Dysregulated Provision of Oxidisable Substrates to the Mitochondria in ME/CFS Lymphoblasts, 2021, Missailidis et al

This paper adds to the evidence base that is in line with those of us who had a rapid and significant improvement on a ketogenic diet, albeit temporary in my case for a few months. While I agree it’s likely not a long-term treatment, patient treatment testimonials can give us some clues to underlying pathophysiology.

 

I agree, I think it’s only some possible clues and for me it didn’t work nearly as well months later, but it was really interesting to experience what happened.

We also still don’t know if all of us diagnosed with ME have the same underlying pathophysiology.

 

Im not gonna derail the thread, but since were taking about supplements in relation to this paper.. In the 7 years ill alcohol has always given me a significantly increased level of functioning. Not just feeling better, but 30 to 40 perc higher level of function. No idea why.. Slowing faulty nervous system signals? Alternative energy source? Occupuing the liver? Blood Flow? not many people with ME experience this though, its usually opposite. In my first year sick i performed at festivals with my band, only possible cause i was tipsy as fuck lol

 

Thinking about this again, many teams have used the Agilent Seahorse to perform mitochondrial function testing in ME/CFS. Blood/plasma have to be removed from the PBMC's before running the test - in fact there is quite a treatment prep if I remember right. When comparing control sample groups to ME/CFS sample groups there has been very little difference found (my view). In order to see a difference they had to use activated T-Cells where the activation process puts the T-cells in a higher energy state.

Karl Morten and Fluge/Mella have looked at cultured muscle cells exposed to patient or control plasma in an energy expenditure test. Karl presented his findings on this but when he followed up with a second cohort the results were not the same and was not sure why this happened (speculated on the blood draw and preparation process..........).

In their previous paper the authors reported on a very significant difference between control and ME/CFS lymphoblasts in their mTORC1 test. However I seem to remember someone telling me about research in PBMC's and there was little significant difference in mTORC1 between healthy control and ME/CFS cells, certainly not the large separation seen by the authors previously.

Other groups have done detailed analysis on ME/CFS patient mitochondria in the past and the results have been a mixed bag.

So, I'm wondering if there is indeed something special about lymphoblasts when derived from ME/CFS patient lymphocytes that is not generally seen in lymphocytes or PBMC's.

It would be nice to hear @DMissa's thoughts about this topic but I suspect he is showing us respect by not commenting on this particular thread so as not to bias comments.

 

I was actually just pretty busy with some other stuff and hadn't come to s4me yet. The paper seemed to be getting some attention on twitter, so I figured there'd be some discussion here, today. I was also now considering not commenting so as not to bias the discussion, but since Hutan said it is not inappropriate, I think I will just to clear up certain questions that haven't been answered, etc. Will still try to keep it as objective as I can.

In the first paper we hypothesised that the other changes seen were compensatory and in response to an inefficiency of ATP synthesis... but the cause-effect relationships have not been disentangled yet. ie: this model of respiratory inefficiency leading to compensatory changes is the current working hypothesis that we are aiming to continually test, revisit, build upon and revise as we learn more about what's going on. But it is not necessarily the case. It is a hypothesis to be tested! We have plans in place to dissect the cause-effect relationships from multiple angles. I don't want people getting too carried away just yet with Complex V! It's very exciting but I am very wary of overhyping anything, you know?

Thanks Hutan! We are still moving forward with ME/CFS work and have a few new projects on the boil. As for me, this has become a personal passion and so I'll be staying in the field as long as I can. Hopefully forever. My PhD is over within the next month so my fingers and toes are crossed that some funding opportunities pan out.

Could be a few things. We will be testing the possibilities . As you suggest, the epigenetics possibilities are interesting.

Indeed!

I would say that lymphoid cells are definitely used because they are easily accessible.

It can be done in other cell types and our lab has other cell types from pwme, but again, that work was slowed by the pandemic. Stay tuned. We are certainly not committed to only one type of sample.

More in the next reply.

In research in general, I think that published lymphocyte respirometry measurements are numerous and can speak for themselves. I'm not going to tell anyone what to think... I would recommend looking at published work (in general, not specifically ME/CFS) and making your own minds up based on reported measurements, remaining cognizant of different approaches using chemical activation, supplied substrates, immortalisation, normalisation and other protocol variations.

Every cell type has pros, cons, and nuances that the researcher and reader both need to be aware of. I'm going to run through a few commonly used examples and important considerations relating to measurements of energy metabolism/mitochondria off the top of my head (in general, not specifically pertaining to ME/CFS work):

Fibroblasts:

• Primary cells.
  
• Programmed senescence means three things:
  1) Fibroblast cultures age and die off within a few dozen passages depending on how "far along" they are.
  2) As they age, their phenotype changes, including energy metabolism.
  3) The older the individual that the cells are isolated from, the further along this path to senescence the cells already are.
• Fibroblasts contact inhibit. That is, when they touch, they basically switch off their proliferative and energetic metabolism. You have to be very careful with this. It's a delicate balance to get the cell density high enough for good signal while not incurring contact inhibition.
  
• Fibroblasts have a more spherical morphology in suspension that is unlike their long, fibrous morphology in vivo or when attached to a matrix in culture. This also affects their metabolism.
• All of these factors can (and must) be controlled at the same time, but it is very difficult and seemingly not always acknowledged.

