Preprint Indistinguishable mitochondrial phenotypes after exposure of healthy myoblasts to myalgic encephalomyelitis or control serum, 2025, Ryback et al

[chillier]

Could you test severe ME patients using the same methodology, or are there any hindrances like pre-analytical stuff like handling and processing in time etc? Thank you!

Yep, so to be clear we did have severe pwME in the study, just relatively few. I believe something like 5 severe out of 67 total. If you just look at the severe people only there is still no difference between them and the healthy controls. To get more would have required at home sampling which we couldn't do for this study.

Sample processing would be slower as you say, but probably not that big of a deal.

 

[Kitty]

Congratulations to the whole team for such a well run and thorough study—and big thanks for making it so accessible for participants with ME/CFS.

 

[hotblack]

Seems very thorough to me so far.

Normalising for cell count is an interesting addition that they didn’t do in Fluge et al (2016). And could explain a lot.

Beyond that sample size is obviously the clearest difference.

This study also seems to use only women which could be a difference?

Fluge said there was a significant difference in amino acid profiles between men and women but I’m unsure if they tested men and women for cellular metabolism. From a brief look at that paper the precise details of the subgroup of 12 cases tested is unclear.

Fluge collected samples at different points, from different studies and there were 200 patients in total. But only 12 used for cellular metabolism tests. So maybe there’s also some potential for variations in sample handling? “The blood samples were collected before intervention (baseline) in three separate clinical trials.“

Fluge was doing a lot of stuff simultaneously and over different groups and timeframes which potentially confuses things, while this focuses clearly on one area and seems to do so very consistently and thoroughly.

 

[ME/CFS Skeptic]

Just read the full text and it is great to see the methodological quality of the study.

They used blinding, randomization, pre-registration of the analysis plan. They graphs are clear, the statistical models are reported explicitly, there's a good overview of potential limitations. No long discussion section, but all straight to the point.

Well done.

 

[forestglip]

Agree with other posters - very well written and thorough.

I wonder if the diagnostic criteria might be a factor. CCC in Fluge et al, but CCC and/or IOM here, so maybe the factor in the blood wasn't necessarily related to ME/CFS, but was related to a comorbidity required by CCC but not by IOM.

 

[Eddie]

Agree with other posters - very well written and thorough.

I wonder if the diagnostic criteria might be a factor. CCC in Fluge et al, but CCC and/or IOM here, so maybe the factor in the blood wasn't necessarily related to ME/CFS, but was related to a comorbidity required by CCC but not by IOM.

I doubt it, I think the vast majority of people that meet IOM criteria would also meet the CCC criteria. Perhaps there is a difference in the cohorts but it wouldn't be a result of using CCC or CCC and/or IOM. These diagnostic categories allow differences between ME/CFS patients even if they were only using one criteria anyways.

 

[Simon M]

This detail would have been my first thought about the discrepancy between findings. It is extremely admirable to have this level of consideration for participants, though it is a shame that it might skew the ability to replicate findings in this way.

I don't know how significant this is. I quite often don't have symptoms at rest, but I always have the problem of minimal exertion exhausting me. In other words, if the problem was that something in my blood was stopping my mitochondria working properly, I would've expected that something in the blood to be there all the time. Though there might well be more of it during PEM.

I don't know if we have any kind of functional measure of participants, e.g. SF 36 physical function score, but I have thought many would have been physically stressed by going to the lab and having the sample taken.

(Sorry, not well enough to have read the paper yet)

 

[hotblack]

I don't know how significant this is. Eight quite often don't have symptoms at rest, but I always have the problem of minimal exertion exhausting me.

Same. I think I’ve discussed this with @jnmaciuch before and our experiences appear to be quite different wrt PEM triggering.

And if the changes in blood were only visible in people after triggering PEM presumably this would then be measuring some other, downstream effect of what happens to us? Still interesting but not the ‘something in the blood’ cause talked about originally?

 

[hotblack]

Pretty much every question that popped into my head while going through this paper was answered in this paper. That gives me faith in the results.

While I have more unanswered questions after going back to Fluge et al 2016. Which I suppose makes sense, Fluge was more exploratory, this was looking at confirmation.

A couple of other notes while listening..

What would the results have looked like if you didn’t correct for cell count?
This seems to be covered in point 4 of the discussion

However, no differences in cell counts were observed between ME and HC, so are unlikely to have contributed to ME serum effects previously observed by Fluge et al.

However I don’t think we have numbers on cell count for Fluge et al? (Another unknown, along with some specifics of the sub-group they tested mentioned in my previous post).

HCs seem to have more variation in OCR (Figure 3A), particularly with lower longer tails, I wonder if this means Fluge could have by chance had a group with lower levels, given the small sample size, while ME seems a little more cohesive/clumped together? Sorry this isn’t statistically very sound, not my strong point, maybe someone who is better at that can see if his is relevant.

