Antibodies to ß adrenergic and muscarinic cholinergic receptors in patients with CFS

[Jonathan Edwards]

Could you explain the reasoning for replicating the antibody findings? It seems like the consensus is that these findings don't necessarily point to anything specific so I am wondering if resources would be better spent elsewhere ? Or is there a thought that this is significant enough to warrent replication ?
Thanks.

They may not be specific but they may be a clue to something related that is specific - but we need to be sure they are a consistent finding first.

 

[BurnA]

Does Dysautonomia International have a forum, by the way, similar to the www.dinet.org dysautonomia forums? And have there been many people there who have been treated with rituximab?





Indeed, where does that leave us? Let's recap:

The 2014 study on 14 patients with POTS found that:
Beta 1 adrenergic receptor autoantibodies were present in all 14 POTS patients,
Beta 2 adrenergic receptor autoantibodies were present half of these patients.

So it looks like beta 1 adrenergic receptor autoantibodies are the most predicative of POTS.


Fluge and Mella's 2015 study looked for autoantibodies to all alpha and beta adrenergic receptor subtypes, but only found beta 2 adrenergic receptor autoantibodies.

So given that Fluge and Mella had four ME/CFS patients with POTS in their cohort, you would have expected that these researchers would find beta 1 adrenergic receptor autoantibodies in these patients, but they did not.

I don't know the answer to this conundrum.

If we accept the results as they are ( which is a leap of faith considering that neither study had large numbers nor has either study been replicated ) then one answer is that the POTS in CFS/ME patients is different to the POTS in non cfs/me patients.

 

[BurnA]

They may not be specific but they may be a clue to something related that is specific - but we need to be sure they are a consistent finding first.

Thanks. Just out of curiosity, what is involved in such a study? Is it a 'simple ' case of sending lots of blood samples through a machine or what level of testing and analysis is involved ?

 

[M Paine]

Could someone please answer a few basic fundamental questions about the adaptive immune system I have?

In the case of an agonistic or antagonistic antibody binding to a receptor, does that binding event initiate a subsequent attack from other immune cells? So might the immune system attack the receptor or the cell? What is the purpose of the antibody if not to identify and destroy foreign antigens/cells?

Are there different kinds of antibodies which signal different types of immune response?

If heathy control subjects have levels of auto antibodies in their blood, am I understanding correctly that they may be producing antibodies against something very similar, which may be bind ineffectively to their own cells receptors (not agonist or antagonist, but instead they are neutral right?). So they don't cause issues, but still are measurable, correct? They may bind effectively to some other foreign, similar epitope (not sure if this is the correct use of "epitope", but I mean a similar protein either by genetics or shape)?

So in this way, technically they are inert auto-antibodies? If testing could differentiate in better detail, presumably researchers would exclude these?

Do infants aquire B-Lympocytes in breast milk? If so, would they also aquire a measurable amount of auto antibodies? Sad thought

 

[Jonathan Edwards]

In the case of an agonistic or antagonistic antibody binding to a receptor, does that binding event initiate a subsequent attack from other immune cells? So might the immune system attack the receptor or the cell? What is the purpose of the antibody if not to identify and destroy foreign antigens/cells?

Good question but the answer is complex. Antibodies have a front end that binds to their antigen and then a back end that can engage complement or Fc receptors. To engage complement and generate membrane attack complex you need either an IgM antibody or at least three and preferrably more IgGs to cross link the initial C1q molecule. Activation of macrophages or engagement of antibody dependent cytotoxicity through Fc receptors also needs cross linking although it varies with the receptor and is very complex.

If a receptor is sitting in a membrane on its own the likelihood is that an antibody binding to it will not cross link anything that leads to these effector mechanisms. If the receptor is in a raft of similar receptors it is more likely to. But even so, if the receptor is one that cycles into the cell when bound the antibody may just get engulfed and destroyed. So some antibodies to receptors may just act as agonists or antagonists. Others may induce damage.

Are there different kinds of antibodies which signal different types of immune response?

Yes. IgG1 and IgG3 antibodies activate complement and Fc receptors best. IgG4 has virtually no signalling function on its back end. IgM is good at fixing complement but does not bind Fc receptors. IgA binds IgA receptors and IgE binds IgE receptors with degranulation of mast cells.

If heathy control subjects have levels of auto antibodies in their blood, am I understanding correctly that they may be producing antibodies against something very similar, which may be bind ineffectively to their own cells receptors (not agonist or antagonist, but instead they are neutral right?). So they don't cause issues, but still are measurable, correct? They may bind effectively to some other foreign, similar epitope (not sure if this is the correct use of "epitope", but I mean a similar protein either by genetics or shape)?

