Recent advances in the understanding and therapy of myasthenia gravis

Abstract

Myasthenia gravis (MG) is a T-cell dependent autoimmune disease mediated by autoantibodies, which mainly target muscle nicotinic acetylcholine receptors (AChR) and cause loss of functional AChRs in the neuromuscular junction. Both MG and its major autoantigen are studied extensively, yet the etiology of the disease remains unclear, although it is known to be associated with the thymus. A genetic predisposition, combined with several unidentified environmental stimuli, likely creates a favorable milieu in which the disease can appear. Current research focusses on elucidating the cellular and molecular pathways of immune dysregulation, which underly MG outburst and progression. Considerable progress has been made concerning the involvement of the thymus, the identification of impaired mechanisms of immune control and the B–T-cell interaction in MG pathogenesis, while the role of chemokines arises as an intriguing new puzzle. Recent findings fueled the development of novel therapeutic approaches with some encouraging, although preliminary, results. This review summarizes recent achievements in the fields of both basic research and therapeutics.

Full text

10.2217/14796708.1.6.799 © 2006 Future Medicine Ltd ISSN 1479-6708 Future Neurol. (2006) 1(6), 799–801 799
REVIEW
Recent advances in the understanding and
therapy of myasthenia gravis
Efrosini Fostieri,
Kalliopi Kostelidou,
Konstantinos Poulas &
Socrates J Tzartos
†
†
Author for correspondence
Hellenic Pasteur Institute,
Department of Biochemistry,
127 Vas. Sofias Avenue,
11521 Athens, Greece
and
Department of Pharmacy,
University of Patras,
26504 Patras, Greece
Tel.: +30 210 647 8842;
Fax: +30 210 647 8842;
tzartos@pasteur.gr
Keywords: autoantibodies,
autoimmunity, autoreactive
T cells, myasthenia gravis,
neuromuscular junction,
nicotinic acetylcholine
receptor, specific
immunotherapy, thymus
Myasthenia gravis (MG) is a T-cell dependent autoimmune disease mediated by
autoantibodies, which mainly target muscle nicotinic acetylcholine receptors (AChR) and
cause loss of functional AChRs in the neuromuscular junction. Both MG and its major
autoantigen are studied extensively, yet the etiology of the disease remains unclear,
although it is known to be associated with the thymus. A genetic predisposition, combined
with several unidentified environmental stimuli, likely creates a favorable milieu in which the
disease can appear. Current research focusses on elucidating the cellular and molecular
pathways of immune dysregulation, which underly MG outburst and progression.
Considerable progress has been made concerning the involvement of the thymus, the
identification of impaired mechanisms of immune control and the B–T-cell interaction in MG
pathogenesis, while the role of chemokines arises as an intriguing new puzzle. Recent
findings fueled the development of novel therapeutic approaches with some encouraging,
although preliminary, results. This review summarizes recent achievements in the fields of
both basic research and therapeutics.
In the autoimmune disease myasthenia gravis
(MG), the major autoantigen is the muscle nico-
tinic acetylcholine receptor (AChR), a postsynap-
tic ligand-gated ion channel of the
neuromuscular junction (NMJ) with the stoichi-
ometry
α
2
βεδ (adult muscle) or α
2
βγδ (fetal and
denervated adult muscle). The anti-AChR
autoantibodies cause the loss of functional AChR
molecules resulting in impaired neuromuscular
transmission, which is reflected clinically with
weakness and fatigability of voluntary muscles.
The clinical and immunopathological features of
MG can be reproduced effectively in an animal
model. Experimental autoimmune MG (EAMG)
is induced actively in animals, most commonly
mice and rats, by immunization with hetero-
logous AChR and is due to antibodies (Abs) raised
against the heterologous AChR, which cross-react
with the autologous receptor molecule.
Experimental data regarding MG is relatively
vast, yet the etiology of the disease remains elu-
sive, although related with thymic abnormali-
ties. Current treatment lacks specificity and is
often accompanied by various side effects.
Hence, ongoing research is focussed on
unraveling the fine cellular and molecular path-
ways underlying the outburst and progression
of the disease. A more comprehensive view of
MG would give rise to specific and sophisti-
cated therapeutic approaches targeted at differ-
ent levels of immune modulation. This review
on MG presents recent achievements in the
fields of basic research and therapeutics.
The mosaic of different myasthenia
gravis phenotypes
MG is a remarkably heterogeneous disease. It can
either remain localized to a single muscle group
(usually, but not necessarily, the extraocular mus-
cles – named ocular MG) or it can spread to other
skeletal muscles (termed generalized). A major
classification of generalized MG is made on the
basis of the presence or absence of anti-AChR Abs
(seropositive or seronegative, respectively).
Seropositive myasthenia gravis
This group, representing approximately 85% of
MG patients, comprises three distinct disease
entities
(Table 1):
• Early-onset MG appears before 40 years of
age, affects mostly women and is commonly
related with thymic follicular hyperplasia;
• Late-onset MG affects people older than
40 years of age with normal or atrophic
thymus and shows a bias for men
[1];
• Paraneoplastic MG associated with thymoma
affects men and women of any age (mostly
between 40 and 60 years of age) equally and is
characterized by the presence of Abs to other
striated muscle antigens, mainly titin and the
ryanodine receptor
[2].
Seronegative MG: the discovery of
anti-muscle specific kinase autoantibodies
Approximately 15% of patients with generalized
MG symptoms have no detectable anti-AChR
Abs in their serum. Despite various evidence
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800 Future Neurol. (2006) 1(6)
pointing to an autoantibody-mediated disease, the
autoantigen in seronegative MG had long
remained unknown. Vincent and colleagues
were the first to identify the muscle-specific
kinase (MuSK), a key player in postsynaptic
NMJ differentiation and AChR clustering
(Figure 1), as the target of the autoimmune
response in a proportion of seronegative MG
patients (
∼40% [3–5]). The presence of anti-MuSK
Abs appears to define a subgroup of seronegative
MG patients with distinct clinical characteristics:
predominantly localized, commonly bulbar, mus-
cle weakness and muscle atrophy, which often
restricts complete response to commonly used
therapies (acetylcholinesterase inhibitors and con-
ventional immunosuppresants)
[6]. The patho-
genic potential of anti-MuSK autoantibodies has
recently been demonstrated by their ability to
induce an MG-like muscle weakness in MuSK-
immunized rats or rabbits
[7,8] by severely hamper-
ing AChR clustering
[8]. Interestingly, a nonim-
munoglobulin (Ig)G plasma factor, which
transiently inhibits the function of AChR mole-
cules without reducing their number, has been
described for seronegative MG patients
[9]. The
unknown factor likely induces a desensitization-
like state acting directly on human AChR,
mimicking an allosteric modulator
[10].
Genetic predisposition to
myasthenia gravis
MG onset involves a genetically favorable back-
ground combined with several unidentified
environmental stimuli. Strong evidence for an
Table 1. Subgroups of seropostive myasthenia gravis patients.
Subtype
of MG
Age of onset Sex Thymic
pathology
HLA
association
Related autoantibodies
Early-onset < 40 years Mostly women Follicular
hyperplasia
B8, DR3 Possibly other tissue-specific Abs.
Late-onset > 40 years A bias for men Normal or
atrophic
DR7 Abs against muscle antigens, mainly titin
and the ryanodine receptor.
Paraneoplastic Usually 40–60
years
Equally men
and women
Thymoma No clear
association
Abs against muscle antigens and
cytokines (e.g., IFN-α and IL-12).
Abs: Antibodies; HLA: Human leukocyte antigen; IFN: Interferon; IL: Interleukin; MG: Myasthenia gravis.
Figure 1. Model showing part of the postsynaptic apparatus, including the
acetycholine receptor, in the neuromuscular junction.
Binding of neuronal agrin to its receptor complex initiates the signaling scaffold leading to AChR clustering
at the neuromuscular junction. MASK and MuSK may form the receptor complex of agrin. RATL remains an
unidentified molecule that likely links MuSK to rapsyn. Rapsyn is required for cross-linking of AChRs and
other post-synaptic components into large aggregates.
AChR: Acetycholine receptor; MASK: Myotube-associated specificity component; MuSK: Muscle-specific
kinase; RATL: Rapsyn-associated transmembrane linker.
Rapsyn
Extracellular
Intracellular
Channel pore
AChR
MuSK
Agrin
MASK
RATL
D
E
G
J
H
D


