Altered Erythrocyte Biophysical Properties in Chronic Fatigue Syndrome, 2019, Saha, Davis, et al

[Andy]

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multi-systemic illness of unknown etiology affecting millions of individuals worldwide. In this work, we tested the hypothesis that erythrocyte biophysical properties are adversely affected in ME/CFS.

We tested erythrocyte deformability using a high-throughput microfluidic device which mimics microcapillaries. We perfused erythrocytes from ME/CFS patients and from age and sex matched healthy controls (n=14 pairs of donors) through a high-throughput microfluidic platform (5μmx5μm). We recorded cell movement at high speed (4000 fps), followed by image analysis to assess the following parameters: entry time (time required by cells to completely enter the test channels), average transit velocity (velocity of cells inside the test channels) and elongation index (ratio of the major diameter before and after deformation in the test channel). We observed that erythrocytes from ME/CFS patients had higher entry time, lower average transit velocity and lower elongation index as compared to healthy controls.

Taken together, this data shows that erythrocytes from ME/CFS patients have reduced deformability. To corroborate our findings, we measured the erythrocyte sedimentation rate for these donors which show that the erythrocytes from ME/CFS patients had lower sedimentation rates. To understand the basis for differences in deformability, we investigated changes in the fluidity of the membrane using pyrenedecanoic acid and observed that erythrocytes from ME/CFS patients have lower membrane fluidity. Zeta potential measurements showed that ME/CFS patients had lower net negative surface charge on the erythrocyte plasma membrane. Higher levels of reactive oxygen species in erythrocytes from ME/CFS patients were also observed. Using scanning electron microscopy, we also observed changes in erythrocyte morphology between ME/CFS patients and healthy controls.

Finally, preliminary studies show that erythrocytes from “recovering” ME/CFS patients do not show such differences, suggesting a connection between erythrocyte deformability and disease severity.

Paywalled at https://www.cell.com/biophysj/fulltext/S0006-3495(18)31946-5

Edit: We have 3 threads on related or the same research by the same team:
Erythrocyte Deformability As a Potential Biomarker for Chronic Fatigue Syndrome, Davis et al (2018)
Altered Erythrocyte Biophysical Properties in Chronic Fatigue Syndrome, 2019, Saha, Davis, et al
Red blood cell deformability is diminished in patients with Chronic Fatigue Syndrome, Saha et al, 2019

 

[Andy]

A lot of discussion about the research behind this paper is here, https://www.s4me.info/threads/eryth...ronic-fatigue-syndrome-davis-et-al-2018.6938/

 

[duncan]

One parasite and one bacterium leap to mind as potential culprits: Babesia and Bartonella, respectively.

 

[rvallee]

This is all well above my meager understanding of biology, but this seems interesting:

Higher levels of reactive oxygen species in erythrocytes from ME/CFS patients were also observed

From Wikipedia (quotes below are all from wiki, the paper is not yet available on sci-hub):

Reactive oxygen species (ROS) are chemically reactive chemical species containing oxygen.

In a biological context, ROS are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis.[3] However, during times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress.

Higher levels is not at the same level as dramatic increase, so cell damage may not occur at slightly elevated concentrations but other consequences of oxidative stress may still play a role.

Egg, or chicken?

Reactive oxygen species are implicated in cellular activity to a variety of inflammatory responses

Could elevated levels but-not-quite-dramatically-increased of ROS explain hyperacusis? High levels seem capable of causing deafness, could slightly elevated levels cause inflammation that make them oversensitive?

They may also be involved in hearing impairment via cochlear damage induced by elevated sound levels

ROSes play a role in pathogen response... hmmm:

In the mammalian host, ROS is induced as an antimicrobial defense. To highlight the importance of this defense, individuals with chronic granulomatous disease who have deficiencies in generating ROS, are highly susceptible to infection by a broad range of microbes including Salmonella enterica, Staphylococcus aureus, Serratia marcescens, and Aspergillus spp.

Interferons...:

Increased levels of ROS potentiate signaling through this mitochondria-associated antiviral receptor to activate interferon regulatory factor (IRF)-3, IRF-7, and nuclear factor kappa B (NF-κB), resulting in an antiviral state

Now I'm curious if it can be checked whether ME patients have faster cell renewal, the production of which would certainly be energy intensive and could explain telomere reduction?

Current studies demonstrate that the accumulation of ROS can decrease an organism's fitness because oxidative damage is a contributor to senescence.

Quite:

In particular, the accumulation of oxidative damage may lead to cognitive dysfunction

Experimental and epidemiologic research over the past several years has indicated close associations among ROS, chronic inflammation, and cancer.[37] ROS induces chronic inflammation by the induction of COX-2, inflammatory cytokines (TNFα, interleukin 1 (IL-1), IL-6), chemokines (IL-8, CXCR4) and pro-inflammatory transcription factors (NF-κB).

