Rituximab Whacks NK Cells in ME/CFS Says Aussie Group

[Murph]

@Gingergrrl

Puringeric singalling refers to signalling with

purines.

ATP and adenosine etc are considered purines, or have purines in their structure.

The story of how ATP, a well-known energy molecule, came to be accepted as a signalling molecule is an interesting one and another good example of how science learns to correct itself: slowly but eventually!

--

As far as I can tell the Naviaux purinergic signalling thing and the Rituximab theory are totally distinct.

Naviaux's theory is basically hypothesising a whole new kind of immune problem (stuck on cell danger response causing hypometabolism) while the rituximab theory depends on one of the best known types of auto-immune response: b cells making autoantibodies.

in theory it could be both (and perhaps there will be a little of each in the final answer, or in subgroups) but neither one looks especially likely right now.

 

[Wonkmonk]

This describes my situation well except that I had extremely high antibodies to EBV (IgM+ and EA+) for 3-4 yrs post Mono (2012).

It would have been so interesting to see if you were given Rituximab in 2012 and had avoided the mold, if that had cured you at the time. Today with all the autoimmunity it might be a more difficult treatment and take much longer.

Last night I ate pizza and chocolate chip cookies with my daughter & niece, with no MCAS meds, and no longer have any concerns about allergic reactions.

 

[Gingergrrl]

Puringeric singalling refers to signalling withpurines. ATP and adenosine etc are considered purines, or have purines in their structure.

Thanks, Murph, and I read your link and still can't manage to understand what a purine is . I'm actually kind of annoyed with myself about this. Is it part of the structure of the cell itself? Also, does "cell signaling" refer to a process that goes on within a single cell or is the way that one cell signals another cell? I apologize these questions are stupid and there are times since getting sick that I wish I had studied science vs. social work so I could more fully understand these journal articles... but I keep trying

As far as I can tell the Naviaux purinergic signalling thing and the Rituximab theory are totally distinct.

Thank you and that was what I thought but wanted to confirm. I completely relate to the Rituximab theory (in my own case) but never related to the Autism/ Dauer/ Hibernation theory (and not sure about the purinergic part until I can understand it better).

Naviaux's theory is basically hypothesising a whole new kind of immune problem (stuck on cell danger response causing hypometabolism) while the rituximab theory depends on one of the best known types of auto-immune response: b cells making autoantibodies.

I know the latter pertains to me re: B-cells creating pathogenic autoantibodies attacking the body but I'm not sure if the "cell danger response" part relates to me.

It would have been so interesting to see if you were given Rituximab in 2012 and had avoided the mold, if that had cured you at the time. Today with all the autoimmunity it might be a more difficult treatment and take much longer.

I actually do not think it would have b/c I don't believe I was dealing with autoimmunity in 2012. I got severe Mono from EBV in March 2012 and it was classic mono (fever for 3-4 wks straight, liver tests off the chart, severe sore throat, tonsils enlarged with abscess, swollen lymph nodes, severe malaise/fatigue, and positive mono blood tests). I was deathly ill for about 7-8 wks and then recovered.

It wasn't until ten months later (Jan 2013) that I got the second virus that was either a new virus or a re-activation of mono and within two weeks, it was gone but I developed severe POTS and never recovered. I do not believe it shifted into autoimmunity until a few years later although I was diagnosed with Hashimoto's (Autoimmune Thyroid) in Oct 2013. But I never had another cold, flu, or fever since Jan 2013 (now over 5 yrs).

 

[Learner1]

I'm not sure if the "cell danger response" part relates to me.

It applies to everyone to some degree. In his 2013 CDR paper, Naviaux lays out a list of things that stress cells and then describes the mechanisms the cells use to react to these dangers. He says that chronic disease results, but that the dangers can be resolved.

The cell danger response (CDR) is the evolutionarily conserved metabolic response that protects cells and hosts from harm.

It is triggered by encounters with chemical, physical, or biological threats that exceed the cellular capacity for homeostasis.

The resulting metabolic mismatch between available resources and functional capacity
produces a cascade of changes in cellular electron flow, oxygen consumption, redox, membrane fluidity, lipid dynamics, bioenergetics, carbon and sulfur resource allocation, protein folding and aggregation, vitamin availability, metal homeostasis, indole, pterin, 1-carbon and polyamine metabolism, and polymer formation.

