propagate the danger signal to the next set of neighboring cells.

So what does eATP do to the cells, other than make them release more ATP? Well, depends on the cell. In this Part 1, I will mostly focus on the immune system.

When a pathogen enters the body, macrophages and dendritic cells (which are in the tissues) detect them through Toll-Like Receptors, becoming activated. They then secrete Il-1β and TNF-α, major cytokines that start the inflammatory response. This secretion process is hugely dependent on co-stimulation by eATP mainly through the P2X7 receptor. eATP also serves as a signal for those cells to migrate to the infection site in the first place.[source] It also is a necessary factor for neutrophils to get there, as it potentiates the Il-8 mediated chemotaxis through the P2Y2 receptors.[source] The secretion of Il-8 from activated monocytes also requires P2Y2 and P2Y6 stimulation in them.[source]
Macrophages and dendritic cells engulf the pathogens, break them down, and present their peptides on MHC proteins to T cells, activating them if their receptors correspond to the presented peptide. T cells express P2X1, P2X4, P2X7 receptors, and Panx1. All of those, with the exception of P2X7, translocate to the immune synapse (the connection between the T cell and the antigen presenting cell), and ATP is released from the T cell, amplifying the activation signal by acting on its own P2 receptors.[source] The activated T cell produces IL-2, which acts on the T cell itself, triggering it's clonal expansion. Presence of eATP outside of the immune synapse, acting on P2X7, can also increase IL-2 secretion in activated T cells.[source] Th cells then activate B cells which produce antibodies, and cytotoxic T cells go killing infected cells in the body. B cells also express purinoreceptors, but the effects of their activation have not been studied.

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?)


The good stuff

ATP is not the only pro-inflammatory molecule, the immune system has a large number of cytokines to regulate itself as well as other Damage Associated Molecular Patterns, and Pathogen Associated Molecular Patterns. In a lot of studies assessing purinergic signaling in immune cells, they found that many immune cell types have two or more pathways for activation of their primary functions - one through purinergic receptor stimulation by ATP, and other through direct contact with a pathogen, or through cytokines. Those pathways are often needed to act in synergy to achieve a full-blown immune activation.[source] Given all this, it is possible, that a "kind of" inflammatory response is perpetuated in ME/CFS by purinergic signaling. There was an infection (or other triggers), which initiated classical immune response, which resolved the infection, and initiated anti-inflammatory immune resolution, but that resolution succeeded only partially. We might not see very elevated cytokines in ME/CFS because that kind of signaling has already been mostly resolved, while eATP pro-inflammatory signaling still persists. Now, obviously those are not fully separate, but it's very possible they're not always completely in sync either. It could be that they're only kinda-connected. This could explain the finding of Montoya's and Mark Davis's cytokine profiling, that more severe patients have higher pro-inflammatory cytokines, but are still not high enough to distinguish from controls.[source] Those severe patients would have stronger eATP-mediated inflammation, which elicits higher cytokine levels, but because that connection is weak, the cytokine levels don't quite raise above the norm.

But why doesn't this happen in healthy people? What's the difference between them and ME/CFS patients? To answer this, there is a simple approach and a complex approach.

The simple approach is to go "Wait, eATP makes cells release more ATP? This is a positive feedback loop. There must be some inhibitory mechanism of this, otherwise the whole immune system would go into runaway endless inflammation". And there is. ATP can be hydrolyzed to AMP and then to adenosine, by enzymes called nucleotidases. Nucleotidases present outside the cell, or ecto-nucleotidases, are indeed expressed by many cells which also have purinergic receptors. The main such ecto-nucleotidase, which seem to be responsible for most of eATP breakdown is NTPDase1, also called CD39.[source1][source2] 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]

The obvious hypothesis from this is that CD39 function is impaired in ME/CFS. Now, I'm unsure of this because I don't know the specifics of the test, but I'm under the impression that this, to some extent, can be tested in Ron's impedance meter. First, you can introduce recombinant CD39 into the plasma and see if it reduces the signal in ME/CFS cells. Second, you can take healthy cells (in healthy plasma) and treat them with the CD39 inhibitor, ARL 67156, and see if the impedance response starts resembling that of ME/CFS. It might be necessary to add extracellular ATP also, to mimic nearby cells secreting it, as it (presumably) happens in ME/CFS. You'd do two variants - one with just eATP, and one with eATP and ARL 67156. Another way might be to add a non-hydrolyzable ATP derivative, which would activate P2 receptors, but won't be hydrolyzed by CD39. The obvious limitation of this is that it only tests white blood cells, and what's happening in the tissues might be a different story.

So, if CD39 activity really was inhibited in ME/CFS, what could be the reason? I see three possibilities, and to talk about them, I first need to talk about Treg cells. T regulatory cells, formerly known as T suppressive cells, are responsible for control and suppression of the immune response. They are also the most confusing little shits ever.[source] To a lot of questions about them, the answer is "data is not conclusive". But we do know that they are the main source of CD39 and CD73 in the immune system, expressing them in higher amounts than other cells.In fact, breaking down ATP to adenosine with those two enzymes is thought to be one of the main ways in which Tregs suppress the immune system.[source1][source2][source3]

So the three possibilities are:

1. Not enough Tregs positive in CD39 and CD73 are being produced.
Not all Tregs express CD39, only a specific CD45RO+CCR6+ effector/memory-like subset of them, also called TREM. Interesting observations have been made in multiple sclerosis, where the overall number of Tregs was not much different from controls, but the amount of Tregs positive in CD39 was much lower in MS patients.[source] It is possible that a similar thing is happening in ME/CFS, perhaps by a different mechanism.