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immunogenetic predisposition to the disease
was provided by the early implication of the
human leukocyte antigen (HLA) complex in
early-onset MG with thymic hyperplasia
(HLA-DR3 and B8) and late-onset MG
patients with anti-titin Abs (HLA-DR7),
whereas for the paraneoplastic MG subset, no
clear HLA association has currently been
described
[11].
The MYAS1 locus
Although the 8.1 ancestral haplotype, including
HLA-A1 and B8 alleles for class I and DR3 for
class II, has been reproducibly associated with
early-onset MG, the exact disease loci remained
unidentified. Garchon and colleagues initially
established a genetic linkage between MG and
an HLA locus, termed MYAS1
[11]. The MYAS1
locus was then mapped to a segment encompass-
ing the distal part of class III and the proximal
part of class I genes, containing the tumor
necrosis factor (TNF)/Lymphotoxin genes but
excluding the cluster of complement factor
genes
(Figure 2) [12]. Interestingly, the MYAS1
interval overlaps with a region implicated
recently in rheumatoid arthritis suggesting a
nonantigen-specific immunoregulatory function
for the 8.1 haplotype
[13].
Three-gene model
In a previous study, MG was associated with a
class II allele (unrelated to the 8.1 haplotype),
DQA1*0101, which encodes the
α-chain of the
class II molecule DQ
[14]. This allele was shown
to interact with a polymorphism of the
CHRNA1 gene, encoding the AChR
α-subunit [14]. Recent evidence supported the
HLA-dependent control of anti-AChR Ab titers
in MG patients with hyperplastic or normal thy-
mus. According to this three-locus model, two
loci, CHRNA1 and HLA-DQA1 interact to
result in high autoantibody titers, whereas the
third locus associated with the 8.1 haplotype
exerts an additive effect, possibly through non-
antigen-specific immune dysregulation
[15].
Whether this third locus is MYAS1 requires
further investigation.
Humoral & cellular immunity &
experimental autoimmune MG
Antigenic structure of acetylcholine receptor
In the 1980s, the identification of the main
immunogenic region (MIR) as the target for the
majority of anti-AChR monoclonal Abs (mAbs)
isolated from AChR-immunized experimental ani-
mals
[16], orientated the immunological research
towards the study of anti-AChR Abs and their
pathogenic mechanisms. It is now well-docu-
mented that the MIR is not a single epitope but
rather comprises a cluster of overlapping confor-
mation-dependent epitopes, with the core
sequence
α67–76, located extracellularly on the
α-subunit of AChR [17], without excluding that
neighboring AChR subunits also contribute to the
region
[18]. The MIR is of major pathological
importance, since anti-MIR mAbs very effectively
induce passively transferred EAMG and cause
AChR loss
[19]. In MG patient sera, approximately
50% of anti-AChR Abs recognize the
α-subunit
[20], while the content of serum anti-AChR Abs in
anti-MIR Abs has been determined only indi-
rectly, through competition studies, as 50–60%
[21]. Considerable serum Ab fractions recognize
various non-
α AChR subunits (at least β, γ and ε),
Figure 2. Schematic map of the human leukocyte antigen complex and localization of
the MYAS1 locus.
Bars at the top indicate the extent of the three main HLA classes. Landmark genes and the two microsatellite
markers that delimit the MYAS1 interval, BAT3 and C3.2.11, are shown.
Cen: Centromere; HLA: Human leukocyte antigen; Tel: Telomere; TNF: Tumor necrosis factor.
(Reproduced from Vandiedonck C, Giraud M, Garchon HJ: Genetics of autoimmune myasthenia gravis: the
multifaceted contribution of the HLA. 25(Suppl.), 6–11 J. Autoimmun. (© 2005) with permission
from Elsevier.
II III I
HLA class
DRB1 C4B TNF B C E A
BAT3 C3-2-11
Loci
cen tel
II III I
HLA class
DRB1 C4B TNF B C E A
BAT3 C3-2-11
Loci
cen tel

