Invest in ME Conference 12: First Class in Every Way
OverTheHills wraps up our series of articles on this year's 12th Invest in ME International Conference (IIMEC12) in London with some reflections on her experience as a patient attending the conference for the first time.
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Ponderings and speculations about purinergic signaling, in pursuit of a unified ME/CFS theory

Discussion in 'General ME/CFS Discussion' started by necessary8, Oct 29, 2017.

  1. Murph

    Murph :)

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    Although I did pick up the paracrine/autocrine/endocrine distinction, I didn't know that. Thanks.

    (There's a risk of making a goof of yourself when you have some data but shaky understanding of the actual systems the numbers refer to - I've certainly taken that risk! Just looking back at my earlier posts in this thread I can see glaring misunderstandings and mistakes. I'm learning at the sort of rapid rate that's probably only possible when you start at the beginning!!)

    Anyway, does cd39 also only operate in the pericellular halo? Surely not, right? So the low serum levels of adenosine may still indicate cd39 shortfall? I am left wondering how the high serum ratios of ATP:AMP:adenosine can be interpreted.

    Also, is there evidence of purinergic receptor downregulation in mecfs? Those Light studies seem to show up-regulation...
     
  2. dreamydays

    dreamydays

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    In this study https://www.ncbi.nlm.nih.gov/pubmed/27001659 it is shown that PWME have increased T-regs compared to controls. I had an instant sustained improvement in symptoms when I started taking cimetidine, which activates Mtor and reduces T-regs by downregulating Foxp3. I then managed to improve again when I eliminated selenium from my supplements, selenium is known to increase T-regs. Any ideas how to further deplete T-regs would be appreciated.
     
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  3. Learner1

    Learner1 Professional Patient

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  4. pattismith

    pattismith Senior Member

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    Thank you for this thread, it is much informative!

    I don't know if this paper is right, but it says that CD39 are also expressed on B cells, and that CD39 expression on Treg cells is genetically determined and affects their immune regulation power (by ENTPD1 gene)...

    I wonder if polymorphism on this gene has significant implication in ME/CFS patient...
     
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  5. Hip

    Hip Senior Member

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    I am the reverse, I made major improvements from high dose (400 mcg daily) selenium.
     
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  6. pattismith

    pattismith Senior Member

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    I think that down regulating FOXP3 may not be a good idea in ME....:

    FOXP3+ T-regulatory (Treg) cells are thought to be dependent on oxidative phosphorylation.
    mice deficient in ND6, a mitochondrial encoded gene of complex I, have deficient Treg function. Furthermore, 50 nM rotenone, a complex I inhibitor, could selectively inhibit Treg suppressive function without affecting Tconv cell proliferation.

    Genetic deficiency of foxp3 induces dysfunction of Treg cells...Genetic deletion of the foxp3 gene and the loss of Treg cells promote the development of autoimmune and inflammatory syndromes...

    We show, for the first time to our knowledge, that nuclear Foxp3 function is sufficient to program upregulation of multiple electron transport components. This increases SRC and OXPHOS activity for multiple substrates, including lipids in T cells. It does this in conditions replete for glucose (20 mM) and amino acids, and independently of exogenous TGFβ or mTOR inhibition, although the latter is an additive signal for OXPHOS. Subsequent increased fatty acid metabolism results in reduced sensitivity to fatty acid–induced apoptosis, which is reversible by inhibition of fatty acid catabolic enzymes. These data imply that Tregs are programmed by Foxp3 to have flexibility in fuel choice, in addition to gaining a survival advantage in environments with elevated fatty acids.

    Human Tregs appear metabolically flexible, switching between glycolysis-only and both glycolysis and fatty acid oxidation upon activation (36).
    In addition to providing energy for cell survival and division, the choice between utilizing glycolytic and oxidative metabolism defines the lineage decision between Th17 cells and Tregs, respectively (13) (reviewed in ref. 37). The decision to oxidize fatty acids for OXPHOS is taken by Tregs in addition to memory T cells (14) and is influenced by nutrient status, via mTOR and AMPK activation as well as pyruvate dehydrogenase kinase 1 (PDHK1) activation, Acetyl-CoA carboxylase 1 (ACC1) expression (13, 14), and PTEN activity (38, 39).
    Increased uptake of fatty acids by Tregs may be explained by our observation of increased protein levels of acetyl coA synthetase (ACSL) and carnitine palmitate transferase 1A (CPT1A) influenced by Foxp3. Our data suggest that, for Foxp3 to drive increased OXPHOS, both direct DNA binding via the FKH domain and possibly indirect DNA binding via recruited cofactors at the ProR are required. TGFβ and inhibition of mTOR are inductive signals for Foxp3 expression but are dispensable for this function of Foxp3.
    We find Tregs and Tact release ATP (data not shown), which can rapidly be converted to antiinflammatory adenosine via CD39 and CD73 (4045). It remains to be demonstrated whether secreted ATP, rather than ATP released from dying cells, represents a significant substrate for membrane-bound ectonucleotidases. A further consequence that we have demonstrated in this study may be intracellular removal of potentially harmful oxidative substrates, such as long chain free fatty acids. We show that Foxp3-expressing cells are relatively resistant to fatty acid–induced death due to removal by β-oxidation.