2. Not enough CD39+ Tregs are being activated. Tregs, like any other T cells, have a T Cell Receptor (TCR) which upon stimulation activates the cell to its primary function. It has been observed in mice, that Tregs exhibit CD39 activity only when activated.[source] It probably works similarly in humans. As for when Tregs become activated, that is where the data starts being non-conclusive[source], so I'll leave it at that.
This could also lead to a lowered number of them, as Tregs highly express the P2X7 receptor, and are very sensitive to high concentrations of eATP. Without the protection of CD39-mediated ATP hydrolysis, they easily die, or their suppresive capabilites are inhibited.[source]

3. Direct obstruction of CD39 activity.

This third option is the most interesting to me, as it ties some stuff together, but I also don't know if this is possible the way I think it is.
Let's go back for a moment to Ron's impedance assay. The plasma switch results indicated that it is something in the blood that is making the cells behave differently than healthy ones. After reading all this you might think that this something is eATP, but it is most probably not. First, I have seen no publications indicating possible endocrine ATP signaling, only short-range paracrine (to neighboring cells) and autocrine (to self). Second, the filtration results showed that this factor in the plasma is larger than 10kDa, which ATP is not.
But then you also have the result that adding suramin "greatly reduced" the sickly response. Suramin is a non-selective P2 antagonist, blocking many of the P2X and P2Y receptors, to varying degrees. So how can a similar response be achieved by blocking ATP signaling as by filtering out large stuff from the plasma?

The answer can be an anti-CD39 antibody. This antibody would be present in ME/CFS blood and would inhibit ATP breakdown, increasing the activation of P2 receptors, opening of Panx1 channels, resulting in loss of ATP from the cell, constant sort-of-inflammation and many of our symptoms. When it is filtered out, CD39 can work and hydrolyze ATP, returning the cells to normal. When P2 receptors are blocked directly, the cells also return to normal.
This would explain why B cell depletion with Rituximab helps (activated B cells produce antibodies) and why TGF-β was one of the only two elevated cytokines in Montoya's and Mark Davis's findings[source]; TGF- β is one of the other ways in which Tregs suppresses the immune system. It might be so, that the Tregs are working hard to resolve the inflammation, but cannot do it fully because CD39 is blocked by an antibody.
This is an idea that the immune system is basically attacking its own stop button. Or rather a part of it.

There are two problems with this hypothesis, and I have no idea if they can be resolved. First, ME/CFS plasma makes healthy cells behave like ME/CFS cells. In this hypothesis the factor this plasma introduces is the anti-CD39 antibody. But for this antibody to do anything at all to the cell, eATP must be already present. And the cells are healthy, from a human without an active infection. Do certain levels of ATP release occur at all times enough for the blockage of CD39 to make a difference? Or was some eATP floating around in the ME/CFS plasma? I don't know, but it seems like a stretch.

The second problem is the humoral immunity profiling ME/CFS study done by Lombardi et al.[source] They used random peptide microarrays to check all the antibodies in the blood and what peptides they bind to. The aminoacid pattern they identified as a common thing was GVALSG. I checked, it doesn't match the sequence of CD39 at all. Now, I don't know the technical limitations of that study and the possibility that it would miss a key antibody.

Recombinant (synthetic) anti-CD39 antibodies are a thing though, so maybe this can be tested in the impedance assay.

There is another similar possibility that doesn't involve CD39 at all, and that is that the P2 receptors are being activated by some other agonist, not ATP, and that agonist also is larger than 10kDa and is our mysterious plasma factor. Maybe it could be an antibody. Just in case, I compared the sequences of all seven P2X receptors to the GVALSG pattern, and the best I got was 3 aminoacids in a pattern. I don't know much about antibody binding, but I find it hard to imagine a sequence of 3 would make for any meaningful binding. As for a non-antibody protein capable of P2X agonism, the only two I've found so far (mostly by accident, as I almost haven't touched this angle yet) are biglycan[source] (43kDa) and Serum Amyloid A[source] (11-14kDa, depending on subtype). (This is a super-long stretch, and probably not related, but I thought I would also mention it - particles of the NLRP3 inflammasome, which is activated in macrophages by eATP to release IL-1β, can also be released extracellularly, and probably are also a DAMP.[source])

Another possibility that I haven't explored in detail, is that the mysterious blood factor upregulates P2 receptor expression in cells, making them more vulnerable to eATP signaling. Alan Light did find upregulated P2X4 and P2X5 expression in ME/CFS patients post-exercise.[source1][source2] This would have the same problem as the anti-CD39 hypothesis in the sense that it doesn't seem to explain healthy cells becoming sickly in ME/CFS plasma.

