*This machine-generated transcript may have errors. If remediation or a manually-generated transcript is needed, please contact NLM Support at https://support.nlm.nih.gov.*

*This machine-generated transcript may have errors. If remediation or a manually-generated transcript is needed, please contact NLM Support at https://support.nlm.nih.gov.*100:01:12,340 --> 00:01:17,030four models have been proposed for myasthenia gravis in recent

years. The first I have listed is generally agreed

to most closely resemble myasthenia gravis in man. This

model arose is an incidental laboratory observation in the course

of basic studies of purified acetylcholine receptor. The second

model, described by taika and colleagues, involves the

passive transfer of myasthenia gravis antibodies into mice. This

model provides the first evidence that human anti receptor antibodies

are pathogenic, and this model should be very valuable

in elucidating the pathogenesis of neonatal myasthenia, where trans

placental transfer of antibodies does occur. The third model

described by Sata, Murty and others, involves

the direct injection of rats with alpha neurotoxins of cobra

venom. This model has been useful in demonstrating that

paralysis of the societal calling receptor molecule itself can mimic

myasthenia gravis clinically and electro physiologically. However, the

mechanisms involved at the level of the acetylcholine receptor molecule

are quite different in mechanism of that of myasthenia gravis

or of experimental autoimmune myasthenia gravis. The fourth model

experimental autoimmune thing I'm itis has been included for historic

completeness. However, this model today remains a subject

of controversy and its relationship to human myasthenia gravis is

not clear. Our understanding of myasthenia gravis today I

was much two basic studies of the chemistry, pharmacology

and physiology of the electric organs of fishers. Like

the Californian torpedo ray, the electric organ of the

torpedo can be seen as a raised present on the

lateral body of the animal. Large amounts of acetylcholine

receptor can be extracted from this organ and purified by

affinity chromatography, which takes advantage of another exotic product

of nature. The alpha neurotoxin of cobra snakes.

The acetylcholine receptor is soluble ized from electric organ membranes

absorbed onto beads to which toxin is conjugated and finally

eluded free from the toxin in purified form 1972 Patrick

and Lindstrom observed by chance that rabbits immunized with purified

acetylcholine receptor became paralyzed. This observation led to the

development of the model experimental autoimmune myasthenia gravis, which

has been studied most extensively in the rat. E

A. M G ensues when rats are immunized

with acetylcholine receptor together with immunological adjuvants. Clinically,

the rats develop weakness and fatigue ability. Pharmacologically,

this weakness can be improved temporarily with pro Stig mean

and the rats demonstrate sensitivity to minute doses of cure

Ari electro physiologically de Krim entitle electro Maya grams are

found. The phenomena of post Titanic facilitation. Post

activation, post activation exhaustion and reduced amplitudes of miniature

end plate potentials are found immunologically antibodies to acetylcholine receptor

of the rats own post synaptic membrane are demonstrable and

there is also cellular immunity to acetylcholine receptor by electron

microscopy. Reduced numbers of toxin binding sites are found

on the post synaptic membrane. The ultra structure of

the post synaptic membrane is highly simplified, and both

egg and the third component of compliment have been demonstrated

on the post synaptic membrane. The next one rats

are injected into the skin with purified acetylcholine receptor,

either that isolated from electric organ of eel or marine

raise or with purified acetylcholine receptor from rat muscle itself

The antigen is combined with complete Freud's adjuvant,

and an injection of potassium vaccine is also given.

This animal received adjuvant only and remains completely free of

weakness. Rats receiving acetylcholine receptor inoculations develop usually two

episodes of weakness. The first occurs one week after

inoculation and is usually transient. This weakness or acute

phase of weakness is followed by a transient stage of

remission and four weeks later, a chronic phase of

weakness ensues, which is usually progressive. The weakness

involves predominantly the four limbs, the head and neck

and the respiratory muscles. This slide shows the posture

which is characteristic of a MG. The animal

has difficulty raising its head or raising its front half

of its body. The cry is very weak.

The muscles of mastication are so weak that the animals

have to be maintained on soft food. However,

when given soft food and water within easy reach,

the animals can survive for up to a year.

