SlamDancin

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Deformation of human red blood cells in extensional flow through a hyperbolic contraction.

Faghih MM, et al. Biomech Model Mechanobiol. 2019.
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Abstract

Flow-induced damage to red blood cells has been an issue of considerable importance since the introduction of the first cardiovascular devices. Early blood damage prediction models were based on measurements of damage by shear stress only. Subsequently, these models were extrapolated to include other components of the fluid stress tensor. However, the expanded models were not validated by measurements of damage in response to the added types of stress. Recent investigations have proposed that extensional stress might be more damaging to red cells than shear stress. In this study, experiments were conducted to compare human red cell deformation under laminar extensional stress versus laminar shear stress. It was found that the deformation caused by shear stress is matched by that produced by an extensional stress that is approximately 34 times smaller. Assuming that blood damage scales directly with cell deformation, this result indicates that mechanistic blood damage prediction models should weigh extensional stress more than shear stress.

Food for though @necessary8
 

SlamDancin

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“In this study, we show that FLCN is a ciliary protein that functions through primary cilia to regulate mTORC1. In response to flow stress, FLCN associates with LKB1 and recruits the kinase to primary cilia for activation of AMPK resided at basal bodies, which causes mTORC1 down-regulation. In cells depleted of FLCN, LKB1 fails to accumulate in primary cilia and AMPK at the basal bodies remains inactive, thus nullifying the inhibitory effect of flow stress on mTORC1 activity. Our results demonstrate that FLCN is part of a flow sensory mechanism that regulates mTORC1 through primary cilia.”


Cilia are also a possible connection between FSS and mTORC1/AMPK
 

Wishful

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No idea. I haven't ever heard of this illness having any sort of comorbidity with ME/CFS so I'm not considering it. If you have any data showing that it has some correlation, link it.
No, I haven't encountered any link between thalassemia trait and ME. I just thought it might be a useful test of your model (or anyone else's involving RBCs), since it provides a real-life experiment with RBCs with different qualities. If the flow rate through microcapillaries was critical for the model, and would predict that thalassemia trait would make certain aspects of ME better or worse, then an actual patient would test the model. I have the trait (small, asymmetrical RBCs), and my ME is certainly non-standard, but I have no way to know whether there's a link.
 

S-VV

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I’m still not sure if RBC have the primary cilia required seemingly to “recept” exosomes. Certain endothelial cells and cells sensing CNS conditions do however and could be vulnerable to oxidative damage from exosomes. If anyone can find any literature saying exosomes can be received by non-ciliated cells I’d appreciate it.
I thought the same thing, but it turns out NADPH Oxidase is a transmembrane protein that "spits out" ROS. However, at least in normal cells, the ROS are propelled inwards, and to conserve electroneutrality, a H+ is pumped out.
 

SlamDancin

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@S-VV Hey thanks for the thoughtful response.

We should pharmacologically remove cilia in the ME/CFS plasma, and if there is still impedance and RBC deformability, then at least we can say they aren’t necessary for transmission of the exosomes’ cargo. That would be an interesting experiment in my opinion which is why I mentioned it to Ron Davis several months ago. Never heard back but FSS>cilia>exosomes>ROS>damaged RBC makes sense to me. My only question is why would only the RBC be damaged by the NOX if it is indiscriminately being spit out into the blood?
 
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I’m sorry but I can’t find anything specific saying exosomes are capable of releasing anything without a cell that’s able to receive its Message. I’m genuinely interested in being shown Incorrect so if you have any sources on this please.
I linked it in my original post as the two most important citations:
https://journals.lww.com/ccmjournal...erived_exosomes_of_septic_individuals.32.aspx
https://www.researchgate.net/public..._NADPH_oxidase_2_complexes_into_injured_axons
Lastly, NOX2 is a specific form of NOX specifically for Phagocytes and so I really don’t think they can be simply discounted as just part of general immune changes when both NOX and IDO1/2 relate directly to Phagocyte activity.
NOX2 does a lot of things in many different cells. There are also 6 other NOX isoforms that are not NOX2, which do many different things. To directly quote a paper on this matter: "Membrane oxidases identical to the phagocytic NADPH oxidase complex (Nox2) have been demonstrated in endothelial cells, fibroblasts, mesangial cells, smooth muscle cells, neurons, and probably are expressed in nearly all cell types "
I understand that you want to draw connections to your theory, and I'm not saying there can't be one. But this alone is a pretty weak argument for it.

