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.
I think this is the second or third paper these researchers have published about Complex V problems, has anyone besides them found problems with Complex V? Seems to me like a good lead.
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?
@DMissa, nice to see another paper out. What is up next for you and the team? Do you have funding?
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.
So, if those differences are there between ME/CFS and controls, in both non-immortalised PBMCs and lymphoblasts, what does that tell us about what is causing the differences? The lymphoblasts will have been frozen, and presumably washed, and cultured, and treated with the EBV, umm, treatment that immortalises them. And presumably new cells are being made by the lymphoblasts, with the new cells also showing the differences. So, what is perpetuating the difference from controls, in the lymphocytes taken from the patients all the way through to new generations of lymphoblast cells?
Is it epigenetic changes? Could it be bits of virus?
(Sorry, I've run out of energy, so haven't read much of the last bit of the paper.)
Could be a few things. We will be testing the possibilities
. As you suggest, the epigenetics possibilities are interesting.
There are quite big differences between patient lymphoblasts and control lymphoblasts in gender (88% female vs 45% female) and age (53 years vs 30 years). No significant effect of age or gender was found, but it's less than ideal. I guess Covid may have made blood collection difficult in 2020 - but perhaps Emerge could help with control recruitment, to try to get better matched control samples going forward?
Indeed!
Do all the researchers use lymphocytes/lymphoblasts because they think cells involved in immunity are going to tell us something that other body cells cannot? Or is it just because it's relatively easy to get hold of samples? Is there any risk of the 'Drunk looking for his keys under the lamp-post' issue? (not to disparage these good teams doing the work of course).
Could this type of work be done in other cell types? (Has it been done another cell types already?). I think a number of us have biopsies of various sorts - could stomach biopsies and the like be used as a source of cells when done for investigative purposes? Or cells from nasal or cheek swabs?
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.
Yes, I do remember that, and was convinced by it. But I have heard other Australian mitochondrial researchers be very dismissive of the idea of using lymphoblasts to investigate ME/CFS - they feel that the cells will be too different from cells taken fresh from the body. When I mentioned the issue of differences in energy being too small to measure accurately in quiescent lymphocytes, they said that was rubbish. These researchers had not done ME/CFS research though, so perhaps they had not experienced the problem of higher rates of death in ME/CFS lymphocytes. I'm trying to reconcile two very different views of the utility of cultured lymphoblasts in this type of research.
Also, I was wondering if other types of cells could usefully be looked at - perhaps they would tell the same, or a different story.
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
.
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.
This is why I am very excited to look at other cell types!
Very happy to see
@DMissa and team collaborating with other researchers. I also appreciate that Sweetman and co in NZ have been attempting to replicate some of their previous work with small cohorts.
Equally exciting!!
