Brad Christman 5/13/2014

Prof. Walter Bio 341

Mechanism of Neuromuscular Junction Inhibition in Myasthenia gravis

Intro to Myasthenia Gravis:

Myasthenia gravis is an autoimmune disorder that interferes with normal

transmission of the neurotransmitter acetylcholine in the neuromuscular

junction. The physiology of the disorder is well documented. Acetylcholine

receptor antibodies (AchR-ab) produced by specific mature B cells circulate in

the blood and target sensitized nicotinic acetylcholine receptors (AchR) on the

postsynaptic membrane of the neuromuscular junction (Lindstrom 1973). In

Myasthenia Gravis the mechanism and pathogenesis of neuromuscular

transmission impairment is still under study however a reduction in the number

of functional AchR’s on the postsynaptic membrane has been documented

(Drachman et. al 1973). At least three antibody-mediated mechanisms have

been proposed to explain AchR impairment: accelerated endocytosis and

degradation of AChR’s, functional blockade of Ach-binding sites, and

complement-mediated damage of the postsynaptic membrane (Vincent 2005).

Adaptive Immune Response & Autoimmunity:

In the adaptive immune response, specific B cells that circulate in the serum of

the blood engulf specific antigens and incorporate a part of them onto their

surface membrane. The display of this antigen fragment bound to its specific

MHC molecule recruits a matching mature Helper T-cell. The binding of these

two cells stimulates the Helper T-cell to secrete specific cytokines that induce

the B cell to multiply and mature into antibody producing plasma cells. These

plasma cells produce specific antibodies that can circulate in the blood

targeting the antigen. This process of the adaptive immune response results in

chemical signaling that allows other immune system cells such as macrophages

and killer T-cells to locate and destroy the targeted antigen (Lodish et al. 2013).

The adaptive immune system may take a period of time before it can adapt and

multiply the antibodies necessary to neutralize a new antigen. However, once

the B cells and Helper T-cells have been adapted, they can stay dormant in

memory cells of the lymph ready to quickly bind to its specific antigen and

multiply if it reappears. This fighting off of specific foreign bodies such as

specific bacteria and virus’s is a vital aspect of our immune system.

Autoimmunity is defined when the antigens being attacked by antibodies of the

adaptive immune response are not foreign antigens but tissues and cells of the

organism (Rose & Bona 1993). Because of this, if Myasthenia Gravis goes

untreated, the immune response of the antibodies can continue attacking the

tissues until the organism as a whole is compromised. As a result of the

pathology of the adaptive immune response and the specific nature of

antibodies, autosensitization of tissue has been recognized as the initial trigger

in autoimmunity; autosensitization has to occur before autoimmune antibodies

are produced. Autosensitization is when the immune response recognizes the

self as a foreign body and initiates the adaptive immune response against it

(Goodnow et al. 2005). Little molecular research has been done on the factors

involved in triggering autosensitization, although environmental factors,

viruses and gene expression have been thought to be possible contributors

(Vincent 2005). In Myasthenia gravis the Thymus is suspected to be a main

player involved in the autosensitization of Ach-R. Ach-R specific T cell lines

can be cloned from the thymus, and cultured thymic lymphocytes

produce AChR-specific autoantibodies. The thymus is the site of T-Cell

maturation. Thymic abnormalities are frequently present and specifically

associated with MG. 30-45% of people with thymomas have MG and 10-15%

of MG patients have thymomas. Because of this a thymectomy has been

employed as a effective treatment option (Meriggioli 2009).

The AChR is an oligomeric membrane protein consisting of five subunits α2, β,

δ and ɛ at the adult end plate. The dominat AchR-ab epitope is directed toward

the main immunogenic region (MIR) on the alpha subunits. However, other

studies suggest that some patients do not have MIR antibodies and some have

antibodies that are directed against regions on different subunits (Vincent

2005). The epitope is the antigen specific binding site of an antibody. Due to

the adaptive/specific nature of antibody production, not everyone’s epitopes are

structurally identical. In this way antigen-antibody binding affinity can change

from one MG case to the other. However, the highly conserved genetic code

for AchR of skeletal muscles results in nearly identical AchR molecular

structure from person to person. In addition

to epitope variability, ChR antibody titres are highly variable among MG

patients, ranging from 0 to more than 1000 nm/L. These titer variations may or

may not correlate with the location and severity of the disease. For example,

the disease can remain localized (e.g. Ocular MG), involve mainly specific

muscle groups (e.g. Bulbar MG) or spread to include most muscles

(Generalized MG). There is still a lot that is not fully understood about the

variability in the pathogenesis of the adaptive immune response in MG.

