Oxidative Stress is a shared characteristic of ME/CFS and Long COVID, 2024, Shankar, Bonilla, Davis et al.

[Turtle]

No problem. I think it's also fair to suggest that I should sometimes read more before commenting. Unfortunately, for this study, not even the reported sex ratios make any sense to me and that doesn't make me enthusiastic to spend my energy to dive further into it.

Being active on this forum one quickly comes to the realisation that the majority of studies are in fact not particularly interesting at all and especially when it comes to LC studies, 95% of the time it suffices to look at patient selection to understand that the study can be discarded as "not interesting at all" (which I'm not suggesting is the case here).

Did you notice that Ron Davis (Stanford) is one of the authors. If mistakes are made they should be corrected, let me clear about that.
But in my opinion Ron Davis deserves a lot more credit than the NIH, given his record on ME/CFS research. And his personal reason: finding a cure for his son, Whitney Dafoe. Dismissing the whole paper because of some misstakes?

 

[Mij]

#MECFS2023 Next Vishnu Shankar, immunologist in Mark Davis’ lab at Stanford is talking about oxidative stress and suppression of mitochondrial ATP levels in MECFS and Long Covid patients, and reactive oxygen species. He was inspired to work on ME by reading The Puzzle Solver book.

September 8, 2023

 

[EndME]

There is reference to a methods section, but I couldn't see one in that file. I searched on 'Methods' and couldn't find the section.

This seems to be in referrence to the "Methods Description" section in the supplementary material (can be downloaded here https://www.biorxiv.org/content/10.1101/2024.05.04.592477v1.supplementary-material), but unfortunately even that section doesn't contain the necessary details. It does state that 60% of LC patients meet IOM criteria for ME/CFS, but nothing about symptomology or severity of the acute infection of the LC group.

 

[richie]

I agree with Jonathan i.e. Oxidative Stress has been claimed to be the cause of all manner of things. If it were true then I'm guessing that DecodeME would turn up clues e.g. genes liked to selenium. Also, rare genetic variant/whole genome sequencing (family) studies should identify cases. The other thing is that presumably there'd be clues re immunology - I vaguely remember something re this from my time as a laboratory technician -- 40 ish years ago!
EDIT - haven't read the paper!

There was a guy in South Africa , Nash Petrovic, who supplied tailored antioxidant and other supps and had some successes among ME diagnosed. Of course the group is heterogenous (I believe anyway) but he may have helped some.

 

[EndME]

Did you notice that Ron Davis (Stanford) is one of the authors. If mistakes are made they should be corrected, let me clear about that.

But in my opinion Ron Davis deserves a lot more credit than the NIH, given his record on ME/CFS research. And his personal reason: finding a cure for his son, Whitney Dafoe. Dismissing the whole paper because of some misstakes?

Of course, I’m very aware of that and I also noticed that one of the authors is Mark Davis who is a world-class immunologist for whom I have a lot of respect and similarly Bonilla is very well-known in the field and has a lot of expertise. All of these authors have my respect and obviously they know far more than I'll ever know about immunology, biology and genetics.

Others with far more expertise in the field have commented on the immunological aspects of the study already. I typically try to look at cohort characteristics, simply because I lack all the necessary background an immunologist has. But given the errors currently present, and even more importantly the lacking patient description, mentioned by @Hutan, which is strictly necessary to judge any LC data, there really is little for me to do and anything left to do seems like it isn't currently worth the energy for me, simply because if you perform a large array of tests on a very small sample set you can always find a positive result here and there simply by luck alone. I cannot tell if any of this data is causative and that's really the only thing that interests me. Should this work be followed-up by a larger study, I would be extremely happy to read it. Similarly if someone findings parts of the paper that are interesting and worth scrutinising I will pay attention to what they are saying.

One should not forget that it’s very possible to be dismissive of a paper, which I don't even think I've been, without being dismissive of the people having written it, any of their previous work or their intentions. To support this with an example: I have a lot of admiration and respect for the all of the work of the great Akiko Iwasaki, but that doesn't change the fact that the LC cortisol claims looked bogus to me.

I view the role of this forum to critically analyse data, independently of ones own beliefs or who the authors are.

