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Anti-purinergic therapy restores itaconate/IFNa (Dr. Phair's theory) and Dr. Prusty's proposed abnormalities in ME/CFS

serg1942

Senior Member
Messages
543
Location
Spain
Hi everybody,

After listening to Dr Prusty's recent interview, and after doing a bit of research, I really wonder why we don't focus more on the anti-purinergic therapies to correct these abnormal pathways.

I already compiled detailed data explaining to Dr. Phair on this thread, how when autistic murine models took suramin, all metabolites abnormalities that would be altered according to his itaconate hypothesis, did normalize, including Kreb's cycle intermediaries, oxidative stress markers such as GSH/GSSG and NADH/NAD+, and at least part of the innate response including the complement protein C1q. In this vein, someone posted on the same thread how suramin has demonstrated to inhibit the synthesis of Interferons, at least of INFbeta:

https://pubmed.ncbi.nlm.nih.gov/29558821/

Regarding Dr Prusty's theory, he explains how herpes virus reactivate and affect plasmatic B1 cells resident in the hematopoietic organs (mainly in the bone marrow), making them to secrete less natural immunoglobulins that would lead to less cleaning of celular debris, what would in turn lead to auto-immunity. An example of this would be low IgM against fibronectin1, high serum fibronectin1 and how all this would combine to cause both mitochondrial fission and hyperfussion (both part of the CDR process as explained by Dr. Naviaux).

So, what's on top of this cascade, viral reactivation or the perpetuated cell danger response caused by extracellular purines?

Well, it seems that when purinergic receptors are inhibited, many infections cannot reactivate/replicate/infect:

For example, when renal human cells are infected in vitro with HHV-6A virus, the infection is halted when a P2X7 antagonist is applied. Here, the intracellular calcium doesn't increase when the anti-purinergic drug is added, probably indicating that the CDR is effectively prevented within the cell:

https://www.frontiersin.org/articles/10.3389/fphar.2020.00096/full

Similarly, when fibroblasts are treated with the anti-purinergic kaempferol, the infection with CMV replication is impaired:

https://www.pnas.org/doi/10.1073/pnas.1907562116

In the same vein, here it is shown how the infection of astrocytes (glial cells) with HHV1 is an ATP-dependent process, necesary for neurons to get infected. Well, suramin shows to effectively inhibit HHV1 replication in astrocytes and neuronal infection. Interestingly, when ATP was inoculated, mimicking the infected astrocytes (which secret ATP to allow neuronal infection), neurons got infected as well. This clearly indicates that purinergic signal is necesary for infection and inflammation of neuroglia and neurons by HHV1:

https://onlinelibrary.wiley.com/doi/abs/10.1002/glia.23895

Many other pathogens seem to need the purinergic signal for the successful infection and replication process such as other viruses, including Zika virus, HIV or Sars-Cov-2, and also bacteria (Pneumococcus), parasites (toxoplasma) and even fungi:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378483/

The protein DRP-1 has also been shown to be key during HHV-6 reactivation, as it directly causes mitochondrial fragmentation. Well, suramin has been shown to inhibit Drp-1 expression as well:

https://jps.biomedcentral.com/articles/10.1007/s12576-019-00666-9

On the contrary, Dr. Prusty explained how the protein mitofusin-1, which maintains the mitochondria fused, decreased when gamma-globulins from ME/CFS patients were inoculated to healthy cells. Well, suramin also showed to increased mitofusin-1 levels in murine hepatocytes treated with LPS:

https://jps.biomedcentral.com/articles/10.1007/s12576-019-00666-9

On the other hand, if Prusty's theory is correct and if purinergic signal was upstream in the patho-physiological process, then gammaglobulins levels should be restored with anti-purinergic therapy. This seems to be true in the adquired autistic murine model developed by Naviaux. In this experiment total immunoglobulins increased by 20%:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596371/

Furthermore, suramin has been shown to reduce renal fibronectin1 expression in rats:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464039/

Note that I don't expect to see easy and fast benefits with anti-purinergic therapies. Actually, I am learning that high levels of purinergic receptors inhibition can be counter-productive and even dangerous. For example, the second study done with suramim in autistic children, showed that doses of 20 mg/kg didn't work, and only doses of 10 mg/kg did work (autistic children saw over 50% of overall improvement after 3 months on suramin).

This would explain why some ME/CFS who have tried suramin at this dose didn't see any benefits.

