Note: Descriptions are shown in the official language in which they were submitted.
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CA 02388659 2002-05-31
PHOSPHOLIPASE A2 EXPRESSION AND ACTIVITY AND USE THEREOF FOR
DIAGNOSIS, PROGNOSTICATION, PREVENTION AND TREATMENT OF NEURAL
INFLA2~1ATORY AND DEMYELINATING DISEASE
FIELD OF THE INVENTION
The invention relates to phospholipase AZ expression and
activity and uses thereof for diagnosis, prognostication,
prevention and treatment of neural inflammatory and/or
demyelinating disease.
BACKGROUND OF THE INVENTION
Etiology and pathogenesis of MS and EAE
Multiple sclerosis is an inflammatory demyelinating
disease, which typically strikes young adults, and is
characterized by demyelinating episodes ranging from
relapsing-remitting to chronic progressive in nature. The
lesions are multi-focal and confined to the central nervous
system (CNS) which includes the brain, spinal cord and optic
nerve. Despite extensive studies, the etiology of the disease
still remains obscure and its pathogenesis is not fully
understood. The consensus is that unknown environmental
agents) initiate the disease in genetically susceptible
individuals. Several genes are thought to be involved in
conferring susceptibility to MS. These include HLA class II
(likely the DR2, DQ6 locus) (Tienari, 1994) and the T-cell
receptor (TCR) genes (Tienari, 1994). However, a definite set
of genetic markers for MS remains unknown. Nevertheless,
genetic factors are thought to be important contributors to
the onset of the disease because MS shows familial clustering
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CA 02388659 2002-05-31
2
and racial differences in risk (Oger and Lai, 1994; Sadovnick
et al., 1996; Ebers, 1996).
A number of environmental factors have also been
suspected in MS, such as viral and bacterial infections.
Elevated antibody titers against a number of viruses have been
reported in the cerebrospinal fluid (CSF) and serum of MS
patients (Allen and Brankin, 1993). However, viruses have not
been detected in the CNS parenchyma in MS.
MS is studied using the established, generally accepted
animal model system of experimental allergic encephalomyelitis
(EAE), in for example rodents such as rats and mice (Ruuls et
al, 1996; Ewing and Bernard, 1998; van der Meide et al, 1998,
Smith et al, 2000 . As with MS, EAE is also more easily
induced in certain strains of mice and rats.
Target autoantigens and Cytokines in MS and FnF
An important clue to the pathogenesis of MS is the
detection of myelin basic protein (MBP)-reactive T-cells in MS
in plaques. Injection of MBP peptides into experimental
animals can induce EAE (Richert et al., 1989; Martin et al.,
1990). Different regions of MBP are encephalogenic in
different animal species, e.g., residues 87-106 in Lewis rats
and SJL mice, and 1-9 in PL/J and B10.PL mice. Strong evidence
for MBP and additional environmental agents in the
pathogenesis of MS comes from studies showing that transgenic
mice expressing TCR specific for MBP spontaneously develop EAE
but only when exposed to a non-sterile environment (Goverman
et al., 1993). Thus, exposure to some infectious agents)
triggers the breakdown of myelin resulting in the availability
of MBP and other myelin components for presentation to the TCR
via antigen-presenting cells. T-cells that secrete interferon
gamma (IFNy) with reactivities to MBP, PLP (proteolipid
protein), MOG (myelin-oligodendrocyte glycoprotein), and MAG
(myelin-associated glycoprotein) have been detected in the CSF
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CA 02388659 2002-05-31
3
of MS patients (Olsson et al., 1990: Sun et al., 1991; Zhang
et al., 1993). Anti-PLP antibodies have been detected in
about 3% of MS patients (Warren et al., 1994), and PLP has
been shown to be encephalitogenic (Tuohy, 1994). Studies also
show that MOG may be as effective as MBP or PLP in the
pathogenesis of MS and EAE (Adelmann et al., 1995; Johns et
al., 1995). Thus, a number of CNS myelin components may serve
as target autoantigens.
It is thought that autoantigen specific T-cells
sensitized in the periphery migrate into the CNS where they
initiate the inflammatory changes leading to CNS tissue damage
and functional impairment (Bansil et al., 1995). EAE can be
induced by injecting mice with MOG or MBP or by the passive
transfer of T-cells from affected animals (Moktarian et al.,
1984; Zamvil et al., 1985). The findings to date may be taken
to indicate that an initial breakdown of myelin by some yet
unknown cause, results in the release of myelin components
which are then presented by antigen presenting cells to T-
cells with receptor specificity for MBP or other myelin
antigens. These interactions result in a variety of immune
cell responses leading to antibody production and
cytotoxicity.
Proinflammatory cytokines such as IFN-Y, TNF-a and (3, IL-
12 and IL-lei also increase in the CNS of rats with EAE
(Issazadeh et al., 1996) and in the brain in MS (Hofman et
al., 1989). IL-2 and IFN-'y mRNA levels were shown to be
increased in CSF cells from SJL/J mice during MBP-induced EAE
(Renno et al., 1994). IFN-y increases the severity of the rate
of relapse in patients with MS (Panitch et al., 1987). TNF-a
and ~i are present in acute and chronic lesions (Hofman et al.,
1989). Furthermore, transgenic mice over expressing TNF-a
develop a chronic inflammatory demyelination (Probert et al.,
1995; Taupin et al., 1997), although other studies on TNF null
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CA 02388659 2002-05-31
4
mice showed similar results (Liu et al., 1998). There are
strong similarities in the pathogenesis of MS and EAE (Ewing
and Bernard, 1998). As such, EAE is a generally accepted
animal model system for MS, and studies on EAE animals have
contributed significantly to the understanding of the
involvement of the cellular and humoral immune responses in MS
(Ewing and Bernard, 1998).
Pathology of MS and h~AE lesions
The CNS lesions in MS and EAE are characterized by
widespread focal lesions particularly in perivascular,
periventricular and subpial white matter. The pathology varies
in acute and chronic lesions. Demyelination is a
characteristic feature of acute MS lesions. However, the loss
of oligodendrocytes in acute lesions is variable (Bruck et
al., 1994; Ozawa et al., 1994). Loss of myelin and
oligodendrocytes is more extensive in chronic stages (Prineas
et al., 1993; Bruck et al., 1994; Ozawa et al., 1994). The
focal lesions also contain inflammatory infiltrates, which
consist of T cells and macrophages. In chronic lesions,
there is a significant increase in the number of antibody
producing plasma cells (Ozawa et al., 1994). CD4+ T-cells are
found at the edge of the lesions, while macrophages are
numerous in and around MS lesions (Traugott et al., 1983; Bo
et al., 1994). Activated T-cells are also present in the
lesion (Hofman, et al., 1986). Many of these changes in
inflammatory cell influx is also seen in EAE lesions in the
CNS (Norton et al., 1990; Ewing and Bernard, 1998). However,
the inflammatory changes in the CNS rather than demyelination
are more prominent in EAE.
Current approaches in EAE and MS therapy
Several experimental approaches have been tested in an
effort to ameliorate EAE symptoms. Most of these involve
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CA 02388659 2002-05-31
immune modulation. These include treatments to block various
chemokines or cytokines. Studies performed in blocking
chemokines involve the development of anti-MIP-la, anti-MCP-1
and anti-IP-10 antibodies for treatment of EAE. When the
5 treatment was given before the occurrence of clinical
symptoms, anti-MIP-la antibodies reduced disease incidence by
80~ and decreased disease severity from a clinical score of
2.6 in untreated mice to a score of about a 0.5 in treated
mice. When this treatment was given after symptoms began, the
severity decreased to a score of 1.25. The anti-MCP-1
treatment only had a minimal effect (Karpus et al, 1995).
Treatment with anti-IP-10 tested in only a small experimental
group decreased incidence by about 65o and reduced the
clinical score to a 0.8 compared to a 3.9 in untreated animals
(Fife et al, 2001).
Treatments used to block cytokines have also been tested.
These involve blocking lymphotoxin, TNF and IFN-Y. In mice
given anti-LT/TNFa antibodies before symptoms appeared reduced
disease severity from a score of 3.9 in controls to a 0.2 in
treated animals (Ruddle et al, 1990). Similar studies using a
TNF binding protein completely prevented EAE in animals
treated before symptoms were seen. When this treatment was
given after symptoms occurred, the treated animals followed a
course from a grade 2 to 0, while control animals went from a
grade 2 to 3 to 1 (Selmaj, et al, 1998). Treatments performed
using anti-IFN-y antibodies actually worsened disease severity
(Leonard et al, 1996).
Other immunomodulatory treatments evaluated were those
done to prevent the actions of macrophages and T cells.
Animals treated with liposomes (C12MDP) to eliminate
macrophages showed a 40a reduction of EAE incidence (Trap, et
al, 1998; Bauer et al, 1995, Huitinga et al, 1990). Disease
severity was also reduced, from a mean clinical score of 3.4
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6
in controls to a 0.8 in treated animals, when treatment was
given before symptoms occurred (Huitinga et al, 1990).
Another method to prevent the actions of lymphocytes is to
prevent their entry into the CNS by blocking adhesion
molecules at the blood-brain barrier. Studies such as these
have been performed using antibodies to ICAM-1, LFA-1 and the
a4 integrin. Animals treated with anti-ICAM1 or anti-LFA 1
did not show a significant effect in disease reduction. When
they were combined, however, their effect reduced a score of
2.5 in control animals to below 0.5 in the treated group
(Kawai et al, 1996). Treatments using anti-a4 integrin
antibodies reduced clinical incidence by 75~ (Yednock et al,
1992) and reduced disease severity from a 1.5 to a 0.5 (Kent
et al, 1995). Other experiments in attempts to block proper
T-cell activation and function were also performed. The use
of the copper chelator, cuprizone, was used to block IL-2
synthesis and therefore T-cell activation. Treated mice
showed decreased disease severity from a score of 4.3 in
controls to a 3.3 in mice treated one week before EAE
induction. Piperonyl butoxide, an insecticide that is known
to deplete T cells delivered before symptoms occurred reduced
disease score from a 4.2 in controls to a 2.2. Animals
treated after symptoms occurred showed reduced severity to 3.7
(Emerson, et al, 2001).
Oral tolerance has also been evaluated as a treatment for
EAE. By feeding animals with myelin antigens, a Th2 response
is elicited while Thl inflammatory responses are reduced. An
80$ reduction of EAE incidence was reported in animals fed MBP
prior to disease induction. In addition, disease severity was
reduced from a maximum score of 4 in control to a 1.4 in
treated rats (Popovich et al, 1997). In mice, disease
severity was reduced from a 1.6 in control to a 0.6 in treated
animals (Meyer et al, 1996). Other methods of switching the
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CA 02388659 2002-05-31
7
Th1 inflammatory cell response to a Th2 cell type response
have also been extensively studied. One such treatment is
with estrogen. It is known that in pregnant women there is a
switch from a Thl to a Th2 response because of increased
levels of this hormone. Mice treated with estrogen showed
about a 30% reduction of EAE incidence, a delay of disease
onset of about 10 days, and a reduction in disease severity
from a 4.5 in untreated animals to a 1.5 (Ito et al, 2001).
Mesopram, a type IV phosphodiesterase-specific inhibitor, has
also been shown to produce a Thl to Th2 switch. EAE was
prevented in rats treated before the onset of symptoms. Mice
treated starting at the first signs of clinical symptoms
showed a reduction of a mean clinical score of 4.7 in control
animals to a 2.7 in treated animals (Dinter, et al, 2000).
Retinoids, which are ligands of the steroid receptor
superfamily, are also thought to favor Th2 cytokine
production. They are also thought to increase TGF(3 secretion,
which is immunosuppressive. When retinamide was given prior
to EAE induction, control animals reached a mean clinical
score of 3 during relapses, while treated animals went up only
to a grade 2 but came down to a 0.75 with no sign of relapse.
When retinamide was given after disease symptoms appeared,
control animals went from a grade 3.5 to a grade 3 with
relapse while treated animals went from a grade 4 to a grade 2
with no relapse (Racke, et al, 1995). Interferon is another
molecule thought to serve an immunomodulatory function.
Treatment of mice with IFN(3 decreases the amount of
relapse/mouse from 2.17 in controls to 1.17 in treated
animals. Disease severity was also reduced. Control animals
progressed from a 3.5 to a 3.8 while treated animals showed a
mean clinical score of 3.0 reducing down to a 2.5 (Yu, et al,
1996) .
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8
Many signaling pathways are involved in the complex
immune reactions seen in EAE and MS. Various kinases are
needed to turn-on many of these pathways. Tyrosine kinases
mediate the activation of various molecules such as TNFa,
prostaglandins (PGE2), and nitric oxide. Tyrosine kinase-
blockers have therefore also been evaluated as a possible
treatment strategy. These studies have shown about a 600
reduction in incidence of EAE. Also, disease severity was
decreased in animals treated before symptoms were seen from a
mean clinical score of 3 in controls to a 0.5 in those which
received the inhibitor. Mice treated after symptoms occurred
reduced severity from a 3 to a 1.5 (Brenner, et al, 1998).
Recent efforts have also focused on decreasing axonal
damage in EAE. One way to do this is to reduce the amount of
oxidative stress. An inhibitor of inducible nitric oxide
synthase (iNOS) given to mice before EAE symptoms appeared
decreased symptoms from a 1.3 mean score in controls to a 0.5
in treated animals (Brenner et al, 1997). Metallothinine (MT)
is thought to protect cells from reactive oxygen species.
Rats treated with MT-II starting at the day of onset of
symptoms reduced the score from a 4.5 in controls to a 2 in
treated animals(Penkowa and Hidalgo, 2000).
Another way to reduce axonal damage is by blocking
glutamate production, which can damage oligodendrocytes and
myelin. Experiments using the AMPA/kainate glutamate receptor
antagonist NBQX reduced severity from a score of 3 in controls
to a 1.5 in treated animals (Smith et al, 2000; Pitt et al,
2000), while MPQX resulted in a greater reduction from a score
of 3 to a 0.8. Treating mice during recovery reduced the
occurrence of a relapse (Smith et al, 2000).
Of these efforts to develop new treatments for MS, only a
few have been approved and are in use. MS therapies currently
being used consist of immunomodulatory drugs such as
corticosteroids, Interferon beta, and Glatiramer acetate.
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CA 02388659 2002-05-31
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Corticosteroids have anti-inflammatory and immunosuppressive
effects, which also transiently restores the blood-brain
barrier (Noseworthy et al, 2000). They shorten the duration
of the relapse and accelerate recovery. Since they are only
effective as a short-term treatment, they are most commonly
used to treat an acute relapse (Anderson and Goodkin, 1998;
Bansil et al, 1995). Further,the responsiveness to
corticosteroids declines over time, and extended use may lead
to adrenal suppression, cardiovascular collapse and
arrhythmias. (C. F. Lacy, L.L. Armsrtong, M.P. Goldman, L.L.
Lance. Drug information hand book 8th Edition, 2001, 549-551).
Interferon(3 has, been used as a therapy for patients with
active Relapsing/Remitting Multiple Sclerosis (RRMS) since the
1980's. It is recently being used for secondary progressive
patients as well. The exact mode of action of this drug is
not yet known. It is thought to play an immunomodulatory role
by suppressing T cell mediated inflammation (Stinissen et al,
1997) . Recombinant IFNj3 is available in 3 drugs: IFN~i-1b
(Betaseron) and two IFN(3-1a preparations (Avonex and Rebif)
(Polman and Uitedehaag, 2000). These drugs reduce rate of
clinical relapse. However, neutralizing antibodies develop
against these drugs rendering them ineffective with time.
Also, flu-like symptoms are a prominent side effect early on
in the treatment.
Glatiramer acetate (copaxone) is a synthetic co-polymer
of tyrosine, glutamate, alanine and lysine, thought to mimic
MBP and thus, block T cell recognition of MBP (Steinman, et
al, 1994). This drug is therefore beneficial in RRMS but not
progressive MS. This drug also decreases the rate of relapse
and appears to be better tolerated by patients than interferon
therapy. Further, treatment with this drug may cause
cardiovascular problems such as chest pain, flushing and
tachycardia, and respiratory problems such as dyspnea. (C. F.
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CA 02388659 2002-05-31
Lacy, L.L. Armsrtong, M.P. Goldman, L.L. Lance. Drug
information hand book 8t'' Edition, 2001, 777-779)
Recently, another drug that has been approved for the use
in RRMS and secondary progressive MS is mitroxantrone. This
5 drug is used to arrest the cell cycle and prevent cellular
division. It is primarily used in leukemias (Rolak, 2001).
In MS it reduces relapse rate and increases the length between
exacerbations. This drug however has long-term side effects
causing cardiac toxicity. Another treatment that has limits
10 to its usefulness is intravenous immunoglobulin. It acts to
alter the immune system in a beneficial way and it has shown
to cut relapses in half (Rolak et al, 2001). However, the
treatments are very expensive.
As discussed above, there are a few moderately effective
treatments for RRMS and secondary progressive MS that have
shown to reduce both the frequency of the disease and severity
of exacerbations. However, problems still exist in treating
MS, and there are still no proven treatments, for example, for
primary progressive MS. There is therefore a continued need
for improved materials and methods for the treatment of
neurodegenerative diseases such as MS.
The area of MS diagnosis is significantly less developed,
as no measurable biochemical/genetic markers of the disease
state exist. As a result, MS diagnosis relies on examining
the pathology of the affected tissue by Magnetic Resonance
Imaging (MRI) methods. MRI is very costly, and as such its
availability is severely limited, typically leading to long
waiting lists for testing. Increased cost also limits
availability of MRI equipment and expertise to larger
communities, thus necessitating travel for those patients
residing elsewhere. Further, to justify performing such a
costly test, patients are chosen which appear to already
exhibit relatively severe symptoms associated with MS, and as
CA 02388659 2002-05-31
11
such this type of diagnosis is performed significantly later
than disease onset, and thus does not provide the opportunity
for earlier detection and treatment. There therefore further
exists a continued need for improved methods and materials for
the diagnosis and prognostication of neurodegenerative
diseases such as MS.
