Discriminatory cytokine profiles predict muscle function, fatigue and cognitive function in patients with ME/CFS. McArdle et al. 2020

John Mac

John Mac

Senior Member (Voting Rights)
Full Title:
Discriminatory cytokine profiles predict muscle function, fatigue and cognitive function in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract
Click to expand...
Myalgic Encephalomyelitis (ME) /Chronic Fatigue Syndrome (CFS) is a severely debilitating and complex illness of uncertain aetiology, affecting the lives of millions and characterised by prolonged fatigue.
The initiating factors and mechanisms leading to chronic debilitating muscle fatigue in ME/CFS are unknown and are complicated by the time required for diagnosis.
Both mitochondrial dysfunction and inflammation have been proposed to be central to the pathogenesis of ME/CFS.
This original and extensive study demonstrated that although there was little dysfunction evident in the muscle mitochondria of patients with ME/CFS, particular blood plasma and skeletal muscle cytokines, when adjusted for age, gender and cytokine interactions could predict both diagnosis and a number of measures common to patients with ME/CFS.
These included MVC and perceived fatigue as well as cognitive indices such as pattern and verbal reaction times.
We employed advanced multivariate analyses to cytokine profiles that leverages covariation and intrinsic redundancy to identify patterns of immune signaling that can be evaluated for their predictions of disease phenotype.
The current study identified discriminatory cytokine profiles that can be sufficiently used to distinguish HCs from patients with ME/CFS and provides compelling evidence that a limited number of cytokines are associated with diagnosis and fatigue.
Moreover, this study demonstrates significant potential of using multiplex cytokine profiles and bioinformatics as diagnostic tools for ME/CFS, potentiating the possibility of not only diagnosis, but also being able to individually personalise therapies.
Click to expand...
https://www.medrxiv.org/content/10.1101/2020.08.17.20164715v1?rss=1

This was a study I took part in.

Papers from the same study.
https://bmjopen.bmj.com/content/bmjopen/7/11/e015296.full.pdf
https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fasebj.31.1_supplement.lb789
 
Last edited:
This is one of the set of studies the Medical Research Council funded in 2012, basically the first time the MRC had funded biomedical research. The ME Association also added in a little money for a study that was part of the project.
 
Only MIP-1β differed significantly (p=0.044) between HC and ME/CFS, after controlling for age and sex.
Click to expand...

However, when taking into account the influence of correlations between cytokines by entering them into a joint model (Table 3 and SI Appendix Table S5), higher plasma values of IL-1β, IL-8, IL-10, IP-10, and RANTES were significantly associated with higher Chalder Fatigue scores i.e. greater perceived fatigue, after adjusting for age and gender and taking into account the influence of other cytokines in the mode and lower plasma values of PDGF, eotaxin, G-CSF, and MIP1α were associated with greater perceived fatigue. Similarly, lower values of PDGF and MIP-1a and higher values of IL-10 and RANTES were associated with lower SF-36 scores (worse physical health)
Click to expand...

The problem with statistical models like this is that they don't tend to replicate.

The sample size was decent, but 92 male patients and 63 female patients seems like an odd ratio.

On the neuropsychological/cognitive testing, patients had the usual poor reaction times and poorer verbal memory, suggesting poor concentration.

Analysis of muscle samples for mitochondrial ROS production, respiration and mitochondrial membrane potential were performed in a subsample of ME/CFS patients (n=7) and HCs (n=6). Figure 2A shows changes in 2-OH-Mito-E + fluorescence over time in muscle fibres from patients with ME/CFS and HCs. Data demonstrated no significant difference in mitochondrial superoxide generated by muscle fibres from patients with ME/CFS and HCs either prior to (Figure 2A) or following (Figure 2B) the addition of the nitric oxide synthetase inhibitor L-NAME. No difference in mitochondrial H2O2 generation was seen either with endogenous or added substrates in muscle bundles from patients with ME/CFS compared with HCs (Figure 2C). Data for citrate synthase activity, mitochondrial respiration, P:O ratio demonstrated no significant changes in mitochondrial number (represented by citrate synthase activity), resting mitochondrial respiration or efficiency of oxidative phosphorylation. (SI Appendix, Figure 2). Similarly, no significant differences in mitochondrial membrane potential were seen in response to oligomycin and the protonophore FCCP oxidative phosphorylation inhibitors (Figure 2D). No evidence for changes in oxidative damage were observed between patients with ME/CFS and HCs, shown by the analyses of protein oxidation, lipid peroxidation and the levels in protein nitration (3-NT) (data not shown)
Click to expand...

Muscle biopsies were analysed for mRNA content of a number of cytokines (eotaxin 1, MIP-1α, TNF-α, IL-6, IL-8, RANTES, MCP-1, KC and IP-10), however, significant increases were only detected in mRNA content of MCP-1 and IP-10 suggesting the specific increase in production of these cytokines by skeletal muscles of patients with ME/CFS (Figure 3). Levels of Atrogin 1 mRNA were also significantly elevated in muscle biopsies of patients with ME/CFS compared with HCs (Figure 3) suggesting an increase in protein degradation processes in skeletal muscles of patients with ME/CFS compared with HCs.
Click to expand...