EBV and mold qualify as cell dangers.

 

[Gingergrrl]

EBV and mold qualify as cell dangers.

Thanks for explaining and in that case, it definitely does pertain to me.

 

[Wonkmonk]

I actually do not think it would have b/c I don't believe I was dealing with autoimmunity in 2012.

But you had chronic active EBV after 2013 and that's what presumably caused the autoimmunity in the long term. Rituximab in that phase could have removed the EBV-infected B-cells and stopped the chronic EBV-infection.

 

[Gingergrrl]

But you had chronic active EBV after 2013 and that's what presumably caused the autoimmunity in the long term. Rituximab in that phase could have removed the EBV-infected B-cells and stopped the chronic EBV-infection.

It's certainly possible (and an interesting idea) but no one knew what was wrong with me in early 2013 and my PCP at that time said that I had "CFS" and that "There was no treatment for CFS". She was nice about it but basically fired me as her patient. It wasn't until much later that year, through my own persistence (and getting rid of that PCP!) that I was diagnosed with both POTS and Hashimoto's Disease. Rituximab was not even remotely on anyone's radar for me until several years later.

Edit: I also saw a naturopath in 2013 who tested me for re-activation of EBV and found that the IgM and Early Antigen (EA) tests were positive. She sold me a million supplements but none of them ended up helping me at that time.

 

[alex3619]

This study has not been published yet. We have to wait to see what the details are.

Its interesting to me for several reasons, though I am sceptical too. My first degree is from Griffith University, though a different campus. My first theory on ME was all about intracellular calcium, via a specific mechanism, and relating to hypoxia. That mechanism was wrong, but I learned a lot from examining it.

One thing that has to be kept in mind is that for many minerals the blood levels and tissue levels, or specific tissue levels, can be different. This also occurs with potassium, in which about half of us were shown to be deficient at an intracellular level ... this research was from the 90s I think. Serum levels are usually normal.

 

[Murph]

Thanks, Murph, and I read your link and still can't manage to understand what a purine is . I'm actually kind of annoyed with myself about this. Is it part of the structure of the cell itself? Also, does "cell signaling" refer to a process that goes on within a single cell or is the way that one cell signals another cell? I apologize these questions are stupid and there are times since getting sick that I wish I had studied science vs. social work so I could more fully understand these journal articles... but I keep trying

I sure hope we don't have to have a deep structural understanding of what a purine is! As far as I'm concerned It's just a name for a type of molecule that floats around in a cell.

Sugars are one type of molecule. Proteins are a type of molecule. This type of molecule is called purines. That's kind of how far my understanding goes!

As far as I know, purines are used for energy inside the cell and for signalling outside the cell. (e.g. the cells leak out ATP when something is going horribly wrong inside, as a cry for help.)

(I think there are also special cases where the cell leaks a signalling molecule so its own sensors on the outside can see it, in this case it's kind of outside signalling, but for your own information. Kind of like talking to yourself.)

The body is constantly doing tons of signalling. Cytokines (another type of molecule! although in this case a functional classifiaciton rather than structural, I think) are a group of signalling molecules. Some happens inside the cell, some outside.

Purinergic signalling is just a tiny subset of all the signalling going on in the body, but it's a very fast-growing field. It seems to be involved in everything, and it certainly looks like it might be relevant to us.

 

[Murph]

Here's the paper. (And in full online)

Small sample and in vitro but the results do look real.

BMC Pharmacol Toxicol. 2018 Mar 27;19(1):12. doi: 10.1186/s40360-018-0203-8.
Rituximab impedes natural killer cell function in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients: A pilot in vitro investigation.
Eaton N1,2, Cabanas H3,4, Balinas C3,4, Klein A3,4, Staines D3,4, Marshall-Gradisnik S3,4.
Author information
Abstract
BACKGROUND:
A recent in vitro pilot investigation reported Rituximab significantly reduced natural killer (NK) cell cytotoxicity in healthy donors. Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) is a debilitating disorder of unknown etiology. A consistent finding is a significant reduction in NK cell cytotoxicity. Rituximab has been reported having questionable potential therapeutic benefits for the treatment of CFS/ME, however, the potential effects of Rituximab on NK cell cytotoxicity in CFS/ME patients are yet to be determined.