Electro physiologically with repetitive nerve stimulation. There is a

detriment of the muscle response. When the stimulation is

prolonged, the phenomena of post activation facilitation is seen

which is followed by post activation exhaustion. These

phenomena are characteristic of myasthenia gravis in man here,

after the injection of prostate mean intraparty. Nearly the

deck criminal which preceded the injection is repaired and this

repair lasts up to three hours and then the detriment

returns by miniature electrodes Studies rats were studied at various

times after a single injection, with the receptor and

achievements for the rats studied for the first seven days

after injection, the amplitude of the miniature end plate

potentials was normal. This is the normal range.

However, from Day seven there was a significant reduction

in amplitude of the maps and even in the period

of clinical remission, the amplitude remained low and continued

to be very low out into the chronic phase of

the disease. Histological e The findings in the acute

phase and chronic phase are quite different. I will

discuss the acute phase first. This shows the hist

o chemical and him a toxic linen ears and appearance

of the end plate region of normal rat skeletal muscle

by Alpha Enough, alas, etait reaction the colonist

eras reaction at the post synaptic membrane can be seen

mhm. This just shows the appearance of intact in

plate morphology. In the acute phase of weakness in

the rat, there is a tremendous infiltration of modern

nuclear inflammatory cells in the region of the post synaptic

membrane. The colonist er is positive material, is

sequestered away from the muscle fiber and is undergoing disintegration

And with higher power magnification, you can see

the nuclei of the modern nuclear cells actually are embedded

in this colonist to raise positive material. The end

plate is being engulfed by inflammatory cells. The next

slide reminds you of the ultra structure of the post

synaptic membrane and the nerve terminal in a rat.

Here is the nerve terminal containing vehicles which are presumed

to hold the A subtle Colleen. Here you can

see mitochondria, the underlying muscle fiber with the membrane

the post synaptic membrane, highly specialist with delicate

folds beneath the nerve ending the synaptic space is very

narrow. This is the Schwann cell here in the

acute phase of myasthenia gravis in the rat. This

whole area of the motor junction is a disaster area

The nerve terminal is quite intact. The muscle

fiber underneath is completely denuded of post synaptic membrane.

Here you can see remnants of the post synaptic membrane

separating the intact nerve terminal from the muscle fiber.

There are fake acidic inflammatory cells here containing debris of

membrane. After Day 12, these inflammatory cells disappear

from the post synaptic region and the nerve terminal comes

back into its position opposite the post synaptic membrane,

which at this time is showing signs of repair.

But continued degeneration also occurs, and at this stage

and into the chronic phase of E. A.

M. G, the ultra structural picture is indistinct

indistinguishable from that of human myasthenia gravis. The

synaptic spaces widened and the post synaptic membrane is highly

simplified. The acetylcholine receptor in the post synaptic membrane

of a normal rat neuromuscular junction can be visualized by

its reaction with a peroxide is conjugated alpha neurotoxin of

the snake venom. But normally the Estelle Colin receptor

is situated at the tops of the folds of the

post synaptic membrane in the chronic phase of a MG

as Dr Engel has also demonstrated for human myasthenia

gravis, the acetylcholine receptor is very or that I

should say, the toxin binding sites on the post

synaptic membrane are greatly reduced in number. Now I

will briefly go over the phases of experimental autoimmune myasthenia

gravis so we can look at some of the immunological

events that are occurring. First of all, if

we concentrate on the preclinical phase of the AMG a

day zero, the rat is injected in the skin

with an emotion of acetylcholine receptor. With adjuvants,

the antigen is drained to the local lymph nose,

where the specific immunological reaction is triggered. By day

four. In the peripheral blood specific immunoglobulins and sensitized

T cells can be demonstrated by day five through seven

Immunoglobulin can be demonstrated on the post synaptic membrane

of the neuro muscular junction, also by skin testing

and by tests of peripheral blood lymphocytes. We know

that there are sensitized thymus derived, or T lymphocytes

both in the blood and in the tissues. The

events which occur at this pre clinical phase leading to

membrane license, which is seen ultra structurally fig acidosis

and chemo taxes. Attracting the inflammatory cells might involve

either activation of the complement system, which does occur

with anti receptor antibodies. It might involve non immune

cells such as B lymphocytes or macrophages, having receptors

for the FC end of immunoglobulins or the chemo.

