A Functional Cell-Based Bioassay for Assessing Adrenergic Autoantibody Activity in Postural Tachycardia Syndrome, 2019, Raj et al

Andy · Jun 26, 2019

Abstract
Background
Activating autoantibodies (AAb) to adrenergic receptors (AR) have previously been reported in patients with postural tachycardia syndrome (POTS). These AAb may contribute to a final common pathway for overlapping disease processes, reflecting a possible autoimmune contribution to POTS pathophysiology. In prior studies, measurement of AAb activity was inferred from costly, low-throughput, and laborious physiological assays. In the present study, we developed and validated an alternative cell-based bioassay for measuring AAb activity in serum by means of pre-treatment with monoamine oxidase (MAO).

Methods
A total of 37 POTS patients and 61 sex-matched healthy control participants were included. Serum was pre-treated with MAO to remove endogenous catecholamines that could falsely inflate AR activation by AAb. A receptor-transfected cell-based bioassay was used to detect presence of α1AR-AAb and β1AR-AAb in serum.

Results
MAO effectively degraded catecholamines as demonstrated by suppression of norepinephrine-induced α1AR activation in POTS (6.4±0.7 vs. 5.5±0.9; P=0.044) and in controls (4.1±0.5 vs. 3.9±0.6; P=0.001). Mean activity values were greater in the POTS vs. controls for α1AR-AAb (6.2±1.2 vs. 5.3±1.0; P<0.001) and β1AR-AAb (5.7±1.8 vs. 4.1±0.9; P<0.001). Compared to controls, more POTS patients were positive for α1AR-AAb activity (22% vs 4%; P=0.006) and β1AR-AAb activity (52% vs. 2%; P<0.001).

Conclusions
The co-presence of norepinephrine in serum samples can artifactually elevate α1AR and β1AR activity, which can be avoided by serum pre-treatment with MAO. Using this novel bioassay, we show that POTS patients have increased α1AR-AAb and β1AR-AAb activity compared to healthy controls in the largest POTS cohort reported to-date.
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Open access at www.sciencedirect.com/science/article/pii/S2589909019300061

wigglethemouse · Sep 19, 2019

This was the commentary posted by Dysautonomia International in June (copied from a Facebook user, so sorry I don't have a source link). Researchers in Oaklahoma carrying out work on alpha-1 & beta-1 adrenergic auto-antibodies had doubts about the test they were working on and spent a long time figuring out that norepinephrine in serum samples can artificially elevate a1AR and ß1AR activity. This is important for others working in the same area.

BIG NEWS! Results from the 2014 Dysautonomia International Conference POTS Antibody Study, our very first conference study, have finally published! And what the researchers found is super important to advancing our understanding of autoimmunity in POTS.

It's a very technical journal article, but to summarize the key points...

1. People with POTS tend to have higher than normal levels of norepineprhine (NE) in their blood, which is a catecholamine that activates the sympathetic nervous system. We already knew this from prior research, but what we didn't have clear evidence of until this study is that NE can cause false positives on adrenergic receptor antibody tests. This makes sense, because NE binds to adrenergic receptors, similar to the way an antibody binds to the receptor.

2. Researchers have always assumed that NE degrades quickly if you don't carefully preserve the blood sample. Since the researchers in this study weren't trying to preserve the NE, they initially assumed it would have degraded before they ran their antibody tests. But it didn't degrade and it messed up their first round of antibody tests (which is why it took long to finish this study, because they had to figure out what was messing up the antibody assays, and how to fix the problem). Finding that NE doesn't degrade as quickly in blood samples as previously thought is really important for POTS research, but is also an important finding that will impact research on other diseases too, because NE and its effect on adrenergic receptors is studied in a lot of other diseases: other forms of dysautonomia, heart rhythm disorders, asthma and allergic disorders, anxiety, PTSD, Parkinson's, etc.

3. After the researchers figured out the NE was messing up the antibody results, they had to develop a way to remove NE from the blood/serum samples before running the antibody test without removing antibodies that may be present. They did this by "washing out" the NE with monoamine oxidase, and enzyme that breaks down NE.

4. After they washed out the NE from the blood/serum samples, they found that a majority of POTS patients had antibodies to one or both of the adrenergic receptors they explored in this study (alpha1 and beta1 adrenergic receptors). This is the largest cohort of POTS patients to date that have been tested for these antibodies (37 patients). Prior smaller cohort studies have found these antibodies in a majority of POTS patients too. And if you have been following our page, you know that other antibodies have also been found in a majority of POTS patients, like angiotensin receptor antibodies.

5. Most people with POTS (and people with related disorders like OI, AAG, NCS, etc.) want to know if they have these antibodies, but to date we haven't had a reliable way to test for these antibodies on a large scale, because the testing done in prior research studies was very expensive, time consuming, and labor intensive. For a test to be widely available on a commercial scale, it has to be accurate, economical and not very labor intensive. The new testing approach developed by the University of Oklahoma lays the groundwork for eventually commercializing this antibody assay and making it available to patients around the world. There will be more research required before that happens, but we are on the right path and making good progress in our quest to identify biomarkers in POTS!

You can find the full journal article here: http://bit.ly/2KFBT9u.

Tremendous thanks to all of the donors and study volunteers who made this possible, and the researchers who worked tirelessly on this project for the past five years! You can help Dysautonomia International advance POTS research even further by making a contribution at CurePOTS.org.
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ahimsa · Sep 24, 2019

@wigglethemouse -

I noticed that the date of the comment you posted is 2014. Is that a typo? Or was that a comment about one of the previous studies?

I did find this more recent blog post on the Dysautonomia International Web Site:

http://www.dysautonomiainternationa...ce-research-study-immune-phenotyping-in-pots/

But I have no idea what the blog post adds to this specific thread since the science is way above my head.

wigglethemouse · Sep 24, 2019

ahimsa said: ↑

I noticed that the date of the comment you posted is 2014. Is that a typo? Or was that a comment about one of the previous studies?
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No, it is not a typo, and not another study. The link you provided has a little more info (relevant section pasted below) that explains "2014 Conference" refers to when and where the samples were collected. The link in the quote below does refer to the paper in this thread.

The second, a study from Dr. David Kem’s lab at the University of Oklahoma confirming a much higher rate of adrenergic receptor antibody activity in POTS patients compared to healthy people (the samples were collected during our 2014 Conference).
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It seems it took 5 years to get good measurements - they eventually figured out that norepinephrine affected the test.

ahimsa · Sep 24, 2019

Thank you @wigglethemouse - my brain was simply unable to absorb the information!

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