 

[Trish]

Our aim here is to enable open discussion of any published science without fear or favour. That way we hope to help each other to understand how research works and whether published results and claims are well founded. It's not about whether we respect individual researchers or not, it’s about what the published work shows. My ideal would be to have the people who do the research joining in the discussion too.

 

[FMMM1]

There was a guy in South Africa , Nash Petrovic, who supplied tailored antioxidant and other supps and had some successes among ME diagnosed. Of course the group is heterogenous (I believe anyway) but he may have helped some.

Interesting, wondering if this was done as a trial i.e. biomarker (indicating oxidative stress), blinded - control group get placebo and trial group supplement?

 

[FMMM1]

I agree with Jonathan i.e. Oxidative Stress has been claimed to be the cause of all manner of things. If it were true then I'm guessing that DecodeME would turn up clues e.g. genes liked to selenium. Also, rare genetic variant/whole genome sequencing (family) studies should identify cases. The other thing is that presumably there'd be clues re immunology - I vaguely remember something re this from my time as a laboratory technician -- 40 ish years ago!
EDIT - haven't read the paper!

My post above was based on working as a technician in a veterinary research laboratory which (among other things) looked at trace minerals/deficiency diseases. Selenium deficiency (which effectively equates to oxidative stress) was one of the two main areas (cobalt/B12 was the other). This was looking at the situation where selenium was severely limited - I guess you could have milder scenarios which are clinically relevant - perhaps sort of an echo of the problem i.e. rather than full blown selenium deficiency/oxidative stress as cause of pathology/disease.

 

[richie]

Interesting, wondering if this was done as a trial i.e. biomarker (indicating oxidative stress), blinded - control group get placebo and trial group supplement?

As far as I know no. I am in the anything that helps camp, but I share any concerns about trumpeting sth as science which is not. about absolute and /or monolithic claims re treatments, aetiologes etc.

 

[InitialConditions]

I get the impression the work on ME/CFS at Stanford is not particuarly well coordinated. I don't see research themes emerging. Are they trying to do to much at once?

The abstract for this paper does not give me faith, and people here have rightly pointed out several errors already.

 

[DMissa]

"Using flow cytometry, we compared mitochondrial bioenergetic parameters in CD19 B cells as well as CD4 and CD8 T cell lymphocyte populations. Sample gating strategy for identifying these lymphocyte populations is shown in Supplementary Figure S1A. Although we did not identify any differences between healthy controls and ME/CFS or LC donors in mitochondrial mass or membrane potential (Supplementary Figure S2), decreases in mitochondrial ATP levels were observed across ME/CFS (1.6x decrease on average; HC v. ME/CFS two-sided t-test CD4 T cells p = 0.0458, CD8 T cells p = 0.0974, CD19 B cells p = 0.356) and LC donors (2.8x decrease on average; HC v. LC two-sided t-test CD4 T cells p = 0.0076, CD8 T cells p = 0.0182, CD19 B cells p = 0.0842), compared to healthy controls (Supplementary Figure S2). Our findings are consistent with a published study showing oxidative phosphorylation defects among PBMCs in CFS patients, compared to healthy controls69"

Unstimulated PBMCs are quiescent and when taken out of the body are also dying (every researcher around the world that I have spoken to who has measured this in time course experiments also finds that they die faster in ME/CFS). Naturally you will see generalised decreases in functional indicators and generalised increases in damaging or dysfunctional indicators in ex vivo disease cells that are falling apart at the seams. ROS could easily be a cause of this death/dysfunction but it could also be an consequence. This needs to be tested directly in a future study.

"Based on our results in Figure 3A, which also detected changes in acylcarnitine metabolism in ME/CFS donors, we compared lipid droplet levels using HCS LipidTox Green Neutral Lipid staining, which stains for neutral intracellular lipid droplets. As shown in Supplementary Figure 4G, we found lower lipid droplet levels in both LC and ME/CFS donors across all measured lymphocyte populations (ME/CFS: CD19 p = 5.19*10-4 , CD4 p = 4.58*10-4 , CD8 p = 3.01*10-4 ; LC: CD19 p = 0.140, CD4 p = 0.0669, CD8 p = 0.0759)."