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as demonstrated directly through the binding of
MG Abs to recombinant extracellular domains
of these subunits expressed in a yeast expression
system
[Kostelidou K et al., Unpublished Results] [22]. Inter-
estingly, in a small portion of MG sera, Abs
against cytoplasmic AChR epitopes are also
detected
[23]; however, their significance
is unknown.
Effector humoral response
In both MG and EAMG, the anti-AChR Ab
response is polyclonal and heterogenic. The anti-
AChR autoantibodies destroy functional AChRs
by complement-dependent lysis of the postsynap-
tic membrane, increased AChR degradation via
antigenic modulation, or less commonly, by direct
inhibition of neurotransmitter binding. However,
the relative significance of these mechanisms in
individual patients has not yet been well-estab-
lished. The key role of complement in MG and
EAMG pathogenesis is corroborated by early and
recent studies demonstrating that complement
blockade/deficiency can protect animals against
induction of EAMG
[24–26]. Alternatively, the
absence of complement regulatory molecules,
such as the decay-accelerating factor (DAF), sig-
nificantly enhances susceptibility to EAMG, since
DAF
-/-
transgenic mice were shown to be signifi-
cantly more prone to EAMG induction than their
DAF
+/+
littermates [27]. Recently, Christadoss and
colleagues provided direct evidence for the impli-
cation of the classical (and not the alternative)
complement pathway in the elicitation of the
actively induced EAMG
[28]. The same group
associated EAMG severity with the level of C1q
conjugated-circulating immune complexes, which
are known to trigger the classical complement cas-
cade
[29], and further demonstrated an involve-
ment of the immune complex receptor Fc
γ
receptor III in EAMG pathogenesis [30].
B & T cell interaction through
costimulation molecules
Autoreactive CD4
+
T cells from MG patients
respond to various synthetic AChR peptides
from different AChR subunits in proliferation
or enzyme-linked immuno-spot (ELISPOT)
assays in the context of major histocompatibil-
ity complex (MHC) class II molecules
[31–33]
and provide help to B cells, thus becoming
essential for anti-AChR Ab production
[34].
Current immunological research addresses the
question of costimulation requirements for the
activation of AChR-specific T cells and
initiation of pathogenic Ab production.
Binding of the CD40 ligand (CD40L) to
CD40 triggers B cells to function as antigen pre-
senting cells (APC) and upregulates expression
of the costimulatory molecules B7-1 and -2,
which interact with CD28 and the cytotoxic
T lymphocyte-associated antigen (CTLA)-4. In
EAMG, CD40–CD40L interaction was found
to be essential for both disease induction and
progression, since CD40L
-/-
mice were com-
pletely resistant to EAMG demonstrating
severely compromised AChR-reactive B-cell and
T-helper (Th)1/Th2 responses
[35] and anti-
CD40L treatment during the course of an ongo-
ing EAMG still suppressed the disease, involving
a downregulation of B7-2 and the Th1 cytokines
interleukin (IL)-12 and interferon (IFN)-
γ and
an upregulation of CTLA-4
[36].
Concerning the B7-mediated signaling,
CD28
-/-
mice were less susceptible, but not com-
pletely resistant, to EAMG induction compared
with wild-type controls
[35], while in vitro blockade
of B7 signaling induced an anergy-like state in
AChR-specific T-cell lines
[37]. Christadoss and
colleagues demonstrated, using knockout mice,
that B7-1 (but not B7-2) deficiency significantly
decreased the anti-AChR Ab-production following
AChR immunization, likely perturbing T- and B-
cell communication rather than suppressing the
primary response to AChR
(Table 2) [38].
Hence, the B7-1 molecule, as well as the
CD40–CD40L interaction, appear to be sine qua
non for the costimulation events leading to the
production of anti-AChR Abs by B cells.
Recently, another member of the CD28 family,
the inducible costimulatory molecule (ICOS) was
implicated in EAMG pathogenesis. ICOS is likely
to be important for downstream stimulation of
the effector T-cell functions (cytokine production
and CD40L expression) that are crucial for the
initiation and maintenance of the B-cell response.
Genetic disruption of ICOS was shown to confer
resistance to actively induced EAMG
[39].
Effect of cytokines on
experimental autoimmune
myasthenia gravis pathogenesis
Cytokines have pleiotropic functions. Depend-
ing on the time, and place, of action they may
exert different effects on immune responses.
Various studies have explored the role of
cytokines on EAMG pathogenesis
(Table 3).
The cytokine IL-12 facilitates EAMG develop-
ment. Resistance of IL-12
-/-
mice to EAMG was
related to both decreased production of comple-
ment fixating anti-AChR Abs and elicitation of a

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Recent advances myasthenia gravis research – REVIEW
stronger anti-AChR Th2 response [40]. Unlike
IL-12, the role of IFN-
γ is less clear – IFN-γ recep-
tor knockouts exhibited significantly lower inci-
dence and disease severity than their wild-type
controls
[41] and IFN-γ deficiency conferred resist-
ance to IFN-
γ
-/-
mice [42]. However, in another
transgenic model of different genetic background,
IFN-
γ disruption did not affect EAMG induction
[40]. The proinflammatory cytokine IL-18 is also
involved in EAMG pathogenesis. IL-18
-/-
mice
were resistant to EAMG
[43], while treatment with
an anti-IL-18 Ab suppressed ongoing EAMG by
upregulating the immunosuppressive Th3
cytokine transforming growth factor
β [44].
The Th2 cytokines IL-4, -6 and -10 have also
been studied. Genetic disruption of IL-10 con-
ferred EAMG resistance to IL-10
-/-
mice [45], while
simultaneous transgenic expression of IL-2 and
-10 increased susceptibility to EAMG
[46]. These
findings suggest that IL-10 facilitates EAMG,
potentially by stimulating B-cell differentia-
tion/proliferation and Ig class switch. IL-6
-/-
mice,
as with the IL-10 knockouts, were significantly less
susceptible to EAMG induction than their wild-
type littermates, demonstrating a significantly
compromised germinal center formation,
decreased serum complement levels and impaired
class switching
[47]. A similarly defective class
switching was observed in AChR-immunized
knockout mice genetically deficient for TNF
receptor p55 and p75
[48] or for TNF-β [49]. These
animals were also resistant to EAMG. Christadoss
and colleagues proposed that a cytokine hierarchy
exists in the development of EAMG, with IL-6,
TNF and IL-18 playing the frontline role
[47]. It
remains to be clarified whether IL-6 and TNF act
in concert or whether one regulates the production
of the other. Genetically-deficient mice were also
used to investigate the role of IL-5 in EAMG.
Upon immunization with AChR, IL-5
-/-
mice
exhibited increased resistance to EAMG, com-
pared with wild-type controls, which was related
with reduced primary anti-AChR lymphocyte
response and reduced C3 complement levels in
muscle extracts
[50].
Unlike the other Th2 cytokines, IL-4 is not a
prerequisite for EAMG induction
[51]. After a sin-
gle immunization with To rp e d o AChR, IL-4
-/-
mice develop a chronic form of EAMG marked by
the persisting presence of anti-mouse AChR Abs
and mouse AChR-responsive CD4
+
T cells [52].
These findings underlie the protective role that
IL-4 possibly exerts by modulating/intercepting
anti-AChR Ab production and EAMG symptoms.
Signal transducer and activator of transcription
(STAT)4 and 6 molecules mediate the differentia-
tion of naive CD4
+
T cells into Th1 and 2 cells,
after interaction with IL-12 or -4, respectively. In
Table 2. The implication of costimulation molecules in experimental autoimmune myasthenia
gravis pathogenesis.
Interaction Experimental approach Mechanism of action Effects observed Ref.
CD40L–CD40
CD40L
-/-
mice
Severely compromised AChR-
reactive B-cell responses,
reduced Th1/Th2 responses
Complete resistance
[35]
Anti-CD40L Ab to rats Downregulation of B7–2, IL-12
and IFN-γ and upregulation
of CTLA-4
Suppression of
ongoing disease
[36]
B7–CD28
CD28
-/-
mice
Decreased AChR-specific Abs
of the IgG1 isotype, switch to
Th1 profile
Reduced susceptibility [35]
CD28 analog in AChR-specific
T-cell lines
Decreased IL-2 production and
lymphoproliferative responses
Anergy [37]
B7–1
-/-
and B7–2
-/-
mice
Significantly decreased anti-
AChR Ab production, likely due
to perturbed T- and B-cell
communication
Resistance [38]
ICOSL–ICOS
ICOS
-/-
Decreased anti-AChR Ab
production, inhibition of
Th1/Th2 differentiation in
response to AChR
Resistance
[39]
Abs: Antibodies; AChR: Acetycholine receptor; CTLA: Cytotoxic T-lymphocyte antigen; ICOS: Inducible costimulatory molecule; Ig: Immunoglobulin;
IL: Interleukin; IFN: Interferon; Th: T helper.

