(MG). - University Hospital Foundation

Today’s Talk: What We Know
About MG, and What We Wish We Knew
•
Some History
•
Clinical features
•
How MG works: the Nerve-muscle junction;
Autoimmunity
•
Origin, and Genetics (GWAS)
•
Treatment Strategies
1672 – 103 yrs before Mozart
uro jam nunc faeminam prudentem, & probam, quae per plures annos
hujusmodi spuriae paralysi non tantum in membris set etiam in lingua
obnoxia fuit; haec per tempus quoddam lebere, & expedite satis loquitur,
post sermones tamen longos, aut illos sestinanter, & laboriose prolatos,
illico sicut piscis obmutescens, amplius ne gry quidem proloqui potest,
porro nec nisi post horam unam, aut alteram vocis usuram ullam recuperat.
I now have under my care a prudent and honest woman, who for many years has
had a spurious paralysis which is obnoxious not only in her limbs but also in her
tongue; This is free for a time, and she is able to speak satisfactorily, but after
sufficiently long or sustained sermons, and laborious speeches, she becomes as
mute as a fish, and is unable to speak, but after an hour she is able to use her
voice and it recuperates.
Physostigmine: N-M Jct
Sir Henry Dale
M. Walker. Lancet 1:1200-1, 1934
Clinical Features of MG
• Muscle weakness.
• Fatigue on repeated contraction.
• Double vision, droopy lids, weakness;
Crisis: Respiration, Swallowing
• No changes in sensation or autonomic
function.
A Little Background
• The Neuromuscular Junction: Site of the
abnormality in MG
A Receptor problem
• The Immune System’s Job:
The body’s police department
Must recognize ~ a Trillion items:
Distinguish good from bad: “LAPD”
Current Concepts of MG
•
Receptor disorder:
AChRs at NM junctions
•
Autoimmune:
Antibody mediated (T Cell
dependent)
•
Ab Mechanisms
Accelerated degradation
Blockade
Comp.-mediated damage
•
Sequenced & Cloned
•
Antigen = AChR
MuSK
Origin:
•
Treatment:
Thymus: Contains AChR &
Immune cells
Genetic Factors (GWAS)
Current – Effective
Future – Specific ?
Unsolved Problems in MG
• What triggers the autoimmune response?
• What keeps it going?
• What can cure it?
Circa 1970
Bungarus Multicinctus
C.Y.Lee 1970
Motor Point Biopsies
Nl
AChRs / jct
Nl
MG
7
Control
3.8 x 10
Myasthenic
0.7 x 10
7
AChE
MG
Labeled with 125I-BuTx
Fambrough, Drachman,
Satyamurti Science 1973
Autoimmune Pathogenesis of MG:
Early Evidence
• Association with other autoimmune
diseases.
(Ian Simpson1960)
• Thymic abnormalities:
Hyperplasia
Thymoma
• Improvement of MG with steroid
treatment.
• EAMG:
Immunization with AChR
produces model of MG.
EAMG
?Cell mediated
Patrick & Lindstrom
Science 1972
Neonatal MG: Clue to Ab-mediated
pathogenesis
Note decremental
response at 5 Hz
MG is Antibody-mediated
Antibody Mediated Mechanisms
in MG
1. Accelerated degradation of AChRs.
2. Blockade of AChRs.
3. Damage to AChRs (Complement).
MG Antibody Accelerates
AChR Degradation
Kao & Drachman
Science 1977
Diagnosis in MG
• AChR antibody
MuSK antibody
• Repetitive nerve stimulation
• SFEMG
• Tensilon test
• CLINICAL FEATURES!
AChR Antibody Titer
• Positive in ~85% of MG patients.
~50% with ocular MG
• In different patients absolute titer does not
correlate with severity.
• In a given patient, change in titer
corresponds to change in severity.
“Antibody Negative” MG
~ 40 to 50 percent of patients with “ocular
MG” have no detectable anti-AChR Ab.
10 to 20 percent of patients with generalized
MG have no detectable anti-AChR Ab
~40% of these have antibodies to MuSK:
(Muscle Specific Kinase) at NM Jct.
Low affinity antibodies : cell-based assay.
Origin of M.G.
• Role of Thymus.
• Viral infection ?EBV (NO)- Poliovirus?
• Abnormal immune regulation.
• Genetic Predisposition!
GWAS
Thymus and the origin of MG
•
Thymus is abnormal in 75% of pts: 85% of these
have “Hyperplasia”. 15% have Thymoma.
•
Thymectomy is beneficial in up to 80% of pts.
•
Myoid (muscle-like) cells express AChRs, in
midst of immunocompetent cells.