There is definitely some research out there saying that cilia are necessary for both biogenesis of exosomes and reception, as in some cases removal of cilia stops exosomes from exerting effect in the biliary system.
You're approaching this subject from a preconcieved notion that exosomes are messengers with dedicated exit and entry systems, and while that might be true, it doesn't prevent the exosomes from doing other things, or doesn't exclude them from laws of physics. An active NOX enzyme inside an exosome will generate superoxide, superoxide dismutes to hydrogen peroxide, which easily crosses the exosome membrane and doesn't care about cilia or anything else like that. Additionally there is some evidence that NOX in exosomes can be membrane-bound, allowing for direct superoxide release outside of the exosome. A good source for all this: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842114/

If RBC were being damaged by NOX exosomes you would expect monocytes to be damaged as well, at least as far as I can tell.
Yes. That is how I explain the nanoneedle results.
That would lead to problems with antigen presenting, T cell responses and phagocytosis.
I would imagine it would possibly lead to some dysfunctions of those cells but it's very hard for me to predict what ones and to which degree.
Also cilia are what sense pH (hypoxia) and FSS In certain subsets of endothelial cells. It makes perfect sense to me that ciliated cells in the vasculature release the NOX as a result of changing FSS and pH.
It doesn't to me. If vascular cells would release NOX exsosomes, in response to increased shear stress, which is a result of reduced RBC deformability, which is a result of NOX exosomes, then that constitutes a self sustaining vicious cycle. You would need a very good explanation for why that doesn't happen all the time in all people, and why would it be different in ME/CFS patients.
With my model, I provided such an explanation - the IDO trap. You didn't.

In regards to cilia. They might mediate the release of NOX exosomes from immune cells, and they might also independently mediate the NO release from vasculature in response to shear stress. Might. I don't know enough about the subject to say with any degree of certainty. But I also don't think it really matters at this stage. If this hypothesis is confirmed, then cilia will become of much larger importance as then we will need to understand how the exosomes are released in the first place. And if we get there I will read a lot of papers about them including all the ones you've linked me (which I do appreciate a lot because I didn't know about their involvement in exosome secretion). That's my final take on cilia, I think.

My only question is why would only the RBC be damaged by the NOX if it is indiscriminately being spit out into the blood?
I never said that. In fact I said the opposite, multiple times.

Please don't take this the wrong way @SlamDancin but it would be very nice if you could perhaps read my hypothesis carefully, including the sources, if you're gonna try to point holes in it (which I do welcome very much, I'm all for discourse). Cause half of this stuff I already said in my initial post, putting a lot of energy in writing it as clearly as possible. And repeating it takes a lot of energy once again, which is a bit of a waste.
Sorry if I come across confrontational, that is not my intention. You've made me double check some assumptions I made and discover some new sources on it, so I do appreciate it. : )
 

SlamDancin

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@necessary8 You’ve been fine. I’m not trying to poke holes in this to make it fall apart but rather because I think you are probably more close to correct than most theories I’ve ever read and I just want to make sure I understand it before I run it up the ladder to whomever will listen. Good work and good explanations, thank you. I’ll re-read the OP before I ask any other questions.

Like I said I’m fully willing to try my best to get this into the hands of someone who can appreciate it and test it. Helps if I can appear to really understand it though And if all questions we will have already considered here first. This is absolutely not a my theory vs your theory type of thing.
 
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@necessary8 I just want to make sure I understand it before I run it up the ladder to whomever will listen
Understandable : )
Perhaps I can explain one more thing more clearly, cause now that I think about it, it might be buried pretty deep in the sources I linked. One of those two main papers I spoke about (the one on sepsis), directly measured ROS present in the medium with those NOX-containing exosomes. Hence we know that they produce and release ROS by themselves.

I often though would large doses of omega oils offset this ?
I don't think so, but I hadn't done the math to know for sure.

Deformation of human red blood cells in extensional flow through a hyperbolic contraction
This is very interesting, I will keep it in mind

My aunt and daughter both have ME. Both have SNPs which if expressed would affect NO levels . Could this be a fine tuning mechanism ?
I could see it being a small cofactor increasing susceptibility.
 
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Addendum 1 - in the light of the Stanford symposium​

It has been brought to my attention that Juan Santiago’s data, which are the most in depth look at erythrocyte deformability in ME/CFS yet, showed no difference between patients and controls. But there is a problem. His experiments were done on RBCs taken out of the plasma and suspended in PBS - a medium which does not contain calcium ions. According to my hypothesis, the deformability would be impaired by oxidative damage to cell-surface Ca-ATPases which results in increased calcium concentration in the cell as it passively diffuses from the plasma to the inside, and the ATPases don’t pump it out fast enough. If you take the cells out of the plasma, and throw them into a solution without calcium, the direction of passive diffusion of calcium reverses - it now flows out of the cells by itself. And the changes to deformability also reverse themselves. Source: https://dm5migu4zj3pb.cloudfront.net/manuscripts/113000/113611/JCI88113611.pdf
As you can see, this paper clearly states that Ca-induced alterations in erythrocyte deformability are completely reversible.
In my opinion, Santiago's experiments need to be repeated in the presence of normal levels of calcium, and possibly all other electrolytes as well. But calcium especially.
I have talked to Santiago directly about this, and he agreed with me.

Additionally, it has also been brought to my attention that the activity of IDO is modulated by its association with oxygen, which I wasn't aware of before. That provides a much more direct way for the RBC deformability to worsen the IDO trap in response to exercise, directly through hypoxia. This could act in conjunction to the nitric oxide mechanism, provided that the trap occurs somewhere in the tissues, somewhere where red blood cells need to pass through capillaries to deliver the oxygen to.
 