(Newson-Davis et al. 1978).

Physiology & Diagnosing:

In muscle innervation, when an action potential (AP) reaches the nerve terminal

of a motor neuron, the depolarization causes an influx of calcium that results in

the release of Ach from thepresynaptic terminal. Ach diffuses across the

synaptic cleft and binds to Ach-R found on the edges of junction folds of the

postsynaptic membrane, resulting in a graded end-plate potential (EPP). Ach-

R’s are transmembrane ligand gated Ion channels that undergo a

conformational change when Ach binds to it. This conformational change

opens an ion channel that allows sodium and potassium ions to diffuse across

the postsynaptic membrane causing a graded potential. If this graded potential

reaches threshold, it creates an action potential in the sarcolemma that goes on

to contract a motor unit via excitation-contraction coupling. Almost always, the

end plate potential triggers an action potential (Plomp et al. 1995).

Normal EPP’s depolarize to a greater level than the AP threshold level. The

difference between these two levels is the safety factor. For Myasthenia gravis

patients, this safety factor is reduced. Worst case, the EPP fails to reach AP

threshold, resulting in no muscle contraction. In diagnosing Myasthenia gravis,

repetitive nerve stimulation (RNS) is one of the first diagnostics employed to

test for MG. This diagnostic test rapidly innervates nerves and takes data on

the EPP’s. In Myasthenia Gravis patients the EPP is decremental, meaning that

the depolarization each graded potential decreases with every innervation. This

is due to the fact that there is a reduced number of active AchR that cannot

handle the rapid influx of Ach that result from rapid innervation (Kothari

2004). AchR’s are found at a normal density of10,000 receptors/micrometer2.

The amount of sodium and potassium the channels allow through their pores,

conductance varies from 50–110 pS, with the conductance depending on the

specific subunit composition as well as the permeant ion (Miyazawa

& Fujiyoshi 2004).

The most specific diagnostic test for Myasthenia Gravis is testing for the

acetylcholine receptor antibodies (AchR-ab). Three libratory studies are

commercially available and may be used when testing for the presence

of AchR-Ab: binding, modulating, and blocking (Plomp et al. 1995). Although

the antibodies circulate throughout the body, often time’s impairments in ocular

movement, speech, swallowing and breathing are most noticeable. Usually

ocular impairment is the first symptom of Myasthenia Gravis. No sensory,

autonomic, or cognition impairment results with the disease. This is due to the

fact that the blood brain barrier prevents the AchR-ab from inhibiting neural

synapses in the central nervous system. The onset of the disorder can vary

greatly between cases. Onset of severe life threatening symptoms also known

as a Myasthenic crisis also varies greatly between cases.

Current Treatments:

There is no specific protocol for the treatment of Myasthenia gravis. Although

in a crises, acetylcholineesterase inhibitor is first used to prevent the breakdown

of acetylcholine in the NMJ. This treatment leads to an increase in the level and

duration of acetylcholine in the NMJ and an increase in the strength of

neuromuscular transmission. In addition, MG patients are placed on

immunosuppressant drugs and a prednisone steroid therapy that decreased the

titer of autoimmune antibodies. This was only a temporary treatment. The next

step in treatment, a long-term solution is plasmapheresis, thymectomy or

intravenous immunoglobulins. The goal of plasmapheresis or plasma exchange

is to remove the circulating immune complexes and AchR-Ab. Patients

undergo a 2-week course of 5 to 6 exchanges. Removing on average 1-Pint of

plasma per exchange. Although the number of exchanges and interval between

them often times must be tailored to each patient, taking into account the

general clinical conditions, severity of MG, and potential side effects (Vincent

2005). Open studies showed that PE was effective in at least 60% to 70% of

treated patients, however this treatment option is temporary, usually lasting

only 4-5 weeks. Con’s to the use of chronic plasma exchange are the need for

good vascular accesses and the obvious effects on several plasma components

being removed. The long-term benefit of plasma exchange is still under study

(Gadjos et. al 2002).