Muscle cells:

• Could be considered the most physiologically relevant tissue for studying myalgias etc.
• Primary cells.
  
• Muscle mitochondrial biogenesis is stimulated with exercise. This means that activity levels are important to control for in muscle studies.
• Invasive to obtain.

Lymphocytes:

• Easily accessible.
• Primary cells.
• Metabolically quiescent, short-lived.
• Activatable by various means:
  -immortalisation: long-term proliferative and metabolically active phenotype, but involves EBV transfection.
  -chemically or immunogenically: metabolically active. Transient effect with kinetics of activation that vary between individuals. ie: Activating and measuring activated lymphocytes from multiple individuals may catch the cells in different phases of the activation curve.

There's more but I think that's enough to give you the picture. There is no "one size fits all" cell type. I think the best approach is to apply them in combination while cognizant of the individual nuances of each, both experimentally and with regard to their roles in the body. Since it's expensive and time-consuming to do all of them, people often start with lymphoid cells to form the basis for subsequent studies using primary cells from other tissues .

This is why I am very excited to look at other cell types!

Equally exciting!!

 

Forever? Oh no you won't @DMissa. What you will be doing is sort this whole mess of a disease out once and for all, and that in the very near future. That an order by the way . No pressure.

Do you think you'll be giving a presentation on this paper at some point, preferably a somewhat dumbed-down version that even the likes of me can understand?

What I did understand from your paper is that you're trying to investigate as carefully, systematically and thoroughly as possible, and that you're presenting your results as factually as possible and without over-hyping them. I really appreciate that approach. Thank you.

 

@Ravn I appreciate the warm words and "gentle" encouragement .

Back on a serious note I gave a short talk about this last year at the IACFS conference but it is now a bit out of date - I was learning how to best approach transcriptome data from home at the time, so the overall interpretation changed somewhat between then and now. If I have a similar opportunity again I'll PM you to let you know. If you have specific questions that you might feel shy with asking in the thread please don't hesitate to PM or email me.

Especially given how many researchers have a personal stake in this (family etc), I'm sure that everyone in the field is trying their absolute hardest to get to the bottom of this disease in as rigorous a way as possible. I can promise you that we are far from the only ones trying to "do it right" for this community of beautiful people.

Edit: will also add that I don't want to interfere with the impartial discussion of the paper, so I'll remain absent from the thread from here unless something urgent is brought to my attention.

 

@DMissa thank you so much for talking to us, thank you for explaining, thank you for your work. Congratulations on your PhD, and this publication.

 

Going back many years before CFS, there was a growing feeling that all the tests for ME were negative because they were all done at rest. I remember one doctor found that qeeg (?) results were abnormal only if they were taken standing up instead of lying down.

The differences in results could well be because patients were at different stages of ME when samples were taken. Studies where the cells are stressed might give the most useful results.

I believe that ME is caused by small changes in lots of cells whereas most disease is caused by large changes in a few cells. Because biology is so dynamic these small changes may not show up well.

 

Hi Daniel, many thanks for all the efforts from you and the rest of the team. In the past, I've recorded a number of videos, using Zoom, with researchers and other people from the ME world, attempting to look smart by asking clever questions about their work that the forum members have suggested, and putting the end result up on our YouTube channel.

Due to my involvement with DecodeME that side of my 'work' has been largely set to one side but if you have any interest in doing something like this then please feel free to PM me here.

 

IO think Karl Morten used muscle cells (& the seahorse - check his talk in New Zealand a few years ago).

 

Don't know much either but transcription means making a protein - post transcription means the protein is altered after transcription. I kind of recall a talk Jonas Bergquist did a few years back i.e. on proteomics. Basically the body make a remarkably few proteins (maybe 40K) but post transcript modification means there are about 4 million kicking around.

 

See also A coordinated multiorgan metabolic response contributes to human mitochondrial myopathy (2023) for related findings in (genetically determined) primary mitochondrial myopathies.

 

Thanks for the link to relevant discussion.

I went down the rabbit hole from your link, and saw some s4me chatter about the unlikelihood that a direct ATP deficiency is at play. TBH I am probably inclined to agree if you ask me that question today, as a guess only... my thinking + interpretation of my old data has changed a little bit since my first papers, but my musings are only fluff until cause-effect relationships etc are experimentally determined - "probable inclinations to agree" even if from myself will not solve the disease (lol) and so we have talented students plugging away at introducing or ameliorating Complex V defects (amongst other things that prior data has shown differences in) in various cellular models and with various strategies, then characterising the phenotypic outcomes to see if they match or overlap with what we see in cells from pwME. We will see! Might take years to come to light because it's a lot of work that's on multiple PhD project scale, but it's definitely being quietly worked on every day by very passionate people.

 

My muscle biopsy showed some degree of mitochondrial dysfunction, but not severe enough to count as proper mitochondrial myopathy.

 

I want to look at muscle but it's just been so practically challenging. Getting enough material to do the extent of in-depth experiments that I do (with muscle biopsies) from a sufficient number of sedentary controls in addition to patients.. is a huge problem. (sedentary controls are absolutely necessary for muscle studies in this space because of how activity affects muscle dynamics + mito content)