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Cellular bioenergetics is impaired in patients with chronic fatigue syndrome

Simon

Senior Member
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Monmouth, UK
Here's a bit more about the Seahorse technology and what it measures, using info from the Seahorse site. First off, it's looking at the body's two main energy pathways, glycolysis and respiration, which you can see in these diagrams (from Seahorse videos).
IMG_1384.JPG

First 55 seconds of this video is good on the underlying biology
  • Glycolysis produces modest amounts of ATP, the cells' main fuel as well as pyruvate, the molecule that feeds mitochondria. Glycolysis produces a lot of protons (acid, effectively, often as lactic acid).
  • Oxidative phosphorylation (or respiration) is the process that takes place in mitochondria, the real fuel factories that burn fuel efficiently, consuming oxygen and foodstuffs and pumping out ATP.
Seahorse measures glycolysis through the protons (acid) and oxidative phosphorylation through the drop in oxygen levels. It's secret is tiny probes that are lowered to trap cells in a micro-chamber of a droplet and it measures activity only in this micro-chamber.

IMG_1381.JPG


But this micro-chamber is only a tiny amount of the fluid in each compartment. After each measurement, the probes are raised, everything mixes together and it can then take a new measurement. This ability to take repeated measures over time is key.

This study measures a bunch of different aspects of oxidative phosphorylation, which it does by injecting different compounds to probe what's really going on. Think of it as an exercise test for mitochondria, revealing what it's doing and how much capacity it has to ramp up activity. The central measures in the study are:

1. Basal respiration, the tickover rate of activity
2. ATP production, which is simply basal respiration less a bit of inefficiency loss (proton leak)
3. Maximal respiration: what the mitochondria can theoretically do using a drug to put the foot to the floor.
4. Reserve capacity: simply maximal respiration less basal respiration (peak capacity less what it's doing, or headroom to increase activity).

Fig 1 from the paper shows an example of how this testing works but I think my version is a bit simpler (the names at the top are the compounds added to put mitochondria through their paces).
mito-test-rig.png


The study looked at glycolysis too, which uses a slightly different detailed methodology, but that found nothing of interest, so I will spare you the details.
 
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Mithriel

Senior Member
Messages
690
Location
Scotland
This is why the 2 day CPET testing is so important. it gives a "whole body" view of what is wrong with us - our aerobic systems are broken and we have to move to the inefficient anaerobic system for the activities of daily life with the result that we have less energy and delayed recovery because you need the aerobic system to mop up the products of anaerobic respiration. It is easily understood, not too technical and opens the way to a treatment - stay within an energy envelope as much as possible.

As a bonus it is a more precise way of defining PEM which is too easily misused to describe what happens to healthy people when they exercise too much. It is also proves the BPS theories wrong and shows why they cause harm to ME patients as opposed to people "tired all the time".

ME has a lot of parallels with diabetes especially in that you can live with it if you manage things within its limitations. Diabetes is when your pancreas cannot produce enough insulin or your cells can't utilise the insulin available so glucose accumulates in the blood.

BUT, they do not know exactly what is happening at a cellular level. That is left to researchers while GPs concentrate on the whole body effects and minimising the harms that can occur as a result of the body processes (not fully understood) which are going on in the background.

We do not need to know the details before we know why we are not working. Individual papers from different research groups are good for giving us details and could lead to cures and treatments so are important but just because they do not all agree at this moment is not a good reason to ignore why we are ill and we must keep pushing that.
 

alex3619

Senior Member
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13,810
Location
Logan, Queensland, Australia
This is why the 2 day CPET testing is so important. it gives a "whole body" view of what is wrong with us - our aerobic systems are broken and we have to move to the inefficient anaerobic system for the activities of daily life with the result that we have less energy and delayed recovery because you need the aerobic system to mop up the products of anaerobic respiration. It is easily understood, not too technical and opens the way to a treatment - stay within an energy envelope as much as possible.
Yes.

Diabetes is tricky though, and I think type 2 is a spectrum disorder or a conglomeration of many different disorders with common pathology. Indeed I suspect many ME patients may be diagnosed with diabetes, but its ME that is driving the faulty sugar metabolism.
 

msf

Senior Member
Messages
3,650
I wanted to say this as well. It's very tempting to look at results like this, and go "Look, this proves we can't produce ATP as well as healthy people! This is why we're tired!", but the key thing a lot of people looking at studies like that are forgetting is cell types. The human body is made of a multitude of different types and suptypes of cells, that do very different things, express different proteins, and have many other differing properties. And yes, the energy metabolizm of most cells consists of the same molecular machinery - glycolysis, pyruvate decarboxylation, beta-oxidation, krebs cycle, electron transport chain. But the regulation of those processes differs from cell to cell. Every time you hear about a discovered intracellular dysfunction, you have to ask the question "okay, what cells exactly?"