    My understanding would be that FOXP3+Treg may have impaired OXPHOS for some reason and this could have a link with a deficit of CD39 expression whether direct or indirect

    Edit: after reading more about Cimetidine, it seems that it can shift Treg into activated Th17 , which may be interesting for ME, but could produce activation of some auto-immune diseases like MS, RA, Psoriasis...

    Edit bis: interesting finding in wikipedia about Loss of Th17 cells in HIV pathogenesis:

    Additionally, the loss of Th17 cells in the intestine leads to a loss of balance between inflammatory Th17 cells and Treg cells, their anti-inflammatory counterparts. Because of their immunosuppressive properties, they are thought to decrease the anti-viral response to HIV, contributing to pathogenesis. There is more Treg activity compared to Th17 activity, and the immune response to the virus is less aggressive and effective.[16]

    Revitalizing Th17 cells has been shown to decrease symptoms of chronic infection, including decreased inflammation, and results in improved responses to highly active anti-retroviral treatment (HAART). This is an important finding—microbial translocation general results in unresponsiveness to HAART. Patients continue to exhibit symptoms and do not show as reduced a viral load as expected.[20] In an SIV-rhesus monkey model, it was found that administering IL-21, a cytokine shown to encourage Th17 differentiation and proliferation, decreases microbial translocation by increasing Th17 cell populations.[17] It is hopeful that more immunotherapies targeting Th17 cells could help patients who do not respond well to HAART.
     
    Last edited: Nov 11, 2017
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  7. Learner1

    Learner1 Professional Patient

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    So how do you tell if your T cells are exhausted?

    http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005177

     
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  8. pattismith

    pattismith Senior Member

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    "This is the first study to show significantly higher levels of VPACR2 receptors, CD4+CD25+Tregs and FoxP3+Treg expression in CFS/ME patients compared to healthy controls."

    "Tregs and CD4+CD73+CD39−T cells were elevated in CFS/ME group in comparison with the non-fatigued controls....
    In T cells, CD73 dampens the release of pro-inflammatory cytokines by inhibiting NFκB activation (78), promoting a Th2 type response. High levels of adenosine or ATP activates a negative feedback process that inhibits neutrophil function and protects against prolonged inflammation or injury (79,80)" (@nandixon quoted this study which is consistent with CD39 deficiency hypothesis)

    And it has been shown that higher Treg expression means generally less Th17 expression, so there might be an imbalance between the two in ME, but I couldn't find any evidence of this imbalance.
    The only anecdotal report I found to support this suspicion was from a PR member @MartinDH here

    in MS, we can see how Tregs, CD39 expression and Th17 is important for the course of the disease, alhtough the immune datas found are not exactely the same as in ME...

    In this study, percentages of CD39(+) Treg and Th17 cells were compared between relapsing-remitting MS patients and controls and were related to the vitamin D status. The Th17 cell population was expanded in about 40% of the MS patients. In MS patients in remission, but not during relapse, a positive association was found between Th17 cell and CD39(+) Treg percentages (r=0.468, p=0.007). Since CD39(+) Tregs have been shown to have Th17 suppressive capacities, we propose that a dysregulated Th17/CD39(+) Treg balance might contribute to disease exacerbation.
    Th17 expansion in MS patients is counterbalanced by an expanded CD39(+) regulatory T cell population during remission but not during relapse. .

    "Despite the fact that CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells) play a central role in maintaining self-tolerance and that IL-17-producing CD4+ T cells (Th17 cells) are pathogenic in many autoimmune diseases, evidence to date has indicated that Th17 cells are resistant to suppression by human Foxp3 + Treg cells. It was recently demonstrated that CD39, an ectonucleotidase which hydrolyzes ATP, is expressed on a subset of human natural Treg cells. We found that although both CD4+ CD25 high CD39+ and CD4+ CD25 high CD39- T cells suppressed proliferation and IFN- production by responder T cells, only the CD4+ CD25 high CD39 + , which were predominantly FoxP3+ , suppressed IL-17 production, whereas CD4+ CD25 high CD39- T cells produced IL-17.
    An examination of T cells from multiple sclerosis patients revealed a normal frequency of CD4+ CD25+ CD127 low FoxP3+ , but interestingly a deficit in the relative frequency and the suppressive function of CD4+ CD25+ CD127 low FoxP3+ CD39+ Treg cells. The mechanism of suppression by CD39+ Treg cells appears to require cell contact and can be duplicated by adenosine, which is produced from ATP by the ectonucleotidases CD39 and CD73. Our findings suggest that CD4+ CD25+ Foxp3+ CD39+ Treg cells play an important role in constraining pathogenic Th17 cells and their reduction in multiple sclerosis patients might lead to an inability to control IL-17 mediated autoimmune inflammation."
     