So what about other inhibitory mechanisms of purinergic signaling, other than ecto-nucleotidases? The only other thing that I found, were some observations that P2X7 activation can inhibit Panx1-mediated ATP release.[source] Yes, this is the reverse of what I said at the beginning. There were two studies that reported this, one on murine cells, other on human HEK293 cells. This is a cell line cultured from embryonic kidney cells, often used in research because it's easy to modify them to do what you want, so they have different properties based on how you "bake them". In this case, Panx1 was artificially introduced to those cells through transfection.

Those observations raise two possibilities. First, that depending on the cell type, P2X7 activation might open or close Panx1 channels. The cell types in which P2X7 activation has been observed as leading to Panx1 channel opening are macrophages[source], astrocytes[source], and enteric neurons[source]. (And maybe more, I just haven't found the papers about it.) The P2X7-mediated Panx1 activation seems to be widely accepted in literature, but citations about it are often not present. It is very possible that in some cells P2X7 activation opens Panx1 and in other types closes them.
The second, more interesting possibility, is that there is some other factor, which decides if P2X7 activation in a cell opens or closes Panx1 channels. A distinction on P2X7 splice variants might be it.[source] Or it might be some yet undiscovered thing, possibly our mysterious blood factor, or something tied to it biochemically. We don't really know.

On this anticlimactic note, I want to conclude Part 1.

Possible future ponderings

What about the complex approach that I mentioned earlier? The complex approach is to understand exactly how does resolution of inflammation work. How does antibody synthesis stop. When are Tregs activated and when are they suppressed. How do Panx1 channels close. How does this all tie together in a system that "knows" when pathogen extermination is over and when it's time to suppress the inflammatory response. Then we can try to find out what in that sequence might have gone wrong in ME/CFS. My thoughts on this will come in Part 2, if I don't get overwhelmed by it. Probably will take me quite a long while.

Now, in the title, I put "in pursuit of a unified ME/CFS theory". That's the goal, but I'm nowhere near it. I feel like before we can talk about even a speculatory unified theory of ME/CFS, four other areas need to be addressed in detail.

First is the connection to the gut. In the gut, weird things happen with the immune system. For example, intestinal CD8+ T cells have higher P2X7 receptor expression than CD8+ (cytotoxic) T cells found in other places of the body. This causes them to be very sensitive to eATP, in a manner similar to Tregs. I wonder what's their CD39 expression like. The digestive system also has its own variation of Tregs to not allow rampant immune reactions to the food passing through it, and to the natural intestinal bacteria. Could it be possible that reduced CD39 activity causes more pathological immune response in the gut, resulting in our digestive symptoms and destroying good bacteria? Maybe. Could it also tie back into why the CD39 activity is lower, in a feedback loop? I don't know. I need to read more.

Second is the connection to the brain. In the nervous system eATP is a neurotransmitter. It also activates microglia, which are basically the nervous system's macrophages. Jarred Younger's research points to microglia activation in ME/CFS and it could also explain some, or all, of our neurological symptoms, as well as the common dysautonomia. There could also be a feedback loop in here.

Third is the connection to exercise and PEM. The exacerbation of symptoms after exercise is a very distinct, hallmark symptom of ME/CFS and is the main thing that makes ME/CFS such a disabling condition. I have yet to see a plausible biochemical model of PEM, and I believe this is a compulsory requirement for any general theory of ME/CFS. The delayed nature of PEM and the fact that mental exercise can also cause it, can be important clues to what it is biochemically.

Fourth is the intracellular stuff. This is the hard part, because of how complex it is, as there are hundreds/thousands of proteins involved in the functioning of any cell. But if we are to tie this to PDH inhibition, glycolysis inhibition, and to the metabolomics findings, we need to understand what does purinergic signaling do inside the cell, how it impacts the cellular metabolism. The impedance assay results I think show that the signal difference is due to insufficient ATP inside the cell to pump out the salt. This is the only thing injecting ATP or pyruvate into the cell, and adding suramin to the plasma have in common - they increase the ATP in the cell. The question is - is the insuffucient ATP due to the loss through pannexin channels, or due to impaired ATP production, or both? If both, are they connected? In which direction? I have some ideas about how to answer those questions with the impedance assay, but I would have to know more about the specifics of it first, because I might have some incorrect assumptions here (Ron, if you're reading this, can I ask you a few questions about the assay?)
Theorizing about intracellular stuff is even more difficult because it will differ depending on cell type, but a common core is very possible. Dr Naviaux certainly seems to think this is the case, because his Cell Danger Response theory is exactly this. I still don't understand many things about it, like why are the metabolomics results in ME/CFS the reverse of an acute CDR. But it certainly makes sense in a lot of ways.

So, as for future continuations of this. The gut and brain connections I will probably explore in detail when I have the time and energy for it, and write part 3 and part 4 about it. The PEM aspect is more difficult to search for existing publications. The best way would be for a specialist in exercise physiology to look over all of the indicated pathways, and tell if something rings a bell as something they know as changing with physical exercise. (If any of you know good papers about cell-to-cell communication during exercise, send them my way) And the intracellular thing... .___. I don't wanna do it. That stuff is hard. But if someone else wants to try, I want to present to them the most fucking confusing paper I have yet found. It states that CD39 (which is outside the cells), makes the cells have less ATP *inside*. Yes, this is exactly opposite to everything I said here. Further, it states that Tregs have lower intracellular ATP that Th cells, and those low levels make cyclophosphamide able to selectively kill Tregs, sparing other T cells. Yes, cyclophosphamide is the drug currently being trialed in Norway (along with Rituximab) as a treatment for ME/CFS based on some case studies showing that it helps. Now, this specific confusion I can resolve, it's just dose-dependent. Tregs are more sensitive to cyclophosphamide, so it only kills them, boosting the immune response (used in cancer), but high-dose cyclo kills all T cells, suppressing the immune response (used in autoimmune disease). As for why the rest makes no sense - I have no idea. It can be an error in the methodology of the study, or the results might be correct and tie into our illness in a clever way that I'm too stupid to understand. Or it might be totally unrelated.