Yes, there are details but that is roughly what we assume.

So in this way, technically they are inert auto-antibodies? If testing could differentiate in better detail, presumably researchers would exclude these?

Absolutely. What we lack are good functional tests for most antibodies. However, for some of these vasoactive receptors there are functional assays - which are the ones that demonstrate agonism or antagonism. Showing that antibodies will trigger inflammation is more tricky to do in a physiological way. In the 1960s Henry Kunkel and his associates did a lot of very good biochemical work on antibodies showing the kinetics of binding to various systems. But that sort of work is very labour intensive and has gone out of fashion. We probably need to get back to it. Unfortunately antibodies have been unfashionable since T cells came on the scene in the 1980s. That may finally be changing though.

Do infants aquire B-Lympocytes in breast milk? If so, would they also aquire a measurable amount of auto antibodies? Sad thought

No, infants cannot make use of maternal B cells because they are foreign and B cells do not survive long in anything but lymphoid tissue anyway. But maternal antibodies are passed in the placenta and I think in milk. So the infant starts life with its mother's antibody repertoire. If the mother has autoantibodies the infant may get autoimmune disease. This occurs with lupus and myasthenia most commonly. The infant may also suffer heart damage from maternal antibodies in Sjogren's syndrome. But because the infant does not generate their own autoantibody producing plasma cells from their own B cells within a matter of days or weeks the effect of maternal autoantibodies disappears.

 

[Hip]

The antibody findings look interesting but the difference between patients and controls does not make me think these are pathogenic antibodies actually causing symptoms. A proportion of controls had similar levels to patients -a smaller proportion, but not an order of magnitude smaller as far as I can see.

Could the autoantibodies found in controls be non-functional antibodies, that neither agonize nor antagonize the receptor?

If I understood correctly, in the Fluge and Mella paper (quoted below) they point out that non-functional antibodies are mostly found in healthy controls:

Various studies analysed function and binding of antibodies against ß AdR and M AChR. In fact most studies on ß AdR antibodies in cardiovascular disease used a cardiomyocyte contraction assay (Wallukat and Schimke, 2014).

Autoantibodies against β1 AdR in cardiomyopathy were shown to affect ligand binding and cardiomyocyte function similar to agonists (Herda et al., 2012).

However, the impact of ß1 AdR antibodies on receptor function is quite complex as antibody binding to different epitopes and heterogeneous effects on receptor traffic and activity including antagonist effects were observed (Schulze et al., 2005 and Turki and Liggett, 1995).

In addition, non-functional antibodies were described mostly in healthy individuals (Bornholz et al., 2014).

EDIT: Ah I see that @M Paine just asked this very same question.

 

[M Paine]

Thank you for your very detailed answer.

I wonder if it's possible to compare the capsid and glycoprotein gene sequences of EBV, CMV, HSV and other commonly associated viruses to look for protein sequences similar to human genes, or narrowed down to glycoprotein sequences perhaps.

I do wonder what mechanisms B-lyphocytes normally enlist to prevent autoimmunity during the mystical process of creating new antibodies. And what, if any, feedback there might be to correct such a situation of having generated auto antibodies.

 

[Jonathan Edwards]

Thank you for your very detailed answer.

I wonder if it's possible to compare the capsid and glycoprotein gene sequences of EBV, CMV, HSV and other commonly associated viruses to look for protein sequences similar to human genes, or narrowed down to glycoprotein sequences perhaps.

I do wonder what mechanisms B-lyphocytes normally enlist to prevent autoimmunity during the mystical process of creating new antibodies. And what, if any, feedback there might be to correct such a situation of having generated auto antibodies.

I am puzzled why people think similarity of sequence would breach tolerance to self. If anything it should ensure poor response to foreign. The mimicry theory has got hold of the communal mind but has no logical coherence to it.

The mechanisms B lymphocytes enlist to prevent autoimmunity are quite well understood. There are known checkpoints in bone marrow, in lymphoid follicles and in T cell zones (with the thymic checkpoint controlling T cell clones). What is interesting is that tolerance depends on agreement between various recognition systems - maybe because none of these alone can be expected to foil all potential Trojan horses. The down side is that being complicated may introduce new ways of 'crashing' the system. We are all familiar with more complicated software having more ways of crashing. The process of making new antibodies is not very mystical now that we have the work of Michael Neuberger on how random variation is generated.