  9. pattismith

    pattismith Senior Member

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    Did you see it is a common pattern with MS, (with deficit in CD4(+)CD25(high)CD39(+)Tregs) ?


    CD39+Foxp3+ regulatory T Cells suppress pathogenic Th17 cells and are impaired in multiple sclerosis.
    Fletcher JM1, Lonergan R, Costelloe L, Kinsella K, Moran B, O'Farrelly C, Tubridy N, Mills KH.
    2009
    Abstract

    Despite the fact that CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) play a central role in maintaining self-tolerance and that IL-17-producing CD4(+) T cells (Th17 cells) are pathogenic in many autoimmune diseases, evidence to date has indicated that Th17 cells are resistant to suppression by human Foxp3(+) Treg cells.
    It was recently demonstrated that CD39, an ectonucleotidase which hydrolyzes ATP, is expressed on a subset of human natural Treg cells.
    We found that although both CD4(+)CD25(high)CD39(+) and CD4(+)CD25(high)CD39(-) T cells suppressed proliferation and IFN-gamma production by responder T cells, only the CD4(+)CD25(high)CD39(+), which were predominantly FoxP3(+), suppressed IL-17 production, whereas CD4(+)CD25(high)CD39(-) T cells produced IL-17.
    An examination of T cells from multiple sclerosis patients revealed a normal frequency of CD4(+)CD25(+)CD127(low)FoxP3(+), but interestingly a deficit in the relative frequency and the suppressive function of CD4(+)CD25(+)CD127(low)FoxP3(+)CD39(+) Treg cells.
    The mechanism of suppression by CD39(+) Treg cells appears to require cell contact and can be duplicated by adenosine, which is produced from ATP by the ectonucleotidases CD39 and CD73. Our findings suggest that CD4(+)CD25(+)Foxp3(+)CD39(+) Treg cells play an important role in constraining pathogenic Th17 cells and their reduction in multiple sclerosis patients might lead to an inability to control IL-17 mediated autoimmune inflammation.
     

    Attached Files:

    Last edited: Nov 12, 2017
  10. Learner1

    Learner1 Professional Patient

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    @pattismith. Is there a commonly accepted lab test or panel for any of this?
     
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  11. pattismith

    pattismith Senior Member

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    I have no idea if these tests are available, those papers are only fondamental research....Maybe in the USA?
     
  12. Learner1

    Learner1 Professional Patient

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  13. pattismith

    pattismith Senior Member

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    yes! it is consistent with Naviaux findings and this is why necessary8 made this passionating unified ME/CFS theory, and came to the conclusion that CD 39 expression might be deficient for us, and that it could explain much of our problems..;

    Her post was far too long for me to read, so It took much time to understand where she was going, but I'm starting to get a bit of the picture too...:)
     
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  14. Learner1

    Learner1 Professional Patient

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    Right! I'm getting the idea, and I read a lot if medical journal articles, but this is wading in very deep for me.

    My oncologist thinks my immune system issues and MCAS could have contributed to my cancer and I can see why in what I'm reading. This all does seem to fit.

    So, how can we test this? Do any exist or do we have to be a lab rat?

    Then, what can be about it? Suramin isn't mentioned in any of the articles I've been reading, but I see other drugs mentioned. And would there be any nutrients?

    Also seems there's a relationship to membrane health, so wondering about lipids...

    Thanks, @necessary8 for getting this started... You, too @pattismith !
     
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  15. pattismith

    pattismith Senior Member

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    I wish I could be a lab rat :lol: as long as nobody would open my head to see what's going on in it...:D
     
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  16. Learner1

    Learner1 Professional Patient

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    Hmm... I did a little more digging. This is a little worrisome, if I'm understanding correctly...
    From
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485743/
     
  17. pibee

    pibee

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    Not that I understand it completely, but interesting article:


    http://mastcellmaster.com/documents...extracellular-mt-DNA-Autoimmun-Rev-8-2013.pdf
     
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  18. dreampop

    dreampop Senior Member

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  19. nandixon

    nandixon Senior Member

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    I've given a possible explanation for this here.
     
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  20. necessary8

    necessary8

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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...


    Ponderings and speculations about purinergic signaling, in pursuit of a unified ME/CFS theory, Part 2 - PEM and intracellular stuff


    Extracellular ATP in skeletal muscles, and how fatigue works

    Skeletal muscles are what we usually think about when we say “muscle” - the muscles we can consciously control, which we use to move around. As it turns out, skeletal muscle cells also have purinoreceptors[source], as well as Panx1 channels[source]. They also express CD39 in high amounts.[source]

    Purinoreceptors also exist in the neurons that connect to the skeletal muscles, and a few years ago Alan Light along with other researchers, hypothesized that the activation of P2X and a few other receptors, in those neurons, constituted the perception of fatigue. They tested this in a fascinating study, where they were able to artificially induce the feeling of fatigue in healthy subjects, by injecting ATP and lactic acid into their muscles.