Maximal voluntary contraction was lower in patients than controls, there was no difference in the generation of maximal involuntary contraction by electrical stimulation between the two groups overall and at any frequency (relative to MVC). Given that this is relative to MVC, this is expected, it simply suggests that patients were producing sufficient maximal effort.

Figure 1C indicates that the rate of loss of torque (rate of fatigue during electrical stimulation to contract) appeared greater in the muscle of patients with ME/CFS compared with HCs and this difference increased with duration of stimulation. However, there was no significant difference between HC and ME/CFS and no group by time interaction.
Click to expand...

No significant difference was seen in the rate of fatigue of muscles when electrically stimulated to contract in patients with ME/CFS (Figure 1C), also suggesting no intrinsic defect in contractile proteins.
Click to expand...

Notably, during the "fatigue resistance test" the amplitude of electrical stimulation was not set at supramaximal levels. Instead set at "30% of the participants MVC force when stimulated at 100Hz".

Looking at the graph, it is indeed curious that it was not statistically different. Given that the two seem to deviate further over time, it is possible that a difference would have emerged if the test went on longer than 2 minutes.

Also note that fatigue during similar tests in muscular dystrophies and peripheral neuropathies have the same results, namely the fatigue is predominantly "central" in origin. The fact that the same pattern is observed in vastly different pathologies suggests that there is substantial coupling between peripheral afferents and "central fatigue", likely mediated through spinal feedback of type 3 and type 4 afferents, the purpose of which is to reduce the rate of unexpected peripheral fatigue (and thus makes force output more predictable to the brain).

https://repository.ubn.ru.nl/bitstream/handle/2066/50860/50860.pdf (2006 thesis by Joke Kalkman. Studies compared fatigue in myotonic dystrophy, facioscapulohumeral dystrophy, and Hereditary Motor and Sensory Neuropathy type I - patients had less peripheral fatigue than health controls)
 
(I worked on this this morning, before running out of energy. I agree with Snow Leopard about the fatiguability study - it looked as though there was or soon would have been a significant difference.)

The authors have covered a lot in this paper. Good to see that they are referring to ME/CFS (mostly - still some use of CFS in places in the paper) and 100% of the cohort reported suffering from PEM.

Here's some commentary about what they found out about just
Muscle function and fatigue in patients with ME/CFS

Voluntary muscle contraction
They note that the people with ME/CFS had lower mean voluntary contraction force (MVC).
Figure 1A
Screen Shot 2020-08-23 at 7.45.02 AM.png
There is a difference in age and gender of the ME/CFS and Healthy control samples - there was some adjustment for that. There's text that suggests that the difference in MVC has a p value of 0.0272 after adjusting for age and gender. So, it's not a particularly strong difference. The difference might just be a product of residual unadjusted effects of the ME/CFS sample having a higher percentage of females, an older mean age and more people with a sedentary lifestyle.

My experience is that my muscle strength is variable - at times I think it's similar to what is typical for a woman my age, and then other times I just can't summon up adequate muscle strength. Walking up steps can be fine one hour and then later take a ridiculous amount of effort. So, I'm happy to assume that the difference in MVC they found is real.


Actin-myosin filament interactions
They report that there were no differences in the contractions in 'isolated skinned muscle fibres'. They stimulated the fibres with electricity and tested by increasing calcium concentrations. The authors felt this proved that the actin-myosin filament interactions were fine.

Involuntary contraction of muscles in lower limb
They found that involuntary contraction of lower limb muscles (by electrical stimulation) produced similar contraction forces in the ME/CFS and healthy control cohorts. Again, there was some unspecified adjustment for age and gender differences in the sample. I didn't see a P value for this. There does look to be a bit of a trend to lower forces in the CFS sample. I wonder what would be found if the ME/CFS people had only been tested when in PEM.

Figure 1B
Screen Shot 2020-08-23 at 8.05.33 AM.png

Muscle fatiguability
They tested fatiguability of involuntary contractions. See figure 1C - (duration on the x-axis), it looks as though the ME/CFS cohort's muscles became increasing less effective over time, relative to the controls. However, the text notes that there was no significant difference and doesn't give a P value. The conclusion is that there is no difference. It makes me wonder if the ME/CFS cohort was mixed, with some people not actually having ME/CFS - or perhaps some had PEM prior to testing and some did not. And also if the adjustment for age and gender was appropriate.

Figure 1C
Screen Shot 2020-08-23 at 9.00.10 AM.png

Atrogen-1 levels
They found lower levels of atrogen-1 in the muscle biopsies of the ME/CFS cohort, compared to the controls, reportedly suggesting an increase in protein degradation processes. This looks like a real difference. The question is, is this something specific to ME/CFS or is it just a product of reduced muscle mass as a result of a sedentary lifestyle. The finding looks worth investigating a bit more.