SUt~IARY OF THE INVENTION
In a first aspect, the invention provides a method of
preventing or treating a neural inflammatory or demyelinating
disease in an animal, said method comprising inhibiting the
activity of a phospholipase AZ in the animal.
In another aspect, the invention further provides a
method for identifying and/or characterizing a compound for
the prevention or treatment of a neural inflammatory or
demyelinating disease, said method comprising assaying the
activity or expression of a phospholipase A2 in the presence of
a test compound,-to identify a compound that inhibits
phospholipase A2 activity or expression, wherein inhibition is
indicative that the test compound may be useful for the
prevention or treatment of a neural inflammatory or
demyelinating disease.
In another aspect, the invention further provides a
method of assessing a neural inflammatory or demyelinating
disease in an animal, said method comprising:
(a) determining a test level of phospholipase A2 protein
or phospholipase A2 encoding mRNA or phospholipase AZ
enzyme activity in tissue or body fluid of the animal;
and
(b) comparing said test level of phospholipase AZ protein
or phospholipase A2 encoding mRNA or phospholipase A2
activity to an established standard; or to a
corresponding level of phospholipase AZ protein or
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CA 02388659 2002-05-31
12
phospholipase A2 encoding mRNA or phospholipase Az enzyme
activity in tissue or body fluid of a control animal; or
to a corresponding level of phospholipase A2 protein or
phospholipase A2 encoding mRNA or phospholipase A2
enzymatic activity in tissue or body fluid obtained from
said animal at an earlier time;
wherein an increase in said test level is indicative of the
neural inflammatory or demyelinating disease. In an
embodiment, the method further comprises the step of assaying
the compounds for activity in the prevention or treatment of a
neural inflammatory or demyelinating disease. In embodiments,
the tissue or body fluid is selected from the group consisting
of blood, plasma, cerebrospinal fluid, endothelia, macrophages
and lymphocytes.
In an embodiment the above-noted method comprises
administering to the animal an effective amount of a
phospholipase AZ inhibitor. In an embodiment the inhibitor is
selected from the group consisting of arachidonic acid
analogues, benzenesulfonamide derivatives, bromoenol lactone,
p-bromophenyl bromide, bromophenacyl bromide,
trifluoromethylketones, sialoglycolipids and proteoglycans.
In further embodiments, the inhibitor is selected from the
group consisting of arachidonyl trifluoromethyl ketone, methyl
arachidonyl fluorophosphonate and palmitoyl trifluoromethyl
ketone.
In an embodiment the method comprises inhibiting the
expression of a phospholipase A2. In an embodiment the method
comprises administering to the animal an effective amount of
an inhibitor of phospholipase A2 expression, such as an
antisense molecule. In an embodiment the antisense molecule
is a nucleic acid that is substantially complementary to a
portion of an mRNA encoding a phospholipase A2. In an
embodiment the antisense molecule is complementary to a
portion of a nucleic acid sequence substantially identical to
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CA 02388659 2002-05-31
13
a sequence selected from the group consisting of SEQ ID NO. 1
and SEQ ID N0. 3. In an embodiment the portion of an mRNA
comprises at least 5 contiguous bases. In an embodiment the
phospholipase A2 is a mammalian phospholipase A2, in a further
embodiment, human phospholipase A2. In an embodiment the
phospholipase Az is a cytosolic phospholipase A2.
In an embodiment the animal is a mammal, in a further
embodiment, a human.
In an embodiment the neural inflammatory or demyelinating
disease is Multiple Sclerosis.
In an embodiment the phospholipase AZ is a cytosolic
phospholipase A2.
The invention further provides uses and commercial
packages (comprising the relevant reagents) and appropriate
instructions to carry out the method) corresponding to the
above-mentioned methods.
Accordingly, in a further aspect, the invention provides
a Use of a phospholipase A2 inhibitor for preventing or
treating a neural inflammatory or demyelinating disease in an
animal.
The invention further provides a use of a phospholipase A2
inhibitor for preparation of a medicament for preventing or
treating a neural inflammatory or demyelinating disease in an
animal.
In a further aspect, the invention provides a commercial
package comprising a phospholipase A2 inhibitor together with
instructions for preventing or treating a neural inflammatory
or demyelinating disease in an animal, or for the preparation
of a medicament for preventing or treating a neural
inflammatory or demyelina'ting disease in an animal.
In an embodiment, the animal is a mammal, in a further
embodiment, a human.
In an embodiment, the neural inflammatory or
demyelinating disease is selected from the group consisting of
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CA 02388659 2002-05-31
14
Multiple Sclerosis, Alzheimer's disease, amyotrophic lateral
sclerosis and stroke.
In an embodiment, the phospholipase AZ is a cytosolic
phospholipase A2.
In an embodiment, the inhibitor is selected from the
group consisting of arachidonic acid analogues,
benzenesulfonamide derivatives, bromoenol lactone, p-
bromophenyl bromide, bromophenacyl bromide,
trifluoromethylketones, sialoglycolipids and proteoglycans.
In an embodiment, the inhibitor is selected from the
group consisting of arachidonyl trifluoromethyl ketone, methyl
arachidonyl fluorophosphonate, palmitoyl trifluoromethyl
ketone.
In an embodiment, the inhibitor is an inhibitor of
phospholipase AZ expression. In an embodiment, the inhibitor
of phospholipase A2 expression is an antisense molecule. In an
embodiment, the antisense molecule is a nucleic acid that is
substantially complementary to a portion of an mRNA encoding a
phospholipase A2. In an embodiment, the antisense molecule is
complementary to a portion of a nucleic acid sequence
substantially identical to a sequence selected from the group
consisting of SEQ ID N0. 1 and SEQ ID N0. 3. In an
embodiment, the portion of an mRNA comprises at least 5
contiguous bases.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l: Schematic illustration of PLAZ enzyme activity.
Figure 2: Endothelial cells in EAE lesions express cPLA2.
Spinal cord tissue of mice with EAE at clinical grades 1-4.
Arrows indicate cPLA2+ elongated cells in EAE lesions. cPLA2
positive cells are seen in grades 1-3. No cPLAZ labeling of
endothelial cells is seen in grade 4 micrograph. Slides were
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CA 02388659 2002-05-31
counterstained with methyl green which gives a grey staining
in the black and white pictures.
Figure 3: Immune cells in EAE infiltrates also express cPLA2.
5 cPLA2+ immune cells in the infiltrates were seen at all
clinical grades. Arrows point to positive immune cells.
Slides counterstained with methyl green.
10 Figure 4: Changes in the number of cPLA2+ endothelial cells in
EAE lesions. High numbers of endothelial cells, between 45%-
85% express cPLA2 during the earlier stages of the disease,
i.e., at grades 1 to 3. The numbers peaked at 85% in grade 3
and reduced to less than 20% at grades 4 and 5.
Figure 5: Changes in the number of cPLA2+ immune cells in EAE
lesions. Between 25$ to 50% of the immune cells in the CNS
infiltrates were cPLA2+ at all clinical grades.
Figure 6: Histogram showing total numbers of immune cells in
EAE lesions. Total number of immune cells infiltrating the
CNS at different clinical grades. The number of cells in EAE
lesions increases at grades 4 and 5.
Figure 7: Total number of cPLA2+ immune cells in EAE lesions
at different clinical grades. The total number of cPLA2
positive immune cells increases at later grades of 4 and 5.
Figure 8: Cell types expressing cPLA2 in EAE lesions
The cell types expressing cPLA2 in the spinal cords were
assessed using double immunofluorescence. GFAP+ astrocytes (row
1), CD34+ endothelial cells (row 2), Mac-1+ macrophages (row 3)
and CD4+ T cells (row 4) were cPLA2+ at and near the EAE
CA 02388659 2002-05-31
16
lesions. Double labelling of these cells is shown in the
column labelled "merge".
Figure 9: Incidence of EAE. In the control group, 200 of
the mice showed clinical signs of EAE induced paralysis. In
contrast to the controls, mice treated with either 2 or 4 mM
AACOCF3 had EAE incidence of 57o and 28~, respectively.
Figure 10: Clinical course of EAE. Graph showing changes in
the clinical course of the disease. EAE was induced in all
groups of mice. Mice in the control group (Ctl) that did not
receive any treatment reached a peak clinical score of almost
3 at days 12-14 during the first paralytic episode. Compared
to the control group, mice treated with 2 and 4 mM AACOCF3 only
reached scores of 1.5 and 0.4, respectively. Furthermore, the
control group relapsed into a second paralytic episode between
days 26 and 34, while the 4mM treated group remained
unaffected.
Figure 11: Effect of delayed (i.e. after the peak of the
first attack of EAE symptoms) PLAZ inhibitor treatment of
mice. Mice that develop a milder form of the disease, i.e.,
reach a mean clinical score of 3, while recovering to a grade
2 on day of treatment, show complete recovery and lack of
subsequent relapses when treated with 4 mM AACOCF3 (treat-gr2).
In contrast control mice that reach a mean clinical score of
3, while recovering to a grade 2 or less after the first
paralytic episode, suffer subsequent paralytic episodes,
reaching a mean clinical score of 2.5 (Ctl-gr2).
Figure 12: Human cPLA2 DNA sequence (GenBank #: M72393; Sharp
et al., 1991).
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CA 02388659 2002-05-31
17
Figure 13: Mouse cPLA2 DNA sequence (GenBank #: M72394;
Nalefski et al., 1994)
DETAILED DESCRIPTION OF THE TNVENTION
Although a variety of environmental factors are thought
to induce the onset of MS in genetically susceptible
individuals, it is proposed herein that these factors likely
trigger the activation of a common mechanism that leads to
infiltration of immune cells into the CNS, neural tissue
damage and myelin breakdown. It is described herein that a
likely candidate that could mediate such a common mechanism is
the enzyme phospholipase A2 (PLA2). One of the metabolic
products of PLAZ is arachidonic acid, which gives rise to
eicosanoids such as prostaglandins, thromboxanes and
leukotrienes that are potent mediators of inflammatory
responses. Another metabolic product of PLA2 is
lysophosphatidylcholine (LPC) which has potent detergent-like
properties. Injection of LPC into the CNS and PNS causes
myelinolysis (Hall, 1993, Jeffery and Blakemore, 1995; Ousman
and David, 2000). LPC is also a chemoattractant for human T
cells and monocytes (Ryborg et al., 1994; Prokazova et al.,
1998). LPC also induces expression of a number of chemokines
and cytokines that are involved in immune cell influx and
activation in the CNS (Ousman and David, 2001). Some of these
cytokines and chemokines are known to induce the expression of
PLA2. Therefore, LPC produced by PLAZ-mediated hydrolysis of
phosphatidylcholine could result in expression of chemokines
and cytokines that induce further expression of PLA2. This
cascade could result in inducing severe inflammation (via
arachidonic acid)and demyelination (via LPC). Blocking
arachidonic acid derivatives such as prostaglandins have been
shown to reduce the severity of EAE (Reder et al., 1994). It
is proposed herein that blocking a more upstream target i.e.,
i .~ i.! ~, i.;~~.i ... ,~, I. ~.. :1 . j,
CA 02388659 2002-05-31
18
PLAZ itself would have a profound effect on the induction and
progression of EAE as it would block the production not only
of arachidonic acid and its derivatives but also the
generation of LPC and LPC-induced chemokine and cytokine
expression.
Phospholipase A2
Phospholipase AZ hydrolyzes the fatty acyl ester bond at
the sn-2 position of glycerophospholipids (Figure 1). The
immediate products of a PLA2-catalyzed reaction are a free
fatty acid (e. g., arachidonic acid), and a lysophospholipid
(e. g., lysophosphatidylcholine). Phospholipase A2 has 2 major
physiological functions: (1) membrane turnovers (2) potent
mediator in the activation of inflammatory processes (Dennis,
1994). Ten different PLA2 have been identified which fall into
two major types: secreted (sPLA2), and cytosolic (cPLA2).
Various forms of PLAZ are found in different tissues and cell
types or are unique to the venom of reptiles and insects.
Secreted PhA2: Several forms of sPLA2 exist all of which have
molecular weights of about 14 kDa. Group IB sPLA2 is the
pancreatic form that is secreted in digestive juices. It is
not expressed in the CNS. Group IIA sPLA2 is produced by many
other cells of the body including neutrophils, thymus, bone
marrow, spleen, astrocytes, Schwann cells, etc., (Kramer et
al., 1989; Komada et al., 1989: Ishizake et al., 1989; Wright
et al., 1989 Murakami et al., 1990: Nakano and Arita, 1990).
Group IIA sPLA2 is detected in exudates from sites of
inflammation or tissue injury such as ascites fluid suggesting
that macrophages are a source (Kramer et al., 1989; Trotter
and Smith, 1986; Forst et al., 1986; Chang et al., 1987;
Seilhamer et al., 1989). Group IIA sPLA2 from various sources
have been purified. It is expressed widely in the brain
i ~ ~ r I,
CA 02388659 2002-05-31
19
(Molloy et al., 1998). Another form of sPLA2, group V is
expressed mainly in the heart, lung and placenta, and in very
low levels in the brain, except in the hippocampus where it
may play a specific physiological role (Molloy et al., 1998).
Group X is found mainly in human leukocytes. Groups IA, IIB
and III are found only in certain venoms, and group IX in the
marine snail (Dennis, 2000). Pro-inflammatory cytokines such
as TNF and IL induce expression of sPLA2 in cultured astrocytes
(Oka and Arita, 1991), chondrocytes (Lyons-Giordano et al.,
1989) and vascular smooth muscle cells, (Nakano et al., 1990;
Arbibe et al., 1997). In addition, human endothelial cells
from the umbilical vein express type II sPLAz when treated with
TNF (Murakami et al., 1993). sPLA2 require millimolar
concentrations of calcium for their activation. US Patent
6,103,469 (Hawkins et al., August 15, 2000) relates to a sPLA2.
The activity of sPLA2 can be blocked by p-bromophenacyl
bromide (Glaser et al., 1993). Other inhibitors are currently
being tested by Eli Lilly in preclinical trials in non-CNS
models of inflammation (Ogata et al., 2001).
Cytosolic PI~A2: Three forms of cPLA2 have been identified in
recent years. The calcium-dependent form of cPLA2 (group IV)
is found in a variety of mammalian cells and tissues (Glaser
et al., 1993). It has a molecular weight of 85 kDa. Group IV
cPLA2 requires micromolar concentrations of calcium and is
widely expressed in the brain (Molloy et al., 1998), as well
as in neutrophils and endothelial cells (Arbibe et al., 1997;
Fujimori et al., 1992; Lautens et al., 1998). It prefers
arachidonic acid at the sn-2 position, which means it is
capable of selectively releasing arachidonic acid (Glaser et
al., 1993). cPLA2 is phosphorylated and its activation
increased by MAP kinase (Lin et al., 1993). Group IV cPLA2 has
been purified from a variety of cellular sources. US Patent
6,242,206 (Choiu et al., June 5, 2001) relates to a cPLA2.
I
I .. ~ ~i J~....6 i ~.~i. ~ ~' ~~
CA 02388659 2002-05-31
cPLA2 expression is increased in neurons in the
hippocampus after transient global ischemia (Owada et al.,
1994). In addition, mice deficient in cPLA2 (group IV) are
resistant to cerebral ischemia (Bonventre et al., 1997) and
5 MPTP neurotoxicity (Klivenyl et al., 1998). Like sPLA2, the
expression of cPLA2 in a variety of cells is increased by pro-
inflammatory cytokines such as TNF, IFN-y, IL-1 and CSF-1
(Hulkower et al., 1992; Goppelt-Struebe and Rehfeldt, 1992;
Lin, Lin and DeWitt, 1992; Xu et al., 1994; Wu et al., 1994).
10 It can be inhibited by arachidonic acid analogues such as
arachadonyl trifluromethylketone (AACOCF3) and methyl
arachidonyl fluorophosphonate (MAFP) (Dennis, 2000: Glaser et
al., 1993). Ross et al., (1995) isolated a 180 kDa calcium-
dependent form of cPLA2 from human brain which could be
15 inhibited by bromophenacyl bromide, as well as, AACOCF3.
Two calcium-independent forms of cPLA2 have also been
isolated from the bovine brain (Hirashima et al., 1992;
Farooqui et al., 1997). The 29 kDa form is inhibited by
sialoglycolipids, and various proteoglycans (Yang et al.,
20 1994). Another 80-85 kDa calcium independent form of cPLA2,
which exists in multimeric form of 300 kDa has been identified
in macrophages. This form can be inhibited by the arachidonic
acid analogue, AACOCF3. Other calcium-independent forms have
been identified in myocardial cells and the brush border of
the intestine (Murakami, Nakatani Atsumi et al., 1997) but
these are not of relevance to the CNS.
The precise physiological role of the various forms of
cPLA2 in the CNS is not known at present. The studies described
herein are particularly interested in the ability of cPLA2 to
induce inflammatory responses via production of arachidonic
acid. This activity of various forms of cPLA2 can be
effectively inhibited by the arachidonic acid analogues AACOCF3
and MAFP (Balsinde et al., 1999). Elevated levels of PLA2 have
been detected in MS tissue in an older study (Woelk and
I ' ~ .. ;I; ~ ~ , ;, I
CA 02388659 2002-05-31
21
Peiler-Ichikawa, 1974), however, this study was done in vitro
utilizing post mortem tissue, and thus provides no indication
of conditions in living tissue. Another study found no change
in the level of secreted PLA2 activity in MS samples versus
controls, and found a decrease in cytosolic PLA2 activity in
samples from MS subjects (Huterer, Tourtellotte and Wherrett,
1995). Furthermore, the downstream products of arachidonic
acid and 5-lipoxygenase, such as leukotriene C4 are elevated in
the CSF of MS patients (Dore-Duffy et al., 1991). Levels of
prostaglandins, which are derived from arachidonic acid by the
action of cyclooxygenase, also correlate with the severity of
MS (Dore-Duffy et al., 1986), and blocking these reduces the
severity of EAE in mice (Reder et al., 1994). In addition,
TNF and IL-(3, which are capable of inducing expression of both
forms of PLAz, are elevated in the CSF of patients with MS
(Hauser et al., 1990).