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STAT4
-/-
mice, all functions stimulated by IL-12
are impaired, including IFN-
γ production by Th1
cells, whereas STAT6
-/-
mice have defective IL-4-
mediated functions, such as B-cell proliferation
and Th2 cell development. A recent study, using
STAT4- or -6-deficient mice, demonstrated that
after AChR immunization, STAT6
-/-
mice were
significantly more susceptible to EAMG than the
STAT4
-/-
and wild-type animals [53]. The latter
suggests a prevalent role for a Th1 cell response in
EAMG pathogenesis, yet the implication of IL-10
and -6 (discussed above) denotes that the anti-
AChR autoimmune response likely involves a
hybrid Th1/Th2 polarization.
Role of the thymus in myasthenia
gravis pathogenesis
As mentioned previously, the thymus has been
implicated in the onset and/or maintenance of
MG due to the frequent coappearance of thymic
abnormalities with MG (50–60% of patients
present with thymic hyperplasia and 10% with
thymoma), and to the generally beneficial effect
of thymectomy on patients’ clinical profile
[54].
The phenotypically distinct entities (early-onset,
late-onset and paraneoplastic MG) may underlie
the existence of several etiological routes for
AChR autoimmunity, manifested with uniform
clinical symptoms. Therefore, hyperplastic thy-
mus and thymoma are generally thought to
contribute differently to disease pathogenesis.
Hyperplastic thymus
A predominant characteristic of thymic hyper-
plasia is the massive B- and T-lymphocyte infil-
tration in the thymic medulla, where both
single AChR subunits and intact AChR mole-
cules are normally expressed by thymic epithe-
lial cells (TEC)
[55] and myoid cells [56]. Plasma
cells, which spontaneously produce anti-AChR
Table 3. The effect of cytokines in experimental autoimmune myasthenia gravis pathogenesis.
Cytokine Experimental approach Mechanism of action Effects observed Ref.
IL-12
IL-12
-/-
mice
Decreased production of
complement fixating anti-
AChR Abs, elicitation of a
stronger anti-AChR
Th2 response
Resistance
[40]
IFN-γ
IFN-γ R
-/-
mice
Low anti-AChR Abs Reduced
susceptibility
[41]
IFN-γ
-/-
mice
(of H-2[b] haplotype)
Reduction of anti-AChR Abs Resistance [42]
IFN-γ
-/-
mice (C57BL6)
No effect [40]
IL-18
IL-18
-/-
mice
Reduction of anti-AChR Abs
and Th1 response
Resistance
[43]
Anti-IL-18 Ab to rats Upregulation of TGF-β,
decreased Th1 response
Suppression of
ongoing disease
[44]
IL-10
IL-10
-/-
mice
Stimulation of B-cell
differentiation/proliferation
and Ig class switch
Resistance [45]
Transgenic mouse model
expressing IL-10 under the
IL-2 promoter
Stimulation of B-cell
differentiation/proliferation
and Ig class switch
Increased
susceptibility
[46]
IL-6
IL-6
-/-
mice
Significantly compromized
germinal center formation,
decreased complement levels,
and impaired class switching
Reduced
susceptibility
[47]
IL-5
IL-5
-/-
mice
Reduced primary anti-AChR
lymphocyte response and C3
complement levels in
muscle extracts
Reduced
susceptibility
[50]
IL-4
IL-4
-/-
mice
No effect
[51]
Abs: Antibodies; AChR: Acetylcholine receptor; IFN: Interferon; Ig: Immunoglobulin; IL: Interleukin; TGF: Transforming growth factor;
Th: T-helper cell.

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Abs, have been isolated from thymic germinal
centers derived from early-onset MG patients
[57], in addition to AChR responsive CD4
+
T
cells, which were shown to contain a poten-
tially auto-reactive subpopulation displaying
increased Fas expression
[58]. Hence, molecules
promoting T- and B-cell survival and migra-
tion are likely related with pathogenesis of
thymic hyperplasia. In fact, the B-cell differenti-
ation/antiapoptotic cytokine IL-6 and the regu-
lator of transepithelial T-cell migration
RANTES, are over-expressed in MG thymic
epithelium. This results from the constitutive
over-expression (by TEC) and activation of the
p38 and extracellular regulated kinase 1/2
mitogen-activated protein kinases (MAPK),
suggesting that a dysfunction in MAPK tran-
scriptional or post-transcriptional control may
initiate alterations in myasthenic thymus
morphology with pathological impact
[59].
TEC and myoid cells may also be implicated in
the primary autosensitization step in MG. Levin-
son and collaborators proposed that an inflamma-
tory reaction in the hyperplastic thymus
modulates the expression of AChR and that of
several immune components (MHC and costim-
ulatory molecules), and facilitates the re-entry
into the thymus of autoreactive CD4
+
Tcells that
escaped central deletion
(Figure 3) [60]. AChR
expression and presentation within this activated
environment, combined with a genetically suscep-
tible background, could be adequate to prime the
AChR-responsive CD4
+
T cells, leading to
autoimmunity
[60]. Evidence provided by Berrih-
Aknin and colleagues supports this hypothesis:
firstly a large number of IFN-
γ- and TNF-α-regu-
lated genes, including MHC II genes, are highly
expressed in the myasthenic thymus, suggesting
the existence of a proinflammatory environment;
and, secondly, AChR expression in the thymus is
significantly susceptible to IFN-γ and TNF-
α
regulation [61]. However, the initial stimulus
inducing the inflammatory environment remains
unidentified, although bacterial and viral agents
are likely candidates.
Apart from the autoimmunity-favorable micro-
environment in the hyperplastic MG thymus,
additional control mechanisms must fail in order
for the disease to develop. Indeed, the regulatory
CD4
+
CD25
+
Tcells, although normal in
number, were found to be severely defective in
their ability to suppress autoreactive CD4
+
Tcells,
and demonstrated a significantly decreased
expression of the Foxp3 transcription factor that
is essential for the regulatory T-cell function
[62].
Interestingly, in the case of thymic hyperplasia
with diffuse B-cell infiltration (thymitis), as well as
in involuted thymus, Toll-like receptor (TLR)4
mRNA levels were shown to be significantly up-
regulated when compared with expression in ger-
minal center hyperplastic thymus and thymoma,
suggesting the existence of a relationship between
innate immunity and MG. An attractive theory
would be that TLR4-mediated signaling is initiated
in response to an exogenous or intrinsic alarm stim-
ulus, which activates the innate immune system,
eventually contributing to autoimmunity
[63].
Thymoma
Thymomas are thymic epithelial tumors fre-
quently associated with paraneoplastic auto-
immunity. Approximately 30% of all
thymoma patients develop MG, their auto-
antibody repertoire comprising Abs against
AChR and other muscle antigens, including
actin, myosin, the ryanodine receptor and
especially titin
[64]. MG-associated thymomas
share several common features:
• Intratumorous thymopoiesis and maturation
of potentially autoreactive T cells
[65];
• Reduced expression of MHC class II
molecules
[66–68];
• Intratumorous enrichment in AChR-respo-
sive T cells
[69] restricted to minority HLA
isotypes, not commonly found in non-
thymomatous MG patients
[70];
• Export of mature T cells to the periphery
[71].
In fact, a strong correlation exists between para-
neoplastic MG and the ability of thymomas to
produce and export mature naive CD4
+
T cells,
given the significantly increased levels of this
T-cell subset in MG-positive compared with MG-
negative thymomas
[72].
Although no myoid cells or intact AChR are
present in thymomas, single receptor subunits have
been detected. However, it is disputable whether
thymomas actively sensitize
[70,71] or just fail to tol-
eralize
[67] the newly-produced auto-reactive
thymocytes against AChR. In any case, given that
B cells are generally rare in thymomas, a humoral
immune response should occur in the periphery.
Interestingly, neutralizing autoantibodies against
IFN-
α and IL-12 are found commonly in paraneo-
plastic MG, potentially affecting Th1 polarization
of the autoimmune response. Anti-IFN-
α Abs
have been isolated from MG thymomas, suggest-
ing that active priming against these cytokines
actually occurs within the thymoma
[73].