Implications for treatment

To close things of, I wanted to touch a bit on treatment, albeit very carefully. Please don't take my words as medical advice.

If rampant eATP signaling is at the core of ME/CFS, then suramin by blocking it could indeed by very beneficial. However. If the reason for this is impaired ATP hydrolysis through ecto-nucleodidases, there is another thing to consider. Suramin, other than its P2 antagonism, was also shown to inhibit ecto-nucleotidases.[source] The question is if this effect is less or more potent than the P2 antagonism. I would imagine less, based on how the autism trial went. But there is also the issue of adenosine not being produced and not activating the P1 anti-inflammatory receptors. Now, I'm not saying that suramin is a bad idea and we shouldn't try it in ME/CFS. My very uninformed guess is that overall the effect would still be positive. But it might not be. So I just wanted to point this out.

There's also couple other interesting things I found in regard to treatment.
Firstly, a couple of years ago, the AstraZeneca pharmaceutical company filed a patent for AZD9056, a potent, orally bioavailable, selective P2X7 inhibitor. It was to be used in rheumatoid arthritis. The drug was tolerated well, but failed to bring any symptomatic change in a phase IIb clinical trial, so it never reached the market. I have no idea if they would be interested in doing this, but what if AstraZeneca could pay for an ME/CFS clinical trial of AZD9056?
Secondly, it seems that the food dye FD&C Blue No. 1, also known as Brilliant Blue FCF, or E133, is a Panx1 inhibitor with neutral effect on P2X7.[source] I don't really know what dose would be needed for it to have any meaningful effect, and if that dose is still known to be safe, but it might be possible to calculate this if we can somehow find out its bioavailability. Just please don't go eating some dangerous amount of this food dye, okay? I'm kinda wary of sharing this last thing in an open forum, but I'm gonna trust you guys to be responsible.
Thirdly, there are some herbs known to be P2 inhibitors. @Jesse2233 beat me to the punch in making a thread about it, as he usually does. There are more than just the two he mentioned though. I might write a post about them after I look more into it.

Thanks for reading.
I think I found something relevant to a very small part of this theory.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489427/ (I believe you posted this paper later in the thread showing the connection between neuropathic pain, microglia, and purinergic signalling).
"Nerve injury often causes debilitating chronic pain, referred to as neuropathic pain, which is refractory to currently available analgesics including morphine. Many reports indicate that activated spinal microglia evoke neuropathic pain. The P2X4 receptor (P2X4R), a subtype of ionotropic ATP receptors, is upregulated in spinal microglia after nerve injury by several factors, including CC chemokine receptor CCR2, the extracellular matrix protein fibronectin in the spinal cord, interferon regulatory factor 8 (IRF8) and IRF5. Inhibition of P2X4R function suppresses neuropathic pain, indicating that microglial P2X4R play a key role in evoking neuropathic pain."

I found this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962576/ which discusses the use of Low Dose Naltrexone in pain conditions and speculates extensively on its role in reducing inflammation via microglia and TLR4.

"Anti-inflammatory effects of LDN in vivo and in vitro
In describing LDN’s clinical utility, it is important to understand the dual physiologic mechanisms of naltrexone and other opioid antagonists. Most clinicians are familiar with naltrexone as a potent and nonselective opioid receptor antagonist and treatment for opioid addiction. Naltrexone, at typical dosages, significantly blocks activity at mu- and delta-opioid receptors as well as (to a lesser extent) kappa-opioid receptors [16]. Because beta-endorphin activity at mu-opioid receptors is associated with endogenous analgesic processes, it may seem counterintuitive to administer naltrexone to individuals with chronic pain, as we might expect the medication to reduce analgesia produced by beneficial endogenous opioid activity.

Naltrexone, however, exerts its effects on humans via at least two distinct receptor mechanisms. In addition to the antagonist effect on mu-opioid and other opioid receptors, naltrexone simultaneously has an antagonist effect on non-opioid receptors (Toll-like receptor 4 or TLR4) that are found on macrophages such as microglia [17]. It is via the non-opioid antagonist path that LDN is thought to exert its anti-inflammatory effects. Microglia are central nervous system immune cells that are activated by a wide range of triggers [18]. Once activated, microglia produce inflammatory and excitatory factors that can cause sickness behaviors such as pain sensitivity, fatigue, cognitive disruption, sleep disorders, mood disorders, and general malaise [19]. When chronically activated, the resulting proinflammatory cascade may become neurotoxic, causing several deleterious effects [20]. Given the wide variety of inflammatory factors produced by activated microglia (e.g., proinflammatory cytokines, substance P, nitric oxide, and excitatory amino acids)" (guessing that glutamate may be included here?) "[21], a range of symptoms and medical outcomes could share the pathophysiological mechanism of central inflammation. Conditions such as fibromyalgia may involve chronic glial cell activation and subsequent production of proinflammatory factors. The hypothesis is indirectly and partially supported by the high degree of symptomatic overlap between fibromyalgia and cytokine-induced sickness behaviors.