A lot of those studies, even Ron's work, use PBMCs. Peripheral Blood Mononuclear Cells are a middle layer of blood, separated by centrifugation. It contains T-cells, B-cells, NK cells, monocytes, and sometimes trace basophils. In other words, immune system cells. Our PBMCs having its energy metabolism impaired doesn't directly explain us feeling fatigue, or not being able to tolerate exercise. Such findings in muscle cells could maybe, kinda explain it (I don't think fully though). Interestingly enough, Fluge and Mella did, in fact, perform those same Seahorse measurements in cultured muscle cells exposed to ME/CFS serum. And the results were the opposite - oxygen consumption was actually increased, not decreased. So I would be careful with assuming that those results here, or any similar studies, can be extrapolated to the whole body. Those results are indicative of an *immune* alteration, if anything. It can be a dysfuntion of the immune cells, a different cell type ratio than the controls, or just immune activation. We don't know.

I wrote about this in my blog on the subject.
 

Simon

Senior Member
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3,789
Location
Monmouth, UK

My notes from reading the full paper. This looks like an important contribution to the growing field of research probing problems in producing energy in mecfs. These preliminary findings need replicating and showing they are unique to mecfs. Some points have already made by others.


The big story
These results are impressive. Even with the very small control sample size (12), these are "strong data, robust to statistical correction". There's remarkable separation between patients and controls, with almost all patients below the lowest control: you don't see that too often:
Mito-big-news.png


(my adaption of figure 5D, maximal respiration in low glucose)

Corrected p values for the key measures, including basal respiration, ATP production, maximal respiration and reserve capacity, were <0.005.

So these findings suggest that (white blood) cells from mecfs patients struggle to produce energy like healthy cells, and in particular can't ramp up energy production to meet increased demand. Which intuitively makes a lot of sense.

Significant issues with this study (most discussed by the authors)

Small sample
: "The primary limitation of this study was sample size which resulted in underpowered analysis", the authors plan to seek larger samples in future. There are only 3 fresh control samples so these particular findings simply reliable.

Questionable controls: Perhaps the biggest issue is the lack of matched controls and the lack of sick controls. @alex3619 mentions specificity, the ability to separate mecfs patients from others that are sick. This is crucial both in clinical practice and for research hoping to find out what's gone wrong in mecfs. That said, it's a widespread problem in the field. (A good time to mention the UK mecfs biobank that has both large sample size and sick (MS) as well as healthy controls.)

The authors say sedentary controls would be better. Activity could plausibly affect mito performance and the authors are considering measuring activity levels in future work.

Need to validate methodology: the authors note the to validate these findings using other well-established measures of mitochondrial performance such as morphology and membrane potential.

Results almost too good? What makes this study so impressive is that the results are so striking, which also makes me nervous, particularly for a study using Fukuda criteria that are unlikely to identify a tightly-defined cohort. So I'd have expected a bit more variation in results, unless, say, inactivity is a major factor driving these results. Maybe I'm being unfair.

One more, for geeks. Fig 3 (frozen samples) and Fig 5 (high glucose) both show data from 12 patients and 38 CFS controls, and the paper suggests they have identical conditions (frozen, 10mM glucose). So I would have expected results to be much more similar than they are (Fig 3 results are consistently lower). Am I missing something?

Some wider questions about probing energy issues in mecfs

Which types of cells are affected? Some people have already commented this is on PBMCs (white blood cells) and the results could be down to one sub-group such as T-cells (I'd be surprised, given how big the overall effect is). The authors did note this, and it's true of any PBMC study - which includes much mecfs work.

Conflicting findings - what's really going on?

This study found that glycolysis is normal in mecfs, while oxidative phosphorylation is the issue, while others have found glycolysis is the issue e.g. Armstrong 2015 metabolomics work, the Fluge/Mella amino acid metabolism study and the unpublished Ron Davis metabolomics work. I think earlier work by Julia NEwton's group has suggested a problem in glycolysis, or at least in pyruvate dehydrogenase at the end of glycolysis.

I gather Fluge/Mella found opposite mitochondrial findings using Seahorse (does anyone have details?). @viggster said these findings are broadly in line with very early results from Seahorse in the NIH intramural study.

