Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls, 2023, Giloteaux et al

Background

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, heterogenous disease characterized by unexplained persistent fatigue and other features including cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Cytokines are present in plasma and encapsulated in extracellular vesicles (EVs), but there have been only a few reports of EV characteristics and cargo in ME/CFS. Several small studies have previously described plasma proteins or protein pathways that are associated with ME/CFS.

Methods
We prepared extracellular vesicles (EVs) from frozen plasma samples from a cohort of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) cases and controls with prior published plasma cytokine and plasma proteomics data. The cytokine content of the plasma-derived extracellular vesicles was determined by a multiplex assay and differences between patients and controls were assessed. We then performed multi-omic statistical analyses that considered not only this new data, but extensive clinical data describing the health of the subjects.

Results
ME/CFS cases exhibited greater size and concentration of EVs in plasma. Assays of cytokine content in EVs revealed IL2 was significantly higher in cases. We observed numerous correlations among EV cytokines, among plasma cytokines, and among plasma proteins from mass spectrometry proteomics. Significant correlations between clinical data and protein levels suggest roles of particular proteins and pathways in the disease. For example, higher levels of the pro-inflammatory cytokines Granulocyte-Monocyte Colony-Stimulating Factor (CSF2) and Tumor Necrosis Factor (TNFα) were correlated with greater physical and fatigue symptoms in ME/CFS cases. Higher serine protease SERPINA5, which is involved in hemostasis, was correlated with higher SF-36 general health scores in ME/CFS. Machine learning classifiers were able to identify a list of 20 proteins that could discriminate between cases and controls, with XGBoost providing the best classification with 86.1% accuracy and a cross-validated AUROC value of 0.947. Random Forest distinguished cases from controls with 79.1% accuracy and an AUROC value of 0.891 using only 7 proteins.

Conclusions
These findings add to the substantial number of objective differences in biomolecules that have been identified in individuals with ME/CFS. The observed correlations of proteins important in immune responses and hemostasis with clinical data further implicates a disturbance of these functions in ME/CFS.

Open access, https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-023-04179-3

 

They use Fukuda criteria (how many would qualify under better criteria?) for the ME group, and compare it against health controls. This definitely needs to be compared to similarly unhealthy non-ME controls.

 

The paper says "All cases met the 1994 CDC Fukuda [28] and/or 2003 Canadian consensus criteria for ME/CFS" but also "All patients who were selected met the 1994 Fukuda definition for ME/CFS.", so it's not particularly clear.

 

To be of any interest micro clots would be in the 5 micron or larger range. I think some of the pictures suggested up to 100 micron. These should be removed in the preparation of 'plasma' from blood by the centrifugation (or sedimentation) of cells (about 5 microns - so 'plasma' pretty much by definition is not going to contain 'micro clots'). I have had to assume that the clots described by Pretorius are newly formed during the incubation process with fluorescent tag - which I think is half an hour at room temperature. At normal calcium levels plasma will of course clot completely in that time.

These vesicles are smaller than half a micron - ten times smaller at least and mostly about 100 times smaller than 5 microns. They would need very powerful centrifugation.

 

https://twitter.com/user/status/1657507560407150594

 

I am never symptom free, am always suffering the effects of ME, so I find this idea that we must be suffering PEM to be odd. PEM is just a part of ME.. but surely we should be able to find a test that shows abnormalities at rest, and not following exertion, also.

 

What @Braganca says still resonates with me, though, because PEM is tricky to pin down. Exertion-related symptom fluctuation is absolutely a thing, and we know it when we see it, but it’s difficult to differentiate with absolute certainty between variations in baseline illness, and variations in the different flavours of PEM (especially between faster and slower onset PEM).

Research which seeks to establish biochemical differences between baseline and PEM states would need to be sensitive to this difficulty.

 

It is also risky to trigger PEM, because that can result in long term worsening.

There is a need to find bio markers that do no harm.

I too feel shitty all the time, no good and bad days.

Edit: I am not slamming researchers for studying PEM nor do I slam patients choosing to enrol in a PEM study- but there are risks and both patients and researchers need to be aware of them and proper follow up should be included in the research to find out how long it took to return to baseline if at all.

 

Serpina5 came up in a multi-omics paper on long covid. And here in a genetic evaluation in me/cfs.

 

In the paper we read :

What the paper does not mention is that PROS1 is a Vitamin K-related protein. The machine learning system I use , suggests that Vitamin K metabolism disruption is an important factor to ME/CFS pathology.

From thread : https://www.s4me.info/threads/machine-learning-assisted-research-on-me-cfs.5015/#post-90238

 

FWIW, that applies to me too, and that's after I managed to accidentally cure my PEM. I still have the same set of symptoms (brainfog, lethargy, aches, etc), and various foods can make them worse, but I don't have PEM making them worse after exertion. I see PEM as a mechanism that affects the mechanisms that lead to ME's other symptoms, rather than causing specific symptoms on its own.

 

Just looked for "PROS1" in the paper & thought about if for 10 seconds ---

"In control samples, Protein S (PROS1) and Fc Receptor Like 3 (FCRL3) were negatively correlated with Vitality"
How did they measure "Vitality"? I'd prefer they'd measured it objectively via actimetry [FitBit type devices] -- Also, how would you get appropriate age & activity matched controls?
If PROS1 & FCRL3 are higher(?)

• in ME, and
• controls with similar activity levels,

then is this a consequence (rather than cause) of inactivity?

 

Brain fog could be getting the better of me, but is there a problem with the references in this paper?

E.g. when they are talking about the previous cytokine level study, reference 26 links to a paper by Nagy-Szakal on fecal metagenomic profiles.

 

They also looked at plasma immune molecules in that study, and many of the authors were listed for both papers, so at a quick look I don't think so.