Both naloxone and naltrexone have been demonstrated to exert neuroprotective and analgesic effects [22]. The neuroprotective action appears to result when microglia activation in the brain and spinal cord is inhibited [23]. By suppressing microglia activation, naloxone reduces the production of reactive oxygen species and other potentially neuroexcitatory and neurotoxic chemicals [24]. The anti-inflammatory effect of opioid antagonists may also extend to the periphery, as evidenced by suppressed TNF-alpha, IL-6, MCP-1, and other inflammatory agents in peripheral macrophages [25]. It should be noted that most animal work has used naloxone, while most human work has used naltrexone (because of its higher oral availability). We cannot discount the possibility that findings from one compound would imperfectly translate to the other.

The hypothesis that naltrexone and naloxone operate via glial cells to exert their beneficial actions is supported by work with dextro-naltrexone. Dextro-naltrexone is a stereoisomer of naltrexone which is active at microglia receptors but has no activity on opioid receptors [26]. Dextro-naltrexone possesses analgesic and neuroprotective properties [27]. Therefore, the analgesic, anti-inflammatory, and neuroprotective effects of naltrexone do not appear to be dependent on opioid receptors.

The majority of work to date has focused on naloxone/naltrexone’s action on microglia TLR4 (e.g., [28]). However, it should be mentioned that the data do not perfectly fit a TLR4 hypothesis [29], and other targets have been proposed, including astrocytes [30] and NADPH oxidase 2 [31]. Other sites of action, including the opioid growth factor receptor (OGFr) [32], are being discovered, raising even more potential mechanisms of action. Given the multiple and varied sites where naltrexone exhibits significant pharmacologic activity, it will be difficult to determine with certainty the paths that are critical for the clinically beneficial effects. This area of research is being vigorously pursued by multiple laboratories."


I'm not at the point where I can understand your whole theory on purinergic signalling but I do believe this might be relevant!!
 

debored13

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in the conclusion of the paper, under the section "Disadvantages of LDN" they state:
"Lack of proper dosage-finding experiments
It is highly probable that 4.5 mg is not the optimal dosage for all individuals with fibromyalgia, as it is rare for any pharmaceutical to have a one-size-fits-all dosage. In addition to obvious variables such as body mass index, individuals may differ in their metabolism, opioid receptor sensitivity, or microglia sensitivity to LDN. It is plausible that individuals who do not respond to 4.5 mg daily may respond to either lower or higher dosages. Other dosing schedules, such as twice a day, have not been explored in clinical studies. For now, the once daily 4.5-mg dosing schedule appears to be used without much critical analysis, as there are no published reports of even basic dose-ranging in human participants. Proper dosing studies need to be performed to determine the therapeutic range of the drug and to identify a process for determining an individual’s optimal dosage. The importance of determining proper dosing strategies is highlighted by animal research that suggests, for example, that while LDN may suppress tumors when used in the typical fashion, it may actually enhance tumor growth when administered more frequently [48].

No hard data on long-term safety
Even though naltrexone has a long history of safe use with a wide range of large dosages, we know very little about the long-term safety of the drug when used chronically in low dosages. The low dosage is often cited as a reason for clinicians and patients to not be concerned about safety. However, we must be open to the possibility that the unique clinical effects possible with the low dosage could also present new health risks. There are no reported serious concerns to date. While inhibition of immune system parameters could theoretically raise the risk of infections or cancer due to decreased immunosurveillance, there have been no reports of such a side effect at any dosage of naltrexone."


This is something I want to bring up--I feel like we'd have better data and better anecdotal evidence on LDN if people knew which dose would be effective for them when they start. There are people who feel better from LDN, and then there is a whole 'nother group which seems like they (quite understandably, mind you) quit early on due to not being able to tolerate the side effects.
I have been giving it a go recently again and while it's not a wonder drug, it is the only thing that has allowed me to even do things like read the above...
 

debored13

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I think I found something relevant to a very small part of this theory.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489427/ (I believe you posted this paper later in the thread showing the connection between neuropathic pain, microglia, and purinergic signalling).
"Nerve injury often causes debilitating chronic pain, referred to as neuropathic pain, which is refractory to currently available analgesics including morphine. Many reports indicate that activated spinal microglia evoke neuropathic pain. The P2X4 receptor (P2X4R), a subtype of ionotropic ATP receptors, is upregulated in spinal microglia after nerve injury by several factors, including CC chemokine receptor CCR2, the extracellular matrix protein fibronectin in the spinal cord, interferon regulatory factor 8 (IRF8) and IRF5. Inhibition of P2X4R function suppresses neuropathic pain, indicating that microglial P2X4R play a key role in evoking neuropathic pain."

I found this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962576/ which discusses the use of Low Dose Naltrexone in pain conditions and speculates extensively on its role in reducing inflammation via microglia and TLR4.