There’s also the issue of using serum or not, the fluid that bathes the cells themselves. Potentially it carries “factors" that could be causing the problems even if the sells themselves are normal. So, I think the Fluge results on seahorse were with serum, and the Ron Davies results too. These new results are serum-free. However, it would be easy enough to test with serum using this tech, and see what happens if you swapped over healthy and patient serum.

Meanwhile, the CPET 2-day exercise studies often find normal day-one results, which doesn't fit well with these results.

Which is a mess in places, or the signs of an early and promising field that needs more work (and funding). I'm going with the latter for now and hope the researchers keep going with bigger and better studies. Don't forget all those UK mecfs biobank samples.
 
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Grigor

Senior Member
Messages
462
Location
Amsterdam
I'm really not sure why she /researchers keeps using FUKUDA. It's almost a waste of time and money really.
As far as I know Fluge and Mella and Armstrong use stricter (ICC) criteria? Not sure.
 

Aroa

Senior Member
Messages
109
Location
Spain
Maureen Hanson talked about her Project "cellular metabolism of immune cells in ME/CFS" at the Discovery Fórum :

Data from T cells (immune regulatory cells) isolated from 20 ME/CFS patients and 20 controls indicate that T cells in the former group are using significantly less of their respiratory capacity. The group is looking for additional quantities of T cells to further explore whether T cell subtypes can be differentiated.

I think this is the Project :
http://neuroimmune.cornell.edu/research/metabolism/
 

aimossy

Senior Member
Messages
1,106
Jo Cambridge from UCL covered some interesting things regarding energy metabolism in B cells at the RME Sweden ME/CFS conference just recently. She gave some snippets of what Chris Armstrong and Fane Mensah (spelling?) were collaborating and working on with metabolomics too including some results, but I am struggling to remember the detail properly now. Here is a link to where she begins to talk about their findings and then goes on to talk about metabolism related things.
 
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pattismith

Senior Member
Messages
3,972
Maureen Hanson talked about her Project "cellular metabolism of immune cells in ME/CFS" at the Discovery Fórum :

Data from T cells (immune regulatory cells) isolated from 20 ME/CFS patients and 20 controls indicate that T cells in the former group are using significantly less of their respiratory capacity. The group is looking for additional quantities of T cells to further explore whether T cell subtypes can be differentiated.

I think this is the Project :
http://neuroimmune.cornell.edu/research/metabolism/

I wonder if these researches are related to the studies I quoted some days ago:

according to the few articles I read, Treg are exhausted in ME (and also MS):

"The pattern of raised IL-2 levels and chronic immune activation with disrupted homeostasis [169,170] coupled with raised levels of Treg cells seen in patients with ME/CFS strongly suggests Treg cell exhaustion as a feature of this disease as well."


and this interesting study shows: "Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells.
These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo."
 

FMMM1

Senior Member
Messages
513
[QUOTE="alex3619, post: 929555, member: 786"]I do think cell sorting should be tried, but I am unsure that it will help. Most of my caveats are technical, so if anyone knows a lot about cell sorting then comment would be good, though you might also need to know a lot about seahorse technology.

First, such cell fractions have further cell fractions within them. I don't think this is a major issue, but it might mean a series of studies is needed.

Second, when you do cell sorting you might change cell chemistry. Various controls would be needed, including cells replaced in patient serum.

Third, when you cell sort you decrease quantity. A lot. So at what quantity will seahorse technology become unreliable?

For me I would first want to know if skin cells or muscle cells have similar issues. Then I would look at cell subfractions if that is viable. However if muscle cells are normal the big question has been answered, and subfraction testing would be about finding more clues. If muscle cells are not normal then we can put subfraction testing off for a while as its no longer so important.[/QUOTE]

Re your comment "if muscle cells are normal" I assume this is the paper by Fluge and Mella (among others) "Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome".

Possibly one way of trying to figure out whether there is an autoimmune response, i.e. causing ME/CFS, would be to try to identify the target. Mark Davis has demonstrated "T cell clonal expansion" and is looking for funding to try to identify the target/targets (check out the recent Stanford Symposium). Hopefully Mark Davis will apply for and get one of the single project grants from NIH. Another option to identify the target may be to try random peptides, see paper titled "Humoral Immunity Profiling of Subjects with Myalgic Encephalomyelitis Using a Random Peptide Microarray Differentiates Cases from Controls with High Specificity and Sensitivity". Problem with the random peptide work seems to be identifying the target protein.

nandixon has pointed out (first page of this thread) that this study may simply be supporting what Mark Davis has found i.e. activation/clonal expansion of T cells. I'm guessing that testing using Seahorse might still be useful i.e. would indicate possible activation/clonal expansion of T cells. Anyone know how much this Seahorse thing costs and the cost of testing (if anyone provides it)?
 