Figure 3c
Screen Shot 2020-08-23 at 10.50.01 AM.png
They also found a larger percentage of small muscle fibres in the ME/CFS cohort relative to the controls.

tl:dr
So, the paper concludes that people with ME/CFS have lower voluntary muscle contractile force, but involuntary force (measured by electrical stimulation) is not different. I don't think this study conclusively proves that, especially under conditions of PEM and longer repeated use.
 
Last edited:
Something to consider is that not all of the participants were included in each sub-study. So, for the muscle testing in vivo, there were only 15 people with ME/CFS. For the muscle biopsy, it varied according to the different tests done, but were only between 7 to 12 people with ME/CFS.
 
Thought I would quote the abstract of Micheal VanElzakkers paper "Neuroinflammation and Cytokines in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): A Critical Review of Research Methods"
Studies seeking to find a peripheral circulating cytokine “profile” for ME/CFS are reviewed, with attention paid to the biological and methodological reasons for lack of replication among these studies.

We argue that both the biological mechanisms of cytokines and the innumerable sources of potential variance in their measurement make it unlikely that a consistent and replicable diagnostic cytokine profile will ever be discovered.
Click to expand...
Paper : https://www.frontiersin.org/articles/10.3389/fneur.2018.01033/full
Thread : https://www.s4me.info/threads/front...-methods-michael-vanelzakker-et-al-2018.6758/

I tried to scan the paper of this thread a few times but could find no detailed description of when they took the blood and how exactly it was processed. Did they use the same equipment and operators for patients and controls and were samples processed together. Montoya went to great lengths in his study to try and even control for centrifuge by alternating samples in slots of the machine.

In Montoya's large Cytokine study paper the "Cytokine Assay" section describes the lengths he went to to ensure good quality. The "Statistical Analysis" section talks about techniques to remove plate effects.

Paper : Cytokine signature associated with disease severity in chronic fatigue syndrome patients
https://www.pnas.org/content/114/34/E7150

"Cytokine Assay" section
A total of 19 plates were used. Each participant’s sample was entered in two replicate wells, and matched sets of ME/CFS cases and healthy controls were always mixed in all plates to reduce confounding case status with plate artifacts. Results were accepted as final (569 samples) if more than 95% of data had a coefficient of variation (CV) <10%. When the CV exceeded 30% (15 samples), the averaging over duplicate wells reduced the technical variance in median fluorescence intensities (FIs) by twofold.

Each plate also contained two wells of internal control and wells to account for generic binding to the beads (CHEX1, CHEX2, CHEX3, CHEX4) unrelated to the target cytokine.
Click to expand...

"Statistical Analysis" section
Consistent preprocessing across cytokines facilitated their comparison and biological interpretation. MFI data were preprocessed (pMFI) for each cytokine through a sequence of averaging over duplicate wells, natural-logarithm transformation to reduce variance heterogeneity, isolation and removal of plate effects, and centering and scaling. Use of population marginal means (61) adjusted for covariates of age, sex, and race (white vs. nonwhite) permitted estimation and removal by subtraction of plate effects that were balanced (i.e., 1:1 rather than 2:1) with respect to control vs. case status. Centering and scaling entailed subtracting the sample mean and dividing by the sample SD.
Click to expand...
 
Can somebody explain figure 2B and the text relating to it?
Analysis of muscle samples for mitochondrial ROS production, respiration and mitochondrial membrane potential were performed in a subsample of ME/CFS patients (n=7) and HCs (n=6). Figure 2A shows changes in 2-OH-Mito-E+fluorescence over time in muscle fibres from patients with ME/CFS and HCs. Data demonstrated no significant difference in mitochondrial superoxide generated by muscle fibres from patients with ME/CFS and HCs either prior to (Figure 2A) or following (Figure 2B) the addition of the nitric oxide synthetase inhibitor L-NAME.
Click to expand...
So the text says no significant difference in muscle fibre generated mitochondrial superoxide, neither with nor without addition of a nitric oxide synthetase inhibitor. Yet the graphs seem to show a difference. Figure 2B shows complete separation of ME from HC from the 40 minute mark, with the trend suggesting even further separation with more time. So why is this not significant?
ME mtROS graphs.JPG
 
cassava7 said:
Just a small typo, there were 29 male patients.
Click to expand...

It is certainly confusing. I had to double check Table 1 to confirm that you were right!

Ravn said:
So why is this not significant?
Click to expand...

The error bars don't suggest statistical significance. The lack of significance is likely due to lack of sample-size and within-group variability.
 
Last edited:
Hutan said:
Something to consider is that not all of the participants were included in each sub-study. So, for the muscle testing in vivo, there were only 15 people with ME/CFS. For the muscle biopsy, it varied according to the different tests done, but were only between 7 to 12 people with ME/CFS.
Click to expand...
Do we know the ratio of male / females tested?
Asking as some studies have suggested that females utilize protein differently - might this not affect muscles and perhaps indicate catabolization ?
 
You must log in or register to reply here.
Back
Top Bottom