REVIEW – Fostieri, Kostelidou, Poulas & Tzartos
806 Future Neurol. (2006) 1(6)
According to a model for paraneoplastic MG
pathogenesis proposed by Marx and colleagues,
thymomas produce and export incompletely
toleralized, and thus potentially auto-reactive,
mature naive T cells
[74]. When exported to the
periphery, the thymoma-derived nontolero-
genic T cells may gradually convert the physio-
logically tolerant T-cell subset into a mixed
autoimmunity-susceptible T-cell repertoire origi-
nating from both the thymus and the thymoma
(Figure 4). In this case also, an as yet unidentified
stimulus must occur to activate the potentially
autoreactive Tcells in order for them to provide
help to autoantibody-producing B cells outside the
thymoma. Interestingly, the anti-IFN-
α and
anti-IL-12 autoantibodies discussed above
could act to facilitate the T-cell dependent
production of anti-AChR autoantibodies
[74].
Role of muscle in myasthenia
gravis pathogenesis
Muscle is the target organ of the autoimmune
response in MG. Numerous studies indicate that
far from staying inert before autoimmune attack,
the muscle actively participates in shaping the
eventual disease symptoms by appropriately adapt-
ing AChR expression and secreting/receiving
immunomodulating messages.
Figure 3. New model for intrathymic pathogenesis of myasthenia gravis related with thymus hyperplasia.
An inflammatory reaction in the hyperplastic thymus modulates the expression of AChR and several other immune components, mostly
MHC and costimulatory molecules, (2) and facilitates re-entry into the thymus of auto-reactive CD4
+
T cells (3) that have escaped central
deletion (1). Enhanced AChR expression and presentation within this activated environment, combined with a genetically susceptible
background, could be adequate to prime the AChR-responsive CD4
+
T cells (4), leading to autoimmunity.
AChR: Acetylcholine receptor; MHC: Major histocompatibility complex; TEC: Thymic epithelial cell.
Based on
[60].
Thymic medulla
P
2. Inflammatory e
4.
AChR
the thymus.

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Recent advances myasthenia gravis research – REVIEW
In the case of antigenic modulation, mRNA lev-
els of muscle AChR subunits increase, as demo-
nstrated both in cell culture and MG patient
muscle biopsies, likely following a mechanism
attempting to counterbalance AChR loss
[75]. In
passively transferred EAMG, muscle AChR
expression is upregulated after the administration
of TNF-
α (but not IFN-γ) [61]. The differential
effect of IFN-
γ on thymic and muscle AChR tran-
scripts likely suggests a different pathway regulat-
ing AChR expression in both the thymus and
muscle. Additionally, upon in vivo or in vitro expo-
sure to anti-AChR Abs, rat skeletal muscle cells
respond by producing several immunomodulating
factors such as the disease-promoting molecules
IFN-γ, IL-1, -15, the monocyte chemoattractant
protein-1 chemokine
[76,77] and nitric oxide that
acts protectively
[78]. A recent study demonstrated
that muscle in EAMG and MG also over-pro-
duces the IFN-γ-inducible protein (IP)-10 chemo-
kine and its receptor, CXCR3. Expression of these
molecules is suppressed in experimental animals
after induction of mucosal tolerance using an
AChR fragment
[79]. Since IP-10 and CXCR3
mediate inflammatory cell recruitment, yet no
signs of inflammation are observed in myasthenic
muscle, their role in disease pathogenesis remains
enigmatic
[79].
Figure 4. Proposed model for pathogenesis of paraneoplastic myasthenia gravis.
Thymomas produce and export nontoleralized, potentially autoreactive mature naive CD4
+
T cells that, upon
entering the periphery, gradually convert the physiologically tolerant T-cell subset into a mixed
autoimmunity-susceptible T-cell repertoire. Within this context, thymoma-derived mature naive CD4
+
T cells
are primed against AChR presented by APCs or DCs (although the site of sensitization or the initiating
stimulus remain unknown) and help autoantibody-producing B cells outside the thymoma.
AChR: Acetylcholine receptor; APC: Antigen presenting cell; DC: Dendritic cell.
Based on
[74].
Thymoma
Periphery
Reduced MHC II expression
Altered MHC II restriction
Impaired negative selection
Immature
Tcell
Mature naive
CD4
+
Tcell
Mature naive
CD4
+
T cells
(from thymoma)
+
Unidentified
stimulus
Activatio
Autoantibody-s
Bcell
Export to
the periphery
Abnormal selection
Mixed autoimmunity-prone
CD4
+
population

REVIEW – Fostieri, Kostelidou, Poulas & Tzartos
808 Future Neurol. (2006) 1(6)
An emerging new scenery for
myasthenia gravis therapeutics
A greater insight into the mechanisms governing
MG autoimmunity lead to the design of novel,
immune-directed therapeutic strategies. These
include both new-generation immunosuppres-
sive agents and purely innovative approaches cre-
ated upon the MG-specific demands.
Current treatment
Current medications control MG by improving
neuromuscular transmission or downmodulating
immune function. Acetylcholinesterase inhibi-
tors, most commonly pyridostigmine (Mesti-
non
®
) and neostigmine (Prostigmin
®
) remain
the first-line of MG treatment. Both enhance
muscle strength by increasing acetylcholine levels
in the NMJ, but their effect often diminishes
after some months of administration.
Immunosuppressive treatment includes corti-
costeroids (mainly prednisolone), which are
usually effective in reducing anti-AChR anti-
body titer, but induce serious side effects (i.e.,
weight gain, hypertension osteoporosis) and
nonsteroidal immunosupressants, usually aza-
thioprine, which inhibit lymphocyte prolifera-
tion, have fewer side effects, but require a
longer period to act
[80]. Azathioprine is most
often used together with corticosteroids. Myco-
phenolate mofetil (CellCept), a potent inhibi-
tor of lymphocyte proliferation, has recently
been introduced in MG treatment and gener-
ally proved effective and well-tolerated
[81]. Tac-
rolimus (IL-2-suppressor) and Rituximab
®
(Ab
against the B-cell surface-marker CD20), which
are widely used in transplantation medicine and
lymphoma treatment, respectively, are also can-
didates for study in MG patients, although
there is some skepticism on their usefulness in
view of their toxicity.
Surgical treatment of MG involves thymec-
tomy. The effectiveness of thymecomy is well-
established for early-onset MG, but its thera-
peutic value is less obvious for late-onset MG
patients, while postsurgical clinical improve-
ment is even less common for thymomatous
MG patients. Controlled prospective studies on
this issue would provide evidence clarifying the
therapeutic benefits of thymectomy for the
three subsets of MG patients
[82].
In the case of severely affected MG patients,
plasmapheresis and administration of intrave-
nous immunoglobulins (IVIGs) produce a
direct, albeit short-term, improvement. Addi-
tionally plasmapheresis presents the severe
drawback of removing protective Abs and
plasma components, thus necessitating the use
of replacement fluids, with the risk of infection
and allergic reactions
[83]. Due to these disad-
vantages, plasmapheresis is mostly considered as
a second-line treatment and IVIG, which pro-
duces significantly fewer adverse effects, is gen-
erally preferred. Although not yet clearly
understood, IVIG action lies on different levels
of the autoimmune response: pathogenic
autoantibody production, complement activa-
tion, cytokine profile and activation/effector
functions of T and B cells
[84].
Experimental approaches
The experimental therapeutic strategies currently
under investigation aim to eliminate the auto-
immune response, whilst bypassing generalized
immunosuppression.
Phospodiesterase inhibitors
Recently, Fuchs and colleagues revealed an
involvement of phosphodiesterases in EAMG
pathogenesis and tested the effect of the phos-
phodiesterase inhibitor pentoxifylline on
EAMG progression. Pentoxifylline administra-
tion during the acute or chronic stage of the dis-
ease repressed EAMG progression. Humoral and
cellular anti-AChR responses were down regu-
lated, phosphodiesterase and TNF-
α expression
were decreased in lymph node cells and muscle,
as well as IL-18, -12 and -10 (in lymph nodes).
Remarkably, expression of the Foxp3 transcrip-
tion factor was upregulated, suggesting an
enhanced function of the CD4
+
CD25
+
regula-
tory T cells. These findings, together with the
fact that phosphodiesterase inhibitors are
already an approved treatment for other disor-
ders, makes them very promising candidates for
MG immunotherapy
[85].
Tolerance induction
Induction of mucosal (oral or nasal) tolerance
using various AChR-derived molecules, (native or
denatured AChR, recombinant AChR fragments,
AChR peptides or altered peptides) has been tested
with some encouraging results. This approach
appears to suppress animal disease by altering the
polarization of the anti-AChR autoimmune
response. The shift in Th-cell subsets greatly
depends on the tolerogen (source, conformation
and size) and the route of administration (mucosal
exposure rather than injection). Fuchs and col-
leagues have demonstrated that mucosal adminis-
tration of recombinant fragments of the human