Anyway, this is above my cognitive level but very interesting. It could potentially explain the mechanism of PEM, when increased oxygen demand can't be met because oxygen can't be transported efficiently, leading to a cascade of effect from oxygen-starved cells.

Can't wait to read the paper and not understand most of it, but it seems promising.

 

[duncan]

It could potentially explain the mechanism of PEM, when increased oxygen demand can't be met because oxygen can't be transported efficiently

Technically that would comport with anemia, just not the PEM part. It's a curious tie-in, nonetheless. Babesia is notorious for hemolytic anemia. I satisfy diagnositcs for both. My RBC's are warped as evidenced by out-of -range small RBCs and abnormal MCV and mhcv etc.

All this reflected in the paper may not have anything to do with a downstream effect from infection, but fun to think about.

As I recall, the import of the paper was LESS deformability, though, or deformability instances, but perhaps I have that wrong.

 

[wigglethemouse]

Paywalled at https://www.cell.com/biophysj/fulltext/S0006-3495(18)31946-5

From Wikipedia (quotes below are all from wiki, the paper is not yet available on sci-hub):

Apparently this is just a short article. The paragraph you see in the link is all there is. There will be no further details in this article.

 

[leokitten]

Didn’t one of the main figures in the paper show significant overlap between ME and HC? Everytime there’s a biomarker paper out it always turns out that there is just too much overlap to be useful and biomarker is more or less pointless beyond adding to the research knowledgebase on ME.

I think the only paper I’ve seen that didn’t have any overlap was MyHill’s mitochondrial energy score (MES)

Chronic fatigue syndrome and mitochondrial dysfunction
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680051/

Look at Figure 4, that’s what we are looking for in a biomarker, composite or otherwise.

 

[wigglethemouse]

Didn’t one of the main figures in the paper show significant overlap between ME and HC?

This result on deformability from the first Davis et al RBC paper shows no overlap. The number of patient was low in this initial study. There was a very clear difference. I've not seen an ME study with such a marked differnce

The second article that this thread is based on mentions a recovering patient looks much better. Les Simpson in his large studies noticed the same. In a longitudinal study RBC's looked more normal when patients were in a "better" phase.

I think the only paper I’ve seen that didn’t have any overlap was MyHill’s mitochondrial energy score (MES)

Chronic fatigue syndrome and mitochondrial dysfunction
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680051/

Look at Figure 4, that’s what we are looking for in a biomarker, composite or otherwise.

Like you I've been impressed with this paper. However nobody over the years has been able to replicate this finding. Most recently Karl Morten from Oxford in his NZ lecture stated that both his team and Julia Newtons team had tried to replicate with funding from the ME association and they had not been able to. In fact he said handling of the blood plays a huge role in the results you get and testing 24hours later like the ATP profile test does bares no resemblance to testing on fresh blood. I recommend watching that presentation.

 

[Amw66]

This result on deformability from the first Davis et al RBC paper shows no overlap. The number of patient was low in this initial study. There was a very clear difference. I've not seen an ME study with such a marked differnce
View attachment 6084
The second article that this thread is based on mentions a recovering patient looks much better. Les Simpson in his large studies noticed the same. In a longitudinal study RBC's looked more normal when patients were in a "better" phase.


Like you I've been impressed with this paper. However nobody over the years has been able to replicate this finding. Most recently Karl Morten from Oxford in his NZ lecture stated that both his team and Julia Newtons team had tried to replicate with funding from the ME association and they had not been able to. In fact he said handling of the blood plays a huge role in the results you get and testing 24hours later like the ATP profile test does bares no resemblance to testing on fresh blood. I recommend watching that presentation.

Had anyone other than Karl Morten tried? Norman Booth pestered MEA for years to get interest and replication.
Whilst disappointing that it could not be replicated, the recent research has found a number of interesting anomalies, and created interest.

 

[wigglethemouse]

Had anyone other than Karl Morten tried?