Mechanism of Myasthenia gravis Inhibition:

The fundamental mechanism and pathogenesis affecting the neuromuscular

junction of MG patients is not yet fully understood. However, the reduction of

available AchR has been documented to cause the defecit in neuromuscular

junction transmission in Myasthenia gravis (Fambrough et al. 1973). Although

proposed Mechanisms behind NMJ inhibition bounced around for over a

century,Fambrough and his colleagues conducted the first studies to verify the

mechanism behind NMJ impairment in Myasthenia gravis. To do this, they

took “motor point” biopsies from MG patients. They incubated the samples

with radio labeled I-a-BuTx, a neurotoxin that binds to AchR at the Ach

binding site. After scintillation counting and autoradiography they found an

80% reduction inAchR’s in MG patients n comparison to non-MG patient

controls. They correlated this reduction in AchR with the marked reductions in

the Motor Endplate potential exhibited by these patients. Following this data,

autoimmunity was proposed and the hunt for the antibody began. “In the most

sensitive radioimmunoassay, which detects antibodies bound to AchR labeled

with 125I-α-bungarotoxin (125I-α-BuTx), elevated titers have were found in 80-

90% of patients with MG”. However, the antibody titer corresponded only

approximately to the clinical status of the patients (Drachmen et. al 1973). The

isolation of these antibodies, experimental employment of 125I-α-BuTx, and the

growing knowledge immunology and physiology allowed researchers to make

great progress in the study of this disease. For example, it was found that the

disease can be passively transferred to experimental animals by daily injections

of purified MG IgG (Toyka et al. 1975). The pathogenic role of AchR-ab was

also verified by the drastic improvement of MG symptoms observed following

plasma exchange (Newsom-Davis et. al 1978). With these discoveries, today

MG meets all the criteria of Witbesky to categorize it as an autoimmune

disease (Rose & Bona 1993). Once the antibody was isolated, studies were

done to test the specific mechanism of AchRreduction in MG patients. The first

mechanism studied and verified was the acceleration of AchR degradation

via endocytosis in response to antibody crosslinking of the receptors known as

antigenic modulation. The receptors linked to antibodies are internalized and

degraded by lysosomal enzymes. This crosslinking was found to reduce the

half-life of AchR’s at the NMJ from about 10 days to about 5 days

(Drachman et. al 1978). The second mechanism studied was complementation

and activation of the membrane attack complex resulting in the destructive

changes in the morphology of the postsynaptic membrane. This mechanism is

most likely the most important because it results in a reduction of the

postsynaptic folds and a reduction in the functional AchR’s and ion channels

required for signal transduction (Arahata 1987). It has also been verified that

there is a compensatory response to the reduction of AchR’s. AchR synthesis

has been shown to increase in AchR turnover studies (Wilson et. al 1983). Also

interesting is the presynaptic motor neurons ability to recognize impaired

transmission and try and compensate by increasing the number of Ach packets

released (Plomp et. al). A functional block of AchR by the antibody may also

play some role in the mechanism of inhibition however this mechanism has

been a matter of controversy.AchR-ab almost never binds specifically to the

Ach binding site however the antibodies have been determined to block I-a-

BuTx, which binds to the Ach binding site (Drachman et. al 1973). There is no

reliable evidence for involvement of the cellular mediated immunity in the

pathogenic mechanisms at the neuromuscular junction in MG. The majority of

electron microscopic studies have not identified apoptosis, macrophages or

natural killer cells indicative of cell initiated immune response (Vincent 2005).

Research Proposal & Approach: What is the

Mechanism of Neuromuscular Junction Inhibition in

Myasthenia gravis?

At least three antibody-mediated mechanisms have been proposed to

explain AchR impairment: accelerated endocytosis and degradation of AChR, functional

blockade of Ach-binding sites, and complement-mediated damage of the postsynaptic

membrane. (Vincent 2005).

Intro: What is the mechanism of Acyteylcholine antibodies (Ach-ab) inhibition of

Acetylcholine Receptors (AchR) receptivity and function? Based on the literature,

inhibition of AchRcould be due to any off the three mechanisms proposed above. My

research proposal includes a comprehensive protocol aimed at better understanding the

mechanism by which the Ach-ab inhibits and destroys acetylcholine receptors in the

neuromuscular junction.