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Recent advances myasthenia gravis research – REVIEW
AChR α-subunit extracellular domain suppressed
ongoing EAMG at the acute and even chronic
phases of the disease, as assessed by clinical score,
decreased anti-AChR Ab titers and reduced lym-
phoproliferation in response to AChR
[86,87],
while Link and colleagues used To r pe do AChR as
the tolerogen
[88]. Mucosal tolerance likely acts by
introducing a transmission from Th1 to a mixed
Th2/Th3 regulation
[86,87]. Alternatively, oral
administration of a syngeneic recombinant frag-
ment of the rat AChR
α-subunit also sup-
pressed ongoing EAMG in rats, although via a
different mechanism (a shift from Th1 to Th2
regulation)
[89]. Regarding the syngeneic frag-
ment use for tolerance induction, Baggi and
colleagues reported a breakdown of tolerance to
a self-rat AChR
α-subunit peptide, leading to
EAMG in rats
[90]. However, this pathogenic
effect is likely to be related to the route of pep-
tide administration, which was injection
instead of mucosal treatment.
T-cell receptor vaccination
As discussed previously, early-onset MG mostly
comprises female patients with thymic hyper-
plasia displaying the HLA-DR3 haplotype. Inter-
estingly, HLA-DR3 positive MG patients were
reported to exhibit a bias for the V
β5.1 TCR in
their thymic CD4
+
T cells and to have elevated
anti-V
β5.1 Abs, which tend to act regulatorily
and are related with low disease severity and posi-
tive prognosis
[91]. These data could provide two
alternatives for TCR vaccination in MG:
•Use of Vβ5.1-derived peptides to boost the
spontaneously-produced response against
autoreactive T cells;
• Administration of anti-Vβ5.1 Abs to directly
suppress autoreactive T-cell function.
In fact, an anti-Vβ5.1 Ab has already been
tested in rat EAMG with positive results
[92],
although application to humans would require
human or humanized anti-Vβ5.1 Abs. However,
there is some skepticism regarding TCR vaccina-
tion for MG treatment, which derives mainly
from the issue of TCR usage restriction in MG
per se, since there are studies challenging the
Vβ5.1 T-cell ‘prevalence’ in the thymus of MG
patients
[93].
Manipulated antigen presenting cells
Different strategies involving manipulated
APCs are currently under investigation. Drach-
man and colleagues used viral vectors to convert
APCs into ‘guided missiles’ that specifically
eliminated AChR-specific T cells. The geneti-
cally engineered APCs effectively present the
α-subunit extracellular domain of AChR and
express Fas ligand to selectively interact with
activated AChR-specific T cells and destroy
them via apoptosis
[94]. Alternatively, dendritic
cells (DCs) from EAMG rats, upon in vitro
exposure to IL-10 and subsequent injection,
hindered ongoing EAMG
[95]. Their action was
related with the downregulation of costimula-
tory molecules and decreased anti-AChR Ab
production, likely through blockade of IL-10
expression. These data create expectations for
trials of modified autologous DCs in humans,
given that DC vaccination is already applied for
other conditions, such as cancers.
Acetylcholinesterase gene silencing using
antisense oligonucleotides
In a recent study, acetylcholinesterase expression
was abrogated using antisense oligonucleotides
targeting the acetylcholinesterase mRNA gene
[96].
After treatment with the acetylcholinesterase gene
antisense oligonucleotides, the clinical profile of
rats with EAMG was significantly improved. In
fact, the antisense approach was shown to be more
effective than pyridostigmine, presenting the addi-
tional advantage of selectively blocking alterna-
tively spliced acetylcholinesterase variants.
Nevertheless, an issue arises of how this method
could be applied in humans.
Protective antiacetylcholine receptor
antibody fragments
The use of nonpathogenic univalent human
anti-AChR Ab fragments that bind to AChR,
thereby preventing the binding of pathogenic
autoantibodies, offers another option for MG
treatment. A number of human or humanized
anti-AChR Ab fragments have already been
developed
[97–100]. Among them, two sets of
human anti-AChR Fabs, one against the MIR
and the other against an immunodominant
region close to the MIR, effectively compete
with a great majority of MG patients sera for
binding to AChR
[97,99]. Their combination
could prove useful for short-term therapy, for
example, in life-threatening crises. Attempts to
prolong their in vivo half-life are in progress.
Anti-AChR Ab-specific plasma clearance
An affinity column bearing the AChR or appro-
priate AChR fragments to selectively withhold
the anti-AChR Abs allowing the return of ‘anti-
AChR Ab-cleared’ plasma to the patient would