LPC mediates chemokine and cytokine expression and immune cell
responses
LPC, another metabolic product of PLA2, in addition to
being a strong myelinolytic agent is also a chemoattractant
for T-cells and monocytes and is mitogenic for macrophages
(Ryborg et al., 1994; Prokazova et al., 1998). It has been
found that injection of LPC into the adult mouse spinal cord
leads to the rapid expression of MCP-l, MIP-la, GM-CSF and
TNF-a as determined by RT-PCR (Ousman and David, 2001). The
expression of these chemokines and cytokines mediates the
rapid influx of T-cells and monocytes into the spinal cord,
and to activation of macrophages (Ousman and David, 2000,
2001). These immune cell changes result in rapid
demyelination at the site of LPC injection within the spinal
cord in 4 days. Previous work of the applicants' laboratory
has shown that LPC also induces increased expression of VCAM-1
and ICAM-1 in blood vessels in the mouse spinal cord, as well
CA 02388659 2002-05-31
22
as, induces a marked opening of the blood-brain barrier
(Ousman and David, 2000). These adhesion molecules are
important in mediating the extravasation of leukocytes into
the CNS parenchyma in EAE and are also expressed in active MS
plaques (Lee and Benveniste, 1999; Sobel, Mitchell and
Fondren, 1990; Raine and Cannella, 1992).
Described herein are experiments to assess the expression
of cPLA2 in EAE lesions in the spinal cord in C57BL/6 mice.
This mouse strain has a naturally occurring null mutation for
the major form of sPLAZ (Group IIA) (Kennedy et al., 1995).
Since EAE can be induced in these mice, it is unlikely that
sPLA2 is the only major inducer of the disease. The expression
of cPLA2 was therefore examined in EAE lesions in the spinal
cord of C57BL/6 mice using immunohistochemical techniques. As
a result, it is shown herein that cPLA2 is indeed expressed in
higher amounts in such lesions. Experiments were then carried
out in which the activity of cPLAz was blocked using a chemical
inhibitor. These experiments revealed that blocking cPLA2
prevents the onset and progression of EAE.
Demonstrated herein is an increase in cPLA2 in and around
EAE lesions in C57BL/6 mice that have a natural disruption in
the sPLA2 gene. The increase in cPLA2 was seen in endothelial
cells and astrocytes, whose processes surround blood vessels.
A high level of expression in endothelial cells was seen just
prior to the highest increase in the influx of inflammatory
cells into the spinal cord. cPLAz expression was also seen in
the T cells and macrophages that accumulate at the site of
immune lesions in the spinal cord. Previous studies have
shown an increase of downstream products of PLA2 action such as
prostaglandins and leukotrienes in the CSF of MS patients
(Gallai et al, 1995; Fretland, 1992), however, a role for PLA2
has not been described prior to the studies described herein.
Animal studies using the EAE model to assess the blocking
effects of these downstream products have been shown herein to
..i .. i~ ,....,~~a~ w~ -Ai ~ ~- i1 - i
CA 02388659 2002-05-31
23
reduce the severity of EAE. A prostaglandin El analogue was
shown to delay onset of EAE by a few days and reduce clinical
severity from a mean grade of 2.23 in controls to 0.7 in
treated rats (Reder et al, 1994). A leukotriene inhibitor,
sulfasalazine, also reduced disease incidence in guinea pigs
(Prosiegel et al, 1990). A COX-inhibitor, piroxicam, was
shown to decrease mean clinical score from a 2.8 in untreated
to a 1.5 in treated rats (Weber and Hempel, 1989). In
addition, a dual COX/5-lipoxygenase inhibitor was shown to
reduce the incidence of EAE (Prosiegel et al, 1989). Provided
herein is direct evidence that the use of PLAZ inhibitors
markedly reduces the incidence and severity of EAE. The
incidence of EAE using AACOCF3 was reduced by 72~ in treated
mice. Also, disease severity was reduced from a mean maximal
clinical score of almost 3 in control mice to 0.4 in treated
mice.
As described herein, the effects of blocking PLA2 activity
are not only immunosuppressive, but also prevent myelin
breakdown. The results described herein demonstrate the
effectiveness of this inhibitor in an animal model of MS.
Therefore, blocking PLAZ directly can be used as a new
therapeutic tool for MS. By blocking PLAZ upstream of the
arachadonic acid metabolites, both the inflammatory cascade
and myelin disruption through LPC will be prevented, leading
to a potentially effective treatment for MS.
Accordingly, in an aspect, the invention provides a
method for the prevention and or treatment of neural
inflammatory and/or demyelinating disease in an animal, the
method comprising modulating (in an embodiment, inhibiting)
the activity and/or expression of a phospholipase AZ (PLAZ) in
the animal. Such a method may comprise administering to the
animal an agent capable of modulating PLAZ activity. In cases
involving an inhibition of PLA2 activity, such an agent is a
~i ~ I ~ i j .,. Ky ~ i~ r ~r
CA 02388659 2002-05-31
24
PLA2 inhibitor. Such administration may in embodiments occur
before, at about the time of, or subsequent to the onset of
the disease. An reagent capable of modulating PLA2 activity"
refers to any compound which when introduced into a system
comprising a PLAZ protein, is capable of altering at least one
aspect of PLA2 activity or function. Such an agent may be a
ligand of a PLA2 protein, such as an agonist or antagonist.
Such an agent may act directly on a PLAz protein or indirectly
by modulating a process or activity, which subsequently
results in the modulation of PLAz activity, or may modulate
PLAZ expression. In certain systems (e.g. in vivo), such an
agent may be a prodrug, which is metabolized to an active form
at or prior to its arrival at the site of action.
In another aspect, the invention provides a method for
the diagnosis and/or prognostication of neural inflammatory
and/or demyelinating disease in an animal, the method
comprising determining a level of PLAZ protein or expression or
activity of a PLAZ in a tissue or body fluid obtained from the
animal.
In embodiments, the disease is multiple sclerosis and
related neural diseases. In further embodiments, the disease
is selected from the group consisting of Alzheimer's disease,
amyotrophic lateral sclerosis and stroke. In an embodiment,
the animal is a mammal, in a further embodiment, a human. In
embodiments, the PLA2 is secreted or cytosolic, calcium
dependent or independent. In an embodiment, the PLAZ is of an
average molecular weight of about l4kDa. In an embodiment,
the PLA2 is of an average molecular weight of about 85kDa. In
an embodiment, the PLAZ is cytosolic PLAZ (cPLA2). In an
embodiment, the PLA2 is a calcium-dependent PLA2. In
embodiments, the PLA2 is a type IV PLA2. In embodiments, the
method comprises the modulation of both a secreted and a
cytosolic PLA2. In embodiments, the PLAZ has an activity that
..i . .,iE-..i-:-t,f .. ~, I , t~ s i1
CA 02388659 2002-05-31
generates as a product (a) arachidonic acid (b) lyso-
phosphatidylcholine or (c) both (a) and (b).
Chemokines and cytokines, are thought to mediate (play a
role in) a variety of disease states. In alternative aspects,
5 the invention relates to methods, uses and commercial packages
for immunomodulation(e.g.immunosuppression) and for diagnosis,
prognostication, prevention and/or treatment of T-cell
mediated diseases, including autoimmune diseases,
inflammation, chronic interstitial lung disease, rheumatoid
10 arthritis, inflammatory bowel disease, Crohn's disease,
ulcerative colitis, allergy, contact hypersensitivity,
psoriasis, systemic lupus erythematosus, osteoarthritis, and
diseases mediated by superantigen toxins such as
staphylococcal enterotoxin B, and toxic shock syndrome toxin
15 1.
"Modulation/modulating" as used herein refers to both
upregulation (i.e., activation or stimulation (e.g., by
agonizing or potentiating)) and downregulation (i.e.
inhibition or suppression (e. g., by antagonizing, decreasing
20 or inhibiting)).
A wide variety of alternative genomic approaches are
available to down-regulate the expression of functional PLA2.
For example, in alternative embodiments, transformation of
cells with antisense constructs may be used to inhibit
25 expression of PLA2. Antisense constructs are nucleic acid
molecules that may be transcribed to provide an antisense
molecule that is substantially complementary to all or a
portion of the mRNA encoding PLA2, so that expression of the
antisense construct interferes with the expression of the PLA2.
In an embodiment, the just noted antisense molecule is
antisense to a DNA sequence coding PLA2, in an embodiment, a
human PLA2. Shown in Figure l2 and SEQ ID N0. 1 is a human DNA
i - ,i ~.i, ,. ~, ~ - ~~ - _ - .
CA 02388659 2002-05-31
26
sequence encoding a cPLA2 (Sharp et al., 1991), with the
putative coding sequence shown in SEQ ID N0. 1 and the
corresponding cPLA2 protein sequence shown in SEQ ID N0. 2.
Shown in Figure 13 and SEQ ID N0. 3 is a mouse DNA sequence
encoding a cPLA2 (Nalefski et al., 1994), with the putative
coding sequence shown in SEQ ID N0. 3 and the corresponding
cPLA2 protein sequence shown in SEQ ID NO. 4. In some
embodiments, antisense constructs of the invention may
therefore encode five or more contiguous nucleic acid residues
substantially complimentary to a contiguous portion a nucleic
acid sequence encoding PLA2, such as an mRNA encoding a PLA2,
or said antisense constructs may encode a sequence of five or
more contiguous nucleic acid residues which are antisense to
the DNA sequences in SEQ ID N0. 1 and/or SEQ ID N0. 3.
Substantially complementary nucleic acids are nucleic
acids in which the complement of one molecule is substantially
identical to the other molecule. Two nucleic acid or protein
sequences are considered substantially identical if, when
optimally aligned, they share at least about 70% sequence
identity. In alternative embodiments, sequence identity may
for example be at least 750, at least 80~, at least 85~, at
least 90~, or at least 950. Optimal alignment of sequences for
comparisons of identity may be conducted using a variety of
algorithms, such as the local homology algorithm of Smith and
Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment
algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443,
the search fox similarity method of Pearson and Lipman, 1988,
Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised
implementations of these algorithms (such as GAP, BESTFIT,
FASTA and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, Madison, WI, U.S.A.). Sequence
identity may also be determined using the BLAST algorithm,
described in Altschul et al., 1990, J. Mol. Biol. 215:403-10
(using the published default settings). Software for
I . I ~--~ ..n.f ~" ~ 41 - - a
CA 02388659 2002-05-31
27
performing BLAST analysis may be available through the
National Center for Biotechnology Information (through the
Internet at ht~ //www.ncbi.nlm.nih.~ov/). The BLAST algorithm
involves first identifying high scoring sequence pairs (HSPs)
by identifying short words of length W in the query sequence
that either match or satisfy some positive-valued threshold
score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighbourhood word
score threshold. Initial neighbourhood word hits act as seeds
for initiating searches to find longer HSPs. The word hits are
extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of
the word hits in each direction is halted when the following
parameters are met: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment. The BLAST program may
use as defaults a word length (W) of 11, the BLOSUM62 scoring
matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci.
USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a
comparison of both strands. One measure of the statistical
similarity between two sequences using the BLAST algorithm is
the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. In
alternative embodiments of the invention, nucleotide or amino
acid sequences are considered substantially identical if the
smallest sum probability in a comparison of the test sequences
is less than about 1, preferably less than about 0.1, more
CA 02388659 2002-05-31
28
preferably less than about 0.01, and most preferably less than
about 0.001.
An alternative indication that two nucleic acid sequences
are substantially complementary is that the two sequences
hybridize to each other under moderately stringent, or
preferably stringent, conditions. Hybridization to filter-
bound sequences under moderately stringent conditions may, for
example, be performed in 0.5 M NaHP09, 7$ sodium dodecyl
sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x
SSC/0.1$ SDS at 42°C (see Ausubel, et al. (eds), 1989, Current
Protocols in Molecular Biology, Vol. 1, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
2.10.3). Alternatively, hybridization to filter-bound
sequences under stringent conditions may, for example, be
performed in 0.5 M NaHP04, 7$ SDS, 1 mM EDTA at 65°C, and
washing in 0.1 x SSC/0.1$ SDS at 68°C (see Ausubel, et al.
(eds), 1989, supra). Hybridization conditions may be modified
in accordance with known methods depending on the sequence of
interest (see Tijssen, 1993, Laboratory Techniques in
Biochemistry and Molecular Biology -- Hybridization with
1~'ucleic Acid Probes, Part I, Chapter 2 "Overview of principles
of hybridization and the strategy of nucleic acid probe
assays", Elsevier, New York). Generally, stringent conditions
are selected to be about 5°C lower than the thermal melting
point for the specific sequence at a defined ionic strength
and pH.
In alternative embodiments, the invention provides
antisense molecules and ribozymes for exogenous administration
to bind to, degrade and/or inhibit the translation of PLAZ
mRNA. Examples of therapeutic antisense oligonucleotide
applications, incorporated herein by reference, include: U.S.
Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No.
5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617,
issued Feb. 11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24,
i1 ;i~..'a,e .~: I,., .~I . 1i
CA 02388659 2002-05-31
29
1992; U.S. Pat. No. 5,004,810, issued Apr. 2, 1991; U.S. Pat.
No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No. 4,806,463,
issued Feb. 21, 1989; U.S. Pat. No. 5,286,717 issued Feb. 15,
1994; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423:
BioWorld Today, Apr. 29, 1994, p. 3.
Preferably, in antisense molecules, there is a sufficient
degree of complementarity to the PLAZ mRNA to avoid non-
specific binding of the antisense molecule to non-target
sequences under conditions in which specific binding is
desired, such as under physiological conditions in the case of
in vivo assays or therapeutic treatment or, in the case of in
vitro assays, under conditions in which the assays are
conducted. The target mRNA for antisense binding may include
not only the information to encode a protein, but also
associated ribonucleotides, which for example form the 5'-
untranslated region, the 3'-untranslated region, the 5' cap
region and intron/exon junction ribonucleotides. A method of
screening for antisense and ribozyme nucleic acids that may be
used to provide such molecules as PLA2 inhibitors of the
invention is disclosed in U.S. Patent No. 5,932,435 (which is
incorporated herein by reference).
Antisense molecules (oligonucleotides) of the invention
may include those which contain intersugar backbone linkages
such as phosphotriesters, methyl phosphonates, short chain
alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages,
phosphorothioates and those with CHZ--NH--0--CHZ, CH2--N (CH3) --
O--CHZ (known as methylene(methylimino) or MMI backbone), CHZ-
-0--N ( CH3 ) --CH2, CH2--N ( CH3 ) --N ( CH3 ) --CH2 and 0--N ( CH3 ) --CH2 --
CH2 backbones (where phosphodiester is 0--P--0--CHZ) .
Oligonucleotides having morpholino backbone structures may
also be used (U. S. Pat. No. 5,034,506). In alternative
embodiments, antisense oligonucleotides may have a peptide
nucleic acid (PNA, sometimes referred to as "protein nucleic
i ~ ~.,~.~iri ... .j~s I ~ y
CA 02388659 2002-05-31
acid") backbone, in which the phosphodiester backbone of the
oligonucleotide may be replaced with a polyamide backbone
wherein nucleosidic bases are bound directly or indirectly to
aza nitrogen atoms or methylene groups in the polyamide
5 backbone (Nielsen et al., 1991, Science 254:1497 and U.S. Pat.
No. 5,539,082). The phosphodiester bonds may be substituted
with structures that are chiral and enantiomerically specific.
Persons of ordinary skill in the art will be able to select
other linkages for use in practice of the invention.
10 Oligonucleotides may also include species which include
at least one modified nucleotide base. Thus, purines and
pyrimidines other than those normally found in nature may be
used. Similarly, modifications on the pentofuranosyl portion
of the nucleotide subunits may also be effected. Examples of
15 such modifications are 2'-O-alkyl- and 2'-halogen-substituted
nucleotides. Some specific examples of modifications at the 2'
position of sugar moieties which are useful in the present
invention are OH, SH, SCH3, F, OCN, 0 (CH2)" NH2 or 0 (CH2) n CH3
where n is from 1 to about 10; C1 to Clo lower alkyl,
20 substituted lower alkyl, alkaryl or aralkyl; C1; Br; CN; CF3 ;
OCF3 ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3 ; S02
CH3; ONOz ; NOZ ; N3; NH2; heterocycloalkyl; heterocycloalkaryl;
aminoalkylamino; polyalkylamino; substituted silyl; an RNA
cleaving group; a reporter group; an intercalator; a group for
25 improving the pharmacokinetic properties of an
oligonucleotide; or a group for improving the pharmacodynamic
properties of an oligonucleotide and other substituents having
similar properties. One or more pentofuranosyl groups may be
replaced by another sugar, by a sugar mimic such as cyclobutyl
30 or by another moiety which takes the place of the sugar.
I1 1 ~~, ,gyp ~ ~I I
CA 02388659 2002-05-31
31
In some embodiments, the antisense oligonucleotides in
accordance with this invention may comprise from about 5 to
about 100 nucleotide units. As will be appreciated, a nucleotide
unit is a base-sugar combination (or a combination of analogous
structures) suitably bound to an adjacent nucleotide unit
through phosphodiester or other bonds forming a backbone
structure.
A number of PLAZ inhibitors have been described. Such
inhibitors include, but are not limited to arachidonic acid .
analogues such as the arachidonic acid analogues AACOCF3 and
MAFP described above, sialoglycolipids, proteoglycans and p-
bromophenyl bromide as noted above, and certain
benzenesulfonamide derivatives (Oinuma et al, 1991; European
patent application No. 468 054). Further, bromoenol lactone
and trifluoromethyl ketones (such as palmitoyl trifluoromethyl
ketone, arachidonyl trifluoromethyl ketone) have been reported
as inhibitors of Ca++-.independent PLA2 (Ackermann et al, 1995)
and cPLA2 (Street et al, 1993) activity as well as
bromophenacyl bromide. Accordingly, the invention further
provides methods and uses of such compounds for the inhibition
of inflammatory and/or demyelinating neural disease, such as
MS and related neurodegenerative disease.
