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

[SNT Gatchaman]

Oxidative Stress is a shared characteristic of ME/CFS and Long COVID
Vishnu Shankar; Julie Wilhelmy; Basil Michael; Layla Cervantes; Vamsee Mallajosyula; Ronald Davis; Michael Snyder; Shady Younis; William H Robinson; Sadasivan Shankar; Paul Mischel; Hector Bonilla; Mark Davis

More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). With no current 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 over 16 healthy controls, 15 ME/CFS, and 15 LC, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we also observed aberrations in ROS clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase levels, and glutathione peroxidase 4 mediated lipid oxidative damage.

Critically, these changes in redox pathways show striking sex-specific trends. While females diagnosed with ME/CFS exhibit higher total ROS and mitochondrial calcium levels, males with an ME/CFS diagnosis have normal ROS levels, but larger changes in lipid oxidative damage. Further analyses show that higher ROS levels correlates with hyperproliferation of T cells in females, consistent with the known role of elevated ROS levels in the initiation of proliferation. This hyperproliferation of T cells can be attenuated by metformin, suggesting this FDA-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients.

Thus, we report that both ME/CFS and LC are mechanistically related and could be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.


Link | PDF (Preprint: BioRxiv) [Open Access]

 

[EndME]

What is "over 16 healthy controls, 15 ME/CFS, and 15 LC" supposed to mean?

I haven't looked at the study at all but there is a large discrepancy between the sex ratios (HC: 46% female, ME/CFS: 77% female, LC: 53% female).

 

[Turtle]

What is "over 16 healthy controls, 15 ME/CFS, and 15 LC" supposed to mean?

I haven't looked at the study at all but there is a large discrepancy between the sex ratios (HC: 46% female, ME/CFS: 77% female, LC: 53% female).

So what?

 

[EndME]

So what?

It might be of relevance since the authors report sex differences in their findings "Particularly striking was that only female patients had elevated ROS levels in ME/CFS and LC, together with the hyperproliferation of lymphocytes after stimulation in culture, whereas both sexes showed evidence of elevated levels of reduced glutathione and lipid oxidative damage." But like I've said I haven't even read the study yet.

 

[Turtle]

It might be of relevance since the authors report sex differences in their findings "Particularly striking was that only female patients had elevated ROS levels in ME/CFS and LC, together with the hyperproliferation of lymphocytes after stimulation in culture, whereas both sexes showed evidence of elevated levels of reduced glutathione and lipid oxidative damage." But like I've said I haven't read the study yet.

Then maybe you could try to read it before you write?

 

[EndME]

Then maybe you could try to read it before you write?

It is very common on this forum for not everybody to have the energy and cognitive abilities to read every single study the moment it comes out. They way I've seen this forum work is that typically a bunch of different observations are made until more definite conclusions can be made.

Since you seem to be commenting on a study and demand that it must be read before doing so, can you explain which results might or might not be driven by general sex differences rather than health status (of course they looked at everything split into M and F as well but the sample sizes for that then might become less reliable even though some of the differences look striking in the graphs)?

 

[Jonathan Edwards]

I am not motivated to read further than the abstract here I am afraid. In the past an abstract was supposed to contain the highest concentration of hard numerical data you could fit in, with minimum of interpretation. The reader could do the interpreting. These days so many papers fill an abstract with speculative interpretation and no data at all. The overall impression is that a vast number of measurements have been made on a rather small sample and the few results that look as if they might support a theory are selected for that purpose.

 

[EndME]

Either I am getting the percentages wrong or there is something wrong about the data and sex ratios:

Sample sizes
16 HC (46.6% Female), 15 ME/CFS (76.9% Female), 15 LC (53.3% Female).

According to the plots 7 out of 16 HC’s are female, however 7/16 is not 46.6% but rather 43.75%, so this would appear to be a mistake (7/15=46.6% so that might have been the mix-up). I hope they didn’t work with this in their statistical analysis.

Similarly I don’t understand how 76.9% of ME/CFS patients could be females (12/15=80% and 11/15=73.3) and according to the plots it looks like it should only be 10 ME/CFS females.

For LC it is clear that 53.3% of LC patients being female would mean 8 patients which is also what the data shows.

I wonder, if they are indeed mistakes, who much these mistakes would influence the results. It's hard for me to understand how they could have gotten the sex ratios wrong in a study that is focused on finding sex differences in M/F, so I'm hoping there is an alternative explanation.

 

[Hutan]

It is very common on this forum for not everybody to have the energy and cognitive abilities to read every single study the moment it comes out. They way I've seen this forum work is that typically a bunch of different observations are made until more definite conclusions can be made.

Yes. I often present my reactions as I read, noting questions that I would like to see answered later on. To read the whole report, making notes as I go and then writing up a succinct synopsis would take me much longer, and I might not ever get to post anything. Think of our process as a reading club, with a bit of a baton passing going on, as some of us have to drop out for a rest.