•
BUT- What triggers immune attack?
Abnormal AChR? (No evidence)
Novel fragment: cleaved by Granzyme B?
?Viral infection: Search for viral DNAEBV? Poliovirus?
Features of Thymus in MG
Thymus is abnormal in 75% of patients:
85% Hyperplasia; 15% Thymoma
Hyperplasia
(Germinal centers)
Myoid cells
Kao & Drachman
Science 1977
What Happens in the Thymus?
• ACh Receptor is cut
•
•
by the immune cells
surrounding it (Granzyme B).
The new fragments look dangerous.
The immune cells attack, and make
antibodies.
Casciola-Rosen et al 2008
J. Neuroimm. 201-2:33
• But what triggers the immune cells to cut
AChR ?
A viral infection?
Do Genes Predispose to MG?
• MG in 6% of family members, some have
only AChR Ab;
• MG patients with other autoimmune
diseases: Thyroid, LE, RA, etc. 23%
• Family members with autoimmune
disease: 29%
• GWAS:Genome wide association study
GWAS Concept
• Different from single-gene disorders
(dominant, recessive, sex-linked).
• In these diseases, multiple genes have
partial roles: “Risk” for developing
disease.
• Environmental factors important as well.
• Unexpected associations.
GWAS Genome-wide association
• Find all the relevant genes.
• Better telescopes – more stars
(SNP Chips like Hubble)
• 700,000 SNPs per chip (genes).
• Collaboration with NIH: B. Traynor
• 15 MG Centers in North America: 1,100 MG
patients (and 4,000 normals).
Participating Centers
U. Alberta, Ca
U. British Columbia, Ca
U.Cal. Davis
Duke U.
Emory U.
U. Illinois
U. Indiana
Johns Hopkins
U. Kansas
U. N. Carolina
Ohio State
St. Louis U.
U. Texas, SW
Wash. U.
U. W. Ontario, Ca.
Z. Siddiqi
J. Oger
D. Richman
D. Sanders
M. Benatar
M. Meriggioli
R. Pascuzzi
D. Drachman
R. Barohn
J. Howard
J. Kissel
H. Kaminski
G. Wolfe
A. Pestronk
M. Nicolle
Analytic Steps
• Saliva: Collect, purify DNA.
• Amplify DNA.
• Fragment DNA
• Add to Chips; Wash off unbound DNA.
• Attach fluorescent label.
• Scan in reader.
• Analyze results in 15 terabyte computer.
Robot
Omni Express Chip (12 samples)
Robot to Pick up & Scan Chips
Scanned Chip: Dots = DNA
Dr. T and 15 Terabyte Computer
The Genomewide Association Study
Probability of
SNP #1 by
chance is
1 in 1 trillion;
SNP #2 is 1
in 100 million
Manolio T. N Engl J Med 2010;363:166-176
Previously Unexpected Associations
Some Examples
Manolio T. N Engl J Med 2010;363:166-176
What may we do with the GWAS
Information?
• Design treatments related to genetic
factors.
• Predict course of MG.
• Evaluate treatments most likely to work.
• Susceptibility in family members.
Myasthenia Was “Gravis”
• Remission
• Improved
13%
26%
• Unchanged
• Worse
• Died
21%
10%
30%
• Bad prognosis
=
61%
Grob, Arch Int Med
1961
The Disney World Test
Treatment of M.G.
•
Anti-Cholinesterase Drugs
"Band Aid“
•
Thymectomy
•
Immunosuppression
•
•
Removal of Antibodies
IV gamma globulin
To improve MG
Thymoma
Steroids
Others
Plasmapheresis
•
High Dose Cytoxan for refractory patients:
"Re-booting the immune system"
•
FUTURE: Specific Immunotherapy.
"Time-Linked" Treatment Plan
• Short-term: Severity, or need for quick result.
Anti-ChE; IVIg; Plasmapheresis
• Intermediate-term:
Take over after short term.
Steroids; Cyclosporine, FK506
• Long-term:
To minimize side effects.
Thymectomy
Imuran
CellCept
Anticholinesterase therapy
• Pyridostigmine (Mestinon)
• Delays hydrolysis of ACh; Prolongs
stimulation.
• Effects only temporary, partial.
• "Band Aid"
Physostigmine
M. Walker. Lancet 1:1200-1, 1934
Thymectomy in Myasthenia Gravis
• Indications:
• Diagnosis
• Pre-op:
• Surgery:
• Post-op:
• Results:
Thymoma
or For clinical benefit
CT or MRI for thymoma
Clinical indications
Optimize; Never emergency
Trans-sternal; ?cervical
? Robotic
Pulmonary care; Anti-ChE
Late –
30% remissions
50% improved
Questions About Thymectomy
1. DOES IT WORK?? Will new study tell?
2. How early after diagnosis?
3. Age: How old?
How young?