"Anti-inflammatory effects of LDN in vivo and in vitro
In describing LDN’s clinical utility, it is important to understand the dual physiologic mechanisms of naltrexone and other opioid antagonists. Most clinicians are familiar with naltrexone as a potent and nonselective opioid receptor antagonist and treatment for opioid addiction. Naltrexone, at typical dosages, significantly blocks activity at mu- and delta-opioid receptors as well as (to a lesser extent) kappa-opioid receptors [16]. Because beta-endorphin activity at mu-opioid receptors is associated with endogenous analgesic processes, it may seem counterintuitive to administer naltrexone to individuals with chronic pain, as we might expect the medication to reduce analgesia produced by beneficial endogenous opioid activity.

Naltrexone, however, exerts its effects on humans via at least two distinct receptor mechanisms. In addition to the antagonist effect on mu-opioid and other opioid receptors, naltrexone simultaneously has an antagonist effect on non-opioid receptors (Toll-like receptor 4 or TLR4) that are found on macrophages such as microglia [17]. It is via the non-opioid antagonist path that LDN is thought to exert its anti-inflammatory effects. Microglia are central nervous system immune cells that are activated by a wide range of triggers [18]. Once activated, microglia produce inflammatory and excitatory factors that can cause sickness behaviors such as pain sensitivity, fatigue, cognitive disruption, sleep disorders, mood disorders, and general malaise [19]. When chronically activated, the resulting proinflammatory cascade may become neurotoxic, causing several deleterious effects [20]. Given the wide variety of inflammatory factors produced by activated microglia (e.g., proinflammatory cytokines, substance P, nitric oxide, and excitatory amino acids)" (guessing that glutamate may be included here?) "[21], a range of symptoms and medical outcomes could share the pathophysiological mechanism of central inflammation. Conditions such as fibromyalgia may involve chronic glial cell activation and subsequent production of proinflammatory factors. The hypothesis is indirectly and partially supported by the high degree of symptomatic overlap between fibromyalgia and cytokine-induced sickness behaviors.

Both naloxone and naltrexone have been demonstrated to exert neuroprotective and analgesic effects [22]. The neuroprotective action appears to result when microglia activation in the brain and spinal cord is inhibited [23]. By suppressing microglia activation, naloxone reduces the production of reactive oxygen species and other potentially neuroexcitatory and neurotoxic chemicals [24]. The anti-inflammatory effect of opioid antagonists may also extend to the periphery, as evidenced by suppressed TNF-alpha, IL-6, MCP-1, and other inflammatory agents in peripheral macrophages [25]. It should be noted that most animal work has used naloxone, while most human work has used naltrexone (because of its higher oral availability). We cannot discount the possibility that findings from one compound would imperfectly translate to the other.

The hypothesis that naltrexone and naloxone operate via glial cells to exert their beneficial actions is supported by work with dextro-naltrexone. Dextro-naltrexone is a stereoisomer of naltrexone which is active at microglia receptors but has no activity on opioid receptors [26]. Dextro-naltrexone possesses analgesic and neuroprotective properties [27]. Therefore, the analgesic, anti-inflammatory, and neuroprotective effects of naltrexone do not appear to be dependent on opioid receptors.

The majority of work to date has focused on naloxone/naltrexone’s action on microglia TLR4 (e.g., [28]). However, it should be mentioned that the data do not perfectly fit a TLR4 hypothesis [29], and other targets have been proposed, including astrocytes [30] and NADPH oxidase 2 [31]. Other sites of action, including the opioid growth factor receptor (OGFr) [32], are being discovered, raising even more potential mechanisms of action. Given the multiple and varied sites where naltrexone exhibits significant pharmacologic activity, it will be difficult to determine with certainty the paths that are critical for the clinically beneficial effects. This area of research is being vigorously pursued by multiple laboratories."


I'm not at the point where I can understand your whole theory on purinergic signalling but I do believe this might be relevant!!

So one caution I'm telling myself about my enthusiasm is that this paper has a lot of disclaimers that some of it is just speculation and that it may not work via the mechanism they propose. But I do think their theory is sound and that more research on Low Dose Naltrexone is warranted. Especially because it has been proven to have qualitatively different effects at low doses than at high doses. They point out, by way of contextualizing that, that at doses 1/10 that of an analgesic dose, morphine has been shown to cause hyperalgesia.


Here's one of the disclaimers:
"An alternate explanation of LDN mechanism
While we believe much data is consistent with that claim that LDN works via novel anti-inflammatory channels, there are alternative compelling explanatory models of the LDN mechanism. The most prevalent hypothesis, advanced by Dr. Ian Zagon and colleagues, states that inducing a small and transient opioid blockade will prompt the body to compensate by upregulating both endogenous opioids and opioid receptors [40]. The opioid upregulation effect of temporary naltrexone or naloxone blockade has been demonstrated multiple times previously [41, 42]. This “opioid rebound” effect could have multiple impacts on health and quality of life, including enhanced endogenous analgesia and repression of critical immune factors [40].

Further research is needed with naltrexone and naloxone stereoisomers to determine the true mechanism of clinical action. In the meantime, we note that both the TLR4 and opioid receptor mechanisms may play a role in LDN action, as the hypotheses are not mutually exclusive."