METHODS:
A total of eight CFS/ME patients (48.63 ± 15.69 years) and nine non-fatigued controls (NFC) (37.56 ± 11.06 years) were included using the Fukuda case definition. Apoptotic function, lytic proteins and degranulation markers were measured on isolated NK cells using flow cytometry following overnight incubation with Rituximab at 10 μg/ml and 100 μg/ml.

RESULTS:
There was a significant reduction in NK cell lysis between CFS/ME patients and NFC following incubation with Rituximab at 100 μg/ml at 12.5:1 and 6.25:1 effecter-target (E:T) ratios (p < 0.05). However, there was no significant difference for NFC following incubation with Rituximab at 10 μg/ml and 100 μg/ml. There was no significant difference between CFS/ME patients and NFC for granzyme A and granzyme B prior to incubation with Rituximab and following overnight incubation with Rituximab at 10 μg/ml. There was a significant decrease in granzyme B in CFS/ME patients compared to NFC with 100 μg/ml of Rituximab prior to K562 cells stimulation (p < 0.05). There was a significant increase in CD107a (p < 0.05) and CD107b expression (p < 0.01) in NFC after stimulation with K562 cells prior to incubation with Rituximab. There was a significant increase in CD107b expression between CFS/ME patients and NFC prior to incubation with Rituximab and without stimulation of K562 cells (p < 0.01). Importantly, there was a significant increase in CD107b following overnight incubation with 100 μg/ml of Rituximab in NFC prior to K562 cells stimulation (p < 0.01).

CONCLUSION:
This study reports significant decreases in NK cell lysis and a significant increase in NK cell degranulation following Rituximab incubation in vitro in CFS/ME patients, suggesting Rituximab may be toxic for NK cells. Caution should be observed in clinical trials until further investigations in a safe and controlled in vitro setting are completed.

KEYWORDS:
Chronic fatigue syndrome; Cytotoxicity; Degranulation; Lytic proteins; Myalgic encephalomyelitis; Natural killer cells; Rituximab

PMID:
29587879
DOI:
10.1186/s40360-018-0203-8
Free full text

 

[Gingergrrl]

Sugars are one type of molecule. Proteins are a type of molecule. This type of molecule is called purines. That's kind of how far my understanding goes!

In that case, I understand it! I wish you had been my science teacher LOL.

Here's the paper in full

Thank you for posting it. Spoiler alert: As I suspected, I did not understand it. What do they mean by "overnight incubation with Rituximab"? Was it given to the person via IV infusion in an appropriate dose or something totally different? I also couldn't find any link to the calcium autoantibodies. And lastly, I didn't understand the connection to NK cell functioning.

 

[Murph]

In that case, I understand it! I wish you had been my science teacher LOL.



Thank you for posting it. Spoiler alert: As I suspected, I did not understand it. What do they mean by "overnight incubation with Rituximab"? Was it given to the person via IV infusion in an appropriate dose or something totally different? I also couldn't find any link to the calcium autoantibodies. And lastly, I didn't understand the connection to NK cell functioning.

The experiments were all in vitro (latin for in glass) meaning they were done in test tubes rather than in patients (which they would call in vivo).

I also don't see the connection to NK functioning, but I don't think anyone knows how it works, they are just reporting that it happens.

 

[Gingergrrl]

Thanks @Murph but I still can't quite figure out what they actually did. I googled "incubation" and am sure it has a different meaning in this study but what would be the incubation period for Rituximab?! What does this even mean?

Also, I thought this study showed a connection to intra-cellular calcium levels as the disease mechanism but I can't even find the word calcium mentioned. Am I missing it?

 

[FMMM1]

Thanks @Murph but I still can't quite figure out what they actually did. I googled "incubation" and am sure it has a different meaning in this study but what would be the incubation period for Rituximab?! What does this even mean?

Also, I thought this study showed a connection to intra-cellular calcium levels as the disease mechanism but I can't even find the word calcium mentioned. Am I missing it?