4. Severity of MG: Ocular? (Schumm et al 1985)
5. Surgical approach?
Immunotherapeutic Agents in
Current Use
• Prednisone
• Imuran (Azathioprine)
• Cyclosporine A (Neoral) Inhibits Calcineurin
& blocks IL2 production.
• Tacrolimus
• CellCept (Mycophenolate mofetil)
• Cyclophosphamide (Low vs High dose)
• IVIg
• Plasmapheresis
CellCept
•
Mycophenolate: Used in transplantation; Now in
autoimmune diseases (MG).
•
Mechanism: Prevents B and T cell proliferation;
Blocks purine synthesis.
•
Slow action: Autoimmune cells must die off.
Does not kill cells.
•
Advantages: Safety. Few side effects.
Watch CBC.
•
Dose: 1 – 1.5 gm bid.
Newer Immunosuppressive
Agents
•
Tacrolimus (Prograf): Like CsA: Inhibits
•
Rituximab: Anti-CD20 on B cells (Not plasma
cells)
•
?Leflunomide: Inhibits pyrimidine
synthesis
•
? Abatacept (CTLA4Ig): Inhibits costimulation
(EAMG studies)
•
Rapamycin:
calcineurin & IL2 production
Inhibits IL2 action.
“Refractory MG”
• Failure of adequate treatment with
conventional agents.
• Unacceptable side effects.
• Co-morbidities preclude conventional Rx.
• Requirement for repeated rescue with
short term treatments.
“Clean Slate” Therapy
HEMATOPOIETIC
STEM CELLS CD34+
EAMG: “Clean Slate”
Combined Rx:
Cytoxan + Irrad.
BMT (autologous)
Just like naïve
rats.
Pestronk & Drachman, 1983
High Dose Cytoxan:
Re-booting
•
For refractory patients only, so far.
•
Mature WBC eliminated by Cytoxan.
•
Bone marrow Stem Cells not killed by Cytoxan.
Their aldehyde dehydrogenase
inactivates Cytoxan.
•
Rapid reconstitution of Immune system with GCSF.
Results in Hi Cy Myasthenic
Patients
•
16 patients treated at JH so far. All severe.
6 months to 10 year follow up.
•
Results:
15- Dramatic improvement: 5 mos. to 10 yrs
1- Treatment-free remission 10 yrs.
7- Response to immunosuppressives not
previously effective.
3- Retreatment after 3, 6, & 10 yr. relapsesspectacular responses; Maintenance
CellCept ?
BUT: All relapsed eventually.
•
Effects of Re-Booting
• Reduces autoantibodies (AChR).
Not
eliminated
• T cells increase (TRECs) despite lack of
thymus.
• Re-booting, not Re-formatting.
• Requires maintenance treatment.
Ideal Therapy of MG
• Specifically delete autoimmune response
to AChR.
• Leave Immune system otherwise intact.
• Non-toxic.
• Long-lasting, permanent, or repeatable.
• The “Guided Missile” is on its way.
Conclusions
• We know more about MG than any other
•
•
autoimmune disease.
Properly treated, 95% of MG patients are
restored to normal lives.
Even “refractory” patients respond.
• BUT: We need to know the genetic factors.
• We wish we knew what triggers MG.
• A specific “cure” is the Holy Grail.
Colleagues
D. Fambrough
A. Pestronk
I. Kao
K. Toyka
E. Stanley
R. Adams
K. McIntosh
B. Wu
J-M. Wu
B. Yang
F. Guarnieri
T. August
A. Miagkov
M. Williams
R. Brodsky
Y. Lu
W. Sun
Endocytosis of Clustered AChRs
Clusters
Freeze-Fracture EM
Pumplin & Drachman
J. Neurosci 1983
Pit
EAMG
• Rabbits, Lewis rats, mice, frogs,
monkeys…
• Immunize with AChR from electric organs
of electric eels or fish.
• Clinical weakness
Cellular responses
Antibody responses
There are Antibodies in
"AChR Ab-Negative" MG
• Patients have reduced AChRs at NM
•
•
•
•
junctions (7/7 cases).
Passive transfer to mice AChRs and
mepps (6/7 cases).
~70% of Ab Neg sera bind to mammalian
muscle in culture.
~40% have anti MuSK antibodies.
Low affinity antibodies.
96 well plate
Robot