    Let’s first review how all of this works in a healthy human. During exercise, ATP is actually released from muscle cells, acting on the P2X receptors to potentiate the calcium influx into the cells which is necessary for muscle contraction.[source] (P2X are ion channels, so their activation opens up a channel for Ca+ to flow in). Lactic acid is also produced and released from those cells. When the exercise is not very strenuous, the extracellular ATP is broken down by CD39, and lactate is moved by blood to other parts of the body to be metabolized. With more intense exercise, those processes cannot keep up, and ATP along with lactic acid accumulates in the extracellular space of the muscles. They reach the muscular neurons, where ATP acts on the P2X, and lactic acid on ASIC and TRPV1 receptors, inducing the feeling of fatigue, and in higher amounts also pain. When the person then goes to rest, ATP and lactic acid are no longer released, and the accumulated amounts can be removed, decreasing the feeling of fatigue with sufficient rest.

    So what would happen if CD39 activity was somehow impaired? Well, the eATP would accumulate much easier in muscles, with very mild exertion having the impact that intense exercise has on healthy people. Sound familiar? Yes, I believe this is a very viable model of how PEM works, or actually its first step.

    The second step is immune activation. Muscle cells are actually in communication with the immune system, through various cytokines, and through eATP as well.[source] For example, when a muscle is injured, macrophages are activated, and more of them are recruited to the site of injury, where they facilitate an almost classical inflammation, with the secretion of IL-1β. The M1-polarized macrophages remove damaged cells and debris, and after 1-3 days they shift towards the anti-inflammatory M2 polarization, aiding in regeneration of muscle tissue.[source] A special subset of Tregs also comes to help at this stage.[source] So that’s injury. What about exercise? Well, it is widely accepted that pretty much the same thing happens with exercise, as muscles get somewhat damaged during it, triggering the same inflammation and repair sequence. There have been a number of studies that demonstrated this. The problem is, those studies have a methodological flaw. To check what’s going on with the immune system in muscles, you have to do a muscle biopsy. Which is an injury. Which triggers the same sequence. And sure enough, it has been recently shown that a lot of the same signs of immune activation can be achieved by just doing muscle biopsies, without exercise.[source] Nevertheless, studies done on blood, and not through muscle biopsies, show that some immune activation is in fact being started by exercise.[source] We just don’t know exactly what the immune system does there, and we don’t know if this activation is just due to muscle damage or not. But it seems that the adaptive immune system (so B cells, T cells) don’t play a major role here.[source1][source2] As for eATP, I haven’t seen it directly demonstrated, but it seems to me a fair assumption that it plays a role in this activation.

    Now back to ME/CFS. My hypothesis is that in our case, due to CD39 impairment, even minor physical exertion leads to immune activation through increased eATP signaling, which then reactivates the adaptive immune system, leading to an increase in the anti-CD39 antibodies produced. As for why the adaptive immunity would activate here, while it doesn’t activate in normal exercise - my guess is that in the muscle tissue of an ME/CFS patient who’s overdoing it, eATP reaches the immune cells in higher amounts than it ever does in healthy people during exercise, making the the immune system behave differently. I believe that the dendritic cell maturation status is important here. Dendritic cells present antigens to T cells and B cells, activating them to their immune function, but they can also present self-antigens (proteins used by the body), and suppressing T cells which react to them, preventing autoimmunity. The process by which a dendritic cell “knows” which antigens are self and which are non-self (and therefore which T cells to activate and which to suppress), are complex and not fully understood yet, but a key factor seems to be their maturation status, with mature DCs being more likely to induce immunity, and immature to induce tolerance.[source] And extracellular ATP was shown to promote DC maturation.[source]
    It’s also possible that other factors released by muscles during normal exercise, prevent DCs from performing antigen presentation. And it might be that in ME/CFS those factors are not released properly for some reason. Those might be classical myokines, such as IL-6, IL-10, IL-15, or it might be something more exotic, like extracellular BCL-2.[source]

    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. The DCs then travel to the lymph nodes, presenting self-antigens as non-self, including CD39, and because we have T-cells and B-cells which react to CD39, their activation increases, producing more anti-CD39 antibodies, and resulting in the exacerbation of symptoms we know as PEM.I believe that this model explains five very important things.
    It explains why PEM is delayed - adaptive immunity activation occurs on similar timeframes as PEM. It explains why it’s felt in the whole body and not just the muscle group that performed the exercise - antibodies reach the whole body by blood. It explains why PEM is basically the whole illness getting worse, including increased susceptibility to further crashes. It explains why often only absolute zero activity constitutes as rest for us - that’s the only point in which eATP hydrolysis exceeds the self-sustaining feedback loop of more ATP release leading to more anti-CD39 antibodies, which lead to more eATP. And finally, it explains why other diseases, where purinergic signaling plays a role, don’t get PEM - there, the eATP released from the muscles during exercise is able to be sufficiently hydrolyzed before it can reach other systems in sufficient quantities to impact their function.
    As a bonus, it might also explain why many patients have muscle twitching when they overexert - it’s from the increased muscle cell stimulation by eATP which potentiates calcium influx.