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Thanks for the feedback, everyone! : )


I did, and got no response.


And that is exactly what happens in the people who are mold-sensitive. They get better if they avoid it. Keep in mind that while mold and CCI were two factors that I was able to guess, there is probably many many more, some of which might be plain impossible to avoid. And most people probably have a combination of multiple factors, not just one. Hence not many full remissions after eliminating just mold (though there are some, I have personally talked to one or two).


Then you are a very, very far outlier.


Not quite. The NOX is not released into the cells. Or actually, it might be, but that's not the point. The point is that while the exosome travels in the blood, it by itself produces and releases ROS around it. So you would actually rather have an antioxidant free in the bloodstream rather than an infiltrating one. The lack of success with vitamins is because they are not nearly potent enough and they dont reach high concentrations in the blood. A mega dose of IV vitamin C might help a little bit, and I do hear some people felt a bit better from that. But the problem is that concentration will drop quickly. You would need to maintain super high concentration for days or even weeks for the body to have time to regenerate the broken ATPases, to actually get remission from it. And as soon as you stopped, the illness would come back right away. This is not a viable approach to treatment.


Good to know!


That is simply impossible. Unless you can break the rules of physics, your muscle will always require a lot of oxygen for activity. Anaerobic energy production is wildly inefficient.

The only actual good solutions to treat this, if this hypothesis is true, is to either block NOX or undo the IDO trap. But we need to test the hypothesis first. It might be very wrong.


https://www.ncbi.nlm.nih.gov/pubmed/30585477
It's this one, right? This is really interesting, thank you for telling me about it. This might add another feedback loop, where the IDO trap taking place further impairs NO clearance. But I don't know how significant of an impact it would have, there are certainly other mechanisms of NO clearance. Still, it's interesting to consider.



Small corrections:
The mechanism for PEM I propose does not involve immune activation, at least not in the classical sense. If that were the case we would be seeing a lot more cytokines. Instead, what I propose happens is that there is a constant ongoing NOX-exosome production by the immune system, which should normally be limited by IDO activity of some subtype of dendritic cells or T cells. And in ME/CFS this limiter is disabled because of the IDO trap. And PEM wroks by the means of NO (which we know for sure is abnormally high in exercise in patients) worsens the IDO trap.

Neither lactate, ATP nor NO can act systemically. They all act locally. NO slightly less locaclly, but still not systemically.
NOX exosomes are required to make this mechanism be systemic, as they can be carried by the blood while shedding ROS in their path, wherever they go.


Bigger correction:
The changes observed by Griffith are genetic. Meaning, you have them your whole life. Meaning, they cannot explain ME/CFS by itself, because patients would also have them before being sick with ME/CFS, and you would detect them in healthy people too. Both nanoneedle results as well as RBC deformability seem to be very specific to patients.
Furthermore, nanoneedle results reverse when you do a plasma switch. This means that something in the plasma is mediating the change. It cannot be mediated by a genetic mutation. This is the reason why a lot of genetic explanations for nanoneedle results simply don't work. Oxidative damage from outside the cell is the first thing I found which offers to explain both nanoneedle as well as RBC deformability data.
What I could see happening is that perhaps the TRPMs work in combination with cell surface Ca-ATPases to achieve some important functions in some immune cells, and hence having both fail results in some important change in ME/CFS. But I haven't really looked into this angle at all yet.


One other reason why I like this hypothesis is because rampant NOX exosomes and IDO trap might be a way to explain why we can see many immune alterations but they are all pretty weird and don't seem to involve much direct cell-to-cell communication (cytokines). Which has been a bit of a mystery.
Wow - I can't say I understand all this, but I think it is fascinating. What about the TRPM SNP effect on or relationship to the NK cell abnormalities? I was reading about this today and trying to wrap my head around it. The immune system is so complex it gives me a headache!

Is it possible that this SNP can be activated by any number of things? Epi-genetically speakng? Including viral infections and mold or toxic exposures? Maybe you have already answered this question and I am not clever enough to understand.

Thanks very much for sharing all this. It's really amazing that you are connecting the dots like this without being a biochemist or an immunologist! Or maybe you are - though I would not want to presume anything about your background.
 
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Wow - I can't say I understand all this, but I think it is fascinating. What about the TRPM SNP effect on or relationship to the NK cell abnormalities? I was reading about this today and trying to wrap my head around it. The immune system is so complex it gives me a headache!

Is it possible that this SNP can be activated by any number of things? Epi-genetically speakng? Including viral infections and mold or toxic exposures? Maybe you have already answered this question and I am not clever enough to understand.
IMHO the TRPM SNP data from Griffith are overplayed a lot. There are many, many findings like these, which have some correlation to the disease but no one has really demonstrated why it could be central to the mechanim. But perhaps one day it will become more significant in the light of other findings. For now, I don't think it is.
SNPs are genetic mutations and you have them your whole life, to the best of my knowledge. But I'm also not the right person to ask about this, my knowledge of genetics is very very sparse.

Sorry I can't give you a better answer.