Hypothesis:

I think the mechanism of Acetylcholine Receptor (AchR) inhibition is somewhat

dependent on the specific form of the Acetylcholine antibodies (Ach-ab). I think the

mechanism of AchRinhibition is dependent on the ratio of receptor bound Ach-ab to

total AchR in neuromuscular junction (NMJ) and therefore the mechanism dynamic

throughout the course of the disease and pathogenesis can vary from case to case.

Research Questions:

-What happens when antibodies bind to receptors?

-Does it stop Ach from binding or does it stop AchR from functioning (opening)?

Or does it result in a loss of receptors by endocytosis or the compliment mediated

immune response?

Approach:

Experiment 1: Is Ach-ab bound to AchR?

Yes: Go to experiment 2&3.

No: Go to alternate explanation.

To determine if AchR-ab’s are bound to AchR’s I want to conduct a binding assay on a

reconstituted frog oocyte. I will use a frog oocyte with AchR’s expressed on its surface. I

will follow the protocol for AchR specific mRNA preparation and insertion. I will

implant these AchR specific mRNA into the oocyte for expression. Once the

frog oocyte with AchR’s is prepared I will do a binding assay using acetylcholine and

fluorescently tagged AchR-ab’s purified from human serum. I will follow the protocol for

fluorescent tagging of antibodies. I will expose the prepared oocytes to the tagged Ach-

ab’s. After each trial, spin down the preparation, remove the soup and wash

the pelleted oocytes two times to remove any excess AchR-ab’s. Remove washed pellet

and conduct microscopy and fluorescent spectroscopy. I will look for AchR-ab binding to

the AchR’s. This binding level will tell us whether or not AchR-ab is binding or not

to AchR’s.

Experiment 2: Does AchR-ab binding inhibit Ach binding?

In this experiment we want to see if Ach-ab binding to Ach affects the ability of Ach to

bind to AchR. To do this, a similar experiment to Experiment 1 will be conducted. The

difference between these experiments is that in experiment 2 I will use 125I-α-

bungarotoxin (125I-α-BuTx), which has been experimentally tested to bind to the Ach

binding site (Drachman et. Al 1973). This will be a two-part experiment. Part 1 will

test 125I-α-BuTx’s ability to bind to AchR’s on its own. Part 2 will test 125I-α-BuTx’s

ability to bind to AchR’s in the presence of Ach-ab’s. Binding ability will be quantified

using liquid scintillation spectrometer and autoradiography. A decrease in isotope return

from part 1 to part 2 following the addition of 125I-α-BuTx’s will indicate Ach-

ab’s inhibition of Ach binding to Ach-R. We will be able to calculate mg of bound 125I-α-

BuTx per Oocyte. Based on recent studies and what is known about MG, a decrease in

isotope detection from part 1 to part 2 should be the result. If not see alternate

explanation. This experiment may need to be conducted w/ 14 C radio labeled Ach as

there might be some steric and affinity differences between Ach and 125I-α-BuTx.

Experiment 3: Does AchR-ab binding decrease Ach binding affinity?

To do this experiment I will conduct an antibody-affinity chromatography experiment. To

do this, I will covalently attach the AchR onto beads. I will then conduct three trials. In

one trial I will add Ach via a neutral buffer to the cylinder. The next trial I will add Ach

and AchR-ab into the cylinder. The last trial I will add the AchR-ab first followed by

Ach. I will conduct this experiment at different flow rates and with different AchR-

ab’s epitopes from different MG patients. After each trial I will quantify the amount and

the type of protein that flowed through using SDS-Page and Coomassie blue staining

with Prism software. Then I will then wash and elucidate the cylinder and quantify the

amount and type of elucidated protein. Based on this experiment, I will be able to see the

change in binding affinity between AchR and Ach when AchR-ab is not bound,

competing for binding and bound. (Lodish et al. 2013).This assay could give you good

results on the competitive nature of Ach and the AchR-ab. It could also give you

interesting results on how Achr to Ach binding affinity is affected by different epitopes of

the antibody.

Experiment 4: Does AchR-ab binding inhibit functionality of AchR’s in signal

transduction?

Yes: Go to Experiment #5

No: Go to alternate explanation.

Does the presence of AchR-ab completely inhibit AchR functioning? Or does it only

partially inhibit due to changes in binding affinity, binding duration or conduction time?

To answer these questions, I will do a patch clamping experiment with the reconstituted

frog Oocyte from experiments 1-3. This experiment will allow me to measure the effects

of Ach-ab’s binding on the functionality AchR. AchR is a ligand gated ion channel that is

known to undergo a conformational change and open when bound to 2 Ach’s. To conduct

a patch clamp experiment, I will have to apply a patch electrode with slight suction to a

region of the Oocyte cell membrane. In addition, I will add an intercellular electrode.