Londinium

Senior Member
Messages
178
New Scientist coverage here: https://www.newscientist.com/article/blood-cells-chronic-fatigue-syndrome-drained-energy/

I think it's paywalled for non-subscribers so I'll just share the opening paragraphs - they're good :)

Thirteen years ago, Cara Tomas was rendered bedbound with chronic fatigue syndrome. It came on suddenly, she says, without warning signs. Even now she has good days and bad days due to the lingering effects of the disease. “A lot of people dismiss it as a psychological disease, which is a big frustration,” she says.

Tomas knows more about CFS than most. A PhD student at Newcastle University in the UK, she has just published a paper demonstrating that white blood cells in people with the disease are as listless as the people themselves often feel. “Now we’ve shown there’s a physiological difference, it could explain the whole-body fatigue shown by patients,” she says.

The finding adds to mounting evidence that the disorder has a biological explanation, and raises the prospects for new treatments and diagnostic tests.

For many years, arguments have raged over whether CFS — also known as myalgic encephalomyelitis, or ME — has a physiological or psychological basis. But the latest research comparing samples of peripheral blood mononuclear cells (PBMCs) from 52 people with the condition and 35 without has reinforced the case for a biological explanation.
 
Messages
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New Scientist coverage here: https://www.newscientist.com/article/blood-cells-chronic-fatigue-syndrome-drained-energy/

I think it's paywalled for non-subscribers so I'll just share the opening paragraphs - they're good :)
Full text, here:
http://www.meassociation.org.uk/201...drome-are-drained-of-energy-04-november-2017/
New Scientist: Blood cells in chronic fatigue syndrome are drained of energy

Thirteen years ago, Cara Tomas was rendered bedbound with chronic fatigue syndrome. It came on suddenly, she says, without warning signs. Even now she has good days and bad days due to the lingering effects of the disease. “A lot of people dismiss it as a psychological disease, which is a big frustration,” she says.

Tomas knows more about CFS than most. A PhD student at Newcastle University in the UK, she has just published a paper demonstrating that white blood cells in people with the disease are as listless as the people themselves often feel. “Now we’ve shown there’s a physiological difference, it could explain the whole-body fatigue shown by patients,” she says.

The finding adds to mounting evidence that the disorder has a biological explanation, and raises the prospects for new treatments and diagnostic tests.

For many years, arguments have raged over whether CFS — also known as myalgic encephalomyelitis, or ME — has a physiological or psychological basis. But the latest research comparing samples of peripheral blood mononuclear cells (PBMCs) from 52 people with the condition and 35 without has reinforced the case for a biological explanation.

Less mighty mitochondria

Across almost all measures of energy capacity, the cells from people with CFS were weaker compared with their healthy counterparts. If other cells are equally compromised, it could explain why people with the condition are often bed- or wheelchair-bound for months, and struggle with even modest physical exertion.

“The CFS cells couldn’t produce as much energy as the control cells,” says Tomas. “At baseline, they didn’t perform as well, but the maximum they could reach under any conditions was so much lower than the controls.”

Tomas and her colleagues measured the efficiency of mitochondria, the energy-generating powerhouses in cells. The mitochondria are the dominant source of energy for all of our cells. The team found that mitochondria in CFS cells can’t produce energy properly.

“We’ve shown definitively that it’s a fault in mitochondria,” says Tomas. “It points directly to a physiological, not psychological disorder.”

Tomas measured the oxygen consumption of cells in comfortable and stressed conditions, to see how well they could raise their game with glucose in short supply, a situation forcing the cells to consume more oxygen to compensate. Even at baseline, control cells consumed twice as much oxygen as the CFS cells. The disparity widened dramatically when the cells were stressed.

Metabolically exhausted

In another test that artificially pushed cells to their maximum capacity starting from baseline, CFS cells could only increase their mitochondrial output by 47 per cent, roughly half the 98 per cent increase achieved by control cells.

The implication is that cells from CFS patients can’t raise their output to meet the energy demands of routine physical tasks.

“These exciting results confirm what others have postulated but not been able to prove, namely that cells of patients with CFS are easily metabolically exhausted when put under any form of stress,” says Stephen Holgate of Southampton General Hospital. “In many ways, this is how patients describe their whole-body experience with CFS.”

“This is a major step forwards, supporting previous studies, which demonstrated that mitochondrial function can be impaired in this illness,” says Karl Morten of the University of Oxford. “A major question now is whether the situation in these white blood cells reflects whole-body mitochondrial dysfunction in patients,” he says.