    So what about the lactic acid?

    Before you say “ME/CFS patients reach anaerobic threshold way easier, so more lactic acid! Plus PDH inhibition! everything fits!”... Well, not really. While it is very widely accepted that lactic acid is a waste product generated by cells switching to anaerobic metabolism, more recent research suggests that it’s main role is very different. It’s actually produced by the muscles during aerobic exercise, and then transported from cell to cell by a complex shuttle system. When one muscle group is under intense exercise, it releases lactate, which is then uptaken by other muscle groups, and converted back to pyruvate, instead of using glucose. After the exercise stops, the lactate is uptaken from the blood by the same muscles that released it, again using it for pyruvate production. Therefore, lactic acid is not a waste product, nor a reliable indicator of insufficient oxygen, but rather an energy fuel buffer and transporter, and the whole concept of anaerobic threshold might not be correct.[source] In addition, I’ve looked at the data from Naviaux’s metabolomic study, and lactic acid in ME/CFS patients is no different than controls. I’ve tried to find any other study that demonstrated it being elevated in ME/CFS, but I think I only found one or two old papers with very small sample sizes. I trust Naviaux’s data way more. Plus there are many other diseases which have fatigue as a symptom, but don’t exhibit lactic acidosis. So how does that work? If fatigue needs lactic acid and eATP to happen, how does it exist in ME/CFS, or other diseases, or even in a flu? I see two possibilities here.First is that the lactic acid is in fact released, but very locally and doesn’t enter the bloodstream. I find this unlikely, as the whole role of lactate is to be transported between different tissues, and this transport goes through blood. But I don’t want to rule out this possibility quite yet. I thought that another argument against it was that it would make PEM localized to the muscles involved in the exercise, which it is not. But the lactic acid doesn’t have to be released by the muscles, it can be released by the immune cells, which seem to happen when they’re activated.[source]
    The second possibility is that there is something else, probably in the blood, imitating the effects of lactic acid on muscle sensory neurons in ME/CFS and probably many other diseases. As to what it could be, I don’t know, but there are some clues.
    There’s an important distinction that I have to finally make here - lactic acid (HLa) in extracellular space exists mostly as two separate things - lactate (La-) and protons (H+). In his experiment, Alan Light injected healthy subjects intramuscularly with lactate, protons, and ATP. He found that if he injects any of the three alone, there is no effect, and if all three are injected together there is fatigue (and in higher amounts pain). What he didn’t try, and what I really wish he tried, are combinations of two. Because it’s possible that only La- or only H+ is needed.
    So why did he choose those three metabolites anyway? Well, because in his research about fatigue, he’s seen three main receptor types indicated - P2X, ASIC, and TRPV1. And he just chose the three metabolites that he knows are released during exercise, and have effects on those receptors. Now, if the three metabolites just corresponded each to the three receptors, it would be all nice and simple. But nothing in biochemistry is ever nice and simple. ASIC stands for Acid Sensing Ion Channels, and they are activated by acidic (low) pH, so basically by increased protons. They are more sensitive to this, when eATP is present too.[source] There is some evidence (albeit a bit questionable quality), that lactate likewise makes ASIC receptors more sensitive to protons.[source] TRPV1 is also activated by protons but here lactate actually inhibits that activation.[source] TRPV1 sensitization is also modulated by other factors, such as PI3K[source] and prostaglandin E2[source]. As for P2X, we know that it’s activated by eATP, but as @dreampop pointed out, this activation is inhibitied by acidic pH (with the exception of P2X2, where it is increased instead). Furthermore, it seems that this inhibition is also modulated by other factors, like various amino acid residues.[source1][source2] Do you see how this is getting really complicated? What’s complicating it even more is the fact that those same receptors are indicated in feeling pain and heat. It seems that the exact ratios of eATP, protons and lactate is what makes us feel those pretty different sensations. This is another thing I really wish Dr Light has tested in his study - different ratios those metabolites. It could tell us a lot about how this works. But as he hasn’t, I can only throw wild speculations. And my main speculation is that the presence of lactate (or its imposter, as I mentioned earlier), by inhibiting acid-mediated TRPV1 activation makes the feeling be fatigue instead of pain. So if I were to look for what it might be instead of lactic acid causing fatigue in ME/CFS and other diseases, I would look for something inhibiting TRPV1, probably originating from the immune system. I’m also starting to think that the balance between CD39 inhibition and whatever is going on with lactic acid might decide where a patient fits on the spectrum between painless ME/CFS and fatigueless fibromyalgia. And why some of us have a lot of pain symptoms while others have none.