This experiment will be conducted in 2 parts. Part 1 will constitute filling the patch

electrode with a current conducting saline solution and a known concentration of Ach.

Part 2 will constitute filling the patch electrode with a current conducting saline solution

along with a known concentration of Ach and Ach-ab. This patch clamp device will

maintain constant voltage across the membrane and measure current flow across

membrane at the tip of patch electrode. This method prevents changing voltage gradients

from inhibiting sodium influx (Lodish et al 2013). This experiment will test the effect

Ach-ab has on sodium influx and thus signal transduction in the postsynaptic membrane.

If the current flow (sodium influx) decreases across the membrane in the presence

ofAchR-ab’s than it can be concluded that AchR-ab’s inhibit the functionality of signal

transduction. For each trial it should be ensured that AchR quantity within the patch

electrode is equal to one. This could be a good experiment to test how different ratios

of AchR-ab to Ach effect signal transduction. Another focus of this study might be to see

whether there is a change in ionic channel open time when AchR-ab is bound. Taking

short interval measurements of depolarization time for each test part will give you this

result.

Alternate Explanation:

No binding of AchR-ab to AchR observed

Based on current research and understanding of MG, it is known that the autoantibody

Ach-ab binds to the autoantigen AchR. So it is expected that Ach-ab present in the

solution of Experiment 1&2 will inhibit Ach binding. There are multiple reasons for these

unexpected results. First, a AchR specific mRNA translation mistake could lead to Ach-

ab’s inability recognizing the expressed AchR’s. Secondly, patients expressing MG

symptoms don’t always have AchR-ab’s. MusK is inhibited by MusK-ab antibodies in

the NMJ. MusK is a signaling protein involved with the development and maintenance of

the NMJ. Without proper signaling by MusK the patency of the NMJ decreases. In 20 %

of MG cases, MG symptoms are caused by auto MuSK antibodies instead of

auto AchR antibodies. If auto MuSK antibodies were taken from a human host instead

of AchR-ab’s and used in experiment 1 or 2 most likely no inhibition of Ach binding

would be seen. Auto MusK antibodies cause MG symptoms by a similar but different

mechanism. I chose to focus on AchR-ab MG in this proposal.

Testing the Mechanisms of AchR inhibition:

Now that we know Ach-ab binding inhibits AchR functionality in vitro study, we need to

determine the mechanism of AchR inhibition in living tissue?

There are 3 proposed mechanisms of Ach-ab inhibition of AchR’s. The first mechanism

proposes that Ach-ab simply blocks the binding site of Ach, preventing Ach binding and

signal transduction. If this were the case Ach-ab binding would occur but no reduction

of AchR’s would be observed. The second mechanism proposes that Ach-ab binding

to AchR’s inducesendocytosis of the postsynaptic membrane. If this were the case, we

would see an increased rate of endocytosis when Ach-ab is bound to Ach-R. The third

mechanism proposes that Ach-ab binding to AchR results in complement mediated

damage of the postsynaptic membrane. If this were the case, Reduction in the number

of AchR’s and overall organization of the postsynaptic membrane would decrease.

Experiment 5:Testing for mechanism #1 (Blocking of binding site)

To test for this mechanism, I will create a primary cell culture of the NMJ cells cooled at

4 degrees Celsius to eliminate degradation and minimize possible antibody

dissociation. They will be treated overnight in the cold with AchR-ab’s and then are

saturated with 125I-α-BuTx’s. The loss of 125I-α-BuTx binding sites in the cultures treated

with AchR-ab is attributable to AchR blockade (Drachman et. al 1973).

