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Rutherford Ph.D. Thesis: Investigating the biochemical basis of muscle cell dysfunction in CFS

Murph

:)
Messages
1,799
The in vitro muscle culture approaches reported in this thesis have enabled the investigation of the biochemical basis of muscle cell dysfunction in patients with CFS/ME. It is possible to conclude there to be no evidence of impaired muscle function in CFS/ME patients. Additionally, there was no impairment found in PDK enzyme function. Therefore, it can be determined that bioenergetic function is normal in CFS/ME patients and cannot be attributed to the excessive peripheral muscle fatigue phenotype frequently exhibited.

I thought the scientific work was absolutely excellent except for the conclusion. Absence of evidence is not evidence of absence. A more suitable conclusion would say something like 'Bioenergetic function did not appear to differ from controls. If the difference exists it could not be located using these methods, on this sample, at this time.'

EDIT I see @daisybell said the same thing as me already and in far more PhD suitable terms! :)
 

daisybell

Senior Member
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1,613
Location
New Zealand
I thought the scientific work was absolutely excellent except for the conclusion. Absence of evidence is not evidence of absence. A more suitable conclusion would say something like 'Bioenergetic function did not appear to differ from controls. If the difference exists it could not be located using these methods, on this sample, at this time.'

EDIT I see @daisybell said the same thing as me already and in far more PhD suitable terms! :)
Ha! That'll be the only time I'm told I've said something in PhD suitable terms!!!!
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
We now have multiple researchers telling us the patient's serum/plasma matters. Putting them in some other medium is significant.
This was my immediate thought.

This study appears to be consistent with the latest research. The muscles are not damaged. What it does not address is what serum factors might be at play. However this thesis is likely to have been well underway and maybe nearly or even completely finished before the importance of serum came to light.

So my tentative conclusion is the thesis might be correct, but also that it was investigating the wrong things. However its important to rule things out, and I think this contributed with that.

I have not read the full thesis. I am still rereading the recent cytokine paper.
 

JaimeS

Senior Member
Messages
3,408
Location
Silicon Valley, CA
But of course that relevant research was not done in the UK (to my knowledge) so therefore rendered irrelevant as a result?

That is correct. Only UK research exists in the UK. Outside of the UK is a research-vortex, that sucks in all relevant research and spits out 'edited' pieces now with titles like Pacing (self-management) shows no improvement over diving off cliffs, and CBT/GET "a thing of beauty". Thus, no researcher in the UK is aware that any other sort of research exists.

...this sounds like the plot of a Who episode.

Likely lack of an established lab protocol or workflow for taking patient samples, processing and then immediately testing them. It's a weakness in practically all biomedical research

Practically all is right. ;) It is one of our biggest research challenges.
 

Snow Leopard

Hibernating
Messages
5,902
Location
South Australia
The in vitro muscle culture approaches reported in this thesis have enabled the investigation of the biochemical basis of muscle cell dysfunction in patients with CFS/ME. It is possible to conclude there to be no evidence of impaired muscle function in CFS/ME patients. Additionally, there was no impairment found in PDK enzyme function. Therefore, it can be determined that bioenergetic function is normal in CFS/ME patients and cannot be attributed to the excessive peripheral muscle fatigue phenotype frequently exhibited.

In vitro (especially cell culture) studies cannot be used to "conclude there to be no evidence of impaired muscle function".

What this study shows is that there is no structural defect of the muscle fibres, no classical "mitochondrial disease" (example: no mitochondrial DNA defect).

The can still be peripheral muscle fatigue if something is interfering with metabolic signalling in vivo. This is possible, as an example, due to autoimmune disease, not due to damage due to inflammation, but due to antibodies interfering with signalling.
 
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Messages
58
Practically all is right. ;) It is one of our biggest research challenges.

I'd originally edited out a comment about how that's been part of what's so compelling about Ron Davis's work - both because that's the area where he really advanced genomics, and also because that's why he himself regularly processes samples late in the evening. I'm really looking forward to conversations about that at the symposium this weekend.
 
Messages
15,786
Why not testing the fresh cells and later the cultured cells?
All of the samples were frozen ones from a fatigue center, as far as I could tell.