How much would the specifics of the target muscle cells change things? There seem to be differences in absolute values compared to Fluge, presumably different people’s myoblasts perform a bit differently? What effects could there be here? Could only some people’s myoblasts be affected? That is, there be something in the blood *and* something in muscle tissue?
This seems partially covered in discussion point 1

While genetic differences in the HSMM cells could have altered their susceptibility to ME serum, it is reasonable to expect that the assay would be generalisable to other healthy HSMMs beyond those used in the original study.

 

[jnmaciuch]

In other words, if the problem was that something in my blood was stopping my mitochondria working properly, I would've expected that something in the blood to be there all the time. Though there might well be more of it during PEM.

Just to clarify—my speculation was that it would be something produced by muscle cells (and possibly other tissues) which may leak out into the blood to physiologically relevant levels only if production is amplified enough. Though again, this is only one speculation, could be many other explanations based in methodological differences.

 

[jnmaciuch]

Just read the full text and it is great to see the methodological quality of the study.

They used blinding, randomization, pre-registration of the analysis plan. They graphs are clear, the statistical models are reported explicitly, there's a good overview of potential limitations. No long discussion section, but all straight to the point.

Well done.

Agree! Definitely deserves the commendation

 

[wigglethemouse]

I'd just like to point out that this study was a replication of an experiment in Fluge et al paper of a decade ago. In simple terms it was an experiment to incubate some purchased model muscle cell samples with 20% PwME or HC serum. The Seahorse analyser has some very specific protocols on the way samples are washed, fed, and measured. I'm happy this work was done.

I'm concerned some are interpreting this study to say that there is nothing wrong with mitochondria of PwME, or there is nothing in patient serum affecting patient cells. That is not what this study shows.

Bhupesh Prusty's "something in the blood" is a completely different experiment using purified IGG from severe patients and microscopy of model cells exposed to that IGG. To be replicated that needs a completely different experiment to this paper.

@DMissa lymphoblast experiments with the Seahorse analyzer are using immortalized patient cells to show a complex V deficiency. While using the same measurement equipment as this paper it is a different experiment on partiular patient cells processed with a completely different protocol.

Stanford Nanoneedle tests measured impedance of very fresh blood exposed to a salt stress. Apparently it was a very tricky experiment to make repeatable and frozen or aged samples would not work. Looks likely there is no way to replicate this exactly but the ME Association have funded some researchers (Surrey?) who are trying with their own particular type of electrical measurement.

At the Mitox conference this year researchers presented a new muscle cell + serum experiment that identified differences in ME/CFS and Long Covid patients (unpublished).


These are just 4 examples of promising work that still needs to be replicated in larger sample sizes and by independant groups.

 

[Hutan]

I'm concerned some are interpreting this study to say that there is nothing wrong with mitochondria of PwME, or there is nothing in patient serum affecting patient cells. That is not what this study shows.

Yes. I think this is a very nice write up of a useful replication study, but it doesn't mean 'there's nothing (troublesome) in the blood', or that 'there is nothing wrong with ME/CFS mitochondria'. I don't think the paper claims either of those things and the title goes some way to making the scope of the paper clear.
"Indistinguishable mitochondrial phenotypes after exposure of healthy myoblasts to myalgic encephalomyelitis or control serum"

I think the title could go further though - only one function of mitochondria was assessed. There are other functions and structural issues too that weren't assessed. So, the 'Indistinguishable mitochondria phenotypes' of the title seems like a rather big claim.

 

[BrightCandle]

The two findings I recall for "something in the blood" have not made any claim about mitochondria that I recall. One was about cellular function under stress changing electrical properties of the cells and the other was in passing the condition to Mice. I don't recall either making a link saying the blood caused any change in the mitochondria, this is a different claim and measure rather than a replication of earlier works.

 

[Jonathan Edwards]

I'm concerned some are interpreting this study to say that there is nothing wrong with mitochondria of PwME, or there is nothing in patient serum affecting patient cells. That is not what this study shows.

If you are referring to my comments on another thread, then that concern would be out of context. I was responding to Sasha's concern that if there was no evidence of a failure of energy - dependent processes in mitochondria or thereabouts then wouldn't we have to conclude that ME/CFS is psychological. Simon M had raised a similar concern around my suggestion that one possibility is that the inability to use muscles is at least in part due to neural inhibitory signals rather than a respiratory metabolic failure - which would have nothing to do with 'psychology' but everything to do with synapses and action potentials.

I merely used Audrey's presentation as an example of how it has been pretty hard to find anything wrong with metabolism that would explain symptoms and disease dynamics. For me, and for others familiar with muscle disease the bigger problem with invoking muscle metabolism in ME/CFS is that it really doesn't fit with the overall clinical picture anyway. It may be that people with ME/CFS tend to have a genetic tendency to suboptimal cell metabolism under certain stresses that might be the basis of some of the findings we have seen but it wouldn't explain either the onset and long term dynamics of the illness and wouldn't actually explain the symptoms very well.

These are just 4 examples of promising work that still needs to be replicated in larger sample sizes and by independant groups.