This idea seems validated from the results of the following study, where rats were given probenecid (a pannexin-1 inhibitor, which is also an anti-purinergic agent) before and after causing them cerebral Ischaemic damage. We see that doses of 1 mg/kg were far more effective than doses of 10 mg/kg.

This is a very intriguing question, as the dose of the probenecid that blocks 50% of the pannexin-1 receptor in vitro is 150 mcrM, and the dose of 1 mg/kg is probably much lower than 7 mcrM according to pharmacokinetic studies on rats. So, perhaps we just need a temporary inhibition of the purinergic receptors to achieve a steady recovery.

Also, there are many purinergic receptors, and we have no idea which ones we need to block and to what degree in order to restore homeostasis.

I am going to try this theory myself. I am going to measure the IL-1 beta levels of my PBMCs after stimulation with LPS and ATP. In humans studies this has been shown to be an accurate way to measure the degree of activation of the inflammasome, which should correlate well with the degree of the purinergic signal.

I will be taking probenecid and brilliant blue FCF dye (a food dye) and then test my hypothesis (I am also considering other pannexin-1 inhibitors such as spironolactone or glycyrrhetinic acid).I will aim to inhibit the IL1-beta secretion about 20-30% only, and see what happens.

(Note that @Hip already calculated that perhaps the brilliant blue dye could reach cells in sufficient amounts to be anti-purinergic:
https://forums.phoenixrising.me/thr...i-purinergic-therapy.52427/page-4#post-936102).

I really think this is going to take time and be a hard process. Healing with ME/CFS always is. I will be also taking GcMAF as an immune stimulator and I hope it can help to eradicate some of the infected cells which are contributing to perpetuate the CDR.

I'll let you guys know how my experiment goes. I'd really like to know your opinions on the subject.

Sergio
 
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Violeta

Senior Member
Messages
3,009
I have tried a low purine diet, both vegan (no beans), vegetarian (dairy, no eggs), a high purine diet (low carb, meat/fish), and a medium purine diet (adequate carbs/some protein from meat, some from dairy) and the difference in viral activation is amazing.

I had eaten vegan for many years, and although I had other problems, I never had a cold sore.

I have tried a high protein diet many times and didn't understand why I would get cold sores and one time I tried low carb is when I got shingles.

Eating vegetarian with dairy/no eggs for protein seems to not be causing any outbreaks, although it doesn't cure the fatigue problem.

Just a thought.
 

Tsukareta

Senior Member
Messages
150
So you think these plasma b cell things could be in some sort of antiviral lockdown due to the continued presence in some form of one of these herpes viruses and by manipulating the 'purinergic signalling' in a precise way it will break this holding pattern and increase the levels of the natural immunoglobulins ? i'm not familiar with the autism model but wouldn't this CDR be a natural behaviour of the cells ? is there something defective in their behaviour that causes this unhealthy state to persist ?
 

Violeta

Senior Member
Messages
3,009
I will be taking probenecid and brilliant blue FCF dye (a food dye) and then test my hypothesis (I am also considering other pannexin-1 inhibitors such as spironolactone or glycyrrhetinic acid).I will aim to inhibit the IL1-beta secretion about 20-30% only, and see what happens.
The discussion of purines is very interesting and especially that they are involved as neurotransmitters.

It will be very interesting to see what type of symptoms the probenecid relieves for you.

"Probenecid is used to treat chronic gout and gouty arthritis. It is used to prevent attacks related to gout, not treat them once they occur. It acts on the kidneys to help the body eliminate uric acid."

One can lower uric acid levels by eating a low purine diet. I am trying to understand why someone would take a drug to lower uric acid instead of lowering intake of purines.
 

Violeta

Senior Member
Messages
3,009
This does open up a whole new area of thought.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133749/

Cancers and virally infected cells actively generate an immunosuppressive microenvironment by secreting purines, especially adenosine,

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226072/

"Viruses will hijack host cellular machinery, including metabolic processes. Here, I provide theory and evidence for targeting the host de novo purine synthetic pathway for broad spectrum anti-viral drug development as well as the pursuit of basic science to mitigate the risks of future novel viral outbreaks."
 

serg1942

Senior Member
Messages
543
Location
Spain
The discussion of purines is very interesting and especially that they are involved as neurotransmitters.

It will be very interesting to see what type of symptoms the probenecid relieves for you.

"Probenecid is used to treat chronic gout and gouty arthritis. It is used to prevent attacks related to gout, not treat them once they occur. It acts on the kidneys to help the body eliminate uric acid."