But like they note at the end of the disclaimer, por que no los dos? I personally have had euphoria from certain doses of low dose naltrexone. I was not going for that effect and it was too much and coincided with a dose that sort of "stimmed me out". But in my mind there's almost no doubt, simply from bioassay, that LDN causes some compensatory upregulation in opioid receptors. the question is whether that's what causes the therapeutic effect, and whether it can be tested more to find more specific markers for decreased inflammation.


I highly recommend reading the full text.
 

debored13

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So one caution I'm telling myself about my enthusiasm is that this paper has a lot of disclaimers that some of it is just speculation and that it may not work via the mechanism they propose. But I do think their theory is sound and that more research on Low Dose Naltrexone is warranted. Especially because it has been proven to have qualitatively different effects at low doses than at high doses. They point out, by way of contextualizing that, that at doses 1/10 that of an analgesic dose, morphine has been shown to cause hyperalgesia.


Here's one of the disclaimers:
"An alternate explanation of LDN mechanism
While we believe much data is consistent with that claim that LDN works via novel anti-inflammatory channels, there are alternative compelling explanatory models of the LDN mechanism. The most prevalent hypothesis, advanced by Dr. Ian Zagon and colleagues, states that inducing a small and transient opioid blockade will prompt the body to compensate by upregulating both endogenous opioids and opioid receptors [40]. The opioid upregulation effect of temporary naltrexone or naloxone blockade has been demonstrated multiple times previously [41, 42]. This “opioid rebound” effect could have multiple impacts on health and quality of life, including enhanced endogenous analgesia and repression of critical immune factors [40].

Further research is needed with naltrexone and naloxone stereoisomers to determine the true mechanism of clinical action. In the meantime, we note that both the TLR4 and opioid receptor mechanisms may play a role in LDN action, as the hypotheses are not mutually exclusive."


But like they note at the end of the disclaimer, por que no los dos? I personally have had euphoria from certain doses of low dose naltrexone. I was not going for that effect and it was too much and coincided with a dose that sort of "stimmed me out". But in my mind there's almost no doubt, simply from bioassay, that LDN causes some compensatory upregulation in opioid receptors. the question is whether that's what causes the therapeutic effect, and whether it can be tested more to find more specific markers for decreased inflammation.


I highly recommend reading the full text.
http://science.sciencemag.org/content/221/4611/671
 

debored13

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Abstract
Naltrexone, an opiate antagonist, had both stimulatory and inhibitory effects, depending on the dosage, on the growth of S20Y neuroblastoma in A/Jax mice. Daily injections of 0.1 milligram of naltrexone per kilogram of body weight, which blocked morphine-induced analgesia for 4 to 6 hours per day, resulted in a 33 percent tumor incidence, a 98 percent delay in the time before tumor appearance, and a 36 percent increase in survival time. Neuroblastoma-inoculated mice receiving 10 milligrams of naltrexone per kilogram, which blocked morphine-induced analgesia for 24 hours per day, had a 100 percent tumor incidence, a 27 percent reduction in the time before tumor appearance, and a 19 percent decrease in survival time. Inoculation of neuroblastoma cells in control subjects resulted in 100 percent tumor incidence within 29 days. These results show that naltrexone can modulate tumor response and suggest a role for the endorphin-opiate receptor system in neuro-oncogenic events.
 

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So remember how I said that in future parts I will explore immune resolution, gut and brain connections, and I will probably *not* write anything about PEM and intracellular stuff? Ahem ahem...
Great write-up and more insight into your theories. Do you have a background in biology? It's impressive that your able to keep all of this together - that executive functioning is perhaps my worst symptom after fatigue.

I love that you brought that Light paper into it. I would have loved for Light to be part of a Center of Excellence, he produces really insightful studies. He did emphasize at the symposium that he didn't think fatigue was the proton/eatp/acid from normal fatigue but more like mitochondrial fatigue or immune fatigue, but he also said he found mitochondrial mutations that no one else has consistently found. I would be shocked (and more than a little scared) if that turned out to be true. We need to start naming these fatigues to differentiate them.

His work also suggests ASIC3 receptor is upregulated in CFS after exercise (he also found il-10 high there, which must've been why I thought it was elevated in CFS). There is also a paper from Ronald Straud from the University of Florida, I don't know if you saw it, sorry if you did, that builds on Light's hypothesis by trapping metabolites in the arm's of PWCFS, thereby forcing them to interact with the receptors more. Fatigue and pain did increase I think. He also did a follow up paper where he blocked the receptors with lidiocaine, and this to some extent, blocked the fatigue. I don't know exactly, I read them a long time ago and am too brain dead to try right now.

Anyway, great work, and take time to rest & recover.
 
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Speaking of muscles and purines, this genetic disorder is surprisingly common and sure seems interesting:

https://en.wikipedia.org/wiki/Adenosine_monophosphate_deaminase_deficiency_type_1

--

Adenosine monophosphate deaminase deficiency type 1, also called myoadenylate deaminase deficiency (MADD), is a recessive genetic metabolic disorder that affects approximately 1–2% of populations of European descent.

Symptoms
Although many people with a defective AMPD gene are asymptomatic, others may have symptoms such as exercise intolerance, muscle pain, and muscle cramping.