In another aspect, the invention relates to the use of a
PLA2 as a target in screening assays that may be used to
identify compounds that are useful for the prevention or
treatment of inflammatory and/or demyelinating neural disease,
such as MS and related neurodegenerative disease. In some
embodiments, such an assay may comprise the steps of
a) providing a test compound:
b) providing a source of enzymatically active PLA2; and
c) measuring PLAZ activity in the presence versus the absence
of the test compound, wherein a lower measured activity in the
presence of the test compound indicates that the compound is
an inhibitor of PLA2 and is useful for the prevention and/or
i ~~~ ~ ~G~ ~~~ a~~ l .I j~ I
CA 02388659 2002-05-31
32
treatment of inflammatory and/or demyel.inating neural disease,
such as MS and related neurodegenerative disease.
In another aspect, the invention relates to the use of a
PLAz as a target in screening assays that may be used to
identify compounds that are useful for the prevention or
treatment of inflammatory and/or demyelinating neural disease,
such as MS and related neurodegenerative disease. In some
embodiments, such an assay may comprise the steps of
a) providing a test compound;
b) providing a source of enzymatically active PLA2;
c) providing a substrate for the PLA2;
d) assaying the activity of the PLAZ on the substrate in
the presence of the compound, to identify compounds that
inhibit the PLA2, wherein said compound is useful for the
prevention or treatment of inflammatory and/or demyelinating
neural disease, such as MS and related neurodegenerative
disease. In an embodiment the substrate is a phospholipid
(e.g. phosphatidylcholine) comprising an arachidonoyl group at
the sn-2 position.
The invention also relates to similar assays based on the
detection on the expression of PLA2 and PLAZ protein levels,
which can be detected fro example by immunoassay methods or
specific labeling methods, or via a reporter-based assay as
noted below.
Such assays may further comprise the step of assaying the
compounds for the reduction, abrogation or reversal of EAE
symptoms. Such assays may be utilized to identify compounds
that modulate expression of the PLA2 gene, or compounds that
modulate the activity of the expressed enzyme.
Screening assays of the invention may also be utilized to
identify and/or characterize a compound for inhibiting
demyelination. Therefore, the invention further provides a
method for identifying and/or characterizing a compound for
inhibiting demyelination, said method comprising assaying the
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CA 02388659 2002-05-31
33
activity of a PLA2 in the presence of a test compound, to
identify a compound that inhibits the PLA2, wherein inhibition
is indicative that the test compound may be useful for
inhibiting demyelination. In an embodiment, the just noted
method may further comprise assaying the compound for
inhibition of demyelination.
The above-noted assays may be applied to a single test
compound or to a plurality or "library" of such compounds
(e.g. a combinatorial library). Any such compounds may be
utilized as lead compounds and further modified to improve
their therapeutic, prophylactic and/or pharmacological
properties for the prevention and treatment of inflammatory
and/or demyelinating neural disease, such as MS and related
neurodegenerative disease.
Such assay systems may comprise a variety of means to
enable and optimize useful assay conditions. Such means may
include but are not limited to: suitable buffer solutions, for
example, for the control of pH and ionic strength and to
provide any necessary components for optimal PLA2 activity and
stability (e. g. protease inhibitors), temperature control
means for optimal PLA2 activity and or stability, and detection
means to enable the detection of the PLA2 reaction product,
e.g. arachidonic acid and/or LPC. A variety of such detection
means may be used, including but not limited to one or a
combination of the following: radiolabelling (e. g. 32P, 1QC,
3H), antibody-based detection, fluorescence, chemiluminescence,
spectroscopic methods (e. g. generation of a product with
altered spectroscopic properties), various reporter enzymes or
proteins (e. g. horseradish peroxidase, green fluorescent
protein), specific binding reagents (e. g.
biotin/(strept)avidin), and others.
The assay may be carried out in vitro utilizing a source
of PLA2 which may comprise naturally isolated or recombinantly
produced PLA2, in preparations ranging from crude to pure.
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CA 02388659 2002-05-31
34
Recombinant PLAZ may be produced in a number of prokaryotic or
eukaryotic expression systems, which are well known in the art
(see for example U.S. Patent No. 5,354,677 [Knopf et al.,
October 11, 1994] for the recombinant expression of cPLA2.
Such assays may be performed in an array format. In certain
embodiments, one or a plurality of the assay steps are
automated.
A homolog, variant and/or fragment of PLA2 which retains
activity may also be used in the methods of the invention.
Homologs include protein sequences, which are substantially
identical to the amino acid sequence of a PLA2, sharing
significant structural and functional homology with a PLA2.
Variants include, but are not limited to, proteins or
peptides, which differ from a PLAZ by any modifications, and/or
amino acid substitutions, deletions or additions.
Modifications can occur anywhere including the polypeptide
backbone, (i.e. the amino acid sequence), the amino acid side
chains and the amino or carboxy termini. Such substitutions,
deletions or additions may involve one or more amino acids.
Fragments include a fragment or a portion of a PLAz or a
fragment or a portion of a homolog or variant of a PLA2.
The assay may in an embodiment be performed using an
appropriate host cell comprising PLA2 as a source of PLA2.
Such a host cell may be prepared by the introduction of DNA
encoding PLA2 into the host cell and providing conditions for
the expression of PLA2. Such host cells may be prokaryotic or
eukaryotic, bacterial, yeast, amphibian or mammalian.
A number of methods for measuring PLA2 activity may be
utilized, such as those described by Reynolds et al. (1994)
and Currie et al. (1994) or in US Patent No. 5,464,754 (Dennis
et al., November 7, 1995).
In another embodiment of the invention, a reporter assay-
based method of selecting agents which modulate PLAZ expression
is provided. The method includes providing a cell comprising
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CA 02388659 2002-05-31
a nucleic acid sequence comprising a PLA2 transcriptional
regulatory sequence operably-linked to a suitable reporter
gene. The cell is then exposed to the agent suspected of
affecting PLA2 expression (e.g. a test compound) and the
5 transcription efficiency is measured by the activity of the
reporter gene. The activity can then be compared to the
activity of the reporter gene in cells unexposed to the agent
in question. Suitable reporter genes include but are not
limited to beta-D galactosidase, luciferase, chloramphenicol
10 acetyltransferase and fluorescent green protein.
"Transcriptional regulatory sequence" is a generic term
that refers to DNA sequences, such as initiation and
termination signals, enhancers, and promoters, splicing
signals, polyadenylation signals which induce or control
15 transcription of protein coding sequences with which they are
operably linked. A first nucleic acid sequence is "operably-
linked" with a second nucleic acid sequence when the first
nucleic acid sequence is placed in a functional relationship
with the second nucleic acid sequence. For instance, a
20 promoter is operably-linked to a coding sequence if the
promoter affects the transcription or expression of the coding
sequences. Generally, operably-linked DNA sequences are
contiguous and, where necessary to join two protein coding
regions, in reading frame. However, since enhancers generally
25 function when separated from the promoters by several
kilobases and intronic sequences may be of variable lengths,
some polynucleotide elements may be operably-linked but not
contiguous. In another embodiment, the construct may comprise
an in frame fusion of a suitable reporter gene within the open
30 reading frame of a PLAz gene. The reporter gene may be chosen
as such to facilitate the detection of its expression, e.g. by
the detection of the activity of its gene product. Such a
reporter construct may be introduced into a suitable system
capable of exhibiting a change in the level of expression of
CA 02388659 2002-05-31
36
the reporter gene in response to exposure a suitable
biological sample. Such an assay would also be adaptable to a
possible large scale, high-throughput, automated format, and
would allow more convenient detection due to the presence of
its reporter component.
The above-described assay methods may further comprise
determining whether any compounds so identified can be used
for the prevention or treatment of inflammatory and/or
demyelinating neural disease, such as MS and related
neurodegenerative disease, such as examining their effects)
on inflammatory cell influx and demyelination in lesions in
the EAE animal model system.
In various embodiments, PLA2 inhibitors, or
pharmaceutically-acceptable salts thereof, may be used
therapeutically in formulations or medicaments to prevent or
treat inflammatory and/or demyelinating neural disease, such
as MS and related neurodegenerative disease. The invention
provides corresponding methods of medical treatment, in which
a therapeutic dose of a PLA2 inhibitor is administered in a
pharmacologically acceptable formulation. Accordingly, the
invention also provides therapeutic compositions comprising a
PLAZ inhibitor and a pharmacologically acceptable excipient or
carrier. The therapeutic composition may be soluble in an
aqueous solution at a physiologically acceptable pH.
The invention provides pharmaceutical compositions
(medicaments) containing (comprising) PLA2 inhibitors. In one
embodiment, such compositions include a PLA2 inhibitor in a
therapeutically or prophylactically effective amount
sufficient to treat inflammatory and/or demyelinating neural
disease, such as MS and related neurodegenerative disease.
The invention further provides a use of a PLA2 inhibitor
or a composition comprising a PLAZ inhibitor for the prevention
and/or treatment of inflammatory and/or demyelinating neural
disease, or for the preparation of a medicament for the
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CA 02388659 2002-05-31
37
prevention and/or treatment of inflammatory and/or
demyelinating neural disease.
A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to
achieve the desired therapeutic result, such as reduction of
inflammatory and/or demyelinating neural disease, such as MS
and related neurodegenerative disease progression. A
therapeutically effective amount of PLA2 inhibitor may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the compound to
elicit a desired response in the individual. Dosage regimens
may be adjusted to provide the optimum therapeutic response. A
therapeutically effective amount is also one in which any
toxic or detrimental effects of the compound are outweighed by
the therapeutically beneficial effects. A "prophylactically
effective amount" refers to an amount effective, at dosages
and for periods of time necessary, to achieve the desired
prophylactic result, such as preventing or inhibiting the rate
of inflammatory and/or demyelinating neural disease, such as
MS and related neurodegenerative disease onset or progression.
A prophylactically effective amount can be determined as
described above for the therapeutically effective amount. For
any particular subject, specific dosage regimens may be
adjusted over time according to the individual need and the
professional judgement of the person administering or
supervising the administration of the compositions.
As used herein "pharmaceutically acceptable carrier" or
"excipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are
physiologically compatible. In one embodiment, the carrier is
suitable for parenteral administration. Alternatively, the
carrier can be suitable for intravenous, intraperitoneal,
intramuscular, sublingual or oral administration.
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CA 02388659 2002-05-31
38
Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersion. The use of such media and agents for
pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into
the compositions.
Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The
proper fluidity can be maintained, for example, by the use of
a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which
delays absorption, for example, monostearate salts and
gelatin. Moreover, the PLA2 inhibitors can be administered in a
time release formulation, for example in a composition which
includes a slow release polymer. The active compounds can be
prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation,
including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as
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CA 02388659 2002-05-31
39
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polylactic,
polyglycolic copolymers (PLG). Many methods for the
preparation of such formulations are patented or generally
known to those skilled in the art.
Sterile injectable solutions can be prepared by
incorporating the active compound (e.g. PLAz inhibitor) in the
required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from
a previously sterile-filtered solution thereof. In accordance
with an alternative aspect of the invention, a PLAZ inhibitor
may be formulated with one or more additional compounds that
enhance the solubility of the PLA2 inhibitor.
In accordance with another aspect of the invention,
therapeutic compositions of the present invention, comprising
a PLAZ inhibitor, may be provided in containers or commercial
packages which further comprise instructions for use of the
PLA2 inhibitor for the prevention and/or treatment of
inflammatory and/or demyelinating neural disease, such as MS
and related neurodegenerative disease.
Accordingly, the invention further provides a commercial
package comprising a PLA2 inhibitor or the above-mentioned
composition together with instructions for the prevention
and/or treatment of inflammatory and/or demyelinating neural
disease, such as MS and related neurodegenerative disease.
n.
CA 02388659 2002-05-31
The positive correlation of PLAZ expression with EAE
indicates that the assessment of the level of PLAZ protein or a
nucleic acid (e. g. an mRNA) encoding PLAZ or PLAZ enzyme
activity is useful for the diagnosis or prognostication of
5 inflammatory and/or demyelinating neural disease, such as MS
and related neurodegenerative disease. PLAz mRNA levels may be
assessed by methods known in the art such as Northern analysis
or RT-PCR (see for example Sambrook et al (1989) Molecular
Cloning: A Laboratory Manual (second edition), Cold Spring
10 Harbor Laboratory Press, Cold Spring Harbor, New York, USA).
The level of PLAZ protein or PLA2 encoding mRNA or PLAz
enzyme activity may be measured in a variety of tissues and
body fluids including but not limited to blood, plasma,
cerebrospinal fluid, endothelial cells, macrophages and
15 lymphocytes.
In an embodiment, the level of PLA2 protein or PLA2
encoding mRNA or PLA2 enzyme activity measured in an animal to
be tested may be compared to an established standard of PLA2
protein or PLA2 encoding mRNA or PLA2 enzyme activity.
20 In an embodiment, the level of PLA2 protein or PLA2
encoding mRNA or PLA2 enzyme activity measured in an animal to
be tested may be compared to a corresponding level of PLA2
protein or PLA2 encoding mRNA or PLAZ enzyme activity measured
in tissue or body fluid of a control animal. In an
25 embodiment, the control animal is an age- and/or weight-
matched animal.
In an embodiment, the level of PLA2 protein or PLAZ
encoding mRNA or PLA2 enzyme activity measured in an animal to
be tested may be compared to a corresponding level of PLA2
30 protein or PLAz encoding mRNA or PLAZ enzyme activity measured
in tissue or body fluid of the same animal at an earlier time,
and such a method is used to prognosticate inflammatory and/or
demyelinating neural disease, such as MS and related
neurodegenerative disease.
i '
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CA 02388659 2002-05-31
41
According to a further aspect of the present invention, a
commercial package is provided for the diagnosis or
prognostication of inflammatory and/or demyelinating neural
disease, such as MS and related neurodegenerative disease in
an animal. The commercial package comprising means for the
assessment of the level of PLA2 protein or PLA2 encoding mRNA
or PLAZ enzyme activity in a tissue or body fluid of the animal
together with instructions for the diagnosis or
prognostication of inflammatory and/or demyelinating neural
disease, such as MS and related neurodegenerative disease.
The invention further relates to the use of anti-PLA2
antibodies for prophylactic, therapeutic, diagnostic and/or
prognostic uses. With regard to therapeutic uses, an anti-PLAZ
antibody may be used which is capable of modulating (e. g.
inhibiting) the binding and/or catalytic activity of a PLA2.
With regard to diagnostic and prognostic uses, an anti-PLA2
antibody may be used for detecting PLA2 and, in embodiments,
quantifying the level thereof, in a sample, such as a tissue
or body fluid and lymphocytes. Such detection may further be
used for imaging methods.
Some anti-PLA2 antibodies have already been described,
such as the anti-cPLA2 utilized in the Examples below . To
prepare such antibodies, a PLA2 or fragment/homolog/variant
thereof may be used to immunize a small mammal, e.g., a mouse
or a rabbit, in order to raise antibodies which recognize a
PLAz. An anti-PLAZ antibody may be either polyclonal or
monoclonal. Methods to produce polyclonal or monoclonal
antibodies are well known in the art. For a review, see
Harlow and Lane (1988) and Yelton et al. (1981), both of which
are herein incorporated by reference. For monoclonal
antibodies, see Kohler and Milstein (1975), herein
incorporated by reference.
Antibodies may be recombinant, e.g., chimeric (e. g.,
constituted by a variable region of murine origin associated
i, ~ n i i
CA 02388659 2002-05-31
42
with a human constant region), humanized (a human
immunoglobulin constant backbone together with hypervariable
region of animal, e.g., murine, origin), and/or single chain.
Both polyclonal and monoclonal antibodies may also be in the
form of immunoglobulin fragments, e. g. , F (ab) ' 2, Fab or Fab'
fragments. The antibodies may be of any isotype, e.g., IgG or
IgA, and polyclonal antibodies are of a single isotype or a
mixture of isotypes.
Anti-PLAz antibodies may be produced and identified using
standard immunological assays, e.g., Western blot analysis,
dot blot assay, or ELISA (see, e.g., Coligan et al. (1994),
herein incorporated by reference). The antibodies are used in
diagnostic methods to detect the presence of a PLA2 in a
sample, such as a biological sample.
Accordingly, a further aspect of the invention provides a
method for assessing an inflammatory and/or demyelinating
neural disease, such as MS and related neurodegenerative
disease, in an animal, based on detecting the presence of a
PLA2 in a biological sample obtained from the animal, by
contacting the biological sample with an antibody capable of
recognizing a PLA2, such that an immune complex is formed, and
by detecting such complex to indicate the presence of PLA2 in
the sample.
Those skilled in the art will readily understand that the
immune complex is formed between a component of the sample and
the antibody, and that any unbound material is removed prior
to detecting the complex. It is understood that such an
antibody is used for screening a sample, such as plasma,
lymphocytes, macrophages, cerebrospinal fluid, urine, saliva,
and endo- or epi-thelia for the presence of PLA2.
For diagnostic applications, the reagent (i.e., an anti-
PLAZ antibody) is either in a free state or immobilized on a
solid support, such as a tube, a bead, or any other
conventional support used in the field. Immobilization is
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CA 02388659 2002-05-31
43
achieved using direct or indirect means. Direct means include
passive adsorption (non-covalent binding) or covalent binding
between the support and the reagent. By "indirect means" is
meant that an anti-reagent compound that interacts with a
reagent is first attached to the solid support. Indirect
means may also employ a ligand-receptor system, for example,
where a molecule such as a vitamin is grafted onto the reagent
and the corresponding receptor immobilized on the solid phase.
This is illustrated by the biotin-(strept)avidin system.