Both Karl Mortens team and a Newcastle team failed to replicate. These teams are considered experts on Mitochondria in the UK. Other places have previously tried to replicate but were not successful. The recent ME Association write up of Karl Mortens talk mentions this replication work.
https://www.meassociation.org.uk/20...disease-pathology-in-me-cfs-21-december-2018/

The late Norman Booth talked about some of the earlier failed replication work on PR
Link : https://forums.phoenixrising.me/threads/mitochondrial-and-energy-metabolism-dysfunction-in-me-cfs-—-myhill-booth-and-mclaren-howard-papers.47488/post-785000

A number of scientists have quoted published results of other studies of neutrophils that appear to disagree with John’s measurements. I have worried about this and have had several discussions with John about the disagreements. I am convinced of John’s integrity and talent. He measures neutrophils when they are still alive and are in a sterilized, aerated environment. Here they are going about their task of sensing the presence of foreign bodies and pathogens using their motility and chemotaxis. They are not in the process of honing in on pathogens or carrying out phagocytosis, which heavily rely on glycolysis. In one of the first steps in the ATP Profile, John inhibits mitochondrial function with sodium azide, but for only 3 minutes. This short period is adequate and also short enough to prevent apoptosis of any of the neutrophils. In some other studies neutrophils have been inhibited with the toxin Rotenone for as long as 6 hours [4]. If any of the cells are still alive they will have to have found some other pathway to provide them with ATP. As of 1st February 2016 John has made a change in his procedure, and now measures mixed leukocytes containing monocytes as well as neutrophils. There are only small differences from neutrophils only. John is not happy to use lymphocytes alone because of the varying subtypes present and the responses to infection.

Many people have also used the commercially available (and very expensive) Seahorse instrument from Agilent to test mitochondrial energy. Ron Davis talked about it at IMEC13 (Cara Tomas at Newcastle has also published on this).
https://www.omf.ngo/wp-content/uplo...ents-ME-CFS-IiME-Research-Conf-May-2018-1.pdf

What gives the best result is using isolated T-cells (blood cell that plays a role in immunity) from patients and then stimulated these T-cells and measuring their energy production.

And that's been pretty reproducible between the healthy controls and ME/CFS patients. if we don't use the stimulation in T-cells its more variable and its also dependent on when they've last eaten, and that's a really difficult thing for the patient to define and they can't eat before they come in.

Ron Davis also has the nanoneedle test he is working on and Karl Morten has reproduced issues with muscle cells. Both have included plasma swap experiments showing something in the blood is affecting energy.

Hopefully we will get more info on the biomarker bake-off at some point then we will see how reliable each test is.
https://www.omf.ngo/wp-content/uploads/2018/11/Edited-Ronald-W-Davis.pdf

16:06 So we're now doing what's called a bake-off. That is we're taking blood samples and running them on all the instruments and looking to see how well they do. This is also to see if we get any inconsistencies and that will help us looking at false positive, false negatives.

EDIT : Here is Cara Tomas Seahorse paper. There is overlap between patients and controls.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186802

 

[leokitten]

@wigglethemouse have Davis and co said what other chronic inflammatory illnesses they will test for deformability? To test if this is specific to ME/CFS or simply a sign of many chronic inflammatory illnesses? I think others have also expressed concern about the test's potential lack of specificity.

 

[leokitten]

This result on deformability from the first Davis et al RBC paper shows no overlap. The number of patient was low in this initial study. There was a very clear difference. I've not seen an ME study with such a marked differnce
View attachment 6084
The second article that this thread is based on mentions a recovering patient looks much better. Les Simpson in his large studies noticed the same. In a longitudinal study RBC's looked more normal when patients were in a "better" phase.

Thank you for pointing this figure out.

 

[wigglethemouse]

@wigglethemouse have Davis and co said what other chronic inflammatory illnesses they will test for deformability? To test if this is specific to ME/CFS or simply a sign of many chronic inflammatory illnesses? I think others have also expressed concern about the test's potential lack of specificity.

I have not read anything about testing other illnesses at this stage. From what I've seen other people post, this test is unlikely to be disease specific, but can be useful to show something biologically is wrong.

Les Simpson tested his own RBC's during infection and noticed changes
Link to Post on PR - https://forums.phoenixrising.me/threads/research-update-from-prof-ron-davis-video.56737/post-959242

Les Simpson did a study on RBC comparing ME to MS
Link to Post on PR - https://forums.phoenixrising.me/thr...er-mohsen-nemat-gorgani-phd.61118/post-994417

Les Simpson wanted to establish a clinic to test red blood cells. If Davis et al can come up with a working test maybe his dream can become reality.
Link : http://www.cfidsreport.com/Articles/researchers/lessimpson.htm

An attempt by Simpson to establish a clinic to test red blood cells did not survive due to lack of funds, a problem experienced by many researchers interested in CFIDS. If Simpson’s ideas are correct, agents which improve blood viscosity/flow may relieve symptoms in CFIDS patients. He hopes to someday see placebo-controlled studies conducted “with the primary objective of determining which agent [haemorheological], if any, has the ability to improve patient well-being.” He continues, “Because it is not possible to increase the diameter of a capillary, treatments should be aimed at increasing red cell flexibility.

 

[leokitten]

I have not read anything about testing other illnesses at this stage. From what I've seen other people post, this test is unlikely to be disease specific, but can be useful to show something biologically is wrong.