It's valid to make a response to the abstract, after all, most readers of the paper will only read the abstract. It's valid to note when the number of participants is vague, as in 'over 16 controls'. There will probably be a good explanation for that wording, but we can get to that. It's also valid to note when groups are not matched well - that can cause all sorts of problems.

We're not aiming for a perfect entire response in the first posts here. Sometimes we misunderstand, and that's okay. Someone can explain and some of us might learn something. And the misunderstandings might give the writers of papers an opportunity to work out how to write clearer better papers.

 

[SNT Gatchaman]

I am not motivated to read further than the abstract here I am afraid.

There's a lot in the paper and possibly you'd need 5 separate papers to have a comprehensive abstract with the summary data in each. There's a lot that's interesting about T cell proliferation scaling with oxidative stress. They suggest that T cells proliferate more readily and that leads to far greater energy consumption (and starvation for other systems). I haven't finished reading but I'd have to wonder if T cell proliferation during the follicular (proliferative) phase of the menstrual cycle may explain cyclical symptom increase.

Can I entice you with the following?

The conversion of lipid peroxides to lipid alcohols is catalyzed by glutathione peroxidase 4 (GPX4), which protects lipids from ROS damage. [...] Comparison of GPX4 mean pixel intensity from immunofluorescence staining across 426 HC, 603 ME/CFS, and 239 LC CD3 T cells shows that LC and ME/CFS donor lymphocytes have higher (1.77x and 1.9x, respectively) GPX4 levels, when compared to T cells from healthy controls (p < 2.22 * 10-16 for both ME/CFS and LC v. HC comparisons, two-sided t-test).

 

[Turtle]

It is very common on this forum for not everybody to have the energy and cognitive abilities to read every single study the moment it comes out. They way I've seen this forum work is that typically a bunch of different observations are made until more definite conclusions can be made.

Since you seem to be commenting on a study and demand that it must be read before doing so, can you explain which results might or might not be driven by general sex differences rather than health status (of course they looked at everything split into M and F as well but the sample sizes for that then might become less reliable even though some of the differences look striking in the graphs)?

I apologise to you @EndME for not getting the purpose of this forum. And I also apologise for upsetting you.
Together with what @Hutan wrote I get it better now.

 

[EndME]

I apologise to you @EndME for not getting the purpose of this forum. And I also apologise for upsetting you.
Together with what @Hutan wrote I get it better now.

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).

 

[Hutan]

Some pedantry about the introduction:

it has been estimated that as many as 65 million individuals may have “Long COVID”, a complex multisystemic condition associated with post-acute sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection (PASC)1.Long COVID spans all ages, affecting even those who have experienced only moderate COVID infections, with 10-12% vaccinated, 10-30% of non-hospitalized, 50-70% of hospitalized COVID-19 infection survivors estimated to endure persistent symptoms after infection1,2, with symptoms affecting individual quality of life and functional status71.

Although the clinical presentation of Long COVID (LC) is highly heterogeneous, with adverse events spanning multiple organ systems from dysrhythmia and higher incidence of cardiac disorders1,3 to neurological and cognitive deficits such as memory loss and “brain fog”2,4, there are several common shared symptoms of LC, including fatigue (estimated pooled prevalence of 47%), shortness of breath (32%), and muscle pain (25%)5,73.
Strikingly, the clinical presentation of LC strongly resembles myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a complex chronic disease estimated to affect between 836,000 to 2.5 million individuals in the US alone7.

I thought people were moving on from Long Covid being one thing, to recognise that there are quite a few reasons for people to have persistent symptoms after Covid-19, with ME/CFS being just one of them. But, these authors aren't recognising that. It contributes to muddiness around the what is going on - what about people who only have mild infection? And the commentary makes it look as though an ME/CFS-like condition is most common in hospitalised survivors.

By meta- analysis, it is estimated that 0.01 to 7.62% of the world population has ME/CFS

I don't think that is helpful. Nearly 8% of the world population with ME/CFS?

Based on three symptoms defined by the National Academy of Medicine criteria7, ME/CFS is characterized by (1) profound fatigue lasting for at least 6 months,

That's not strictly correct. The IOM/NAM SEID criteria is

A substantial reduction or impairment in the ability to engage in pre-illness levels of activity (occupational, educational, social, or personal life) that:

1. lasts for more than 6 months
2. is accompanied by fatigue that is:
   1. often profound
   2. of new onset (not life-long)
   3. not the result of ongoing or unusual excessive exertion
   4. not substantially alleviated by rest

It's the impact on function that is the core thing lasting more than 6 months. There is fatigue too, and it's often profound, but it's not necessarily profound all of the time. I think writing it in the way this paper does gives a misleading impression of ME/CFS. We've heard recently of some people with ME/CFS who, in the first months of their illness, did not recognise what they had from the descriptions of chronic fatigue.

Fatigue is the principal hallmark of ME/CFS and also one of the most common symptoms among LC2,5,9 patients.

And they say it again.

Since fatigue implies over and/or misallocation of cellular energy on disease processes,

I don't think fatigue necessarily implies that. I think some of the statements about cytokines are a bit arguable - I don't think we really have solid evidence on cytokines. I don't think we have seen much that is credible on serotonin either.