This is a commercial dye so details about specificity and structure are scarce but one thing to bear in mind is that many neutral lipid stains are not specific to LDs and if you do whole-cell fluorescence measurements with them you will pick up signal from other cellular neutral lipids as well, not just LDs. Plasma membrane is like 40% neutral lipid or something significant like that from memory btw. This is why the chemists we worked with in the LD paper are making alternatives that are intended to be more specific. Non-LD background is a huge issue with a lot of neutral lipid dyes. I wish they did some LD imaging to complement this or that more information about the dye was available from Thermo.

Would have been good to see the kinetics of T cell stimulation between individuals compared given that the measurements were taken at the same 5 day time point. You can have cells from one individual start waning in response earlier than others particularly with this length of time from what I understand.

"We suggest that this lymphocyte dysfunction, driven by oxidative and mitochondrial damage, acts as an “energy sink”, much as an active infection does, draining the body of available energy and giving rise to debilitating fatigue and other sequelae."

Do people with ME/CFS gain less weight with comparable dietary intake? Do they have altered blood glucose? (genuinely asking, I don't know off the top of my head)

 

[Hutan]

decreases in mitochondrial ATP levels were observed across ME/CFS (1.6x decrease on average; HC v. ME/CFS two-sided t-test CD4 T cells p = 0.0458, CD8 T cells p = 0.0974, CD19 B cells p = 0.356) and LC donors (2.8x decrease on average; HC v. LC two-sided t-test CD4 T cells p = 0.0076, CD8 T cells p = 0.0182, CD19 B cells p = 0.0842), compared to healthy controls (Supplementary Figure S2). Our findings are consistent with a published study showing oxidative phosphorylation defects among PBMCs in CFS patients, compared to healthy controls69"

That decreased level of mitochondrial ATP in ME/CFS and LC sounds interesting and large. A '1.6x decrease' seems an odd way to express it - '0.6 of HC' would be clearer. Although they say that this result is consistent with another study, we have seen studies where mitochondrial DNA is not different. I too am concerned that this is all just a function of treatment and storage time of the samples.

we found lower lipid droplet levels in both LC and ME/CFS donors across all measured lymphocyte populations (ME/CFS: CD19 p = 5.19*10-4 , CD4 p = 4.58*10-4 , CD8 p = 3.01*10-4 ; LC: CD19 p = 0.140, CD4 p = 0.0669, CD8 p = 0.0759)."

Again, interesting and statistically significant. Thanks for the information about the staining and good luck to your chemists working on better ones.

Do people with ME/CFS gain less weight with comparable dietary intake?

No, I don't think we have any evidence of that. I don't think the metabolic chamber work that was part of the NIH study found anything notable. Some people with severe ME/CFS are very thin, but I think that's a function of the difficulty of getting food in and digested.

Do they have altered blood glucose?

No, that's not a characteristic of ME/CFS. In fact it might almost be the opposite; I haven't heard of many/any people with ME/CFS and any type of diabetes. ?

 

[DMissa]

Thanks Hutan, that confirms my gut suspicions but I wasn’t certain enough to comment. Good to know.

 

[Hutan]

Just on my diabetes +ME/CFS comment, the MEA has a note on that https://meassociation.org.uk/medical-matters/items/diabetes-mecfs/#
where they say

Although the evidence is only anecdotal, we do receive fairly regular reports about people with ME/CFS going on to develop type 2 diabetes. I suspect that type 2 diabetes may be slightly more common in some groups of people who have ME/CFS. I don’t think this is hormone related – because there is no evidence of pancreatic disease in ME/CFS. It is much more likely to be an effect of the changes in lifestyle that often occur as a result of having ME/ CFS – decreased levels of activity and weight gain in particular.

There is, incidentally, no sound research evidence to suggest that type 1 diabetes (insulin requiring) is more common in ME/CFS. But it would be interesting to do some proper research on the incidence of type 2 diabetes and ME/CFS – so we might start with an ME Association website survey. One of the main symptoms of diabetes is fatigue – so having both ME/CFS and diabetes will almost certainly exacerbate fatigue levels.

 

[Deanne NZ]

@DMissa & @Hutan - Re the question on PwME gaining less weight with comparable dietary intake I can add that this is the case for my son & at least a couple of other teen & young adult males according to the mums in a FB group. I realise it is only anecdotal but I did calculate my son’s intake for several days & it was 3,700 - 4,000 to maintain his weight. Perhaps there is a subset of tall & very skinny males for who this is an issue, although they were much younger at onset and in my son’s case average height & weight for age at that time.