Taxes and cider toxicity might involve T cells themselves,

specifically sensitized, wandering by chance into the region of

the neuromuscular junction, releasing them for kinds. The

final pathway we see as the acute phase, which

occurs 8 to 11 days after immunization. At this

stage, the animals a week electro physiologically there is

evidence of the innovation. Inflammatory cells are pouring into

this post synaptic area, and there is a decrease

in the amount of receptor. At this stage.

I should emphasize that a human equivalent of the acute

phase of experimental myasthenia gravis has not been described yet

But in the experimental animals, everything has been

amplified by our use of adjuvants to initiate the auto

immune process. During the stage of recovery, the

cells disappear, the animals are in clinical or emission

and there is a transient increase in the total amount

of acetylcholine receptor in the animals. Between days 15

and 25 the level of specific anti anti muscle recep

to antibody in the serum is increasing. There is

repair of membrane, the post synaptic membrane, and

at this stage, the amount of acetylcholine receptor is

reducing. The chronic phase of the AMG is indistinguishable

from human myasthenia gravis ultra. Structurally, there is

a similar weakness. There is impairment of neuromuscular transmission

There are high levels of IgG anti receptor antibody

in the serum and in muscle. There is a

reduction in the amount of total amount of acetylcholine receptor

and there is demonstrable on the post synaptic membrane

both immunoglobulin G and the third component of compliment

The next slide. Yeah, how briefly review

some of the properties of antibodies to acetylcholine receptor.

First of all, the antibodies to receptor bind to

antigenics sites on the acetylcholine receptor molecule outside of the

societal calling binding site of the molecule. I'll explain

this further in the next slide. Secondly, we

know that antibodies in vitro can impair the electro physiological

responses of acetylcholine receptors. When a Seattle calling itself

is applied iron to. Theoretically, this activity of

antibody is independent of the action of Compliment. Thirdly

we know that antibodies to acetylcholine receptor can transfer

E AMG in vivo. This can be done by

passively transferring antibodies from the serum of an immune rat

to a normal rat or, as has been done

by Taika and his colleagues by taking antibodies from the

serum of patients with myasthenia gravis and injecting them into

mice. Fourthly, we know from human studies that

antibodies to acetylcholine receptor can cross the human placenta and

presumably responsible for the syndrome of neonatal myasthenia. Fifthly

we know that antibodies to acetylcholine receptor, both

experimentally induced and those occurring spontaneously in humans, can

activate the complement system we've mhm. There have been

many different reports of the occurrence of antibodies to acetylcholine

receptor in patients with myasthenia gravis. The essay is

reported in the literature have employed different principles. And

superficially, these reports might appear confusing. So I

think it's worth of spending a few minutes to look

at the principles of the different essays employed so that

we can then explain some of the apparent discrepancies in

the literature. First of all, the Radio Immuno

essay is employed to detect antibody to acetylcholine receptor have

for the most part employed the principle of radio labeling

the acetylcholine receptor protein by by using the alpha bungalow

toxin from Cobra venom, which has been itself labeled

with radioactive iodine. Now, if we picture the

acetylcholine receptor protein as a block, there is one

binding site for a subtle calling on this receptor molecule

When it interacts with the radioactive toxin, the

receptor itself becomes radio labelled now to detect antibodies.