 

[Eddie]

There's a lot in the paper and possibly you'd need 5 separate papers to have a comprehensive abstract with the summary data in each. There's a lot that's interesting about T cell proliferation scaling with oxidative stress. They suggest that T cells proliferate more readily and that leads to far greater energy consumption (and starvation for other systems). I haven't finished reading but I'd have to wonder if T cell proliferation during the follicular (proliferative) phase of the menstrual cycle may explain cyclical symptom increase.

How much energy does it take for T cells to proliferate? It is plausible to suggest that the proliferation of a certain type of immune cell has a non-negligible impact on overall energy use? I have always found it hard to believe that this type of energy consumption could be anywhere near the energy required to move or think.

 

[Hutan]

We also focused on immune cells because they are a critical consumer of host energy and regulator of host metabolism. Specifically, the immune system accounts for 15-20% daily energy expenditure in humans18,19.
This usage is thought to increase to 25%, during a serious infection, highlighting the significant energy expenditure required for maintenance, activation, and proliferation of immune cells13-14.

That's interesting, I didn't know that. (Edit, like Eddie, I find it a bit hard to believe that's true for e.g. an active adult or a growing child.)


In the Results section, there is this about participant selection and source of the blood samples:

To profile bioenergetic parameters, we obtained healthy control, ME/CFS, and LC PBMCs. ME/CFS and LC donor PBMCs from the ME/CFS Collaborative Research Center and the Stanford Post-Acute COVID Syndrome clinic, respectively. Healthy control patient PBMCs were obtained from the Stanford Blood Bank, where donors were screened using a medical history questionnaire30. ME/CFS patients, including patients meeting the National Academy of Medicine (NAM) ME/CFS criteria before and after the start of the COVID-19 pandemic, were diagnosed by a physician using the National Academy of Medicine7 and Canadian Consensus criteria29. LC donors were diagnosed using the combination of the Center for Disease Control17 criteria for “Long COVID or post-COVID conditions” and a symptom and functional status questionnaire. Together, these measures capture whether patients report new symptoms four or more weeks after COVID infection and incorporates the Post-COVID 19 Functional Status (PCFS) Scale27. Additional details related to patient characteristics in each group are included in the methods section.

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.

The control blood seems to have come from a biobank, whereas the ME/CFS and Long Covid blood seems to have been collected specifically for the study. I'm not sure about that, I'm just basing that on the above paragraph. That raises questions about the type and length of storage - maybe that accounts for differences in some of the subsequent investigations? e.g. T-cell proliferation.

I'm not clear about the potential for overlaps in the ME/CFS and Long Covid groups. The ME/CFS includes patients who met the NAM criteria before and after the pandemic, but that leaves the door open for patients who didn't meet the NAM criteria before and after the pandemic to also be included. Perhaps some of them didn't have ME/CFS before the pandemic and only developed it after Covid-19? I don't know whether any of the LC donors meet ME/CFS criteria or if the LC donor group includes people who have, for example, only persistent loss of taste or difficulty breathing.

 

[Trish]

The sample sizes, especially once they are split into male and female subsets, are so small it's hard to know what to make of these results. I hope they will repeat with much larger cohorts. I wonder whether they could look at the effect of female hormones, and specifically where in the menstrual cycle the woman is, and whether there a difference between pre and post menopause, and whether HRT or other hormone treatments being taken at the time affect the results.

 

[Jonathan Edwards]

Can I entice you with the following?

Not really. I have heard people burbling on about oxidative stress for about 45 years now and have not seen anything that suggests it is of any great relevance to anything.

If you have good quality data you can put them in an abstract. If you have too much data that usually means that you are using a scattershot approach and have no real idea why you are measuring. Important findings do not nee all these complicated subsidiary analyses.

The idea that somehow oxidative status should be crucial to T cell dysfunction is to me pretty implausible. Immunology turns on much more specific things by and large.

 

[Jonathan Edwards]

I don't think fatigue necessarily implies that.

Indeed, the statement referred to seems excessively naive.

 

[Jonathan Edwards]

That's interesting, I didn't know that.

The references 18 and 19 are bot to some guy called Straub, probably a rheumatologist, who I have never heard of, claiming to be able to explain to the rest of physicians how to understand inflammatory disease in terms of energy balance. Nature Reviews Rheumatology is a fluff journal that takes anything that pays I think. I am pretty sure this is just some oddball's speculation.

And anyway none of this makes sense. When I go on a skiing holiday I probably increase my metabolic demand, with six hours maximal effort in freezing temperatures, by 50-70 or even 100%. I feel well exercised at the end of the day but I don't feel 'fatigued' in the sense that ill people do. Human beings can easily accommodate an extra 60% metabolic demand without feeling in any way unwell. The whole thing seems have been dreamt up by someone who has no real grasp of medical science.

If, in contrast, you inject a few micrograms of endotoxin into someone they will feel dreadfully fatigued despite not doing anything much And if you block TNF in RA fatigue melts away within a hour or so. Not a lot of time for proliferation there!

 

[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!