Experiment 6: Testing for Mechanism #2 (Induction of Endocytosis)

To test whether or not muscle cells induce endocytosis in response to Ach-

ab bound to AchR, I would again use a primary muscle/nerve cell culture experiment. I

would set it up similar to Experiment #5 only I would culture the cells in fluorescent

media to follow endocytosis. I would microinject the Ach-ab to the NMJ and then

stimulate the release of Ach into the NMJ with a microelectrode. I would conduct

multiple trials of this experiment with varying intervals and time spans of nerve

innervations’. I might try random fast, random slow, repetitive fast, repetitive slow and

no innervations. I would take fluorescent microscopic images of the muscle cells at

standard time intervals and calulate the rate of fluorescent vacuole formation. This would

be possible because the fluorescent die on the outside of the cell would be brought into

the cell and easily observed following edocytotic vacuole formation. This experiment

would be conducted with and without Ach-ab added. If the fluorescent vacuole formation

increased in response to Ach-ab in the NMJ then induction of endocytosis could be a

potential mechanism for AchR signal transduction inhibition. To get further accuracy in

your results, you could repeat the study using C14 tagged anti-AchR-ab antibodies.

Thisimmunohistochemistry technique allows you to track the Achr-abs and see if they are

being internalized via endocytosis. This would show you if membrane sections

containing Ach-abbound to AchR were specifically targeted for endocytosis or not. This

experiment might also provide you with insight into the effect of

motor nueron stimulation on the induction ofendocytosis.

Experiment 7: Testing for Mechanism #3 (Complement mediated damage of the

postsynaptic membrane)

Because of the complex/dynamic/heterogeneous environment of the mammalian

body, I think it is only appropriate to study the adaptive immune response/complement in

a live animal study. To test the third mechanism of AchR inhibition, I want to conduct a

study using mice with experimentally induced Myasthenia gravis. I will follow

documented literature on inducing MG in mice (Toyka et al. 1975). Once MG is induced,

I will conduct an observational histology experiment. This procedure for this experiment

will constitute removing thin tissue samples of muscle and nerve cells. These tissue

samples will be fixed via the snap freezing protocol and examined under a microscope.

Each tissue sample would be examined for the number of AchR’s, the

integrity/organization of the postsynaptic membrane, the presence of immune cells

(Helper T-cells, B cells, Macrophages etc.) and most importantly the presence

of cytolytic membrane attack complexes. The cytolytic membrane attack complex is a

direct indication of immune system complementation. Complement is the process by

which a group of constitutive serum proteins bind to microbial or fungal surfaces, thereby

activating a proteolytic cascade that culminates in the formation of

the cytolytic membrane attack complex (Lodish et. al 2013). Disorganization in the

postsynaptic membrane folds, reduction in the number of AchR’s, and the presence of

immune cells in particular cytolyticmembrane attack complexes are all indicators of

complement mediated damage of the postsynaptic membrane. If these indicators are

identified compliment mediated damage could be concluded as the primary mechanism

of AchR signal transduction inhibition. It might be useful to dye or tag specific aspects of

the tissue samples in order to better observe/quantify specific molecules of interest such

as Ach-ab, AchR or the membrane attack complex. Observations on tissue samples from

experimental induced MG mice will be cross-referenced with tissue samples of normal

mice. This experiment sets up well to look at the NMJ at various stages of the disease.

These different stages in the disease often correlate with differing titers of the antibody.

Therefore the effect different titers have on the mechanism of NMJ inhibition can be

observed. A specific epitope of AchR-ab can be induced in mice, and the changes in

these specific epitopes can be studied in regards to the NMJ inhibition mechanism.

Observing different combinations of AchR-ab epitopes may also be enlightening.

Conclusion:

In conclusion, this series of experiments will seek to discover the mechanism by which

MG antibodies inhibit NMJ signal transmission. Much is already known about the

mechanisms of inhibition that can and do exist in MG patients. However, the variation in

the pathogenesis of the disease between MG patients is still not fully understood. The

dynamic nature of the mechanism of NMJ inhibition may be an explanation for the

variations in MG pathogenesis. Variations in the affinity of AchR-ab epitopes and

variations in the ratio of receptor bound Ach-ab to total AchR in the NMJ I hypothesize

to be the determining factors in the NMJ inhibition mechanism. My research is set up to

see the effects that changes in antibody affinity and antibody concentration have on

verified mechanism of NMJ inhibition. My research proposal could result in clinical trials

for the study of MG pathogenesis if strong correlations were made between specific

inhibitory mechanisms and specific AchR-ab epitopes and antibody concentrations. If

these correlations remained consistent following clinical trials, this relationship between

the mechanism and the variable factors of the disease could lead to the personalization of

the MG treatment plan. For example, the epitope could be determined, the ratio of

receptor bound Ach-ab to total AchR in the NMJ could be quantified and just like that

you know the mechanism of NMJ inhibition that is causing that patients symptoms.

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