One can lower uric acid levels by eating a low purine diet. I am trying to understand why someone would take a drug to lower uric acid instead of lowering intake of purines.
I don't want to lower uric acid. It just happens that probenecid, which has antipurinergic properties, also lowers uric acid levels. So this is an unwanted affect.

This subject is indeed fascinating.
If you want to learn more about all this, I encourage you to listen to Dr Naviaux's talks, such as this one:


And if you want to delve into the nitty gritty of this, I recommend Naviaux's last paper. It is a a hard reading as it's very technical, but the gist is easy to get:

https://www.sciencedirect.com/science/article/pii/S1567724923000351?via=ihub
 

serg1942

Senior Member
Messages
543
Location
Spain
So you think these plasma b cell things could be in some sort of antiviral lockdown due to the continued presence in some form of one of these herpes viruses and by manipulating the 'purinergic signalling' in a precise way it will break this holding pattern and increase the levels of the natural immunoglobulins ? i'm not familiar with the autism model but wouldn't this CDR be a natural behaviour of the cells ? is there something defective in their behaviour that causes this unhealthy state to persist ?
CDR is indeed a very ancestral antiviral metabolic state, which in all diseases is chronically activated, and all metabolic and immunologic alterations can be explained from this chronic CDR state.

A very good question is if this state is preventing us from getting, for example, infected, and if getting out of this state would be counter-productive. This doesn't seem to be the case, as children treated with Suramin get better without no evident sign of infections. It seems therefore that the system could be erroneously stuck on this hypometabilic state, and we need to help it to get back to a healthy homeostasis.

And yes, Dr Prusty theory of ME/CFS is exactly that: chronic herpes virus infection makes cells to get into chronic CDR, and low natural gamma-globulins would be one of the immunological alterations typical of this state.

And, after doing some research it seems to me that by lowering the extracellular ATP, this vicious cycle perhaps could be corrected.
 
Messages
53
I don't want to lower uric acid. It just happens that probenecid, which has antipurinergic properties, also lowers uric acid levels. So this is an unwanted affect.
This is interesting. I have CFS and also gout/high uric acid, which I am controlling with allopurinol. Can you suggest some links to read up on probenecid in relation to CDR?
 

SlamDancin

Senior Member
Messages
557
This is interesting. I have CFS and also gout/high uric acid, which I am controlling with allopurinol. Can you suggest some links to read up on probenecid in relation to CDR?
Any side effects from Allopurinol?
 

Murph

:)
Messages
1,800
Purinergic signalling is a great topic. Key purine is also the key energy molecule: ATP. If we're signalling with it, we're not making energy with it.

I want to say 3 things about how it may apply.

1. sleepiness is brought on by the accumulation of adenosine. adenosine is part of the purine metabolism, it gets made as we break down other purines. There's some signals of adenosine being too high in mecfs in the old naviaux metabolite paper. Are we leaking too much atp and building up adenosine too easily /too soon? is that why we need naps?!

2. @necessary8 has a long thread on purinergic signalling that's worth a look. The basic idea is that cells use ATP for energy, so when they spit them out as a danger signal, it's a highly credible signal. They wouldn't spit it out unless they had to. Then the body perhaps fails to break down those ATP (via cd38) so they signal for longer than they are supposed to. Also, ATP doesn't hit the circulation, it just stays in a "halo" around the cell it came from, signalling to nearby cells and back to the cell itself. So you can't expect blood tests to find it.
https://forums.phoenixrising.me/thr...rsuit-of-a-unified-me-cfs-theory.55801/page-6

3. My own obsession is vasodilation and POTS and how that ties in with me/cfs. If blood vessels aren't dilating properly to let red blood cells through, one way they signal to the blood vessel is by squirting out ATP.
This happens mechanically: they just get squashed by trying to jostle down the narrow tube. Out comes ATP. Seems to me blood vessel dilation has gone wrong in pots-mecfs. Whether the red blood cells have stiff membranes so no atp is coming out, or whether the vessels are unresponsive so red blood cells are exhaustign their atp load, I don't know. but I bet there's something going on there.

in summary i'm very keen for more purinergic research!!
 

datadragon

Senior Member
Messages
404
Location
USA
sleepiness is brought on by the accumulation of adenosine. adenosine is part of the purine metabolism, it gets made as we break down other purines. There's some signals of adenosine being too high in mecfs in the old naviaux metabolite paper. Are we leaking too much atp and building up adenosine too easily /too soon? is that why we need naps?!