REVIEW – Fostieri, Kostelidou, Poulas & Tzartos
810 Future Neurol. (2006) 1(6)
overcome the major disadvantage of plasma
exchange, namely the indiscriminate removal of
protective Abs together with the pathogenic
ones. Such a column was introduced by
Takamori and colleagues who used the To rp ed o
AChR
α-subunit synthetic peptide 183–200,
containing part of the acetylcholine binding site
[101]. However, the immunoadsorption efficiency
of such columns bearing small, nonhuman, syn-
thetic peptides, is likely inadequate, as the
majority of anti-AChR Abs in MG patients are
highly conformation dependent.
Our group has developed a specific Ab-
apheresis therapy for MG, through the con-
struction of ‘immunoadsorbent’ columns car-
rying AChR fragments of native-like
conformational features. We have successfully
expressed the extracellular domains (ECDs,
amino acids 1–210) of the human AChR
α, β,
γ and ε subunits in water-soluble form using
the yeast Pichia pastoris expression system
[22,102]. Subsequent immobilization of each
ECD on an insoluble carrier (CNBr-Sepha-
rose) led to the construction of ‘immuno-
adsorbent’ columns, which were tested for the
feasibility of selective ex vivo elimination of
patients’ anti-AChR Abs. Immunoadsorption
experiments performed with
α-ECD-Sepha-
rose demonstrated that, on average, 35% of
autoantibodies were adsorbed from a set of 64
random MG positive sera
[22,103]. Preliminary
experiments with each of the non-
α-ECD-
Sepharoses and the same set of sera yielded dif-
ferent average immunoadsorption percentages
of autoantibodies by each ECD as follows:
22% by
β-ECD, 21% by γ-ECD and 18% by
ε-ECD, while the combination of all four sub-
units (
α, β, γ, ε) demonstrated that they could
act in an additive fashion or could at least
remove a considerably higher percentage of
autoantibodies than that of any individual
immunoadsorbent
(Figure 5) [Kostelidou et al., Unpub-
lished Data]
. These results suggest that the com-
bined use of all subunits for the preparation of
an immunoadsorbent column could indeed
result in the specific removal of the majority of
autoantibodies from a large portion of MG
sera. A drawback of this approach is that elimi-
nation of the anti-AChR Abs may result in
compensatory overproduction of novel Abs.
Yet such overproduction should be milder than
that occurring in plasmapheresis, where most
Igs are removed, including possible T-cell
inhibitory Igs. The characterization of the
properties of the
α-ECD-immunoadsorbent
column (which are currenly being evaluated on
other ECD-Sepharose matrices and appear to be
consistently preserved) suggested that a matrix
(Sepharose) column with approximately 5 mg of
immobilized polypeptides would suffice to
immunoadsorb most of the anti-AChR autoanti-
bodies from a typical MG serum in a short period
of time. The column could be recycled, thus ren-
dering this technique a feasible, antigen-specific,
therapeutic approach
[22,103].
Conclusion
During the previous decade, we witnessed a turn
of the immunological research on MG towards
the study of the immunological mechanisms,
cellular and molecular, which trigger/maintain
the disease and determine its severity.
Overall, significant progress has been made,
especially regarding MG etiology. A severely
impaired function of the CD4
+
CD25
+
regulatory
T cells to suppress autoreactive CD4
+
T cells was
identified, while the hyperplastic thymus appears
to be involved in MG pathogenesis providing a
hyperactive proinflammatory microenvironment
that is prone to breakage of tolerance to AChR.
What is now greatly anticipated is the identifica-
tion of the stimulus creating this autoimmunity-
nesting milieu. Interestingly, muscle, the target-
organ of the anti-AChR response, appears to
actively contribute in shaping the final MG
symptoms by appropriately adapting AChR
expression and secreting/receiving immuno-
modulating messages, such as chemokines. The
role of these molecules in MG is puzzling, since
chemokines are mainly recruiters of inflammatory
cells, yet no signs of inflammatory cell infiltration
are observed in myasthenic muscle.
Based on recent findings, several approaches
that aim to selectively control the anti-AChR
response without suppressing the overall
immune system, have been rationally designed
Although still experimental, encouraging results
for some of them (phosphodiesterase inhibitors,
manipulated autologous DCs and anti-AChR
Ab-specific apheresis) create expectations for
future trials in humans.
Future perspective
Several issues regarding MG etiology remain
obscure, most importantly the triggering agent.
Although viral or bacterial antigens have been
implicated occasionally, no definite associations
were established. Additionally, muscle-secreted
chemokines have appeared recently in MG
bibliography, but their role is unidentified. The

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Recent advances myasthenia gravis research – REVIEW
involvement of chemokine signaling in MG is a
new, open field, which may soon reveal novel
aspects of disease pathogenesis.
It is obvious that not every therapeutic stra-
tegy that has proven successful in experimental
models will necessarily be effective in humans.
However, recent data from several experimen-
tal approaches likely justifies the initiation of
carefully designed clinical trials in human
patients. Given the low prevalence of the
disease, joint approaches involving patients
from multiple countries will likely be required
and the creation of an international MG registry
could prove very useful to promote this
move. Pentoxifylline seems a strong candidate
for clinical trials, given its encouraging results in
EAMG animals, and already being an approved
medication for several other conditions (mostly
circulatory disorders).
Additionally, our studies demonstrate that
ex vivo serum clearance from anti-AChR Abs
using specific plasma exchange is an effective and
feasible approach that could be applied relatively
easily in clinical practice. Further optimization
of ongoing experimental approaches will provide
new options for specific MG treatment.
Figure 5. Immunoadsorption of anti-acetycholine receptor antibodies from myasthenia gravis sera using
CNBr-sepharose immobilized extracellular domains.
Samples from myasthenia gravis sera (20 fmoles) were incubated with excess of Sepharose-immobilized extracelllular domains (ECDs)
(1 g). The unbound anti-acetylcholine receptor antibodies present in the supernatants were measured by RIA and the percentage of ECD-
absorbed anti-AChR Abs was calculated. Each bar shows the percentage of absorbed Abs by the specified ECD. (A) Immunoadsorption
of anti-AChR Abs from 64 MG sera using Sepharose-ECD. Sera are sorted in decreasing percentage of immunoadsorption
[103]. (B)
Immunoadsorption of anti-AChR autoantibodies from 7 MG sera, using immobilized
α, β, γ and ε-ECDs (first four columns) or a
combination of all the four subunits (last column). The combined use of all ECDs consistently removes more Abs than any individual ECD,
although this is often lower than the sum of individual ECD columns (derived from
[22]).
0
10
20
30
40
50
60
70
80
90
100
58
147
10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 61 64
Sera
A
0
10
20
30
40
50
60
70
80
1234567
B
Sera
% Immunoadsorption
% Immunoadsorption
D
E
J
H
Total