Not sure if I'm answering your question but check this paper by the Griffith's group* "Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels".

"DISCUSSION
Previous investigations have reported significant reductions in NK cell cytotoxic activity in CFS/ME patients, and the current investigation supports those findings 16. The current investigation also confirms our previous results of significantly reduced TRPM3 receptors on NK cells as well as significantly reduced intracellular Ca2+ mobilization in isolated NK cells 16. The current investigation showed inhibition of the ER Ca2+/ATPase pump and depletion of intracellular Ca2+ stores followed by PregS‐activated TRPM3 increased cytotoxic activity in NK cells from CFS/ME patients (Fig. (Fig.66)."

I assume that in this paper* (and presumably the previous paper referred to in this extract) demonstrates low intracellular calcium; I guess the question is are low levels the cause or a consequence.

Regarding incubation with rituximab. I assume all you do is work out the concentration of the drug (i.e. amount present in patients blood) and add that amount to NK cells and see what happens. If rituximab is toxic then the number of cells which die is greater than in the control sample. Here's an extract from the paper [https://bmcpharmacoltoxicol.biomedcentral.com/articles/10.1186/s40360-018-0203-8]:
"Rituximab Treatment
NK cells were treated with concentrations of 10 μg/ml as previously reported [41] and 100 μg/ml of RTX (Rituxan, Genentech, CA, USA) and incubated for 24 h at 37 °C with 5% CO2 in RPMI-1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, USA).

Natural Killer Cell Cytotoxicity Assay"

The Griffiths group use publicly accessible journals; the technical stuff re how you handle the cells appears to be there (section titled "Natural Killer Cell Cytotoxicity Assay") so I assume we can accept the results (unless Alex, i.e. those who know the technical stuff, challenge it).

Again, the question appears to be so what if rituximab kills NK cells. Rituximab didn't work in the ME/CFS phase 3 trial. Am I missing something e.g. that the toxicity of rituximab (i.e. killing NK cells) explains cases where it works?

 

[Cort]

Rituximab works by killing B-cells, which are a central part of the immune system. So to me it's not surprising at all to hear that wiping them off would in some ways impair the immune system, because (from my limited understanding) those B-cells are not existing for no reason. Probably there is a trade-off, if for example an uncontrolled EBV infection or some other factor makes those B-cells malfunction, then the individual is probably better off without them. But if the problem is elsewhere than in the B-cells, then I would assume Rituximab could make patients worse.

I remember early on it was suggested that RItuximab could be effecting NK cells and that could help account for it's success in some patients. Note that Fluge/Mella not co-authors of this paper and I wouldn't be surprised if they weren't following NK cell functionality in the participants. If so it'll be interesting to see what they say.

 

[Gingergrrl]

Not sure if I'm answering your question but check this paper by the Griffith's group* "Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels".

@FMMC1 So it sounds like there are two totally separate papers about Rituximab from Griffith Uni and one is about CA+ Channels and one is about NK Cells (or is this incorrect)? I am struggling to figure out what they are talking about but it is often hard for me to know if this is b/c the papers are lacking in substance vs. I just do not have a strong enough science background to understand them.

The Griffiths group use publicly accessible journals; the technical stuff re how you handle the cells appears to be there (section titled "Natural Killer Cell Cytotoxicity Assay") so I assume we can accept the results (unless Alex, i.e. those who know the technical stuff, challenge it).

I agree and am hoping that @alex3619 can interpret this all for us

Again, the question appears to be so what if rituximab kills NK cells. Rituximab didn't work in the ME/CFS phase 3 trial. Am I missing something e.g. that the toxicity of rituximab (i.e. killing NK cells) explains cases where it works?

I truly am not sure and still don't understand what they meant re: an "incubation period" for Rituximab? Do they mean how long it takes to kill the B-cells (which can be quick) or how long it takes to know if the person is a responder, or something else? This just seems like a very strange term to use unless it has a completely different meaning?