Here is a link of Adenosine A3 receptor and cgmp/Nitric Oxide also to 5-HT

The adenosine A3 receptor, also known as ADORA3, is an adenosine receptor, but also denotes the human gene encoding it. Adenosine A3 receptors regulate serotonin transport via nitric oxide and cGMP. Activation of an A3 adenosine receptor results in an increase of 5-hydroxytryptamine (5HT) uptake in RBL cells, due to an increase in maximum velocity (Vmax). The A3 adenosine receptor-stimulated increase in transport is blocked by inhibitors of nitric oxide synthase and by a cGMP-dependent kinase inhibitor. In fact, compounds that generate nitric oxide (NO) and the cGMP analog 8-bromo-cGMP mimicked the effect of A3 receptor stimulation, suggesting that the elevation in transport occurs through the generation of the gaseous second messenger NO and a subsequent elevation in cGMP. https://pubmed.ncbi.nlm.nih.gov/7525554/

These complex sleep/wake data of 5-HT1A ligands suggest that 5-HT1A receptor activation may increase waking, increase slow wave sleep or increase REM sleep depending on where the 5-HT1A receptors are located within the central nervous system. https://pubmed.ncbi.nlm.nih.gov/9886141/ and another https://magazine.caltech.edu/post/serotonin-sleep

Recent data suggest that interleukin-1-induced enhancement of non-rapid eye movement sleep is mediated, in part, by the serotonergic system. To determine if sleep changes induced by interleukin-1 are mediated by a specific serotonergic receptor subtype, we evaluated interleukin-1 effects on sleep in rats pretreated with the 5-hydroxytryptamine (serotonin)-2 receptor antagonist ritanserin. Ritanserin (0.63 mg/kg, intraperitoneally) by itself did not alter sleep-wake behavior, although it did reduce cortical brain temperature. Interleukin-1 (5 ng, intracerebroventricularly) enhanced non-rapid eye movement sleep, suppressed rapid eye movement sleep, and induced a moderate febrile response. Pretreatment with ritanserin completely blocked the febrile response to interleukin-1 and abolished the interleukin-1-induced enhancement in non-rapid eye movement sleep that occurred during postinjection hours 3-4, without altering interleukin-1 effects on rapid eye movement sleep. The present data suggest that serotonin may partially mediate interleukin-1 effects on sleep by interacting with 5-hydroxytryptamine (serotonin)-2 receptors. These results also suggest that interactions between the serotonergic system and interleukin-1 may be important in regulating sleep-wake behavior. https://pubmed.ncbi.nlm.nih.gov/10408622/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568388/
 
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SlamDancin

Senior Member
Messages
557
“Results: We report that Meloxicam (MXC) inhibits intracellular DNA-, but not RNA-induced immune responses. We find that MXC inhibits the phosphorylation of STING by examining in different cells with various DNA stimulations. We further find that MXC significantly dampens the expression levels of interferon-stimulated genes (ISGs) by using DNA 3' repair exonuclease 1 (TREX1)-deficient cell, an experimental model for self-DNA-induced autoimmune disease. Importantly, we demonstrate that MXC could promote the survival in Trex1-/- mouse model for Aicardi-Goutières syndrome (AGS).”

https://pubmed.ncbi.nlm.nih.gov/37120570/

Not sure there is evidence of classical DNA induced autoimmunity in ME/CFS but Suramin also inhibits STING/ISGs and I’m about to try to see if I get any added benefit of Meloxicam over Ibuprofen
 

datadragon

Senior Member
Messages
404
Location
USA
@serg1942,

For example, when renal human cells are infected in vitro with HHV-6A virus, the infection is halted when a P2X7 antagonist is applied. Here, the intracellular calcium doesn't increase when the anti-purinergic drug is added, probably indicating that the CDR is effectively prevented within the cell:

https://www.frontiersin.org/articles/10.3389/fphar.2020.00096/full

For future reference if needed:

P2X7 receptor (P2X7R) is required for secretion of IL-1, and can be blocked by divalent cations such as magnesium (Mg). We demonstrated that Magnesium sulfate is efficacious in blocking IL-1-mediated-inflammation in HUVECs via downregulation of P2X7Rs on HUVECs https://pubmed.ncbi.nlm.nih.gov/31493768/ P2X7R is involved in the progression of atherosclerosis by promoting NLRP3 inflammasome activation. Stimulation with oxidized low-density lipoprotein (oxLDL) upregulated P2X7R, NLRP3 and interleukin (IL)-1 expression. Oxidized low-density lipoprotein (oxLDL) promotes THP-1 macrophage production and the release of interleukin-1 (IL-1) by activating the purinergic 2X7 receptor (P2X7R)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway. P2X7R knockdown by siRNA suppressed NLRP3 inflammasome activation by inhibiting the PKR phosphorylation mediated by oxLDL. P2X7R plays a significant role in the development of atherosclerosis and regulates NLRP3 inflammasome activation by promoting PKR phosphorylation.
https://pubmed.ncbi.nlm.nih.gov/25761252/