    At this point, I want to make something absolutely crystal clear. The reason I think P2X antagonists could be beneficial as treatments is because they would inhibit the release of more ATP, and the immune reactivation. NOT because they would prevent the neurons from sensing large eATP levels, disabling the preceptory signal of fatigue. The only reason we’re looking at how that signal is conducted, is to search for clues on what’s causing it. Disabling that signal without fixing the underlying issue could be catastrophic. So I don’t want you guys looking for ASIC and TRPV1 antagonists now, okay? It can end really badly.


    Where this model falls apart a little bit

    ...is the connection to the brain. While I haven’t looked at purinergic signaling in the nervous system in detail yet, we appear to have a much more fundamental problem here. Antibodies don’t cross the blood-brain barrier. They’re way too big. And while size isn’t the only factor deciding whether a molecule can pass this barrier or not, the biggest thing known to date to be able to pass, is CINC-1 at 7800Da[source], it in itself being an exception more than the rule, with most molecules being way smaller. By comparison, the smallest class of antibodies, IgG, is around 150000Da, about 19 times larger. So it seems very unlikely for our hypothetical anti-CD39 antibody to be able to affect the nervous system. This problem is also not limited to my hypothesis - we know that the blood factor Ron identified with his impedance assay is bigger than 10000Da, so no matter what it is, it’s probably not passing the blood-brain barrier. And yet we have neurological symptoms, and they interact with PEM in both ways - they get worse after physical exercise, and also mental exertion can cause PEM.

    There are probably a number of ways to make this piece of the puzzle to fit as well, but I first need to learn more about the anatomy and physiology of the nervous system, before I can talk about them in detail, just to make sure I’m not saying stupid shit. So this is mostly a subject for future parts. For now, I will just quickly mention two interesting solutions that immediately come to my mind. First is our hypothetical lactic acid imitator, that I mentioned earlier. Maybe this one is smaller, and can act on the brain. And second, is the idea that maybe the anti-CD39 antibodies act on the endothelial cells of the blood-brain barrier itself, causing increased ATP release from them. This ATP itself then seeps into the brain (ATP is pretty small), acting on microglia and/or neurons, which are relatively close to those endothelial cells.


    A quick stab at cellular metabolism

    Yes, the intracellular stuff. To start, I wanted to talk about the metabolomics results here, because I believe those to be the most important finding in ME/CFS to date. Firstly, because unlike Seahorse testing, or the impedance assay, which can only be done on isolated cells, metabolomics gives us an overall picture of metabolism in the whole body. Each set of cells do their own thing, breaking some things up and building other things, and they dump the side products of those reactions into the blood, to be taken up and further metabolized by a different set of cells, or to be filtered out by kidneys and excreted with urine. So by measuring those side products in the blood, metabolomics gives us a very good idea of how the whole body in general is doing. The second reason I believe those findings in ME/CFS are extremely important, is because the alterations in metabolism which they showed are huge. They are not barely significant, or visible only on average, they very clearly distinguish patients from controls, sometimes differing by orders of magnitude. They are also present in all patients, albeit with some differences.

    And I might have found a way for CD39 impairment alone to explain those results.

    In his paper on ME/CFS metabolomics, dr Naviaux noted that all of the pathways found to be abnormal are regulated by the availability of NADPH or the redox state of a cell, and hypothesized that the key signal for those abnormalities to occur, is the rise of dissolved oxygen in the cell.[source] This is in regard to his theory of the cell danger response, which I want to talk about here, but to do that, first a very quick and simplified review of cellular energy production, just so we’re all on the same page.
    First, glucose is broken down to pyruvate through glycolysis, producing some ATP and NADH, but not much. Then, pyruvate is transformed into Acetyl-CoA, by the pyruvate dehydrogenase complex. Fatty acids are also transformed into Acetyl-CoA, in the process of beta-oxidation. Acetyl-CoA then enters the krebs cycle, where it’s metabolized, producing lots of NADH, by adding electrons to NAD+. Those electrons are then picked up by the electron transport chain (turning the NADH back to NAD+), to be then added to oxygen, producing water. This process pumps protons across the inner mitochondrial membrane, creating an electrical gradient, which powers ATP synthase, the enzyme that attaches a phosphate group to ADP, producing ATP. ATP is then broken down to ADP and AMP by everything in the cell that needs energy to work. Here is a diagram of all that:
    [​IMG]

    So back to cell danger response. This is basically a theory, that many things dangerous to a cell, be it pathogens, heavy metals or other factors, will “steal” electrons from this process, leaving less NADH for the electron transport chain. This means that the oxygen cannot be fully utilized, and so it accumulates in the cell, activating all sorts of enzymes that prepare the cell for fighting the invader and limiting its spread to other cells.[source]

    Pathogens are still biological organisms, they are made of the same building blocks that we are, and during an infection often steal ours, to replicate themselves. So it would make sense for cells detecting a pathogen to limit their metabolism, making it harder for the pathogen to replicate.