A more suitable conclusion would say something like 'Bioenergetic function did not appear to differ from controls. If the difference exists it could not be located using these methods, on this sample, at this time.'
Frankly I think even that would be misleading. Many of the results were quite different for patients versus controls, and would have been significant at those levels if there were a sample size bigger than 1-5. So it seems very dishonest to use these results to claim that there's nothing to see in the muscles.

At most it shows a lack of power to draw any conclusions whatsoever, but I think it would be more accurate to say that the results tending toward significance should be repeated with larger samples. It may have been a useful vehicle for the researcher getting her degree, but the lack of power made it scientifically useless from the outset.
 

msf

Senior Member
Messages
3,650
That is correct. Only UK research exists in the UK. Outside of the UK is a research-vortex, that sucks in all relevant research and spits out 'edited' pieces now with titles like Pacing (self-management) shows no improvement over diving off cliffs, and CBT/GET "a thing of beauty". Thus, no researcher in the UK is aware that any other sort of research exists.

Yes, because as we know, the Great British Firewall stops researchers accessing Pubmed.
 

msf

Senior Member
Messages
3,650
I haven´t studied it closely, but it wouldn´t seem to be a complete nul result:

´Glycolytic function was assessed via a combination of extracellular flux analysis (XF) and through the measurement of cellular L-lactate concentration. XF analysis revealed extracellular acidification rate (ECAR) measurements for all glycolytic ii parameters to be comparable in CFS/ME patient muscle samples when compared to controls. Additionally, DCA did not alter ECAR in either group. L-lactate concentration was elevated at rest of post-EPS in CFS/ME cells compared to controls. DCA did not modify L-lactate concentration in either sample group.´

Are they saying that the cellular lactate levels were high, but extracellular levels weren´t? I can´t be bothered to read the whole thing, as there seem to be certain design flaws that reduce the study´s relevance.
 

msf

Senior Member
Messages
3,650
Ok, apparently I could be bothered (a bit).

I think I detect either foul play or at least a conclusion being altered for some reason.

If you look at the quote in my last post, it seems (in poor English) to say that there was a difference in L-lactate levels in cells either at rest or post-EPS, or possibly both (as I said, it is very poorly written).

But when you go to the relevant chapter of the thesis, you find this:


´5.3.1.2 Myoblast L-lactate measurement

L-lactate concentration was measured in CFS/ME (n=5) and control (n=4) myoblast samples. At baseline (0μM DCA) CFS/ME and control myoblasts, samples did not exhibit significantly different L-lactate concentrations. Similarly, DCA treatment (40μm) did not induce any significant effect on either group. Displayed in Figure 5.6.´



and this:


´5.3.1.3 Myotube L-lactate measurement at rest and post-EPS
L-lactate concentration was measured in CFS/ME (n=4) and control myotubes (n=3) at rest and following 24-hours EPS. When CFS/ME myotubes were compared to controls at rest and following EPS there was no significant difference in L-lactate concentration at baseline (0μM) or following DCA (40μM) treatment. Similarly, EPS stimulation exhibited no significant alteration in L-lactate concentration when compared to resting levels in both samples at baseline and following DCA treatment. See figure 5.7.´




Again, the second paragraph is not written well at all. This opaqueness may be intentional, though. If you look at the graph, there does seem to my untrained eye to be a difference between cases and controls after exercise. I am statistically-illiterate, but if you look at the graph, cases go from approx 1.6-7 to 2.5 pmols/well after exercise, whilst controls go from approx 1.8 to 1.9 (the same trend is observed in samples treated with DCA). How is this not a significant difference? Perhaps the author does not think that it is a significant (or politically savvy) comparison, but then why do they seem to mention it in abstract?

Another interesting difference:


´Although, lactic acidification work did not impact upon O2.- generation, a cytotoxicity assay revealed CFS/ME myoblasts to exhibit greater viability than control cells following treatment with lactic acid. This was an interesting finding as it suggests the CFS/ME patient cells to tolerate substantial acidification, although it is difficult to determine the mechanism behind this occurrence. However, as previously described several studies have revealed profound 112
intramuscular acidosis in CFS/ME patients [Jones et al. 2012; 2010], it is possible as a consequence that cells have developed an improved buffering capacity to cope with excessive acidification.´



Finally, this gem:


´It is important to note that contrasting patient bio-energetic responses to exercise exhibited with in vivo studies may be due to the level of patient engagement, which is a key limitation of that mode of investigation. For example, Jones et al. [2012] reported CFS/ME patients to fall into 2 distinct categories in relation to Phosphocreatine (PCr) depletion in response to exercise. The first group demonstrated normal bio-energetic dysfunction, exhibiting PCr depletion to a comparable level to controls when exercising at the same level of MVC. Conversely, the second group exhibited low-level PCr depletion and no exercise induced acidosis as a consequence. The authors postulated this effect to be evidence of some form of exercise avoidance behaviour. In contrast, the in vitro exercise model utilised in the present study eliminates the need for patient compliance in the exercise protocol and enables the bio-energetic function of all patient muscle samples to be examined equally following the same EPS strategy.´


In the context of that last paragraph, I have no qualms about summarising most of the conclusions that can be drawn from the paper as ´it´s in the blood, stupid!´
 
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msf

Senior Member
Messages
3,650

msf

Senior Member
Messages
3,650
I just found this:

´Dichloroacetate is a small molecule of 150 Da (see structure in Figure 3) explaining in part the high bioavailability of this drug and the fact that it can penetrate into the traditional chemotherapy sanctuary sites, including the brain. In vitro, DCA activates PDH by inhibition of PDK at concentration of 10–250 μM or 0.15–37.5 μg ml−1 in a dose-dependent fashion (Stacpoole, 1989). To date, four different isoforms of PDK have been identified that have variable expression and sensitivity to the inhibition by DCA (Sugden and Holness, 2003). The isozyme constitutively expressed in most tissues and with the highest sensitivity to DCA is PDKII; in our published preclinical work we showed that PDK2 inhibition with siRNA completely mimicked DCA effects (Bonnet et al, 2007).´

I think people have suggested that PDK4 is the problem; so perhaps that explains DCA´s lack of effect.

Calling all PDK/DCA experts, calling all PDK/DCA experts...
 
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msf

Senior Member
Messages
3,650
This made me think, has anyone suggested a trial of DCA in ME patients yet?

´A large number of children and adults have been exposed to DCA over the past 40 years, including healthy volunteers and subjects with diverse disease states. Since its first description in 1969 (Stacpoole, 1969), DCA has been studied to alleviate the symptoms or the haemodynamic consequences of the lactic acidosis complicating severe malaria, sepsis, congestive heart failure, burns, cirrhosis, liver transplantation and congenital mitochondrial diseases. Single-arm and randomised trials of DCA used doses ranging from 12.5 to 100 mg kg−1 day−1 orally or intravenously (reviewed in (Stacpoole et al, 2003)). Although DCA was universally effective in lowering lactate levels, it did not alter the course of the primary disease (for example sepsis).´

It seems to have some nasty effects in MELAS though, so I wouldn´t suggest that anyone tries it themselves.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567082/
 
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15,786
But this study seems to show some lactate problems in the muscle cells themselves which are not regulated by PDK (or at least not affected by DCA). Anyone got any ideas about what might be causing this?
Chronic exposure to lactate might be causing changes in the cells themselves? That might explain why they seem less bothered by lactate or abnormal pH, from what I understand.
 

msf

Senior Member
Messages
3,650
Sure, I was just wondering what those changes might be. Some change in the mitochondria? To be consistent with the study though, it has to be one that is not related to PDK, or glycolysis. Or perhaps the study is just wrong.
 
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RogerBlack

Senior Member
Messages
902
Remember, these are cultured cells, grown on from patient cells, and induced to divide.

Only certain defects will pass through to the daughter cells from the cloned ones.
Muscle cells do not in general in the body get replaced.
They are not routinely refreshed, and will normally be there for the life of the infection with CFS, so one hit can affect all muscle cells, forever.
If you then go on and culture cells from these patient muscle cells, only certain defects will pass through to the offspring, and it's a big step to assume that cultured cells will inherit any defect.

In a purified cell culture, there will be almost no antibody action going on, and clearly no influence of the rest of the body.

Washing with patient (and control) serum would at least tell us more.

I'd love to see this experiment, but washed with patient serum from a patient having a good day, and having a very PEM affected day, and having just done exercise that did in fact produce PEM (but had not at the time the sample was taken).