Jo Cambridge and I wrote a review in Immunology in 1999 where we set out what we thought might be the competing signals pro and contra tolerance or immunity in the context of various autoantibody responses. Rheumatoid factor is probably the easiest one because there is an obvious route to 'crashing' the system. If the signalling end of an Ig molecule acts as antigen then all sorts of strange things can be predicted in terms of complement and Fc receptor (both positive and negative signalling subtypes) engagement. Presumably our Ig repertoire has evolved to evade this built in crash as best it can, but it has to make a trade off against other sorts of crashes. I think we probably understand the crash mechanism for a lot of autoantibodies at a basic level. The difficulty is that because there are trillions of possible antibody species the precise way things go wrong in any individual will be one of an almost infinite number of variations on a theme.

 

[Hip]

On this issue of functional and non-functional autoantibodies:

If it is difficult to determine whether anti-muscarinic or anti-adrenergic receptor autoantibodies are functional or not (ie, whether or not they have an agonist or antagonist action on the receptor), looking to see of ME/CFS patients have other conditions that have been linked to the presence of these autoantibodies might be a more reliable way to determine whether the autoantibodies are functional.

The remarkable thing about POTS is how simple and clear cut it is to diagnose. Essentially, if your heart rate increases by 30 bpm or more when you stand up from a relaxed lying down position, then you have POTS (OK, there are some caveats, but that's the essence of it). So you can make an accurate POTS diagnosis very easily, even at home.

Orthostatic hypotension is also easy to diagnosis, if you have a blood pressure meter. I believe in ME/CFS, it's more delayed orthostatic hypotension that patients have (where the drop in blood pressure can take up to 10 minutes to appear).

Of course this makes the assumption that anti-muscarinic or anti-adrenergic receptor autoantibodies are behind POTS and OH, but if they are, then the presence of these conditions in a patient would imply the autoantibodies are functional.

 

[M Paine]

I am puzzled why people think similarity of sequence would breach tolerance to self. If anything it should ensure poor response to foreign. The mimicry theory has got hold of the communal mind but has no logical coherence to it.

I'm sorry, I can't quite follow what is meant here. Is it correct that your view is that the process for obtaining autoimmunity does not involve similar human and pathogen gene sequences?

The reason I made the connection was the streptococcus aureus protein coat genetic sequence which was found to be 85% similar to the ß adrenergic receptor(s). My understanding of this was that antibodies may have been created potentially against this pathogen which have affinity to our own cell receptors. Is that not a likely scenario?

In any case, I would point out that my own understanding of Immunology is basic, so when I refer to the process as mystical, I purely meant from my own point of view.

 

[Jonathan Edwards]

I'm sorry, I can't quite follow what is meant here. Is it correct that your view is that the process for obtaining autoimmunity does not involve similar human and pathogen gene sequences?

The reason I made the connection was the streptococcus aureus protein coat genetic sequence which was found to be 85% similar to the
ß adrenergic receptors. My understanding of this was that antibodies may have been created potentially against this pathogen which have affinity to our own cell receptors. Is that not a likely scenario?

In any case, I would point out that my own understanding of Immunology is basic, so when I refer to the process as mystical, I purely meant from my own point of view.

You are right. My view is that autoimmunity is very unlikely to involve the immune system being confused by an antigen from a microbe that is similar to self. This idea has been around for fifty years but there is essentially no support for it except in Guillain Barre syndrome. The 85% similarity between a bacterial peptide sequence and a host peptide sequence is very unsurprising, since there are tens of thousands of host proteins and millions of microbe proteins. And it is hard to see why it would be relevant anyway. If the body makes a B cell whose antibody binds to both it will die because anything recognising self is deleted. If it only recognises the bacterial protein it will survive but then it will not be an autoantibody. A theory that suggests that these rules suddenly do not apply needs to explain why they suddenly do not apply!! I am afraid that immunologists are mostly simple souls and many of them do not seem to have thought that through.

 

[Jonathan Edwards]

You might say but surely immunologists debate this sort of thing and come to an agreement about what is a good theory and what is not. The truth is that in my career as a clinical immunologist I never came across the sort of debate we have here on PR in public. A few friends and I might debate like this in the bar in the evening, but at meeting of 3000 delegates I would tend to find about four people interested in that. One of the reasons I like PR is that I can go on having the arguments we used to have in the lab on Friday night after work was finished. Most of the other labs thought we were a bit nuts I think, but they envied the way we got through the chardonnay.

 

[M Paine]

Thank you for clarifying, as a person with a rudimentary understanding of the mechanics of the adaptive immune system, such a straight forward idea for attaining autoimmunity is very attractive. If for no other reason than that it is a simple enough concept to understand.

 

[Hip]

I wonder if the reason that Fluge and Mella did not find any beta adrenergic receptor autoantibodies in the 4 POTS patients in their study relates to the different subtypes of POTS?

The 2014 POTS study which found alpha 1, beta 1 and beta 2 adrenergic receptor autoantibodies in POTS patients does not mention which POTS subtype the patients had.