    What doesn’t make sense is that Naviaux’s initial paper on CDR predicted that it would result in metabolic changes pretty much opposite to the ones he later observed in ME/CFS. What I really would like to do, is to go over every major metabolic abnormality in ME/CFS one by one, look at all the pathways involved, and try to make some sense of this. But unfortunately, this is incredibly complex, and I don’t have enough energy to properly do that. So for now, I’m just gonna believe Naviaux that higher dissolved oxygen is the trigger for the metabolic changes, and work from there.

    For this to occur, there needs to be a discrepancy in the oxygen supply to a cell, and the available NADH for converting that oxygen to water. An impairment in CD39 can cause this by two mechanisms. First, an increased Panx1 activation means not only the release of ATP, but also NADH, as it too can pass through the hemichannel. So this means less NADH in the cell, for the electron transport chain to use. Second, is the fact that extracellular ATP actually increases oxygen supply to skeletal muscle cells, by acting on the purinoreceptors of the endothelial cells, inducing local vasodilation.[source1][source2] This is because during intense exercise, the energy metabolism of skeletal muscles works at a higher rate, needing more oxygen. But in our hypothetical model of ME/CFS, CD39 impairment causes disproportionately large accumulation of eATP. This would presumably cause an increased oxygen supply while the energy metabolism and NADH turnover not actually being upregulated that much, therefore creating the discrepancy I mentioned earlier, rising dissolved oxygen levels, triggering CDR and causing the metabolic changes.

    If you’re wondering why I think the energy metabolism is not upregulated there, let’s look at how it’s regulated. I’ll start with an example. When the muscles are at rest and not using much ATP to contract, ATP concentration rises in the cell. The ATP molecules actually bind to phosphofructokinase, the enzyme that catalyzes the third step of glycolysis, and inhibit its activity. When the muscles contract, they use up the ATP, decreasing this process, and also causing the AMP levels to rise. AMP also binds to phosphofructokinase, but it instead increases its activity, upregulating glycolysis. It works like this at pretty much every stage of cellular energy metabolism - glycolysis, pyruvate decarboxylation, krebs cycle - the key enzymes that catalyze those reactions, are regulated by the ratios of ATP/ADP, ATP/AMP or NADH/NAD+. Sometimes there’s an extra step, or the intermediates are also regulators to prevent accumulation, but overall those ratios are what’s important.

    So in my ME/CFS model, when we have a higher accumulation of eATP in response to exercise that is not really intense in any way, the energy metabolism is not being upregulated, because ATP is not being used a lot. It is lost through Panx1 channels, yes. But ADP and AMP are also lost, so the ratio doesn't actually change. The same goes for NADH and NAD+. The only thing that directly upregulates energy metabolism, that can be caused by increased purinergic signaling, is calcium influx. But the actual intracellular concentration of calcium in muscle cells is very tightly regulated by the sarcoplasmic reticulum which can either absorb or release large amounts of calcium on a millisecond timescale, in response to signals from motor neurons. This is just my guess, but I think the increased calcium influx from purinergic signaling in intense exercise is just to give the sarcoplasmic reticulum more calcium to work with, and not to circumvent it (although that in extreme cases it might also happen to some degree, hence the muscle twitching).

    So my view is that overall the increased purinergic signaling in skeletal muscle would just upregulate the oxygen supply, without a corresponding upregulation in the energy metabolism. I can be wrong here though, those processes are complex and I’m far from knowing them all. But I think it is a viable explanation for how CD39 impairment could cause the metabolic abnormalities in ME/CFS. Now, my hypotheses here are mostly about skeletal muscle cells. It might work differently in other organs, but skeletal muscles are actually the body’s largest organ, and one very active metabolically, so if we have large metabolic changes, it seems very likely that they are involved. They probably don’t explain all of the abnormalities, but I can’t really discuss that in detail without the aforementioned tedious process of looking at all the pathways involved one by one. So it’s basically just a preliminary idea.

    There could also be another feedback loop here. Dr Naviaux thinks that a cell going into CDR would release more ATP. And while I haven’t found any experimental proof of a specific pathway that would cause Panx1 activation when dissolved oxygen rises, it would make a lot of sense from a functional point of view, for a cell that sensed danger to warn its neighbours.