To that end, Tomas is currently taking samples of muscle cells and testing them in the same way as the blood cells. “It would be good if we could get this repeated in muscle cells,” she says. “It’s important the patient population know we are looking into this. Patients sometimes think no one cares, but we do have interest, and want to find out what’s going on.”
 

FMMM1

Senior Member
Messages
513
I wanted to say this as well. ---------------- Every time you hear about a discovered intracellular dysfunction, you have to ask the question "okay, what cells exactly?"

A lot of those studies, even Ron's work, use PBMCs. Peripheral Blood Mononuclear Cells are a middle layer of blood, separated by centrifugation. It contains T-cells, B-cells, NK cells, monocytes, and sometimes trace basophils. ------------Those results are indicative of an *immune* alteration, if anything. It can be a dysfuntion of the immune cells, a different cell type ratio than the controls, or just immune activation. We don't know.

It's nice the UK is coming around to doing actual research in ME/CFS. But this doesn't really tell us anything new about the illness mechanism, in my opinion.
-----

This is also why I was excited when Ron said he wanted to use pluripotent stem cells, because that way you can test all the cell types you want (with limitations, but still).

Check out Maureen Hanson's presentation at the recent Community Symposium on the Molecular Basis of ME/CFS, sponsored by OMF, at Stanford University.

She presented data on "total T cells" showing the same altered mitochrondrial metabolism (oxidative phosphorylation in mitochrondria). The study also used the Seahorse analyser.

It would be interesting to see data from people with Lymes disease and fibromyalgia i.e. diseases with overlapping symptoms.

Being able to demonstrated an activated immune system seems significant to me. Currently doctors don't seem to have any biomedical test for ME/CFS so if this could be developed into a test then it would be start. The question is is this the downstream effect of the disease and if so what's the upstream cause - autoimmunity? How do we check for autoimmune targets (couple of possible options - see Mark Davis's talk on T cell expansion and this paper "Humoral Immunity Profiling of Subjects with Myalgic Encephalomyelitis Using a Random Peptide Microarray Differentiates Cases from Controls with High Specificity and Sensitivity").
 

alex3619

Senior Member
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13,810
Location
Logan, Queensland, Australia
One issue I have with the possibility this is only found in a subset of WBCs such as T cells is the CPET data does not make sense if a problem is only in immune cells, or just T cells, or any other immune subset. While there is not a good match with CPET data yet, the CPET data suggests the problem is more widespread, which would be accounted for if muscle were also affected, or quite possibly every single tissue type. However what we do not know is whether or not there are multiple problems, and some think there are. If so then muscle might have a different problem. I doubt it but it really might be that complex.
 

FMMM1

Senior Member
Messages
513
-----

In other words, this study may be confirming that a population of T cells is inappropriately activated - rather than there being some bodywide OXPHOS dysfunction that is also occurring in other cells like muscle, liver, etc (although deranged cytokine signaling from the activated T cells can potentially cause mitochondrial dysfunction in those other cells).

Whether this is the case can perhaps be determined using the method the authors suggest themselves at the end of the paper ---fluorescence activated cell sorting :

Reader beware - I'm not a scientist:

Alex,
possibly
1) this study showing impaired mitochondrial energy production in T cells; and
2) Fluge Mella's study showing that muscle cells in ME/CFS are fine i.e. provided you bath them in plasma from normal people (and vice versa) - high molecular weight blood borne factor (antibody?) switching metabolism in ME/CFS;
are both correct.

E.g. if nandixon is correct, and this paper is in effect only showing activated T cells, then that could still mean that mitochondrial energy production in muscle cells in ME/CFS is affected by a blood borne factor. In fact the activated immune system could, as nandixon suggests, be triggering the production of whatever is turning off energy production in muscle cells.

Maybe what we need is to replicate this cellular energy study using activated T cells and un-activated T cells, preferably from the same patient. I don't know if the fluorescence cell sorter the authors refer to can separate activated T cells, and un-activated T cells, but if they can then that seems to be a reasonable study. If you can't separate activated T cells out then there are possibly indirect ways to test whether this is due to activated T cells. E.g. possibly by producing activated T cells and thereby looking at the relationship between % T cells activated and mitochondrial energy production.

Haven't heard anything about Ron Davis's search for the blood borne factor (antibody?).

Mark Davis said that the the "immune system is getting simpler all the time --- because were understanding it".

Maybe we need money to help the scientists to identify the blood borne factor, repeat this study i.e. cellular energy in T cells, and fund Mark Davis work on clonal expansion. Then we might be able to understand a little more.
 
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