Alternatively, a peptide tail is added chemically or by
genetic engineering to the reagent and the grafted or fused
product immobilized by passive adsorption or covalent linkage
of the peptide tail.
Such diagnostic agents may be included in a commercial
package or kit which also comprises instructions for use. The
reagent is labeled with a detection means which allows for the
detection of the reagent when it is bound to its target. The
detection means may be a 'fluorescent agent such as fluorescein
isocyanate or fluorescein isothiocyanate, or an enzyme such as
horseradish peroxidase or luciferase or alkaline phosphatase,
or a radioactive element such as lzSI or 5lCr.
A further aspect of the present invention is a diagnostic
imaging method, which comprises introducing into a biological
system, an anti-PLAZ antibody, which is used in conjunction
with an appropriate detection system to identify areas where
PLA2 is present or absent.
The invention further relates to the role of PLA2 in a
variety of in vitro and in vivo inflammatory and/or
demyelinating neural disease systems, such as MS and related
neurodegenerative disease model systems, such as the EAE model
system, and the use of such systems for inflammatory and/or
demyelinating neural disease, such as MS and related
neurodegenerative disease research. Accordingly, the
invention provides a variety of in vitro and in vivo model
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CA 02388659 2002-05-31
44
systems for the study of the mechanisms of the development and
progression of inflammatory and/or demyelinating neural
disease, such as MS and related neurodegenerative disease, and
for the development and characterization of materials and
methods for the prevention, treatment, and/or diagnosis of
inflammatory and/or demyelinating neural disease, such as MS
and related neurodegenerative disease. In an embodiment, such
a system comprises a mutation or disruption in a PLA2 gene or
other means of PLAZ inactivation. In embodiments, the PLAZ
gene encodes a PLA2 which is cytosolic or secreted, calcium
dependent or independent. In an embodiment, the PLA2 is a
cytosolic PLA2. In an embodiment, both copies of the gene are
mutated or disrupted. The system may comprise a transgenic
non-human mammal, such as a rodent, such as a mouse.
Applicants have determined that immune cell influx and
demyelination at neural lesions correlate with PLA2 expression
and activity. Accordingly, the invention further provides a
method of inhibiting immune cell influx and demyelination at
neural lesions in an biological system, via inhibiting the
activity and/or expression of a PLAz in said system. The
invention further provides a use of a PLA2 inhibitor for the
inhibition of immune cell influx and/or demyelination at
neural lesions in a biological system, or for the preparation
of a medicament for the inhibition of immune cell influx
and/or demyelination at neural lesions in a biological system.
The invention further provides a method of assessing immune
cell influx and/or demyelination at neural lesions in a
biological system, the method comprising:
(a) determining a test level of PLA2 protein or PLA2
encoding mRNA or PLA2 enzyme activity in said systems and
(b) comparing said test level of PLAZ protein or PLA2
encoding mRNA or PLA2 activity to an established standard;
or to a corresponding level of PLAZ protein or PLAz
encoding mRNA or PLA2 enzyme activity in a control system;
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CA 02388659 2002-05-31
or to a corresponding level of PLAZ protein or PLAZ
encoding mRNA or PLAZ enzymatic activity determined in
said system at an earlier time;
wherein an increase in said test level is indicative of immune
5 cell influx and/or demyelination at neural lesions.
The invention further provides a commercial package
comprising a PLAZ inhibitor together with instructions for
inhibiting immune cell influx and/or demyelination at neural
lesions. The invention further provides a commercial package
10 comprising means for the assessment of the level of PLA2 or
PLAZ encoding mRNA or PLA2 enzyme activity in a biological
system together with instructions for assessing immune cell
influx and/or demyelination at neural lesions in biological
system.
15 In embodiments, the above noted biological system is a
mammal, in a further embodiment, a human.
Although various embodiments of the invention are
disclosed herein, many adaptations and modifications may be
made within the scope of the invention in accordance with the
20 common general knowledge of those skilled in this art. Such
modifications include the substitution of known equivalents
for any aspect of the invention in order to achieve the same
result in substantially the same way. Numeric ranges are
inclusive of the numbers defining the range. In the claims,
25 the word "comprising" is used as an open-ended term,
substantially equivalent to the phrase "including, but not
limited to". The following examples are illustrative of
various aspects of the invention, and do not limit the broad
aspects of the invention as disclosed herein.
EXAMP?~ES
Example 1: Materials and Methods
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CA 02388659 2002-05-31
46
Generation of EAE: EAE was induced in female C57BL/6 mice (18-
20g) by subcutaneous injections of 50~g of myelin
oligodendrocyte glycoprotein (MOG3s-ss-MEVGWYRSPFSRVVHLYRNGK
[SEQ ID N0. 5] ) (Sheldon Biotechnology Centre, McGill
University) in Complete Freund's Adjuvant (Incomplete Freund's
adjuvant containing lmg heat inactivated Mycobacterium
tuberculosis (Difco Labs)). An intravenous injection of 200
ng of pertussis toxin (List Biologicals) was also administered
on days 0 and 2. The mice were monitored clinically for EAE
symptoms daily using the following 5-point scale:
Grade 0 = normal (no clinical signs).
Grade 1 = flaccid tail.
Grade 2 = flaccid tail and mild hindlimb weakness (fast
righting after mice are placed on their backs).
Grade 3 = flaccid tail and severe hindlimb weakness (slow
righting after mice are placed on their backs).
Grade 4 = flaccid tail and hindlimb paralysis.
Grade 5 = flaccid tail, hindlimb paralysis plus forelimb
weakness/moribund.
Immunohistochemistry: The mice at different clinical grades
were deeply anesthetized and perfused via the heart with 0.1 M
phosphate buffer (pH 7.2) followed by perfusion with 40
paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The
spinal cords of the mice were post- fixed for an hour in the
same fixative, and then cryoprotected overnight in 30~ sucrose
in phosphate buffered saline (PBS). Cryostat sections (14~,m)
of cross sections of the cervical, thoracic and lumbar spinal
cord were incubated in 0.1% H202 to remove endogenous
peroxidases, and then blocked in O.lo Triton-X 100 and 2%
normal goat serum for 5 hours. Tissues were then incubated
with an antibody against cPLA2 (polyclonal rabbit anti-cPLA2 -
Santa Cruz Biotech) overnight. Tissue sections were then
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CA 02388659 2002-05-31
47
incubated with a biotinylated goat anti-rabbit antibody and
then washed and incubated with the avidin-biotin complex
conjugated to horseradish peroxidase (Vectastain kit). The
staining was visualized using diaminodibenzidine (Sigma) using
protocols described previously (Ousman and David, 2000).
Sections were counterstained with 3~ methyl green, and then
dehydrated in ethanol. The slides were cover slipped in
Permount.
Double Immunofluorescence: Cryostat sections of tissue
obtained by perfusion as described above were blocked in 0.1%
Triton-X 100 and 2o normal goat serum and then incubated
overnight with an antibody against cPLA2 (same as that
described above) combined with either antibodies specific for
astrocytes (mouse anti-GFAP- Sigma), endothelial cells (rat
anti- CD34- BD PharMingen), T cells (rat anti CD4-
PharMingen), or macrophages (monoclonal antibody Mac-1).
Tissue sections were then washed and incubated with a
biotinylated goat anti rabbit secondary antibody combined
with the appropriate goat anti-rat/mouse rhodamine-conjugated
secondary antibody. Tissue sections were then washed and
incubated with fluorescein-conjugated steptavidin. The slides
were washed and cover slipped in phenylenediamine containing
mounting medium.
Quantification: Counts were done using an ocular grid. For
the immunoperoxidase stained sections, two cPLA2+ cell types
were counted: round cells (immune cells in the infiltrate at
and near EAE lesions) and elongated cells (endothelial cells).
Three levels of the spinal cord (cervical, thoracic and
lumbar) were quantified for 3 animals in each grade (1-5).
Counts were made on three sections at least 45~m apart. The
positive cells were taken as a percentage per lesion.
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CA 02388659 2002-05-31
48
Treatment of EAE-induced mice with cPLA2 inhibitors: EAE was
induced in C57BL/6 mice as mentioned above. At days 0 and 2 a
501 intravenous injection of either 2mM or 4mM arachidonyl
trifluoromethyl ketone (AACOCF3-Cayman Chemicals) diluted in
1% DMSO buffer was administered. This was followed on
alternated days by intraperitoneal injections of 2001 of the
same inhibitor at 2 or 4 mM concentrations until day 24. The
mice were scored clinically based on the scoring system
described above. Monitoring was done in a blinded fashion so
that the person doing the scoring was unaware of the treatment
groups.
Example 2: Expression of cPLA2 in the spinal cord in EAE
The expression of cPLA2 in EAE was assessed in the C57BL/6
mouse strain, which has a naturally occurring null mutation
for sPLA2 group IIA (32), the major form of sPLA2 in the CNS.
Therefore, if PLAZ plays a role in the onset of MOG-induced EAE
in C57BL/6 mice, it has to be mediated mainly by cPLA2. By the
immunoperoxidase technique, increased expression of cPLA2 was
observed at the site of EAE lesions in the spinal cord. The
labeling occurred in endothelial cells (Figure 2), as well as
immune cells in the CNS inflammatory infiltrates (Figure 3).
The percentage of cPLA2+ endothelial cells ranged from 70% to
85% between clinical grades 1 - 3, and decreased to about 200
at clinical grades 4 and 5 (Figure 4).
The percentage of cPLA2+ round cells in the immune cell
infiltrates in EAE lesions remained at around 30%-50% in all
clinical grades (Figure 5). However, since the total number
of cells in the infiltrates increases with increasing clinical
grades and with higher inflammatory scores (Figure 6), the
total number of cPLA2 cells in the spinal cord increased with
increasing severity of the disease (Figure 7). Double-
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CA 02388659 2002-05-31
49
immunofluorescence labeling studies indicate that T cells,
macrophages and astrocytes near EAE lesions express cPLA2
(Figure 8). These results show that the highest number of
cPLAz+ endothelial cells are seen at grades 1-3 which precedes
the period of highest influx of inflammatory cells at grade 4.
Example 3: Blocking with a cPhA2 inhibitor prevents the onset
of EAE
To assess if cPLA2 is important for the onset of EAE we blocked
it using a chemical inhibitor. C57BL/6 mice were treated with
the cPLA2 inhibitor AACOCF3 on the day of immunization and on
day 2 with 50 ~1 of 2 or 4 mM AACOCF3 intravenously, followed
by intraperitoneal injections of the inhibitor (200 ~,l at 2 or
4 mM) on alternate days until day 24. Mice were monitored
clinically using the scoring scale described above. Treatment
with the inhibitor resulted in a remarkable reduction in the
onset and progression of EAE. 1000 of the vehicle-treated
control mice got EAE, while 57$ of the 2mM treated and only
280 of the 4mM treated groups got EAE (Fig. 9). The
progression of the disease was also markedly reduced as shown
in figure 10. Vehicle-treated controls reached an average
maximum clinical score of 2.9 at 12-14 days, while the 2mM and
4mM treated groups reached scores of 1.5 and 0.4, respectively
(Fig. 10). Unlike the controls, which relapsed into a second
paralytic episode between days 25-34, mice treated with 4mM
AACOCF3 remained unaffected (Fig.lO). The analysis was carried
out blind, so that the person doing the clinical scoring was
unaware of the treatment groups. The treatment is well
tolerated in that the animals do not show any side-effects.
The body weight and food-intake of treated mice were
unaffected compared to controls at 35 days after induction of
EAE. These results provide very strong evidence that blocking
PLAZ has a profound effect in the prevention of EAE.
~i , . ~u i ~ 7 vk~ v~~ ~ - 4~ ~i-..
CA 02388659 2002-05-31
Example 4: Delayed Treatment of EAE-induced mice with a cPLA2
inhibitor
5 Materials and methods:
EAE was induced in C57BL/6 mice as described above. A 501
intravenous injection of either 4mM AACOCF3 diluted in 1% DMSO
containing buffer or vehicle (1% DMSO containing buffer) was
10 administered on days 14, 16, 18 and 20 after induction of EAE,
when animals began to remit. The mice were scored clinically
in a blinded fashion as mentioned above.
Results:
Blocking with cPLA2 inhibitor prevents further relapse: To
assess if cPLA2 plays an important role in the progression of
EAE, it was blocked using the chemical inhibitor AACOCF3
described above. EAE induced C57BL/6 mice were given a
delayed treatment with the cPLA2 inhibitor on the day the
animals began to remit (day 14). The animals were given a
one-week treatment ( indicated by arrows in Figure 11) and
were monitored in a blind fashion using the clinical scoring
scale described above. The treated animals could be divided
into two groups: those that received treatment starting at day
14 that were at clinical grades of 3 and 4 , and those that
were at a grade of 2. The former group showed a
chronic/primary progressive. form of the disease and were
unaffected by the treatment regime and were not different from
control groups. These groups peaked at a mean clinical score
of 3.5. The animals in the treated and untreated control
groups fell to a grade 2.6 and cycled back up to a score of
3.3. Vehicle treated animals progressed to a more severe
form, reaching a mean clinical score of 3.8 (Figure 11). In
~.~,'w I~, . .;I~ ...d~ I ;I r ;;
CA 02388659 2002-05-31
51
contrast, animals that received treatment on day 14 that had
a clinical score of 2 had a remarkable reduction in the
progression of the disease. Although these animals peaked to
a mean clinical score of grade 3 prior to treatment, they
progressively dropped down to a grade 0.3 (Figure 11). Their
advance into a second relapse was prevented. In contrast,
untreated control animals that also showed a clinical score of
2 on day 14, peaked to a score of about 3.0, then remitted to
a score of between 1-2 but progressed into a second paralytic
episode (score of 2.5)and remained thereafter at a score of
about 2Ø This clinical picture indicates a
relapsing/remitting form of the disease. These results
therefore provide very strong evidence that initiating
treatments to block cPLA2 after the peak of the first paralytic
episode can prevent the occurrence of subsequent paralytic
episodes in relapsing/remitting forms of the disease. It is
possible that the chronic/primary progressive forms of EAE
could be alleviated by higher doses or more prolonged
treatment with the inhibitor.
All references cited herein or in the references section
below are herein incorporated by reference.
i I L' 'I ~ dr ~ ,~ I
CA 02388659 2002-05-31
52
Ackermann et al. (1995) Inhibition of macrophage Ca(2+)-
independent phospholipase A2 by bromoenol lactone and
trifluoromethyl ketones. J Biol Chem. 270:445-50.
Adelmann, M., Wood, J., Benzel, I., Fiori, P., Lassmann, H.,
Matthieu, J.M. et al., (1995). The N-terminal domain of the
myelin oligodendrocyte glycoprotein (MOG) induces acute
demyelinating experimental autoimmune encephalomyelitis in the
Lewis rat. J. Neuroimmunol. 63: 17-27.
Allen, I., and Brankin, B. (1993). Pathogenesis of multiple
sclerosis: the immune diathesis and the role of viruses. J.
Neuropathol. Exp. Neurol. 52: 95-105.
Andersson PB, Goodkin DE. (1998) Glucocorticosteroid therapy
for multiple sclerosis: a critical review. J Neurol Sci.
160(1):16-25.
Arbibe, L., Vial, D., Rosinski-Chupin, I., Havet, N., Huerre,
M., Vargaftig, B.B. and Touqui, L. (1997). Endotoxin induces
expression of type II phospholipase A2 in macrophages during
acute lung injury in guinea pigs: involvement of TNF-alpha in
lipopolysaccharide-induced type II phospholipase A2 synthesis.
J. Immunol. 159: 391-400.
Balsinde, J., Balboa, MA., Insel, PA. And Dennis, EA (1999)
Regulation and inhibition of phospholipase A2. Annu. Rev.
Pharmacol. Toxicol. 39: 175- 189.
Bansil, S., Cook, S.D. and Rohowsky-Kochan, C. (1995).
Multiple sclerosis: immune mechanism and update on current
therapies. Ann. Neuol. 37 (S1):587-5101.
i i ;i
CA 02388659 2002-05-31
53
Bauer J, Huitinga I, Zhao W, Lassmann H, Hickey WF, Dijkstra
CD. (1995) The role of macrophages, perivascular cells, and
microglial cells in the pathogenesis of experimental
autoimmune encephalomyelitis. Glia. 4:437-46.
Bignami, A. and Ralston, H.J.III (1969). The cellular
reaction to Wallerian degeneration in the central nervous
system of the cat. Brain Res. 13: 444-461.
Bonventre, J.V., Huang, Z., Taheri, M.R., 0'Leary, E., Li, E.,
Moskowitz, M.A. and Sapirstein, A. (1997). Reduced fertility
and postischemic brain injury in mice deficient in cytosolic
phospholipase A2.
Brenner T, Boneh A, Shohami E, Abramsky 0, Weidenfeld J.
(1992) Glucocorticoid regulation of eicosanoid production by
glial cells under basal and stimulated conditions. J
Neuroimmunol. 40 (2-3) :273-9.
Brenner T, Brocke S, Szafer F, Sobel RA, Parkinson JF, Perez
DH, Steinman L. (1997) Inhibition of nitric oxide synthase for
treatment of experimental autoimmune encephalomyelitis. J
Immunol. 158 (6) :2940-6.
Brenner T, Poradosu E, Soffer D, Sicsic C, Gazit A, Levitzki
A. (1998) Suppression of experimental autoimmune
encephalomyelitis by tyrphostin AG-556. Exp Neurol.
154(2):489-98.
Bruck., W., Schmied, M., Suchanek, G., Bruck, Y., Breitschopf,
H., Poser, S., Piddlesden, S. and Lassmann, H. (1994).
Oligodendrocytes in the early course of multiple sclerosis.
Ann. Neuol. 35: 65-73.
~~ ~ ~I~ , .~~ I ;I f I
CA 02388659 2002-05-31
54
Chang, H.W., Kudo, I., Tomita, M. and moue, K. (1987).