    Just to turn things on their head

    If that last pathway does in fact exist, I think it’s also possible to connect the dots in a different way here. If there is something that prevents glycolysis from being properly upregulated in muscle cells during exercise, it would lead to the krebs cycle also not being upregulated. If the oxygen supply, on the other hand, is properly upregulated, this would create the same disconnect between the amount oxygen in the cell, and the available NADH, triggering CDR. This would then open Panx1 channels via the aforementioned hypothetical pathway, and release ATP, upregulating oxygen supply even more, potentiating the effect in a feedback loop and reactivating the immune system.
    To explain the delayed and full-body nature of PEM, as well as rituximab results, the problem in glycolysis upregulation would probably be caused by an autoantibody.
    I might be wrong about this, but I’m under the impression that this is the model Ron is leaning towards.
    I don’t think it explains PEM as well as CD39 inhibition, because if there is ongoing rapid release of ATP caused by CDR, us using our muscles for the very mild activities that trigger PEM in us, should not make a big difference in the amount of ATP released.
    But there might be some other reason why it does, that I haven’t thought of, so I don’t want to completely cross out this model either.
    As for what it could be preventing glycolysis upregulation, I don’t know enough to give you a comprehensive list of possibilities, but I will mention one of them that I find interesting. The rate of glycolysis is mainly regulated by ATP/AMP ratio. Not by ATP/ADP ratio. And yet muscles when contracting, turn ATP to ADP. So where does the AMP come from? Well, there is an enzyme called adenylate kinase, which takes two ADP molecules, removes a phosphate group from one of them, and puts it on the other one. This creates one ATP molecule, and one AMP molecule. So what would happen if adenylate kinase didn’t work correctly in the muscles? Presumably, when they would use up the ATP, more ADP would accumulate, not being properly converted to AMP, and the upregulation of glycolysis would suffer.
    This is just a loose idea, I haven’t looked at the other pathways which would be impacted by this, to check if everything is consistent with what we currently know about ME/CFS. But I just wanted to throw it out here.


    Possible ways to test all this

    First of all, if we do have an anti-CD39 antibody in our blood, this should be pretty easy to test directly.
    The problem is that it doesn’t have to be to CD39 exactly. It can be an antibody to a nuclear receptor which influences the expression of CD39. Or the expression of P2 receptors. Or Panx1 channels. Or it can be a combination of antibodies to a combination of nuclear receptors. Or an antibody to some other cofactor that I haven’t yet thought about. Or it might even be different things in different patients.

    What I would really like to measure, is the extracellular ATP in muscle tissue, but I have no idea if technology capable of checking that exists. Maybe some kind of spectroscopy scanning through muscles in vivo? Or maybe we really need nanobots for this, as Ron suggested. I honestly have no idea, but if I could measure anything, this would be the thing I would want to measure the most.

    As I mentioned earlier, measuring dye uptake by cells is a common method of assessing the state of hemichannels in them, and I think we really should do this in ME/CFS PBMCs in contact with plasma, and in cultured muscle cells exposed to ME/CFS plasma, possibly also under energetic demand.

    And finally, as @nandixon already pointed out, there are research mice with CD39 genetically deleted. Now, if my theory is correct, a CD39 null mice would not be an accurate model of ME/CFS, because the CD39 activity in them would be constantly zero, and not changing in response to exercise. Other than PEM, they might either have all the impairments we have but more severe because the CD39 is deleted, or maybe because it’s not present from birth, some other compensatory mechanisms occured, which don’t activate when it’s inhibited by an antibody, and the mice are not doing as bad as we are. Another thing is that ME/CFS seems to need a trigger. What would happen if we infected CD39 mice with say, an Epstein-Barr virus? Or a common cold? Would they die? Develop severe autoimmunity? Or would they end up in some pseudo-ME/CFS? If they don’t die, it would be interesting to see how they tolerate exercise. You can’t exactly ask a mouse if it’s tired, but you can give it rewards for running, and see how long until it gives up on it. Further, my hypothesis is that if CD39 is impaired, exercise can trigger self-antigen presentation by dendritic cells. This could be tested in those mice, perhaps by inducing in them some well-known autoimmune disease that has to do with muscles, and measuring if autoantibody production increases with physical exercise.
    Another take on questionable animal models, might be injecting normal mice with recombinant anti-CD39 antibodies. But this also has the problem, that it might not necessarily be an antibody exactly to CD39 that we’re dealing with in ME/CFS, but to something related.

    I’m also starting to wonder if it would be possible (and safe), to inject ME/CFS patients intramuscularly with recombinant CD39 itself. Or maybe with a different ecto-nucleotidase, which wouldn’t be blocked with the antibodies. It wouldn’t cure anyone, but if it caused some symptomatic relief that would prove eATP really is accumulating in those muscles. There’s a lot to consider here in terms of safety though, and I don’t feel qualified to make judgements on this.


    Future ponderings

    …will probably be more about lactate, myokines, energy metabolism, and the brain. I have a feeling it’s all connected in an important way, but I can’t quite put my finger on it yet, as the interactions are very complex, often going both ways, and trying to speculate which outweighs which can be problematic. I have a whole list of papers to read, to try putting it all together, but I don’t know if or when I will succeed, so I didn’t want to delay releasing Part 2 with this, because I think it still brings enough interesting stuff as it is.


    To wrap things up, I just wanted to remind everyone, that there is a reason I titled this “ponderings and speculations” - I am constructing those theories based on dozens of assumptions that might not be true, and while I try to step carefully and double-check myself, this stuff is hard, complicated, and sometimes the data is questionable as well. Any and all of my ramblings might be wrong, and the only reason I don’t use the word “hypothetical” in every sentence, is because it would get annoying.


    Thanks for reading.

    (Also, I had a crash yesterday, just before finishing this, so forgive me if I take a few days before answering questions, as I need to rest and recover)
     

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