REVIEW – Fostieri, Kostelidou, Poulas & Tzartos
812 Future Neurol. (2006) 1(6)
Acknowledgements
Original work of the authors was supported by grants from the
QoL program of the European Commission, the Muscular
Dystrophy Association, the Association Française contre
les Myopathies and the Greek General Secretariat of Research
and Technology.
Executive summary
Background
• Myasthenia gravis (MG) is a T-cell-dependent autoimmune disease-mediated by autoantibodies, mostly against muscle nicotinic
acetylcholine receptor (AChR), a ligand-gated ion-channel of the neuromuscular junction (NMJ).
• The anti-AChR autoantibodies cause loss of functional AChR molecules at the NMJ, resulting in weakness and fatigability of
voluntary muscles.
• A small fraction of MG patients have antibodies (Abs) to the muscle-specific kinase (MuSK), a key-player molecule in NMJ
differentiation, rather than to the AChR.
The phenotypic heterogeneity of myasthenia gravis
• Ocular MG: where the disease remains localized to the extraocular muscles.
• Generalized MG: where the disease spreads to other skeletal muscles. Depending on the presence or absence of anti-AChR Abs,
it is subdivided in seropositive or seronegative MG, respectively.
• Seropositive MG (~85% of MG patients) comprises: (i) early-onset MG (age <40 years) affects mostly women and is commonly
related with thymic hyperplasia; (ii) late-onset MG (age >40 years) affects people with normal or atrophic thymus; and (iii)
paraneoplastic MG associated with thymoma (age 40–60 years old) characterized by the presence of Abs to other striated muscle
antigens, mainly titin.
• Seronegative MG represents approximately 15% of patients, approximately 40% of which have autoantibodies against MuSK of
probable pathogenic significance.
Genetic predisposition to MG
• Early-onset MG has been associated with the human leukocyte antigen (HLA)-DR3 and B8, and late-onset MG with the HLA-DR7
(for paraneoplastic MG, no clear HLA association has been described).
• Recently, a genetic linkage was established between MG and the MYAS1 HLA locus. Additionally, an HLA-dependent control of
anti-AChR Ab titers in MG patients with hyperplastic or normal thymus was proposed.
Antigenic structure of acetylcholine receptor
• The main immunogenic region (MIR) of the AChR is a major target of anti-AChR Abs from experimental animals and MG patients.
It comprises a cluster of overlapping conformation-dependent epitopes, with the core sequence α67–76, located extracellularly
on the α-subunit of AChR.
• The MIR is of major pathological importance in experimental autoimmune MG, since anti-MIR monoclonal Abs very effectively
induce passively transferred experimental autoimmune myasthenia gravis and cause AChR loss. A major pathological significance
of MIR is also suggested in MG.
• Antibodies to other AChR subunits are also present in MG sera.
Effector humoral response in MG
• Among the three Ab-mediated pathogenic mechanisms of AChR loss in NMJ, namely complement-dependent lysis of the
postsynaptic membrane, increased AChR degradation via antigenic modulation and direct inhibition of neurotransmitter binding,
the classical complement pathway likely plays the major pathogenic role.
B- & T-cell interaction through costimulation molecules
• Autoreactive CD4
+
T cells from MG patients recognize various AChR epitopes in the context of major histocompatibility complex
(MHC) class II molecules and help B cells, becoming essential for anti-AChR Ab production.
• CD40–CD40 ligand interaction and B7-mediated signaling, especially the B7-1–CD28 interaction, are essential both for EAMG
induction and progression. The inducible costimulatory molecule is also implicated in EAMG pathogenesis.
The effect of cytokines on EAMG pathogenesis
• Interleukin (IL)-12 and -18 facilitate EAMG development, while the role of interferon (IFN)-γ is less clear. The T-helper (Th)2
cytokines IL-10, -6 and -5 also promote EAMG pathogenesis, while IL-4 likely acts protectively.
• Overall, the Th1 cell response has a predominant pathogenic role in EAMG (and possibly MG), yet the involvement of IL-10 and -6
in disease pathogenesis may denote the implication of a mixed Th1/Th2 anti-AChR autoimmune response.

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Recent advances myasthenia gravis research – REVIEW
Hyperplastic thymus & MG pathogenesis
• Hyperplastic thymus is characterized by: (a) massive B- and T-lymphocyte infiltration in the thymic medulla, where AChR is
normally expressed by thymic epithelial cells and myoid cells, and (b) the presence of AChR-responsive CD4
+
T cells and plasma
cells spontaneously producing anti-AChR Abs.
• It likely offers an autoimmunity-susceptible hyperactive inflammatory environment over-expressing IFN-γ- and tumor necrosis
factor (TNF)-α-regulated molecules (AChR, MHC and costimulatory molecules) and facilitating the return of autoreactive CD4
+
Tcells that escaped central deletion. The initial stimulus inducing this inflammatory environment remains unidentified.
• Regulatory CD4
+
CD25
+
T cells in hyperplastic thymus, although normal in number, are severely defective in their ability to
suppress autoreactive CD4
+
T cells, as evaluated with a specific mitogenic assay.
Thymoma & MG pathogenesis
• MG-associated thymomas are thymic epithelial tumors characterized by: (a) intratumorous enrichment in AChR-resposive T cells;
(b) intratumorous thymopoiesis and maturation of potentially autoreactive T cells; and (c) ability to export mature T cells to
the periphery.
• It is still unclear whether thymomas actively sensitize the newly-produced autoreactive thymocytes against AChR or just fail to
effectively toleralize them.
• Neutralizing autoantibodies against IFN-
α and IL-12 commonly found in paraneoplastic MG suggest active priming against these
cytokines within the thymoma.
The role of muscle in myasthenia gravis pathogenesis
• Muscle actively participates in shaping the eventual disease symptoms by adapting AChR expression and secreting/receiving
immunomodulating messages.
• Muscle AChR mRNA levels are upregulated in cell cultures and MG patients as a result of AChR loss due to antigenic modulation,
and in passively transferred EAMG after administration of TNF-
α (but not IFN-γ).
• Upon in vivo or in vitro exposure to anti-AChR Abs, rat skeletal muscles respond by producing several immunomodulating factors,
such as the disease-promoting molecules IFN-
γ, IL-1, -15 and several chemokines, or nitric oxide that acts protectively.
Current myasthenia gravis treatment
• Current medications control MG by improving neuromuscular transmission or downmodulating the immune system
function. They include acetylcholinesterase inhibitors and steroid or nonsteroid immunosuppressants, while in severely
affected patients, administration of intravenous immunoglobulins or plasmapheresis are recommended. Surgical MG treatment
consists in thymectomy.
Experimental therapeutic approaches
• The aim of experimental therapeutic strategies is to eliminate the autoimmune response without suppressing the overall immune
system.
• Administration of phosphodiesterase inhibitors, induction of oral or nasal tolerance using AChR fragments, vaccination with T-cell
receptor peptides or manipulated autologous dendritic cells, administration of AChR-protective Ab fragments and use of a plasma
exchange approach for selective anti-AChR Ab clearance provide encouraging results.
Conclusion
• During the last decade, immunological research on MG has focused on studying the immunological mechanisms, cellular and
molecular, which trigger/maintain the disease and determine its severity.
• Although significant progress has been made regarding MG etiology, the initial disease-inducing signal remains unidentified,
while several other issues, for example the role of chemokines in MG pathogenesis, are open for investigation.
• Promising results from several experimental therapeutic approaches create expectations for future trials in humans.
Executive summary
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Affiliations
• Efrosini Fostieri, PhD
Hellenic Pasteur Institute, Department of
Biochemistry, 127 Vas. Sofias Avenue,
11521 Athens, Greece
fostieri@pasteur.gr
• Kalliopi Kostelidou,
PhD
Hellenic Pasteur Institute, Department of
Biochemistry, 127 Vas. Sofias Avenue
11521 Athens, Greece
k.kostelidou@pasteur.gr
• Konstantinos Poulas,
PhD
Department of Pharmacy, University of Patras,
26504 Patras, Greece
kpoulas@upatras.gr
• Socrates J Tzartos,
PhD
Hellenic Pasteur Institute, Department of
Biochemistry, 127 Vas. Sofias Avenue, 11521
Athens, Greece
and,
Department of Pharmacy, University of Patras,
26504 Patras, Greece
Tel.: +30 210 647 8842;
Fax: +30 210 647 8842;
tzartos@pasteur.gr