Purification and characterization of extracellular
phospholipase A2 from peritoneal cavity of caseinate-treated
rat. J. Biochem. 102: 147-154.
Chen, S. and Bisby, M.A. (1993). Impaired motor axon
regeneration in the C57/BL/Ola mouse. J. Comp. Neurol.
333:449-454.
Currie et al. (1994) Phosphorylation and activation of Ca(2+)-
sensitive cytosolic phospholipase A2 in MCII mast cells
mediated by high-affinity Fc receptor for IgE. Biochem J.
304:923-8.
Dennis, E. (1994) Diversity of group types, regulation, and
function of phospholipase A2. J. Biol. Chem. 269: 13057-
13060.
Dennis, EA (2000) Phopholipase AZ in eicosanoid generation.
Am. J. Respir. Crit. Care Med. 161: S32-535.
Dinter H, Tse J, Halks-Miller M, Asarnow D, Onuffer J, Faulds
D, Mitrovic B, Kirsch G, Laurent H, Esperling P, Seidelmann D,
Ottow E, Schneider H, Tuohy VK, Wachtel H, Perez HD. (2000)
The type IV phosphodiesterase specific inhibitor mesopram
inhibits experimental autoimmune encephalomyelitis in rodents.
J Neuroimmunol. 108(1-2):136-46.
Dore-Duffy, P., Donaldson, J.O., Koff, T., Longo, M. and
Perry, W. (1986). Prostaglandin release in multiple
sclerosis: correlation with disease activity. Neurology 36:
1587-1590.
i . G 6~~ ~~ I I . I
CA 02388659 2002-05-31
Dore-Duffy, P., Ho, S.Y. and Donovan, C. (1991).
Cerebrospinal fluid eicosanoid levels: endogenous PGD2 and
LTC4 synthesis by antigen-presenting cells that migrate to the
central nervous system. Neurology 41: 322-324.
5
Ebers, G.C. (1996). Genetic epidemiology of multiple
sclerosis. Curr. Opinion Neurology 9: 155-158.
Emerson MR, Biswas S, LeVine SM. (2001) Cuprizone and
10 piperonyl butoxide, proposed inhibitors of T-cell function,
attenuate experimental allergic encephalomyelitis in SJL mice.
J. Neuroimmunol. 119(2):205-13.
Ewing, C. and Bernard, C.C. (1998). Insights into the
15 aetiology and pathogenesis of multiple sclerosis. Immunology
& Cell Biology 76: 47-54.
Farooqui, A.A., Yang, H.C., Rosenberger, T.A. and Horrocks.
L.A. (1997). Phospholipase AZ and its role in brain tissue.
20 J. Neurochem. 69: 889-901.
Fife BT, Kennedy KJ, Paniagua MC, Lukacs NW, Kunkel SL, Luster
AD, Karpus WJ. (2001) CXCL10 (IFN-gamma-inducible protein-10)
control of encephalitogenic CD4+ T cell accumulation in the
25 central nervous system during experimental autoimmune
encephalomyelitis. J Immunol. 166(12):7617-24.
Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon,
I. and Heinrikson, R.L. (1986). Structural and functional
30 properties of a phospholipase A2 purified from an inflammatory
exudate. Biochemistry 25: 8381-8385.
Fretland DJ. (1992) Potential role of prostaglandins and
leukotrienes in multiple sclerosis and experimental allergic
i i t I d y "~ i1 ~ ~~ ' i-
CA 02388659 2002-05-31
56
encephalomyelitis. Prostaglandins Leukot Essent Fatty
Acids.45(4):249-57.
Fujimori, Y., Murakami, M., Kim, D.k., Hara, S. et al.,
(1992). Immunochemical detection of arachidonoyl-preferential
phospholipase A2. J. Biochem. 111: 54-60
Gallai V, Sarchielli P, Trequattrini A, Franceschini M,
Floridi A, Firenze C, Alberti A, Di Benedetto D, Stragliotto
E. (1995) Cytokine secretion and eicosanoid production in the
peripheral blood mononuclear cells of MS patients undergoing
dietary supplementation with n-3 polyunsaturated fatty acids.
J Neuroimmunol. 56(2):143-53.
Glaser, K.B., Mobilio, D., Chang, J.Y. and Senko, N. (1993).
Phospholipase AZ enzymes: regulation and inhibition. Trends in
Pharmacol. 14: 92-98.
Goppelt-Struebe, M. and Rehfeldt, W. (1992). Glucocorticoids
inhibit TNF alpha-induced cytosolic phospholipase A2 activity.
Biochem Biophys Acta 1127: 163-167.
Goverman, J., Woods, A., Larson, L., et al., (1993).
Transgenic mice that express a myelin basic protein-specific
T-cell receptor develop spontaneous autoimmunity. Cell 72:
551-560.
Hall, S.M. (1989). Regeneration in the peripheral nervous
system. Neuropathol. & Appl. Neurobiol. 15: 513-530.
Hall, S.M. (1993). Observations on the progress of Wallerian
degeneration in transected peripheral nerves of C57BL/Wld mice
in the presence of recruited macrophages. J. Neurocytol.
22:480-490.
i o i ;i i
CA 02388659 2002-05-31
57
Harlow, E. and Lane, D (1988) Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Hauser, S.L., Doolittle, T.H., Lincoln, R., Brown, R.H. and
Dinarello, C.A. (1990). Cytokine accumulations in CSF of
multiple sclerosis patients: frequent detection of
interleukin-1 and tumor necrosis factor but not interleukin-6.
Neurology 40: 1735-1739.
Hirashima, Y., Farooqui, A.A., Mills, J.S. and Horrocks, L.A.
(1992) . Identification and purification of calcium-
independent phopholipase AZ from bovine brain cytosol. J.
Neurochem. 59: 708-714.
Hofman, F.M., von Hanwehr, R.I., Dinarello, C.A., et al.,
(1986). Immunoregulatory molecules and IL-2 receptors
identified in multiple sclerosis brain. J. Immunol. 136:
3239-3245.
Hofman, F.M., Hinton, D. R., Johnson, K., Merrill, J.E. (1989) .
Tumor necrosis factor identified. in multiple sclerosis brain.
J.Exp. Med. 170: 607-612.
Huitinga I, van Rooijen N, de Groot CJ, Uitdehaag BM, Dijkstra
CD. (1990) Suppression of experimental allergic
encephalomyelitis in Lewis rats after elimination of
macrophages. J Exp Med. 172(4):1025-33.
Hulkower, K.I., Hope, W.C., Chen, T., Anderson , C.M., Coffey,
J.W. and Morgan, D.W. (1992). Interleukin-1 beta stimulates
cytosolic phospholipase A2 in rheumatoid synovial fibroblasts.
Biochem Biophys Res Commun. 184: 712-718.
I '- ~u I ~ I .j, 9 .~.,i I '. Fl
CA 02388659 2002-05-31
58
Huterer, S.J., Tourtellotte, W.W. and Wherrett, J.R. (1995).
Alterations in the activity of phospholipase A2 in postmortem
white matter from patients with multiple sclerosis.
Neurochem. Res. 20: 1335-1343.
Ishizaki, J., Ohara, 0., Nakamura, E., Tamaki, M. et al.,
(1989). cDNA cloning and sequence determination of rat
membrane-associated phospholipase A2. Biochem Biophys Res
Commun 162: 1030-1036.
Issazadeh, S., Lorentzen, J.C., Mustafa, M.I., Hojeberg, B.,
Mussener, A. and Olsson, T. (1996). Cytokines in relapsing
experimental autoimmune encephalomyelinitis in Da rats:
persistent mRNA expression of proinflammatory cytokines and
absent expression of interleukin-10 and transforming growth
factor-beta. J. Neuroimmunol. 69: 103-115.
Ito A, Bebo BF Jr, Matejuk A, Zamora A, Silverman M, Fyfe-
Johnson A, Offner H. (2001) Estrogen treatment down-regulates
TNF-alpha production and reduces the severity of experimental
autoimmune encephalomyelitis in cytokine knockout mice. J
Immunol . 167 ( 1 ) : 542-52 .
Jeffery, N.D. and Blakemore, W.F. (1995). Remyelination of
mouse spinal cord axons demyelinated by local injection of
lysolecithin. J. Neurocytol. 24: 775-781.
Johns, T.G., Kerlero de Rosbo, N., Menon, K.K., Abo, S.,
Gonzales, M.F. and Bernard, C.C. (1995). Myelin
oligodendrocytes glycoprotein induces a demyelinating
encephalomyelitis resembling multiple sclerosis. J. Immnol.
154: 5536-3341.
I 6 ~ a ~ d. I ,I rt ~; I
CA 02388659 2002-05-31
59
Karpus WJ, Lukacs NW, McRae BL, Strieter RM, Kunkel SL, Miller
SD. (1995) An important role for the chemokine macrophage
inflammatory protein-1 alpha in the pathogenesis of the T
cell-mediated autoimmune disease, experimental autoimmune
encephalomyelitis. J Immunol. 155(10):5003-10.
Kawai K, Kobayashi Y, Shiratori M, Sobue G, Tamatani T,
Miyasaka M, Yoshikai Y (1996) Intrathecal administration of
antibodies against LFA-1 and against ICAM-1 suppresses
experimental allergic encephalomyelitis in rats. Cell
Immunol . 171 ( 2 ) : 262-8 .
Kennedy, B.P., Payette, P., Mudgett, J., Vadas, P.,
Pruzaniski, W., Kwan, M., Tang, C., Rancourt, D.E., and
Cromlish, W.A. (1995). A natural disruption of group II PLAZ
gene in inbred mouse strains. J.Biol. Chem. 270: 22378-
22385.
Kent SJ, Karlik SJ, Cannon C, Hines DK, Yednock TA, Fritz LC,
Horner HC. (1995) A monoclonal antibody to alpha 4 integrin
suppresses and reverses active experimental allergic
encephalomyelitis. J Neuroimmunol. 58(1):1-10.
Klivenyl, P., Beal, M.F., Ferrante, R.J., Andreassen, O.A.,
Wermer, M., Chin, M-R. and Bonventre, J.V. 1998). Mice
deficient in group IV cytosolic phospholipase A2 are resistent
to MPTP neurotoxicity. J. Neurochem. 71: 2634-2637.
Kohler G. and Milstein C. (1975) Continuous cultures of fused
cells secreting antibody of predefined specificity. Nature
256:495-497.
~. 4 ~~~ 6G d,1 :I , I
CA 02388659 2002-05-31
Komada, M., Kudo, I., Mizushima, H., Kitamura, N. and moue,
K. (1989). Structure of cDNA coding for rat platelet
phospholipase A2. J. Biochem. 106: 545-547.
5 Kramer, R.M., Hession, C., Johansen, B., Hayes, G. et al.,
(1989). Structure and properties of a human non-pancreatic
phospholipase A2. J. Biol. Chem. 264: 5768-5775.
Lautens, L.L., Chiou, X.G., Sharp, J.D., Young, W.S., Sprague,
10 D.L., Ross, L.S. and Felder, C.C. (1998). Cytolsolic
phospholipase A2 (cPLA2) distribution in murine brain and
functional studies indicate that cPLA2 does not participate in
muscarinic receptor-mediate signalling in neurons. Brain Res.
809: 18-30.
Lee, S.J. and Benveniste, E.N. (1999). Adhesion molecule
expression and regulation on cells of the central nervous
system. J. Neuroimmunol. 98: 77-88.
Leonard JP, Waldburger KE, Goldman SJ. (1996) Regulation of
experimental autoimmune encephalomyelitis by interleukin-12.
Ann N Y Acad Sci. 795:216-26.
Lin, L.L., Lin, A.Y. and DeWitt, D.L. (1992). Interleukin-1
alpha induces the accumulation of cytosolic phospholipase A2
and the release of prostaglandin E2 in human fibroblasts. J.
Biol. Chem. 267: 23451-23454.
Lin, L.L., Wartmann, M., Lin, A.Y., Knopf, J.L., Seth, A, and
Davis, R.J. (1993). cPLA2 is phosphorylated and activated by
MAP kinase. Cell 72: 269-278.
Liu, J., Marino, M.W., Wong, G., Grail, D., Dunn, A.,
Bettadapura, J., Slavin, A.J., Old, L. and Bernard, C.C.
61 1i I'~ I ~I ~ I
CA 02388659 2002-05-31
61
(1998). TNF is a potent anti-inflammatory cytokine in
autoimmune-mediated demyelination. Nature Medicine 4: 78-
8326.
Lyons-Giordano, B., Davis, G.L., Galbraith, W., Pratta, M.A.
and Arner, E.C. (1989). Interleukin-1 beta stimulates
phospholipase A2 mRNA synthesis in rabbit articular
chondrocytes. Biochem Biophys Res Commun. 164: 488-495.
Martin, R., Jaraquemada, D., Flerlage, M., et al., (1990).
Fine specificity and HLA restriction of myelin basic protein-
specific cytotoxic T-cell lines from multiple sclerosis
patients and heathy individuals. J. Immunol. 145: 540-548.
Meyer AL, Benson JM, Gienapp IE, Cox KL, Whitacre CC. (1996)
Suppression of murine chronic relapsing experimental
autoimmune encephalomyelitis by the oral administration of
myelin basic protein. J Immunol. 157(9):4230-8.
Mokhtarian, F., McFarlin, D.E. and Raine, C.S. (1984).
Adoptive transfer of myelin basic protein-sensitive T cells
produces chronic relapsing demyelinating disease in mice.
Nature 309: 356-358.
Molloy, G.Y., Rattray, M. and Williams, R.J. (1998). Genes
encoding multiple forms of phospholipase AZ are expressed in
rat brain. Neurosci. Lett. 258: 139-142.
Murakami, M., Kudo, I., Natori, Y. and moue, K. (1990).
Immunochemical detection of Oplatelet type0 phospholipase A2
in the rat. Biochem Biophys Acta 1043: 34-42.
Murakami, M., Kudo, I. and moue, K. (1993). Molecular nature
of phospholipase A2 involved in prostaglandin 12 synthesis in
human umbilical vein endothelial cells. Possible participation
~~G~a~~~~~ , ~i f ~ It
CA 02388659 2002-05-31
62
of cytosolic and extracellular type II phospholipase A2. J.
Biol. Chem. 268: 839-844
Murakami, M., Nakatani, Y., Atsumi, G., moue, K. and Kudo,
I. (1997). Regulatory functions of phospholipase A2.
Critical Reviews in Immunol. 17: 225-283.
Nalefski, E.A., Sultzman, L.A., Martin, D.M., et al. (1994)
Delineation of two functionally distinct domains of cytosolic
phopholipase A2, a regulatory Ca(2+)-dependent lipid binding
domain and a Ca(2+)-independent catalytic domain. J. Biol.
Chem. 269:1823-1849.
Nakano, T. and Arita, H. (1990). Enhanced expression of group
II phospholipase A2 gene in the tissues of endotoxin shock rat
and its suppression by glucocorticoid. FEBS Let. 273: 23-26.
Nakano, T., Ohara, O., Teraoka, H. and Arita, H. (1990).
GroupII phospholipase A2 mRNA synthesis is stimulated by two
distinct mechanisms in rat vascular smooth muscle cells. FEBS
Lett. 261: 171-174.
Norton, W.T., Brosnan, C.F., Cammer, W. and Goldmuntz, E.A.
(1990). Mechanisms and suppression of inflammatory
demyelination. Acta Neuropathologiae Experimentalis 50: 225-
235.
Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG.
(2000) Multiple sclerosis. N Engl J Med. 28343(13):938-52.
Ogata, K., Jin, B., Taniguchi, M., et al., (2001) Attenuation
of ishemicia and reperfusion injury of canine livers by
inhibition of type II phospholipase A2 with LY329722.
Transplantation 71: 1040-1046.
I ~ I,li~ ~lil II I
CA 02388659 2002-05-31
63
Oger, J. and Lai, H. (1994). Demyelination and ethnicity:
experience at the University of British Columbia Multiple
Sclerosis Clinic with special reference to HTLV-1-associated
myelopathy in British Columbian natives. Ann. Neurol. 36: S22-
24.
Oka, S. and Arita, H. (1991). Inflammatory factors stimulate
expression of group II phospholipase A2 in rat cultured
astrocytes. Two distinct pathways of the gene expression. J.
Biol. Chem. 266: 9956-9960.
Olsson, T., Wei, Z.H., Hojeberg, B. et al., (1990).
Autoreactive T lymphocytes in multiple sclerosis determined by
antigen-induced secretion of interferon-D. J. Clin. Invest.
86: 981-985.
Oinuma et al. (1991) Synthesis and biological evaluation of
substituted benzenesulfonamides as novel potent membrane-bound
phospholipase A2 inhibitors. J Med Chem. 34:2260-7.
Ousman, S. and David, S. (2000). Lysophosphatidylcholine
induces rapid recruitment and activation of macrophages in the
adult mouse spinal cord. Glia 30: 92-104.
Ousman, S. and David, S. (2001). MIP-la, GM-CSF, and TNF-a
control the immune cell response that mediates rapid
phagocytosis of myelin from the adult mouse spinal cord. J.
Neurosci. 21: 4649-4656.
Owada, Y., Tominaga, T., Yoshimoto, T. and Kondo, H. (1994).
Molecular cloning of rat cDNA for cytosolic phospholipase A2
and the increased gene expression in the dentate gyrus
I I 1i ~ I'~ I LI I
CA 02388659 2002-05-31
64
following transient forebrain ischemia. Mol. Brain Res. 25:
364-368.
Ozawa, K., Suchanek, G., Breitschopf, H., Bruck, W., Budka,
H., Jellinger, K. and Lassmann, H. (1994). Patterns of
oligodendroglia pathology in multiple sclerosis. Brain 117:
1311-1322.
Panitch, H.S., Hirsch, R.L., Schindler, J., et al., (1987).
Treatment of multiple sclerosis with gamma interferon:
exacerbations associated with activation of the immune system.
Neurology 37: 1097-1102.
Penkowa M, Hidalgo J. (2000) Metallothionein I+II expression
and their role in experimental autoimmune encephalomyelitis.