There’s a major problem with this approach since many (or most) PwME have comorbidities or other diseases which are autoimmune and/or inflammatory. I also have psoriasis for example, which might significantly contribute to reduced erythrocyte deformability.

While they wouldn’t necessarily have to show that reduced deformability is specific to ME, they would possibly have to show that it isn’t significant in cohorts of patients without ME that have common ME comorbidities, as well as other major diseases (such as psoriasis which isn’t a common ME comorbidity).

So this test might not be helpful to show there is something biologically wrong that isn’t attributed to a comorbidity or other existing disease.

 

[wastwater]

Could a negative test be developed for example most patients with ME are said to react badly to alcohol are there any other safer more common substances to try
Include unusual reactions as well

 

[wigglethemouse]

Could a negative test be developed for example most patients with ME are said to react badly to alcohol are there any other safer more common substances to try
Include unusual reactions as well

Alcohol tolerance is an interesting subject - Searching "erythrocyte alcohol" brings up lots of interesting papers e.g.
The Effect of Alcohols on Red Blood Cell Mechanical Properties and Membrane Fluidity Depends on Their Molecular Size

These results are in accordance with available data obtained on model membranes. They document the presence of mechanical links between RBC deformability and near-surface membrane fluidity, chain length-dependence of the ability of alcohols to alter RBC mechanical behavior, and the biphasic response of RBC deformability and near-surface membrane fluidity to increasing alcohol concentrations.

So if our RBC's are already impaired it makes sense that alcohol would worsen the situation.

Les Simpson hoped that if we have a good clinical test for RBC issues that correlates to "bad" and "better" days as his research seemed to indicate, then we can track treatments to see what effects they have on RBC's. Is the treatment the helping or hindering.

I think one of the reasons there is so little Big Pharma interest in our disease is the fact that there is no biomarker to track to biologically see if their drugs help. Even such simple things as does for example Ubiquinol help is really hard to decide (note : Nancy Klimas is testing this supplement in GWI phase II clinical trial). My dream is that maybe an RBC deformability test would help facilitate clinical trials of treatments in ME. Of course an awful lot of work is needed to prove this marker tracks with illness level, and it's probably going to take NIH level funding if not more to really quantify.

I enjoyed this talk from last years #CMRC2018 conference about research from the perspective of a father of two kids with ME/CFS and a Pharmaceutical company employee - it is given by Dr Mark Jones from UCB Pharma. He describes the importance of having biological markers that can be tracked during drug development and clinical trials. That's why finding those markers is so important for our research field.


It is also interesting to hear how research work has changed significantly in the last few years due to the abundance of big data and availability of computing power, and how patient involvement really helps.

 

[leokitten]

Could a negative test be developed for example most patients with ME are said to react badly to alcohol are there any other safer more common substances to try
Include unusual reactions as well

The strong alcohol intolerance symptom isn’t consistently present over time.

I had very strong and sudden alcohol intolerance after getting ME, and it remained that way for about 2 years. Immediate vomiting, nausea, strong hangover after drinking just a fraction of a glass of wine.

But then it started to gradually reduce as the years went on, all the while I’ve continued to get worse. I still have some intolerance if I drink beyond two glasses of wine in one sitting, but the symptoms are different than long ago, it mostly just causes PEM.

 

[Milo]

I had very strong and sudden alcohol intolerance after getting ME, and it remained that way for about 2 years. Immediate vomiting, nausea, strong hangover after drinking just a fraction of a glass of wine

I think people need to specify what they mean by alcohol intolerance. Thank you for sharing this @leokitten.

For me i never ever thought of drinking alcohol in the first few years of my illness. I already felt horrible, so why add further load to an already overburdened body?

These days (10 years later) I can tolerate a little bit of wine, like half a glass.

 

[Mithriel]

While the biochemistry of alcohol intolerance is interesting and could give insight into the disease, the real significance is easy to overlook.

ME and CFS are so often dismissed as a form of depression or a neurotic fear of exercise, yet in our society it is accepted to cliche level that someone like that uses alcohol to cope.

The fact that we do not self medicate with alcohol - managing to tolerate some wine hardly counts when you think about it, it is more of a confirmation of alcohol intolerance than otherwise.

I believe this is proof that something physiological is going on in our bodies and for that reason should be more prominent.

 

[Snow Leopard]

Brenu et al note no difference in deformability using a different methodology.

Saha et al note that some patients were taking various medications such as LDN, Rituxan, Vitamin D etc and this could well affect the results, especially given the small sample size. Secondly, no sensitivity of specificity of the association is discussed, presumably because the test of this association as a biomarker needs to be done as a replication.

Altered morphology has been noted before, but of uncertain specificity or signifiance. https://me-pedia.org/wiki/Red_blood_cell