The marginal vitamin B6 deficiency appears to relate to an increased risk of inflammation-related diseases, Recent studies have revealed that vitamin B6 treatment increases cardiac levels of imidazole dipeptides (eg, carnosine, anserine, and homocarnosine), histamine, and -aminobutyric acid (GABA) and suppresses P2X7 receptor-mediated NLRP3 inflammasome. These modulations may implicate possible cardioprotective mechanisms of vitamin B6. These modulations may also be involved in the underlying mechanisms through which vitamin B6 suppresses oxidative stress and inflammation. (the active pyridoxal 5 phosphate (P5P) NOT pyridoxine which lowers active b6) https://link.springer.com/article/10.1007/s00394-021-02665-2
 

datadragon

Senior Member
Messages
404
Location
USA
@SlamDancin @serg1942

PPAR-a inhibits STING. timing is important where inflammation is actually very important in early stages of viral infection (Type I interferon mediates an important innate immune response against viral infection by directly inhibiting viral replication.) and if you inhibit it too early there can be higher viral titers when challenged with influenza A virus etc during the early stages of infection, but it IS beneficial to inhibit in a later stage if the virus was able to replicate and was at that later stage causing inflammation because you did not have a good immune response early on, as inhibiting in later stages experienced less tissue damage to lungs. .

Metabolic Control of Viral Infection through PPAR-α Regulation of STING Signaling. PPAR ligands (activators) are immunosuppressive and can increase susceptibility to infection therefore it appears should not be increased greatly in early infection. PPARs mechanisms of action are diverse and include repression of NFκB and AP-1 DNA binding, regulation of nitric oxide, inhibition of dendritic cell maturation, reduction of cytokine expression by effector T cells, and inhibition of leukocyte recruitment to sites of inflammation. Activation of PPAR-α with specific agonists increased herpesvirus replication and reactive oxygen species (ROS) production. ROS inhibited activation of stimulator of interferon (STING), an ER adapter that induces type I interferon downstream of cytoplasmic DNA recognition. Although high ROS induces inflammasome activation and cytokine production, we found that ROS inhibited interferon production. Treatment of mice with a clinically relevant agonist of PPAR-α increased herpesvirus replication and pathogenesis, comparable to levels observed previously in type I interferon receptor knockout mice. These findings show that activated PPAR-α regulates immunity to cytoplasmic DNA, inhibits interferon production, and increases susceptibility to viral infection (in early stages of infection). Moreover, these results demonstrate that ROS inhibit STING activation and induction of interferon https://www.biorxiv.org/content/10.1101/731208v3

STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation.
STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. https://pubmed.ncbi.nlm.nih.gov/34986331/

Butyrate is a lipid produced by intestinal bacteria that can regulate inflammation throughout the body. Here we show for the first time that butyrate influences the innate antiviral immune response mediated by type I IFNs. A majority of antiviral genes induced by type I IFNs were repressed in the presence of butyrate, resulting in increased virus infection and replication in cells. We found that butyrate increases cellular infection and virus replication in influenza virus, reovirus, and human immunodeficiency virus infections. In sum, the net effect of butyrate on infection with three divergent RNA viruses was an increase in cellular infection and replication. Further exploring this phenomenon, we found that addition of butyrate to cells deficient in type I interferon (IFN) signaling did not increase susceptibility to virus infection. Accordingly, we discovered that butyrate suppressed levels of specific IFN stimulated gene (ISG) products in human and mouse cells. Butyrate did not inhibit IFN-induced phosphorylation of transcription factors STAT1 and STAT2 or their translocation to the nucleus, indicating that IFN signaling was not disrupted. Rather, our data are suggestive of a role for inhibition of histone deacetylase activity by butyrate in limiting ISG induction including RIG-I. Global transcript analysis revealed that butyrate increases expression of more than 800 cellular genes, but represses IFN-induced expression of 60% of ISGs https://www.biorxiv.org/content/10.1101/2020.02.04.934919v1
 
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