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

[Andy]

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

 

[Creekside]

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.

 

[Andy]

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.

 

[Hutan]

However, a number of assays, such as neuroimaging [8], distinguish Gulf War Illness and ME/CFS.

The reference for that is
Baraniuk JN. Review of the midbrain ascending arousal network nuclei and implications for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), Gulf War Illness (GWI) and Postexertional Malaise (PEM). Brain Sci. 2022
For me, the jury is still out on that one. The symptoms for GWI and ME/CFS are so similar that I feel it is likely that there is some overlap. I see we don't have any commentary on the forum thread for the Baraniuk paper yet.

 

[Hutan]

49 ME/CFS cases; 49 controls
Maureen Hanson's Cornell lab and the Lipkin and Hornig Columbia lab - looks like the latter had the samples and the former had the money to analyse them.

The samples had previously been analysed for plasma proteins and cytokines, with patient data attached; this analysis looked at proteins and cytokines in the extracellular vesicles and then looked for relationships between all the data.

Thrombin (611 U/ml) (System Bioscience, Palo Alto, CA, USA) was added and samples were incubated for 5 min at room temperature to remove fibrinogen, centrifuged at 10,000 ×g for 5 min, and the supernatant was collected.

Extracellular vesicles were purified from plasma samples from ME/CFS patients and healthy individuals by precipitation and their size and concentrations analyzed by Nanoparticle Tracking Analysis (NTA) to investigate whether there were differences between clinical groups. All nanoparticles purified were smaller than 500 nm, most of them being in the typical exosome size range of 30–130 nm

I'm just wondering if that tells us anything about the existence of microclots. I think the story with microclots is that there are fibrinogen clumps that don't dissolve with normal procedures. Would we expect researchers who are just going about their normal business, processing the plasma, to see the microclots also, or is there a special procedure that makes them visible?

 

[Hutan]

They looked at the data and removed the data associated with patients who had a high number of outlier values. That (removing outliers at the patient level) seems like a good way to get more homogeneous samples. Obviously there's a limit to it.

The resulting q-values suggested that two ME/CFS patients presented outlier profiles not initially suspected by their clinical features and therefore should be removed from the EV cytokines dataset as they represented 43% and 50% of outliers respectively (21 and 24 outliers out of 48 cytokines).

They seemed to treat the four datasets differently though (EV proteins and cytokines; plasma proteins and cytokines), and I'm not sure that makes sense. If the basis for removal is that the patient may not be an ME/CFS patient, then I would have thought you'd take out their data from all of the analyses.

 

[Hutan]

IL-2 in the EVs was reported as being higher in ME/CFS, and that was the strongest difference in the EV cytokines. But, there was a lot of overlap.

On the EV cytokines - good to see some studies agreeing with each other with their findings

In contrast, we identified 17 EV cytokines that distinguish patients and controls with adjusted p-values of less than 0.2, all higher in ME/CFS subjects. Out of these 17 proteins, the majority (10 out of 17) are known to be pro-inflammatory cytokines/chemokines (TNFα, IL1β, CXCL8, CXCL1, IL15, CCL7, IL17, CCL5, IL1α and IL1R1), 5 are related to adaptive immunity (IL2, CSF2, IL3, IL4 and IL7), IL12p40 has anti-inflammatory properties and NGFβ is both pro- and anti-inflammatory. Higher levels of pro-inflammatory cytokines are in line with previous reports [43–45].

25]. The most significant difference was IL2 (q = 0.007, Fig. 3). IL2 is a secreted cytokine produced by activated CD4 + and CD8 + T lymphocytes and promotes strong proliferation of activated B-cells and subsequently immunoglobulin production. It plays a pivotal role in regulating the adaptive immune system by controlling the survival and proliferation of regulatory T-cells. IL2 levels were found to be higher in cerebrospinal fluid [46] and plasma from ME/CFS patients [47]. The higher levels of IL2 found in EVs in the present study might be part of a specific immune response in ME/CFS. A number of cytokines/chemokines which were observed to be dysregulated are either produced by B cells or are also B cell regulators (e.g. CXCL1 and CXCL12).

 

[Jonathan Edwards]

Would we expect researchers who are just going about their normal business, processing the plasma, to see the microclots also, or is there a special procedure that makes them visible?

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.

 

[Robert 1973]

 

[Braganca]

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.

 

[Trish]

I think the aim is to find out what biochemical differences there are both when in PEM and not, and how each compares with healthy people before and after an exercise challenge intended to trigger PEM. There's no suggestion that pwME are ever symptom free.

 

[Shadrach Loom]

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.

 

[Milo]

because PEM is tricky to pin down

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.

 

[dreampop]

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

 

[mariovitali]

In the paper we read :

Higher levels of another protein associated with hemostasis, PROS1, is correlated with poorer health in the controls but has no significant association with the ME/CFS cohort (Fig. 7a). PROS1, also known as Protein S, is a well-known regulator of hemostasis, with important anti-coagulant effects [62]. The fact that it has no correlation with health of ME/CFS patients may reflect disturbed control of hemostasis in the disease.

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

Primary target is still Liver function. A "Liver Stressor" such as a Virus, Medications or even prolonged Stress may disrupt Liver function which in turn disrupts key metabolic pathways that include Bile Acid Metabolism, Endoplasmic Reticulum Stress, Phagocytosis, Vitamin K Metabolism (list not inclusive)

 

[Creekside]

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

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.

 

[FMMM1]

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?

 

[katrinapears]

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.

 

[Andy]

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.

 

[Tom Kindlon]