Glia.32(3):247-63.
Pitt D, Werner P, Raine CS. (2000) Glutamate excitotoxicity
in a model of multiple sclerosis. Nat Med. Jan;6(1):67-70.
Polman CH, Uitdehaag BM. (2000) Drug treatment of multiple
sclerosis. West J Med. 173(6):398-402.
Popovich PG, Yu JY, Whitacre CC. (1997) Spinal cord
neuropathology in rat experimental autoimmune
encephalomyelitis: modulation by oral administration of myelin
basic protein. J Neuropathol Exp Neurol. 56(12):1323-38.
Prineas, J.W., Barnard, R.O., Kwon, E.E., et al., (1993).
Multiple sclerosis: remyelination of nascent lesions. Ann.
Neurol. 33: 137-151.
I., ', ~ ~ Il 6 al I ~I - I
CA 02388659 2002-05-31
Probert, L., Akassoglou, K., Pasparakis, M., Kontogeorgos, G.
and Kollias, G. (1995). Spontaneous inflammatory
demyelinating disease in transgenic mice showing central
nervous system-specific expression of tumor necrosis factor
5 alpha. Proc. Natl. Acad. Sci. USA 92: 11294-11298.
Prokazova, N.V., Zvezdina, N.D. and Korotaeva, A.A. (1998).
Effect of lysophosphatidylcholine on transmembrane signal
transduction. Biochemistry 63: 31-37.
Prosiegel M, Neu I, Mallinger J, Wildfeuer A, Mehlber L, Vogl
S, Hoffmann G, Ruhenstroth-Bauer G. (1989) Suppression of
experimental autoimmune encephalomyelitis by dual cyclo-
oxygenase and 5-lipoxygenase inhibition. Acta Neurol Scand.
79(3):223-6.
Prosiegel M, Neu I, Vogl S, Hoffmann G, Wildfeuer A,
Ruhenstroth-Bauer G. (1990) Suppression of experimental
autoimmune encephalomyelitis by sulfasalazine. Acta Neurol
Scand. 81(3):237-8.
Racke MK, Burnett D, Pak SH, Albert PS, Cannella B, Raine CS,
McFarlin DE, Scott DE. (1995) Retinoid treatment of
experimental allergic encephalomyelitis. IL-4 production
correlates with improved disease course. J Immunol.
154(1):450-8.
Raine, C.S. and Cannella, B. (1992). Adhesion molecules and
central nervous system inflammation. Semin. Neurosci. 4: 201-
211.
Reder, AT., Thapur, M., Sapugay, AM and Jensen, AM. (1994)
Prostaglandins and inhibitors of acrachidonate metabolism
I ~, ad I ~I I ~I
CA 02388659 2002-05-31
66
suppress experimental allergic encephalomyelitis.
J.Neuroimmunol. 54: 117-127.
Reichert, J.R., Robinson, E.D., Deibler, G.E., et al., (1989).
Human cytotoxic T-cell recognition of a synthetic peptide of
myelin basic protein. Ann. Neuol. 26: 342-346.
Renno, T., Lin, J.Y., Piccirillo, C., Antel, J. and Owens, T.
(1994). Cytokine production by cells in cerebrospinal fluid
during experimental allergic encephalomyelitis in SJL/J mice.
J. Neuroimmunol. 49: 1-7.
Reynolds et al. (1994) 1-Hexadecyl-2-arachidonoylthio-2-deoxy-
sn-glycero-3-phosphorylcholine as a substrate for the
microtiterplate assay of human cytosolic phospholipase A2.
Anal Biochem 217:25-32.
Rolak LA. (2001) Multiple sclerosis treatment 2001. Neurol
Clin. 19(1):107-18.
Ross, B.M., Kim, D.K.., Bonventre, J.V. and Kish, S.J. (1995).
Characterization of a novel phospholipase Az activity in human
brain. J. Neurochem. 64: 2213-2221.
Ruddle NH, Bergman CM, McGrath KM, Lingenheld EG, Grunnet ML,
Padula SJ, Clark RB (1990) An antibody to lymphotoxin and
tumor necrosis factor prevents transfer of experimental
allergic encephalomyelitis. J Exp Med. 1990 172(4):1193-200.
Ruuls et al. (1996) J. Immunol. 157:5721-5731.
Ryborg, A.K., Deleuran, B., Thestrup-Pedersen, K. and
Kragballe, K. (1994). Lysophosphatidylcholine: a
k ~ i I ~ I , it'v ~ ~ ~ '. ~~ '.
CA 02388659 2002-05-31
67
chemoattractant to human T lymphocytes. Arch. Dermatol. Res.
286: 462-465.
Sadovnick, A.D., Ebers, G.C., Dyment, D.A. and Risch, N.J.
(1996). Evidence for genetic basis of multiple sclerosis.
The Canadian Collaborative Study Group. Lancet 347: 1728-
1730.
Seilhamer, J.J., Pruzanski, W., Vadas, P., Plant, S. et al.,
(1989). Cloning and recombinant expression of phospholipase
A2 present in rheumatoid arthritis synovial fluid. J. Biol.
Chem. 264: 5335-5338.
Selmaj K, Walczak A, Mycko M, Berkowicz T, Kohno T, Raine CS.
(1998) Suppression of experimental autoimmune
encephalomyelitis with a TNF binding protein (TNFbp)
correlates with down-regulation of VCAM-1/VLA-4. Eur J
Immunol. 28 (6) :2035-44.
Sharp, J.D., White, D.L., Chiou, X.G. et al. (1991) Molecular
Cloning and Expression of human Ca2+-sensitive cytosolic
phospholipase A2. J. Biol. Chem. 266: 14850-14853.
Smith T, Groom A, Zhu B, Turski L. (2000) Autoimmune
encephalomyelitis ameliorated by AMPA antagonists. Nat Med.
6(1):62-6.
Sobel, R.A., Mitchell, M.E. and Fondren, G. (1990).
Intracellular adhesion molecule-1 (ICAM-1) in cellular immune
reactions in the human central nervous system. Am. J. Pathol.
136: 1309-1316.
Steinman L, Miller A, Bernard CC, Oksenberg JR. (1994). The
i ~ ~~ ~~ ~n ~i i
CA 02388659 2002-05-31
68
epigenetics of multiple sclerosis: clues to etiology and a
rationale for immune therapy. Annu Rev Neurosci. 17:247-65.
Stinissen P, Raus J, Zhang J. (1997) Autoimmune pathogenesis
of multiple sclerosis: role of autoreactive T lymphocytes and
new immunotherapeutic strategies. Crit Rev Immunol. 17(1):33-
75.
Street et al. (1993) Slow- and tight-binding inhibitors of the
85-kDa human phospholipase A2. Biochemistry 32:5935-40.
Sun, J.B., Olsson, T., Wang, W.Z., et al., (1991).
Autoreactive T and B cells responding to myelin proteolipid
protein in multiple sclerosis and controls. Eur. J. Immunol.
21: 1461-1468.
Taupin, V., Renno, T., Bourbonniere, L., Peterson, A.C.,
Rodriguez, M. and Owens, T. (1997). Increased severity of
experimental autoimmune encephalomyelitis, chronic
macrophage/microglial reactivity, and demyelination in
transgenic mice producing tumor necrosis factor-alpha in the
central nervous system. Eurp. J. Immunol. 27: 905-913.
Tienari, P. (1994). Multiple sclerosis: multiple etiologies,
multiple genes? Annals of Med. 26: 259-269.
Tran EH, Hoekstra K, van Rooijen N, Dijkstra CD, Owens T.
(1998) Immune invasion of the central nervous system
parenchyma and experimental allergic encephalomyelitis, but
not leukocyte extravasation from blood, are prevented in
macrophage-depleted mice. J Immunol. 161(7):3767-75.
I I II I , ~''~ I ~I I
CA 02388659 2002-05-31
69
Traugott, U., Reinherz, E.L., Raine, C.S. (1983). Multiple
sclerosis: distribution of T cell subsets within active
chronic lesion. Science 219: 308-310.
Trotter, J. and Smith, M.E. (1986). The role of
phospholipases from inflammatory macrophages in demyelination.
Neurochem. Res. 11: 349-361.
Tuohy, V.K. (1994). Peptide determinants of myelin proteolipid
protein (PLP) in autoimmune demyelinating disease: a review.
Neurochemical Res. 19: 935-944.
van der Meide et al. (1998) J. Neuroimmunol. 84:14-23.
Warren, K.G., Catz, I., Johnson, E. and Mielke, B. (1994).
Anti-myelin basic protein and anti-proteolipid protein
specific forms of multiple sclerosis. Ann. Neurol. 35: 280-
289.
Weber F, Hempel K. (1989) Protection against experimental
allergic encephalomyelitis with complete Freund's adjuvant is
unaffected by prostaglandin synthesis inhibition. Int Arch
Allergy Appl Immunol. 89(2-3):242-5.
Woelk " H. and Peiler-Ichikawa, K. (1974) On the activity of
phospholipase AZ compared with 1-alk-1'-enyl-2-acyl and 1-
alkyl-2-glcerophosphatides in multiple sclerosis. J. Neurol.
207: 319-326.
Wright, G.W., Ooi, C.E., Weiss, J. and Elsbach, P. (1989).
Purification of a cellular (granulocyte) and an extracellular
(serum) phospholipase A2 that participate in the destruction
of Escherichia coli in a rabbit inflammatory exudate. J.
Biol. Chem. 265: 6675-6681.
lin. i 1i ~~-~~i i~~l ~ ~~ '.
CA 02388659 2002-05-31
Wu, T., Levine, S.J., Lawrence, M.G., Logun, C., Angus, C.W.
and Shehamer, J.H. (1994). Interferon-gamma induces the
synthesis and activation of cytosolic phospholipase A2. J.
5 Clin. Invest. 93: 571-577.
Xu, X.X., Rock, C.O., Qui, Z.H., Leslie, C.C. and Jackowski,
S. (1994). Regulation of cytosolic phospholipase A2
phosphorylation and eicosanoid production by colony-
10 stimulating factor-1. J. Biological Chem. 269: 31693-31700.
Yang, H-C., Farooqui, A.A., and Horrocks, L.A. (1994).
Effects of glycosaminoglycans and glycosphigolipids on
cytolsolic phospholipase A2 from bovine brain. Biochem. J.
15 299: 91.
Yelton D.E. and Scharff M.D. (1981) Monoclonal Antibodies: a
powerful new tool in biology and medicine. Ann. Rev. Biochem.
50:657-680.
Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L,
Karin N (1992) Prevention of experimental autoimmune
encephalomyelitis by antibodies against alpha 4 beta 1
integrin. Nature 356(6364):63-6.
Yu M, Nishiyama A, Trapp BD, Tuohy VK. (1996) Interferon-beta
inhibits progression of relapsing-remitting experimental
autoimmune encephalomyelitis. J Neuroimmunol. 64(1):91-100.
Zamvil, S., Nelson, P., Trotter, J., Mitchell, D., Knobler,
R., Fritz, R. and Steinman, L. (1985). T-cell clones specific
for myelin-basic protein induce chronic relapsing paralysis
and demyelination. Nature 317: 355-358.
'~, - d I I.al ~, ,1'~, ~ . ~I l
CA 02388659 2002-05-31
71
Zhang, Y., Burger,D., Saruhan, G., et al., (1993). The
lymphocyte response against myelin-associated glycoprotein and
myelin basic protein in patients with multiple sclerosis.
Neurology 43: 403-407.
I,I,I L~I 41 I
CA 02388659 2002-05-31
72
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MCGILL UNIVERSITY
(ii) TITLE OF INVENTION: PHOSPHOLIPASE A2 EXPRESSION AND ACTIVITY AND
USE
THEREOF FOR DIAGNOSIS, PROGNOSTICATION,
PREVENTION AND TREATMENT OF NEURAL
1O INFLAMMATORY AND DEMYELINATING DISEASE
(iii) NUMBER OF SEQUENCES: 5
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: 3400 - 1000 DE LA GAUCHETIERE ST. W.
(C) CITY: MONTREAL
(D) STATE: QC
(E) COUNTRY: CANADA
(F) ZIP: H3B 4W5
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
2 5 (A) APPLICATION NUMBER: CA
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
3 O (B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 85827-52
35 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514)-954-1500
(B) TELEFAX: (514)-954-1396
4 O (2) INFORMATION FOR SEQ ID NO.: l:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 2846
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
4 5 ( D ) TOPOLOGY
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE
5O (A) NAME/KEY: CDS
(B) LOCATION: (126)..(2375)
(C) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:
CTGAAAAAGG ATCCTGACTG AAAGCTAGAG GCATTGAGGA GCCTGAAGAT TCTCAGGTTT 60
55 TAAAGACGCT AGAGTGCCAA AGAAGACTTT GAAGTGTGAA AACATTTCCT GTAATTGAAA 120
CCAAA ATG TCA TTT ATA GAT CCT TAC CAG CAC ATT ATA GTG GAG CAC CAG 170
Met Ser Phe Ile Asp Pro Tyr Gln His Ile Ile Val Glu His Gln
1 5 10 15
Li'~;~ ~I~I fl I
CA 02388659 2002-05-31
73
TAT TCC CAC AAG TTT ACG GTA GTG GTG TTA CGT GCC ACC AAA GTG ACA 218
Tyr Ser His Lys Phe Thr Val Val Val Leu Arg Ala Thr Lys Val Thr
20 25 30
AAG GGG GCC TTT GGT GAC ATG CTT GAT ACT CCA GAT CCC TAT GTG GAA 266
Lys Gly Ala Phe Gly Asp Met Leu Asp Thr Pro Asp Pro Tyr Val Glu
35 40 45
CTT TTT ATC TCT ACA ACC CCT GAC AGC AGG AAG AGA ACA AGA CAT TTC 314
Leu Phe Ile Ser Thr Thr Pro Asp Ser Arg Lys Arg Thr Arg His Phe
50 55 60
AAT AAT GAC ATA AAC CCT GTG TGG AAT GAG ACC TTT GAA TTT ATT TTG 362
Asn Asn Asp Ile Asn Pro Val Trp Asn Glu Thr Phe Glu Phe Ile Leu
65 70 75
GAT CCT AAT CAG GAA AAT GTT TTG GAG ATT ACG TTA ATG GAT GCC AAT 910
Asp Pro Asn Gln Glu Asn Val Leu Glu Ile Thr Leu Met Asp Ala Asn
80 85 90 95
TAT GTC ATG GAT GAA ACT CTA GGG ACA GCA ACA TTT ACT GTA TCT TCT 458
Tyr Val Met Asp Glu Thr Leu Gly Thr Ala Thr Phe Thr Val Ser Ser
100 105 110
ATG AAG GTG GGA GAA AAG AAA GAA GTT CCT TTT ATT TTC AAC CAA GTC 506
Met Lys Val Gly Glu Lys Lys Glu Val Pro Phe Ile Phe Asn Gln Val
115 120 125
ACT GAA ATG GTT CTA GAA ATG TCT CTT GAA GTT TGC TCA TGC CCA GAC 554
Thr Glu Met Val Leu Glu Met Ser Leu Glu Val Cys Ser Cys Pro Asp
130 135 190
CTA CGA TTT AGT ATG GCT CTG TGT GAT CAG GAG AAG ACT TTC AGA CAA 602
3 5 Leu Arg Phe Ser Met Ala Leu Cys Asp Gln Glu Lys Thr Phe Arg Gln
145 150 155
CAG AGA AAA GAA CAC ATA AGG GAG AGC ATG AAG AAA CTC TTG GGT CCA 650
Gln Arg Lys Glu His Ile Arg Glu Ser Met Lys Lys Leu Leu Gly Pro
4 0 160 165 170 175
AAG AAT AGT GAA GGA TTG CAT TCT GCA CGT GAT GTG CCT GTG GTA GCC 698
Lys Asn Ser Glu Gly Leu His Ser Ala Arg Asp Val Pro Val Val Ala
180 185 190
ATA TTG GGT TCA GGT GGG GGT TTC CGA GCC ATG GTG GGA TTC TCT GGT 746
Ile Leu Gly Ser Gly Gly Gly Phe Arg Ala Met Val Gly Phe Ser Gly
195 200 205
GTG ATG AAG GCA TTA TAC GAA TCA GGA ATT CTG GAT TGT GCT ACC TAC 794
Val Met Lys Ala Leu Tyr Glu Ser Gly Ile Leu Asp Cys Ala Thr Tyr
210 215 220
GTT GCT GGT CTT TCT GGC TCC ACC TGG TAT ATG TCA ACC TTG TAT TCT 842
Val Ala Gly Leu Ser Gly Ser Thr Trp Tyr Met Ser Thr Leu Tyr Ser
225 230 235
CAC CCT GAT TTT CCA GAG AAA GGG CCA GAG GAG ATT AAT GAA GAA CTA 890
His Pro Asp Phe Pro Glu Lys Gly Pro Glu Glu Ile Asn Glu Glu Leu