We know Riux is toxic (to kill the B-cells) so maybe their point is just that it can kill NK cells, too? But if so, I am not sure what this tells us? I am a responder due to my high levels of autoimmunity and in my case, my doctors feel it is the presumed mechanism of zero B-cells equals no new autoantibodies. How NK cells fit into all of this, is beyond me. I'm still trying to understand the calcium channelopathy connection (which if proven to be a bio-marker, makes me question my diagnosis all over again).

I remember early on it was suggested that RItuximab could be effecting NK cells and that could help account for it's success in some patients. Note that Fluge/Mella not co-authors of this paper and I wouldn't be surprised if they weren't following NK cell functionality in the participants. If so it'll be interesting to see what they say.

But if Ritux kills NK cells, like it does B-cells, how would this be helpful for certain patients? I'm asking genuinely b/c I do not understand it! I would be curious what Fluge & Mella have to say as well.

 

[FMMM1]

@FMMC1 So it sounds like there are two totally separate papers about Rituximab from Griffith Uni and one is about CA+ Channels and one is about NK Cells (or is this incorrect)? I am struggling to figure out what they are talking about but it is often hard for me to know if this is b/c the papers are lacking in substance vs. I just do not have a strong enough science background to understand them.
I agree and am hoping that @alex3619 can interpret this all for us

Response- Many of us struggle with this stuff which is where folks like Alex (previously a notable scientist) and Cort are particularly useful. The Griffith's group are all about this being a calcium problem (calcium channelopathy -linked to obscure receptors referred to in their papers). Cort has done good reviews of this groups work and they are easier to understand

I truly am not sure and still don't understand what they meant re: an "incubation period" for Rituximab? Do they mean how long it takes to kill the B-cells (which can be quick) or how long it takes to know if the person is a responder, or something else? This just seems like a very strange term to use unless it has a completely different meaning?

Response- Here's an extract from the paper:
"Natural Killer Cell Cytotoxicity Assay
NK cytotoxic activity was conducted as previously described --- incubated --- for 4 h at 37 °C ---."
I.e. they added rituximab to the N/K cells and incubated them at body temperature; result dead N/K cells


We know Riux is toxic (to kill the B-cells) so maybe their point is just that it can kill NK cells, too? But if so, I am not sure what this tells us? I am a responder due to my high levels of autoimmunity and in my case, my doctors feel it is the presumed mechanism of zero B-cells equals no new autoantibodies. How NK cells fit into all of this, is beyond me. I'm still trying to understand the calcium channelopathy connection (which if proven to be a bio-marker, makes me question my diagnosis all over again).
But if Ritux kills NK cells, like it does B-cells, how would this be helpful for certain patients? I'm asking genuinely b/c I do not understand it! I would be curious what Fluge & Mella have to say as well.

Response- I agree i.e. I've no idea what this tells us; apart from the fact that rituximab can kill N/K cells at certain concentrations. Did the patients receive these doses in the phase 3 rituximab trial?

Not sure if I've managed the multiple quotes well! I've just inserted "Response" into your text.

 

[Malea]

@Wonkmonk I'm afraid that it's quite obvious, but why would someone from group one not benefit from antivirals?

(Because the infections themselves are not the primary problem anymore but the autoimmune reactions are? But if the autoimmune reactions were originally caused by a virus, wouldn't to fight off the virus with antivirals could get someone out of the overreactive-autoimmune-mode?)

 

[FMMM1]

Regarding autoimmunity; there's an article on the simmaron research website which may be of interest [title - "Hope for an ME/CFS Autoimmune Subset: A German Researcher Steps Forward"]. You may have come across Carmen Scheibenbogen's work on autoimmune antibodies to β adrenergic and muscarinic cholinergic receptors.

 

[Wonkmonk]

I'm afraid that it's quite obvious, but why would someone from group one not benefit from antivirals?

(Because the infections themselves are not the primary problem anymore but the autoimmune reactions are? But if the autoimmune reactions were originally caused by a virus, wouldn't to fight off the virus with antivirals could get someone out of the overreactive-autoimmune-mode?)

It would probably depend on the case. In a case like @Gingergrrl, there titers are no longer suggestive of active infections, and so antivirals probably wouldn't (and in her case didn't) help (anymore).

But trying an antiviral might still be an option, especially if a Fam/Valacycovir-sensitive virus is suspected.