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

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?
 
Can someone tell me the significance of all the significant up and downregulations in the gene pathways? Seemed to be a lot, but I dont know much about proteomics/transciptomics

Edit: Mentioned here i guess: "A feature of our results is the striking difference in the pattern of expression changes at the RNA and protein levels. The proteomics revealed a broad pattern of elevated ex-pression of proteins involved in alternatives to glycolytic provision and catabolism of ox-idisable substrates for mitochondrial respiration. By contrast, the levels of transcripts en-coding these proteins were, in many cases, either unchanged or decreased. This is an un-expected but important insight into the underlying cytopathological mechanisms of ME/CFS. It suggests that the overall pattern of dysregulation in ME/CFS cells is a result of a network of normally homeostatic pathways, including competing antagonistic elements like elevated mTORC1 and AMPK activities, that regulate gene expression and metabo-lism at the transcriptional, translational and posttranslational levels [65]. The major path-ways we found to be dysregulated in this way are β-oxidation of fatty acids, glutamine metabolism, branched-chain amino acid catabolism and proteasomal protein degradation."
 
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Hutan said:
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?
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In his 2019 Australian conference talk Dr Fisher said he has used Lymphoblasts in several past mitochondria studies - one might have been parkinsons I think. He described that they continue to grow and you can siphon of a portion to do more studies so that what you are comparing is from the same source and not another patient time point.

So for example they found Complex V issues on patient sample ABC1. At a later date they can take some more cells from this ABC1 sample and do proteomics and metabolomics. The results from the various experiments can be correlated due to being based on the the same cells with the same properties. You don't need to keep going back to the same patient and ask for another blood draw at a different time point, and where the cells may have different characteristic.

I hope I understand and remembered that right?
 
Mitochondrial complex V is also called ATP synthase. A decline in its function seems like it would be a big deal (especially if this affects all cells). It's not the only source of energy in the body but it's a big one.
 
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.
 
Just got through the paper.. Learned quite a bit by reading up on the side. Its very thorough, probably one of the more thorough ive read in this field.

I like that they have concrete hypothesizes, not only "more research on X is warranted".

Id say its time to replicate these findings by an independent group, and also look at other cells.
 
Hutan said:
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.
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I've wondered too about what the immortalisation process does, and is that a key part of the experiment.

Bhupesh Prusty has taken model cells cultured in patient and control plasma and then exposed them to H1N1 & HSV1. Most cells cultured in patient plasma showed a strong antiviral response if I remember right. I wonder if the process of exposing the patient lymphocytes to EBV programs an antiviral response in the lymphocytes of patients that then stays with the cells as they become lymphoblasts and that antiviral response is stronger than with controls........
 
Williams and Ariza at Ohio State University have been studying EBV in ME/CFS for quite some time. I wonder if they would have any ideas about looking for differences in antiviral properties of the cell such as quantifying herpes virus proteins/dUTPases in the lymphoblast culture. Or maybe that will not be relevant when the cells are washed after the immortalisation................ Just guessing/brainstorming
 
borko2100 said:
That's how I experience it. When I do aerobic activities I feel lactate build up, this feeling comes up extremely quick, like my body immediately switches to some inefficient energy burning mode. ...
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It may well feel like so, but note that they say in your quotation, it would not reach statistical relevance, in my understanding they say, they found a(nother) tendency, or in a likely interpretation, it may well be a downstream-effect,

as
Michiel Tack said:
The methods and various pathways are a bit too complex for me to understand but this seems to be the main conclusion/hypothesis behind it:
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quotes:

Instead of an impaired capacity to undertake glycolysis, our results suggest that changes in ME/CFS lymphoblast metabolism might be driven by dysregulated energy stress signaling
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Hutan said:
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).
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Because peripheral blood is very easy to sample and in blood you basically only have white blood cells, red blood cells, and platelets. The latter two aren’t useful to study in this context, so lymphocytes are selected from WBC. They could’ve selected other WBC types though not sure if there’s any value add there.
 
Michiel Tack said:
Thanks for the interesting comments and background. What about diabetes, obesity, heart disease: is there reliable evidence that diet works there as a treatment?
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The difficulty is that it's hard to do studies on diet and these types of diseases as it takes so long for them to develop. We know from multiple large observational studies that a whole-food type diet pattern is associated with lower risk of diabetes, heart diease, being overweight and other lifestyle-related diseases, but there are many confounding factors and the data on what people eat is not the most reliable (self report food frequency questionnaires).

There have been long-term RCTs to look at the effect of diet on hard clinical outcomes such as heart disease, but in five-ten years time can you really trust that the control group has not changed their diet? Or that the intervention group still follow the dietary advice as instructed at the start of the study?

Dietary changes can improve insulin resistance and blood lipid values, and reduce weight and inflammatory markers, but not necessarily in a clinically relevant manner. People do change their diet and can reduce their medication or even stop using it alltogether, but nutritional science has its problems when it comes to reliable evidence all the same ;)

Off topic I guess :) Maybe bring it back a bit by saying that high glutamate and low glutamine may be a risk factor for diabetes and cardiovascular disease. In one study on mediterrenean diet, the association between high glutamate and increased risk of cardiovascular disease disappeared following the dietary intervention (went on for some years), even if glutamate levels were not changed significantly.
 
Marky said:
I was curious what exactly they meant with "energy stress signalling"
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Cells are stressed when they are low on energy/ATP, leading to signalling cascades to increase energy/ATP production.

They found an increase of proteins involved in several pathways that can increase energy/ATP, compared to how these pathways are utilized in the control cells.
 
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Marky said:
I was curious what exactly they meant with "energy stress signalling"
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... completely right.

my understanding was, that they meant an altered energy utilization for any purpose not known right now (maybe a response to an supposed infection or within wound healing pathways; or a counter for that). This would not be a genuine ansewere to impaired glycolysis (which they say indeed, in Michel Tacks quote, would not be the case).

But maybe they mean that an impaired energy utilization elsewhere affects lymphoblasts? - (for a technical reason I am not prepared to read the paper rn)
 
Midnattsol said:
Cells are stressed when they are low on energy/ATP, leading to signalling cascades to increase energy/ATP production.

They found an increase of proteins involved in several pathways that can increase energy/ATP, compared to how these pathways are utilized in the control cells.
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Thanks, thats probably what they meant
 
Trish said:
I'm curious about how anyone would find out they were sensitive to proline, since it is an amino acid prevalent in a wide range of foods.
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It wasn't easy. I knew that something in high-protein foods was making my symptoms worse. Peanut and soy butter had the same effect, so it wasn't meat-only. Then I remembered that I had some gelatin in my cupboard. That was the worst trigger yet, and it's essentially amino acids, and three of those--glycine, proline and alanine--were 15-20x higher than the ratios in peanuts, so they were my top suspects. I then checked the AA content of other foods that I did or didn't react to, which allowed me to cross off some of the other AAs, such as lysine. Next step was to establish the level needed to trigger the symptoms. Two strips of bacon didn't cause problems, but four did.

One issue that led to confusion was that I tested those other foods with bread or pancakes, since 'those are safe!'. Nope, the amounts of wheat-based food was just under my threshold for triggering symptoms. Once I figured that out, I found that the amount of bacon that triggered my symptoms contained ~1400 mg of proline. I then took my hypothesis of '1400 mg of total proline will trigger symptoms' and tested it with several other foods. They all matched expectations. Whole wheat flour is particularly rich in proline, so there goes a big part of my dietary choices. Meat too. :(

Here's a useful tool for determining amino acid content: https://tools.myfooddata.com/protein-calculator It doesn't list all foods, but I still found it useful.
 
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