EFFECTS


Failure to deaminate the AMP molecules has three major effects. First, significant amounts of AMP are lost from the cell and the body. Second, ammonia is not freed when the cell does work. Third, the level of IMP in the cell is not maintained.

  • The first effect—the loss of AMP—is mostly significant because AMP contains ribose, a sugar molecule that is also used to make DNA, RNA, and some enzymes. Though the body can manufacture some ribose and obtain more from RNA-rich sources such as beans and red meat, this loss of ribose due to MADD is sometimes sufficient to create a shortage in the body, resulting in symptoms of severe fatigue and muscle pain. This outcome is especially likely if the individual regularly exercises vigorously or works physically over a period of weeks or months.
  • The second effect, the absence of ammonia, is not well understood. It may result in a reduction of the amount of fumarate available to the citric acid cycle, and it may result in lower levels of nitric oxide (a vasodilator) in the body, reducing blood flow and oxygenintake during vigorous exercise, though this may be offset by increased levels of adenosine, another vasodilator.[11]
  • The third effect, the reduction in IMP, is also not well understood. It may somehow result in a reduction in the amount of lactic acidproduced by the muscles, though serum lactate is typically slightly elevated with MADD

--

I am surprised we don't hear more talk of this as an exclusionary diagnosis.
 
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debored13

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This is great work man
Speaking of muscles and purines, this genetic disorder is surprisingly common and sure seems interesting:

https://en.wikipedia.org/wiki/Adenosine_monophosphate_deaminase_deficiency_type_1

--

Adenosine monophosphate deaminase deficiency type 1, also called myoadenylate deaminase deficiency (MADD), is a recessive genetic metabolic disorder that affects approximately 1–2% of populations of European descent.

Symptoms
Although many people with a defective AMPD gene are asymptomatic, others may have symptoms such as exercise intolerance, muscle pain, and muscle cramping.

EFFECTS


Failure to deaminate the AMP molecules has three major effects. First, significant amounts of AMP are lost from the cell and the body. Second, ammonia is not freed when the cell does work. Third, the level of IMP in the cell is not maintained.

  • The first effect—the loss of AMP—is mostly significant because AMP contains ribose, a sugar molecule that is also used to make DNA, RNA, and some enzymes. Though the body can manufacture some ribose and obtain more from RNA-rich sources such as beans and red meat, this loss of ribose due to MADD is sometimes sufficient to create a shortage in the body, resulting in symptoms of severe fatigue and muscle pain. This outcome is especially likely if the individual regularly exercises vigorously or works physically over a period of weeks or months.
  • The second effect, the absence of ammonia, is not well understood. It may result in a reduction of the amount of fumarate available to the citric acid cycle, and it may result in lower levels of nitric oxide (a vasodilator) in the body, reducing blood flow and oxygenintake during vigorous exercise, though this may be offset by increased levels of adenosine, another vasodilator.[11]
  • The third effect, the reduction in IMP, is also not well understood. It may somehow result in a reduction in the amount of lactic acidproduced by the muscles, though serum lactate is typically slightly elevated with MADD

--

I am surprised we don't hear more talk of this as an exclusionary diagnosis.
 

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I'm excited about this thread. I had to lower my LDN dose and am very tired, and also sleep deprived from too high of an LDN dose so I can't absorb much at the moment. but will return to it. excited to see how LDN fits into this theory
 

voner

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Speaking of muscles and purines, this genetic disorder is surprisingly common and sure seems interesting:

https://en.wikipedia.org/wiki/Adenosine_monophosphate_deaminase_deficiency_type_1
.............

I am surprised we don't hear more talk of this as an exclusionary diagnosis.
I am homozygous for this deficiency. @Valentijn maintains a database of 23andme data from ME/CFS patients and when I submitted my data to her she told me that out of 50 patients that she had in her database, I was the only person that was homozygous for AMPD1 deficiency.
 

dreampop

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Some thoughts:

About proton/eatp/la fatigue; I wonder if enough activation through different parts of the body, or through the body as whole elicits central fatigue (as opposed to just localized fatigue).

I can't believe Straud bends over backwards to call it "central sensitization". Ugh. 1) His study isn't suggestive of central sensitization. 2) It's the peripheral nervous system when the nervous system is involved. 3) Probably most of the metabo receptors aren't even next to neurons.

Could CD-39 autoantibodies (going for endothelial cells) mess with the blood brain barrier? The recent GWI/CFS mRNA study, one of the abnormal ones in CFS regulated the BBB.

We have cerebral hypoperfusion in the midbrain, and the Japanese PET study showed microglia activation is overlayed on exact same area. Any ideas on what the heck is going on there.
 
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dreampop

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how did i miss that xanthines are purines... does this mean that caffeine could have an effect on cfs? or theobromine
Not, really, they antagonize adenosine receptors, which are downstream of the problem OP is suggesting. I tolerate caffeine fine, though many don't.
 

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Of course, many thanks for this interesting, well thought, and well researched theory. That was a lot of work to do and many here appreciate your efforts.

Are you or will you be able to theorize about any possible autoimmune drugs that may target specific mechanics of your theory in an attempt to lessen symptoms and offer some improvement (but not cure)? I'm sure there are some autoimmune or rheumatology drugs out that may help with your theory.