It is possible that each subtype of POTS may have its own characteristic autoantibodies (and I guess it's possible that some subtypes might not even be autoimmune).

The POTS subtypes are:

Low Flow POTS (similar to the hyperadrenergic POTS proposed by Streeten)

Low blood flows, increased arterial resistance. 20% of POTS patients are low flow. Most low flow POTS patients are female.

Two forms of low flow POTS:
• Elevated angiotensin II variety (almost always exclusively female).
• Norepinephrine transporter (NET) deficiency variety (more equal in terms of sexes).

Normal Flow POTS

Normal blood flows and normal arterial resistance.

High Flow POTS (similar to neuropathic POTS)

High blood flows, decreased arterial resistance.

Decreased release of norepinephrine responsible for constricting the arteries.

 

[EtherSpin]

Maybe that would explain the subgroup of ME patients who have PEM from cognitive activities but not from physical exertion.

wow! I've not heard of that combination before. PEM from concentration for extended periods yes, PEM from physical activity yes BUT I assumed the former was because when concentrating we may be stressing out, forgetting to sit/lay down (for people with OI) heart racing, tensing parts of our bodies (or pacing about the floor if it were me!) .

I know quite a few people who do indeed get PEM with its big delays that beggar belief and who fulfill all the CFS sets of criteria anyone could name but who seem to have no cognitive fog at all *unless* they are in the middle of a crash from physical activity.
these people get baffled by seeing other CFS sufferers struggling to do filing,sorting,calculation,reciting , reading etc and get astonished when I tell them I simply *cannot* read books no matter how relevant and helpful they are for CFS, if the narrative is spread across more than a page and needs memory to retain relevant ideas from a few pages back for integration into the whole picture of that chapter then its game over for me / us !

 

[snowathlete]

how do we get more broad antibody checking and discovery going in MECFS? It seems to me that we have evidence this would be worth the effort.

 

[Snow Leopard]

You are right. My view is that autoimmunity is very unlikely to involve the immune system being confused by an antigen from a microbe that is similar to self. This idea has been around for fifty years but there is essentially no support for it except in Guillain Barre syndrome.

My question to @BurnA, @M Paine and others who are proposing molecular mimicry, is why do we have to assume that because an infection is a clear trigger for a particular autoimmune illness (e.g. Guillain Barre syndrome) that it must be due to molecular mimicry?


GBS is associated with Antiganglioside antibodies and I don't see why self tolerance (by B-cells) could not have been bypassed n a similar way to other autoimmune diseases, whereby one of the viral proteins binds to a complementary self molecule and is presented it to the immune system in an aberrant way.

This of course begs the questions - which foreign antigens, the kinetics etc, but it seems plausible.

It doesn't tie in to molecular mimicry, which as I think I have said, does not actually make sense. If the immune system is tolerant to self then the novel presence of something similar to self is no reason to change that. The more similar it is the more you would expect it to be ignored. What does make some sense is if a newly encountered foreign molecule has the ability to bind to some complementary self molecule and present it to the immune system in an aberrant way. That would make sense in coeliac disease where gliadin presumably binds to tissue transglutaminase and presents it to the immune system in such a way that anti-transglutaminase B cells can be expanded to make antibody. Except that in coeliac disease there is not much evidence for the antibodies appearing at the time of first encounter of wheat. So even there it looks as if one has to look for a different rate limiting step.

 

[Jonathan Edwards]

GBS is associated with Antiganglioside antibodies and I don't see why self tolerance (by B-cells) could not have been bypassed n a similar way to other autoimmune diseases, whereby one of the viral proteins binds to a complementary self molecule and is presented it to the immune system in an aberrant way.

I agree, but Hugh Willison provided evidence that there WAS cross reactivity with a particular bacterial protein. Of course that may have been a red herring. The story involved animal model systems as well.

 

[BurnA]

My question to @BurnA, @M Paine and others who are proposing molecular mimicry, is why do we have to assume that because an infection is a clear trigger for a particular autoimmune illness (e.g. Guillain Barre syndrome) that it must be due to molecular mimicry?

I wasn't proposing anything @Snow Leopard
Just wondering where the molecular mimicry concept might fit in. Seems like the right person answered you in any case.

This story caught my attention although they point out that the paper has been retracted.
https://med.stanford.edu/news/all-n...-stem-from-molecular-mimicry-study-finds.html

 

[msf]

Ooh, a bit of role-reversal! Prof. Edwards making the argument (with reservations) for molecular mimicry. Does the fact that quite a few pathogens seem to be able to cause GBS (including flu apparently) mean that molecular mimicry may be more common than is commonly thought?