To be honest, if everyone thinks they are looking for something in a biomedical field, you are almost certain to get at least his number of people saying they have sort of found it. There are probably at least as many studies saying they have found retroviruses. I have never been able to establish what the Stanford group thought they were measuring with their impedance set up or what the response to salt was supposed to indicate. It looked an awful lot like an artefact of the culture system. I find the Prusty and Mortem studies not much easier to interpret. The immortalised cell studies looked interesting but seem most likely to indicate a genetic predisposition. Since a lot of people with ME/CFS are healthy and active, or even competitive athletes, before the illness onset that makes it difficult to link such a predisposition to the illness without some other major factor explaining what changes at onset and thereafter.

The idea that ME/CFS is based on a failure of respiratory metabolism in cells really dates back to Ramsay's claim of fatiguability and Behan's claim of mitochondrial changes - which were never confirmed. Ramsay actually suggested an electrophysiological change and probably thought the illness was neurological, but again, no formal deficits were confirmed.

Jo Cambridge spent the first decade of her research career studying muscle cell pathology. She became known as the lab expert on muscle inflammatory disease in the UK. She has recently been involved in metabolic studies in ME/CFS and thinks there is strong evidence for a shift in substrate utilisation but she does not think that the symptoms of ME/CFS are to do with a metabolic failure in muscle. It doesn't add up, at least not in any simple way.

 

[hotblack]

Audio version of the paper available at this link.

There’s a few bits that sound a bit off but I found it very listenable and it’s how I digested the paper. I hope others find it useful (any comments probably best in this thread to not distract from the science here)

It may be a bit difficult to follow some of the technical methodology and results, although tbh that’s probably the case in text for those of us not familiar with the topic!

And if you want to just listen to just the Abstract, Introduction and Discussion you get the overview I think.

 

[DMissa]

The immortalised cell studies looked interesting but seem most likely to indicate a genetic predisposition.

We are looking at the epigenome of the cell lines at the moment. Could be something in the areas not wiped by the EBV

She has recently been involved in metabolic studies in ME/CFS and thinks there is strong evidence for a shift in substrate utilisation but she does not think that the symptoms of ME/CFS are to do with a metabolic failure in muscle

I think that the bulk of the evidence for this shift is pointing to it being observable predominantly in immune cells. I am privy to under-wraps information that supports this. Whether a cause or consequence of eg: abnormal response of particular immune cell subsets to signal (or aberrant signal)... is another question. If a consequence it would explain an immune cell preponderance (of the shift). If affecting the function of eg: T or B cells (whether upstream or whether due to another upstream factor) that would be a plausible link to the clinical picture.

To be honest, if everyone thinks they are looking for something in a biomedical field, you are almost certain to get at least his number of people saying they have sort of found it.

I agree that this happens. In this case I don't suspect that it is happening, because the particular specifics are concordant across assays, groups, countries, cell subsets. For example I cannot recall a study finding that fatty acid b-oxidation (whether measured directly or inferred by expression data) was unchanged or reduced in any immune cell subset derived from people with ME/CFS. In every case that I can recall the result was elevation. I do speak from a position informed by authoritative knowledge of the relevant techniques and biology, as well (ie: I don't say this from just skimming papers and matching the directions of the pictures, but from a decade of doing these kinds of assays and from asking the people who have done them at conferences about the details of their protocols and cell handling, or any relevant assays they might have done off the grid. lol. Woe betide anyone caught sitting next to me at a conference dinner who has done seahorse etc).

I think the title could go further though - only one function of mitochondria was assessed. There are other functions and structural issues too that weren't assessed. So, the 'Indistinguishable mitochondria phenotypes' of the title seems like a rather big claim.

I think it's fine, just nonspecific, but fitting these particular specifics into a title could be unwieldy. The abstract is specific enough.

 

[DMissa]

Woe betide anyone caught sitting next to me at a conference dinner who has done seahorse etc).

Hah, foreshadowing. I have questions for the authors @chillier (does audrey also have an account?). The tone might appear direct but I'm just rapid-firing questions while reading the paper during a brief window of time, so please don't read into it. (I'm not mean, please don't take the ridiculous number of questions that way )

If you feel these are better answered in private please email me instead of replying, I don't want to wrongfoot anyone in public. I just figured since the authors are active on the forum they might prefer to discuss here

edit: removed and sent in email, the extreme volume of highly technical questions is probably not appropriate to put you through answering on a forum (lol!! )

 

[butter.]

Simon M had raised a similar concern around my suggestion that one possibility is that the inability to use muscles is at least in part due to neural inhibitory signals rather than a respiratory metabolic failure -

'Neural inhibitory signals’ could be, at least in part, downstream of respiratory metabolic failure in neural tissue?

Maybe somewhat related, hasn't increased brain lactate in people with ME been replicated?

 

[hotblack]

removed and sent in email, the extreme volume of highly technical questions is probably not appropriate to put you through answering on a forum

But if people do want to share any questions and answers I’m sure we’d all be as interested as we would be confused