The antibody might, for example, be added before

the radioactive toxin is added. In this case,

if antibody, which I've represented as an inverted Y

here, is added before the toxin, if that

antibody is directed to antigenics determinants on the receptor molecule

in the region of the binding site for a subtle

Colleen, one would expect that the antibody would prevent

the radioactive toxin from gaining access to the receptor molecule

This activity has in fact been described. It

was first described by almond and apple using cell globalized

as Tell Colin Receptor, which had been prepared from

day innovated rat muscle. However, when acetylcholine receptor

was purified by Dr John Lindstrom from normal human muscle

this activity of the antibody could not be demonstrated

Therefore, we know that the androgenic determinants expressed

on Dean elevated muscle a slightly different from those exposed

on junction all receptor from normal human muscle. However

when essays employee receptor which is actually insight you

in frozen sections of human muscle such as has been

described by Dr Bender and his colleagues, application of

antibody to frozen sections of neuromuscular junctions have demonstrated the

blocking of the binding of radioactive toxin in up to

70% of patients. So the demonstration of the inhibition

of binding of toxin by antibody depends on the source

of receptor and on its micro environment, whether it's

in membrane or whether it's soluble list. Another immuno

essay employees the labelling of soluble is human muscle receptor

Before the addition of antibody in this essay,

the toxin binding site is already occupied before the antibody

is added after addition of antibody, a second goat

anti human immunoglobulin is added. This antibody reacts with

the human antibody in the reaction mixture, and if

that human antibody is already attached to the receptor molecule

the radioactive toxin will be precipitated in the final

immune precipitation. If the human antibody is not directed

to the acetylcholine receptor, no radio activity will be

precipitated by the second anti human antibody. The next

slide gives you an illustration of the detection of antibody

in a large population of human subjects. Using that

second antibody method for detection, the antibody is expressed

in terms of the moles of radioactive toxin, which

were precipitated in the antibody essay. And it's important

to note that the tighter of antibody is expressed on

a log scale. Looking at a large number of

non my aesthetic patients, including normals, Eaton Lambert

syndrome, a Maya trophic, lateral sclerosis, polymer

bursitis, other neurological diseases and non neurological diseases,

including other autoimmune diseases like systemic Lupus erythematosus, thyroid

itis. There was no significant amount of antibody in

the myasthenia gravis population. Antibody was detected in 87

percent of patients. There was, however, no

correlation with the severity of the disease and the tighter

of the antibody. So far, this essay for

anti receptor antibody has proven a very useful ancillary test

for the diagnosis of myasthenia gravis. The next slide

illustrates a patient studied by Dr Casey and his colleagues

at UCLA, where a baby born of a my

aesthetic mother had at birth severe neonatal myasthenia, the

tighter of the mothers antibody has shown here again on

a log scale, the mother had a very high

tighter of anti receptor antibody. The baby at birth

also had a high tighter of anti receptor antibody.

The non my aesthetic values cut off. At this

point, the baby gradually improved clinically from its transient

episode of neonatal myasthenia. At three months, the

baby was clinically quite improved. He still had detectable

antibody in his serum. When seen at five months

the baby no longer had any significant amount of

anti receptor antibody in his serum. The new information

that we have gained from studying an animal animal model

of myasthenia gravis have been, firstly demonstration of the

pathogenesis itty of immune responses to acetylcholine receptor. Secondly

new diagnostic tests are evolving to help in distinguishing

cases of myasthenia gravis, which might otherwise be a

diagnostic problem. Thirdly, we're gaining some insight into

the role of the famous in myasthenia gravis. For

example, we do know that the theme is is

essential in the experimental animal, for the production of

antibody to acetylcholine receptor and for the onset of impairment

of neuromuscular transmission. It has also been reported by

Mittag and his colleagues that the famous of myasthenia gravis

patients in some instances does contain antibody to ask,

Tell Colleen Receptor. Fourthly, we now have some

evidence that the complement system might be involved in human

myasthenia gravis, since we do know that antibodies to

acetylcholine receptor can activate the complement system and in the

animal model, we know that compliment is involved in

the impairment of neuromuscular transmission in vivo and in the

model involving passive transfer of human antibodies to the mouse

When complement levels were low, the impairment of

neuromuscular transmission was less. Finally, I would like

to summarize the clinical significance of auto immunity to acetylcholine

receptors. First of all, the antibody tests provide

an ancillary test for the diagnosis of myasthenia gravis,

and as we saw, it can provide a useful

monitor of neonatal myasthenia gravis. The antibody tests might

provide an indicator of fetal risk. It is not

yet known whether antibody tests are going to be a

valuable monitor of therapeutic response. I have not discussed

the essays of cellular immunity, but lymphocyte transformation essays

employing peripheral blood lymphocytes of patients with myasthenia gravis have

been reported by Richmond and his colleagues to show some

correlation with the severity of myasthenia gravis. A.

Bronski and his colleagues in Israel have reported that the

degree of in vitro lymphocyte transformation with purified acetylcholine receptor

has correlated with the effect of steroid therapy in patients

with myasthenia gravis. Finally, when we look to

the future, we can hope that our new knowledge

of the pathogenesis of myasthenia gravis might lead to more

specific therapy for the auto immune basis of myasthenia gravis

Thank you.