 

[Amw66]

@DMissa & @Hutan - Re the question on PwME gaining less weight with comparable dietary intake I can add that this is the case for my son & at least a couple of other teen & young adult males according to the mums in a FB group. I realise it is only anecdotal but I did calculate my son’s intake for several days & it was 3,700 - 4,000 to maintain his weight. Perhaps there is a subset of tall & very skinny males for who this is an issue, although they were much younger at onset and in my son’s case average height & weight for age at that time.

Yes, that's the case for my daughter too- so females also .
Re diabetes - given there seem to be issues with glycolysis, and the body work around may have different response re male/ female metabolism any poll would be interesting to see if there is a split manifest.

I think also that looking at liver function is a neglected area - both for detox, but also as the liver modulates many processes- many of which may play a part in ME/CFS - gluscose metabolism, hormone synthesis, immune response etc; the liver gut axis may be important given hints at microbiome dysbiosis too.

From a recent dip into diabetes for MIL, there seems to be a number of T2 diabetics who are insulin dependent ( i have heard 25% mooted , but don't know the source for this), so nothing is ever as clear cut as it may seem.

 

[Jaybee00]

Was this ever published? Close to a year now since posting the preprint.

 

[Utsikt]

Was this ever published? Close to a year now since posting the preprint.

It’s not on his list at Google Scholar, only the preprint.

 

[SNT Gatchaman]

Now published in PNAS —

Oxidative stress is a shared characteristic of ME/CFS and Long COVID
Shankar, Vishnu; Wilhelmy, Julie; Curtis, Ellis J.; Michael, Basil; Cervantes, Layla; Mallajosyula, Vamsee; Davis, Ronald W.; Snyder, Michael; Younis, Shady; Robinson, William H.; Shankar, Sadasivan; Mischel, Paul S.; Bonilla, Hector; Davis, Mark M.

ABSTRACT
Over 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition marked by fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). With no clinically approved treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions.

By studying bioenergetic characteristics of peripheral blood lymphocytes in 25 healthy controls, 27 ME/CFS, and 20 LC donors, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, especially in the memory subset. Using a combination of flow cytometry, RNA-seq, mass spectrometry, and systems chemistry analysis, we observed aberrations in reactive oxygen species (ROS) clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase protein levels, and glutathione peroxidase 4–mediated lipid oxidative damage.

Strikingly, these redox pathways changes show sex-specific trends. While ME/CFS females exhibit higher total ROS and mitochondrial calcium levels, males have normal ROS levels, with pronounced mitochondrial lipid oxidative damage. In females, these higher ROS levels correlate with T cell hyperproliferation, consistent with the known role of elevated ROS in initiating proliferation.

This hyperproliferation can be attenuated by metformin, suggesting this Food and Drug Administration (FDA)-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Moreover, these results suggest a shared mechanistic basis for the systemic phenotypes of ME/CFS and LC, which can be detected by quantitative blood cell measurements, and that effective, patient-tailored drugs might be discovered using standard lymphocyte stimulation assays.

SIGNIFICANCE
More than 65 million individuals worldwide are estimated to have Long COVID (LC), wherein individuals after infection report persistent fatigue, postexertional malaise, and other symptoms resembling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). With no clinically approved treatments or diagnostic markers for these conditions, there is an urgent need to define the molecular underpinnings. By studying bioenergetic characteristics of immune cells in healthy controls, ME/CFS, and LC donors, we find lymphocytes from ME/CFS and LC donors exhibit elevated oxidative stress. Due to excess oxidative stress and consequent mitochondrial damage, ME/CFS and LC donor lymphocytes consume excess host energy, contributing to debilitating fatigue and other sequelae.


Link | PDF | Proceedings of the National Academy of Sciences [Open Access]

 

[wigglethemouse]

Note on Funding, reformatted for ease of reading.

We are grateful for funding by the
* National Institute of Allergy and Infectious Diseases (U19-AI057229, 5R01AI139550) and the
* Howard Hughes Medical Institute to M.M.D., along with funding from the
* NIH to M.S. (5RM1HG00773510).
* We also acknowledge funding from the Khosla family gift fund.