240 245 250 255
ILI.I ip~I LI
CA 02388659 2002-05-31
74
ATG AAA AAT GTT AGC CAG AAT CCC CTT TTA CTT CTG ACA CCA CAG AAA 938
Met Lys Asn Val Ser His Asn Pro Leu Leu Leu Leu Thr Pro Gln Lys
260 265 270
GTT AAA AGA TAT GTT GAG TCT TTA TGG AAG AAG AAA AGC TCT GGA CAA 986
Val Lys Arg Tyr Val Glu Ser Leu Trp Lys Lys Lys Ser Ser Gly Gln
275 280 285
1O CCT GTC ACC TTT ACT GAT ATC TTT GGG ATG TTA ATA GGA GAA ACA CTA 1034
Pro Val Thr Phe Thr Asp Ile Phe Gly Met Leu Ile Gly Glu Thr Leu
290 295 300
ATT CAT AAT AGA ATG AAT ACT ACT CTG AGC AGT TTG AAG GAA AAA GTT 1082
Ile His Asn Arg Met Asn Thr Thr Leu Ser Ser Leu Lys Glu Lys Val
305 310 315
AAT ACT GCA CAA TGC CCT TTA CCT CTT TTC ACC TGT CTT CAT GTC AAA 1130
Asn Thr Ala Gln Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys
2 0 320 325 330 335
CCT GAC GTT TCA GAG CTG ATG TTT GCA GAT TGG GTT GAA TTT AGT CCA 1178
Pro Asp Val Ser Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro
340 345 350
TAC GAA ATT GGC ATG GCT AAA TAT GGT ACT TTT ATG GCT CCC GAC TTA 1226
Tyr Glu Ile Gly Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu
355 360 365
TTT GGA AGC AAA TTT TTT ATG GGA ACA GTC GTT AAG AAG TAT GAA GAA 1274
Phe Gly Ser Lys Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu
370 375 380
AAC CCC TTG CAT TTC TTA ATG GGT GTC TGG GGC AGT GCC TTT TCC ATA 1322
Asn Pro Leu His Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile
385 390 395
TTG TTC AAC AGA GTT TTG GGC GTT TCT GGT TCA CAA AGC AGA GGC TCC 1370
Leu Phe Asn Arg Val Leu Gly Val Ser Gly Ser Gln Ser Arg Gly Ser
400 405 410 415
ACA ATG GAG GAA GAA TTA GAA AAT ATT ACC ACA AAG CAT ATT GTG AGT 1418
Thr Met Glu Glu Glu Leu Glu Asn Ile Thr Thr Lys His Ile Val Ser
420 425 430
AAT GAT AGC TCG GAC AGT GAT GAT GAA TCA CAC GAA CCC AAA GGC ACT 1466
Asn Asp Ser Ser Asp Ser Asp Asp Glu Ser His Glu Pro Lys Gly Thr
435 440 445
GAA AAT GAA GAT GCT GGA AGT GAC TAT CAA AGT GAT AAT CAA GCA AGT 1514
Glu Asn Glu Asp Ala Gly Ser Asp Tyr Gln Ser Asp Asn Gln Ala Ser
450 455 460
TGG ATT CAT CGT ATG ATA ATG GCC TTG GTG AGT GAT TCA GCT TTA TTC 1562
Trp Ile His Arg Met Ile Met Ala Leu Val Ser Asp Ser Ala Leu Phe
465 470 475
AAT ACC AGA GAA GGA CGT GCT GGG AAG GTA CAC AAC TTC ATG CTG GGC 1610
Asn Thr Arg Glu Gly Arg Ala Gly Lys Val His Asn Phe Met Leu Gly
480 485 490 495
I I If ~ ~li I 4'I ~ I
CA 02388659 2002-05-31
5
TTG AAT CTC AAT ACA TCT TAT CCA CTG TCT CCT TTG AGT GAC TTT GCC 1658
Leu Asn Leu Asn Thr Ser Tyr Pro Leu Ser Pro Leu Ser Asp Phe Ala
500 505 510
ACA CAG GAC TCC TTT GAT GAT GAT GAA CTG GAT GCA GCT GTA GCA GAT 1706
Thr Gln Asp Ser Phe Asp Asp Asp Glu Leu Asp Ala Ala Val Ala Asp
515 520 525
10 CCT GAT GAA TTT GAG CGA ATA TAT GAG CCT CTG GAT GTC AAA AGT AAA 1754
Pro Asp Glu Phe Glu Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys
530 535 540
AAG ATT CAT GTA GTG GAC AGT GGG CTC ACA TTT AAC CTG CCG TAT CCC 1802
15 Lys Ile His Val Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro
545 550 555
TTG ATA CTG AGA CCT CAG AGA GGG GTT GAT CTC ATA ATC TCC TTT GAC 1850
Leu Ile Leu Arg Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp
2 0 560 565 570 575
TTT TCT GCA AGG CCA AGT GAC TCT AGT CCT CCG TTC AAG GAA CTT CTA 1898
Phe Ser Ala Arg Pro Ser Asp Ser Ser Pro Pro Phe Lys Glu Leu Leu
580 585 590
CTT GCA GAA AAG TGG GCT AAA ATG AAC AAG CTC CCC TTT CCA AAG ATT 1946
Leu Ala Glu Lys Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile
595 600 605
GAT CCT TAT GTG TTT GAT CGG GAA GGG CTG AAG GAG TGC TAT GTC TTT 1994
Asp Pro Tyr Val Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe
610 615 620
AAA CCC AAG AAT CCT GAT ATG GAG AAA GAT TGC CCA ACC ATC ATC CAC 2042
Lys Pro Lys Asn Pro Asp Met Glu Lys Asp Cys Pro Thr Ile Ile His
625 630 635
TTT GTT CTG GCC AAC ATC AAC TTC AGA AAG TAC AAG GCT CCA GGT GTT 2090
Phe Val Leu Ala Asn Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val
4 0 640 645 650 655
CCA AGG GAA ACT GAG GAA GAG AAA GAA ATC GCT GAC TTT GAT ATT TTT 2138
Pro Arg Glu Thr Glu Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe
660 665 670
GAT GAC CCA GAA TCA CCA TTT TCA ACC TTC AAT TTT CAA TAT CCA AAT 2186
Asp Asp Pro Glu Ser Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn
675 680 685
CAA GCA TTC AAA AGA CTA CAT GAT CTT ATG CAC TTC AAT ACT CTG AAC 2234
Gln Ala Phe Lys Arg Leu His Asp Leu Met His Phe Asn Thr Leu Asn
690 695 700
AAC ATT GAT GTG ATA AAA GAA GCC ATG GTT GAA AGC ATT GAA TAT AGA 2282
Asn Ile Asp Val Ile Lys Glu Ala Met Val Glu Ser Ile Glu Tyr Arg
705 710 715
AGA CAG AAT CCA TCT CGT TGC TCT GTT TCC CTT AGT AAT GTT GAG GCA 2330
Arg Gln Asn Pro Ser Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala
720 725 730 735
I i1 ~" ~I'~ I N
CA 02388659 2002-05-31
76
AGA AGA TTT TTC AAC AAG GAG TTT CTA AGT AAA CCC AAA GCA TAG 2375
Arg Arg Phe Phe Asn Lys Glu Phe Leu Ser Lys Pro Lys Ala
740 745
TTCATGTACT GGAAACGGCAGCAGTTTCTGATGCTGAGGCAGTTTGCAATCCCATGACAA2435
CTGGATTTAA AAGTACAGTACAGATAGTCGTACTGATCATGAGAGACTGGCTGATACTCA2495
AAGTTGCAGT TACTTAGCTGCATGAGAATAATACTATTATAAGTTAGGTTGACAAATGAT2555
GTTGATTATG TAAGGATATACTTAGCTACATTTTCAGTCAGTATGAACTTCCTGATACAA2615
1O ATGTAGGGATATATACTGTATTTTTAAACATTTCTCACCAACTTTCTTATGTGTGTTCTT2675
TTTAAAAATT TTTTTTCTTTTAAAATATTTAACAGTTCAATCTCAATAAGACCTCGCATT2735
ATGTATGAAT GTTATTCACTGACTAGATTTATTCATACCATGAGACAACACTATTTTTAT2795
TTATATATGC ATATATATACATACATGAAATAAATACATCAATATAAAAAT 2846
(2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 749
(B) TYPE: amino acid
2 O (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens
2 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
Met Ser Phe Ile Asp Pro Tyr Gln His Ile Ile Val Glu His Gln Tyr
1 5 10 15
Ser His Lys Phe Thr Val Val Val Leu Arg Ala Thr Lys Val Thr Lys
30 20 25 30
Gly Ala Phe Gly Asp Met Leu Asp Thr Pro Asp Pro Tyr Val Glu Leu
35 40 45
35 Phe Ile Ser Thr Thr Pro Asp Ser Arg Lys Arg Thr Arg His Phe Asn
50 55 60
Asn Asp Ile Asn Pro Val Trp Asn Glu Thr Phe Glu Phe Ile Leu Asp
65 70 75 80
Pro Asn Gln Glu Asn Val Leu Glu Ile Thr Leu Met Asp Ala Asn Tyr
85 90 95
Val Met Asp Glu Thr Leu Gly Thr Ala Thr Phe Thr Val Ser Ser Met
loo 105 110
Lys Val Gly Glu Lys Lys Glu Val Pro Phe Ile Phe Asn Gln Val Thr
115 120 125
Glu Met Val Leu Glu Met Ser Leu Glu Val Cys Ser Cys Pro Asp Leu
130 135 140
Arg Phe Ser Met Ala Leu Cys Asp Gln Glu Lys Thr Phe Arg Gln Gln
145 150 155 160
Arg Lys Glu His Ile Arg Glu Ser Met Lys Lys Leu Leu Gly Pro Lys
165 170 175
Asn Ser Glu Gly Leu His Ser Ala Arg Asp Val Pro Val Val Ala Ile
180 185 190
i ~ i, n'~ i ~i i
CA 02388659 2002-05-31
77
Leu Gly Ser Gly Gly Gly Phe Arg Ala Met Val Gly Phe Ser Gly Val
195 200 205
Met Lys Ala Leu Tyr Glu Ser Gly Ile Leu Asp Cys Ala Thr Tyr Val
210 215 220
Ala Gly Leu Ser Gly Ser Thr Trp Tyr Met Ser Thr Leu Tyr Ser His
225 230 235 240
Pro Asp Phe Pro Glu Lys Gly Pro Glu Glu Ile Asn Glu Glu Leu Met
245 250 255
Lys Asn Val Ser His Asn Pro Leu Leu Leu Leu Thr Pro Gln Lys Val
260 265 270
Lys Arg Tyr Val Glu Ser Leu Trp Lys Lys Lys Ser Ser Gly Gln Pro
275 280 285
2 0 Val Thr Phe Thr Asp Ile Phe Gly Met Leu Ile Gly Glu Thr Leu Ile
290 295 300
His Asn Arg Met Asn Thr Thr Leu Ser Ser Leu Lys Glu Lys Val Asn
305 310 315 320
Thr Ala Gln Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro
325 330 335
Asp Val Ser Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr
340 345 350
Glu Ile Gly Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu Phe
355 360 365
Gly Ser Lys Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn
370 375 380
Pro Leu His Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu
385 390 395 400
Phe Asn Arg Val Leu Gly Val Ser Gly Ser Gln Ser Arg Gly Ser Thr
405 410 415
Met Glu Glu Glu Leu Glu Asn Ile Thr Thr Lys His Ile Val Ser Asn
420 425 430
Asp Ser Ser Asp Ser Asp Asp Glu Ser His Glu Pro Lys Gly Thr Glu
935 440 445
Asn Glu Asp Ala Gly Ser Asp Tyr Gln Ser Asp Asn Gln Ala Ser Trp
450 455 460
Ile His Arg Met Ile Met Ala Leu Val Ser Asp Ser Ala Leu Phe Asn
465 470 475 480
Thr Arg Glu Gly Arg Ala Gly Lys Val His Asn Phe Met Leu Gly Leu
485 490 495
Asn Leu Asn Thr Ser Tyr Pro Leu Ser Pro Leu Ser Asp Phe Ala Thr
500 505 510
~:~i ~ I~i~ v ~~
CA 02388659 2002-05-31
78
Gln Asp Ser Phe Asp Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro
515 520 525
Asp Glu Phe Glu Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys
530 535 540
Ile His Val Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu
545 550 555 560
Ile Leu Arg Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe
565 570 575
Ser Ala Arg Pro Ser Asp Ser Ser Pro Pro Phe Lys Glu Leu Leu Leu
580 585 590
Ala Glu Lys Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp
595 600 605
2 0 Pro Tyr Val Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys
610 615 620
Pro Lys Asn Pro Asp Met Glu Lys Asp Cys Pro Thr Ile Ile His Phe
625 630 635 640
Val Leu Ala Asn Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Pro
645 650 655
Arg Glu Thr Glu Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp
660 665 670
Asp Pro Glu Ser Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn Gln
675 680 685
Ala Phe Lys Arg Leu His Asp Leu Met His Phe Asn Thr Leu Asn Asn
690 695 700
Ile Asp Val Ile Lys Glu Ala Met Val Glu Ser Ile Glu Tyr Arg Arg
705 710 715 720
Gln Asn Pro Ser Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala Arg
725 730 735
Arg Phe Phe Asn Lys Glu Phe Leu Ser Lys Pro Lys Ala
740 745
(2)
INFORMATION
FOR
SEQ
ID
NO.:
3:
(i) SEQUENCE CHARACTERISTICS
5O (A) LENGTH: 2787
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (109)..(2355)
(C) OTHER INFORMATION:
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CA 02388659 2002-05-31
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 3:
GGCACAGAGA AGCCTGAGGA TTCTCATTTA ACTCTGGGAA CTGCTTCAAG AAGCTACAGT 60
ACCATAGAAG ACCTGGGAAG TGTGAGAATT TCTGCAACTG GGACCAAA ATG TCT TTC 117
Met Ser Phe
1
ATA GAT CCT TAT CAG CAC ATT ATA GTG GAA CAC CAG TAC TCC CAT AAG 165
Ile Asp Pro Tyr Gln His I1e Ile Val Glu His Gln Tyr Ser His Lys
5 10 15
TTT ACT GTT GTG GTT CTA CGT GCC ACC AAA GTA ACC AAG GGG ACC TTT 213
Phe Thr Val Val Val Leu Arg Ala Thr Lys Val Thr Lys Gly Thr Phe
20 25 30 35
GGC GAT ATG CTG GAC ACT CCA GAT CCT TAT GTG GAA CTT TTC ATC TCT 261
Gly Asp Met Leu Asp Thr Pro Asp Pro Tyr Val Glu Leu Phe Ile Ser
40 45 50
ACA ACC CCT GAC AGC AGG AAG CGA ACG AGA CAC TTC AAT AAT GAT ATA 309
Thr Thr Pro Asp Ser Arg Lys Arg Thr Arg His Phe Asn Asn Asp Ile
55 60 65
2 5 AAC CCC GTG TGG AAT GAG ACC TTT GAG TTC ATT TTG GAT CCT AAT CAG 357
Asn Pro Val Trp Asn Glu Thr Phe Glu Phe Ile Leu Asp Pro Asn Gln
70 75 80
GAA AAT GTT TTG GAG ATC ACA CTG ATG GAT GCC AAC TAC GTC ATG GAT 405
Glu Asn Val Leu Glu Ile Thr Leu Met Asp Ala Asn Tyr Val Met Asp
85 90 95
GAA ACC CTA GGC ACA GCT ACA TTC CCT GTA TCT TCA ATG AAA GTG GGA 453
Glu Thr Leu Gly Thr Ala Thr Phe Pro Val Ser Ser Met Lys Val Gly
100 105 110 115
GAG AAG AAA GAA GTC CCT TTT ATT TTC AAC CAA GTC ACT GAA ATG ATT 501
Glu Lys Lys Glu Val Pro Phe Ile Phe Asn Gln Val Thr Glu Met Ile
120 125 130
CTG GAA ATG TCT CTG GAA GTT TGT TCA TGC CCA GAC CTA CGG TTC AGC 549
Leu Glu Met Ser Leu Glu Val Cys Ser Cys Pro Asp Leu Arg Phe Ser
135 140 145
ATG GCA CTG TGT GAT CAG GAG AAA ACT TTC AGA CAG CAG AGG AAA GAG 597
Met Ala Leu Cys Asp Gln Glu Lys Thr Phe Arg Gln Gln Arg Lys Glu
150 155 160
AAC ATA AAA GAG AAC ATG AAG AAA CTT TTG GGT CCA AAA AAG AGT GAG 645
Asn Ile Lys Glu Asn Met Lys Lys Leu Leu Gly Pro Lys Lys Ser Glu
165 170 175
GGG CTT TAT TCC ACA CGT GAT GTG CCG GTG GTG GCC ATT TTG GGT TCA 693
Gly Leu Tyr Ser Thr Arg Asp Val Pro Val Val Ala Ile Leu Gly Ser
180 185 190 195
GGT GGG GGT TTC CGG GCC ATG GTG GGA TTC TCT GGT GTG ATG AAG GCA 741
Gly Gly Gly Phe Arg Ala Met Val Gly Phe Ser Gly Val Met Lys Ala
200 205 210
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CA 02388659 2002-05-31
8~
CTG TAT GAG TCG GGG ATT TTG GAC TGT GCT ACA TAC ATT GCT GGT CTT 789
Leu Tyr Glu Ser Gly Ile Leu Asp Cys Ala Thr Tyr Ile Ala Gly Leu
215 220 225
TCT GGA TCC ACA TGG TAC ATG TCA ACC TTG TAC TCT CAC CCC GAT TTT 837
Ser Gly Ser Thr Trp Tyr Met Ser Thr Leu Tyr Ser His Pro Asp Phe
230 235 240
1O CCA GAG AAA GGT CCC GAG GAG ATT AAT GAA GAG CTA ATG AAA AAT GTC 885
Pro Glu Lys Gly Pro Glu Glu Ile Asn Glu Glu Leu Met Lys Asn Val
295 250 255
AGC CAC AAC CCT CTC TTA CTT CTT ACA CCA CAG AAA GTT AAA AGA TAC 933
Ser His Asn Pro Leu Leu Leu Leu Thr Pro Gln Lys Val Lys Arg Tyr
260 265 270 275
GTT GAG TCT TTA TGG AAG AAG AAA AGT TCT GGC CAG CCT GTC ACC TTT 981
Val Glu Ser Leu Trp Lys Lys Lys Ser Ser Gly Gln Pro Val Thr Phe
280 285 290
ACT GAC ATC TTT GGG ATG CTA ATA GGA GAA ACA CTA ATT CAA AAT AGG 1029
Thr Asp Ile Phe Gly Met Leu Ile Gly Glu Thr Leu Ile Gln Asn Arg
295 300 305
ATG AGC ATG ACC CTG AGT AGT TTG AAG GAA AAG GTC AAT GCC GCC CGG 1077
Met Ser Met Thr Leu Ser Ser Leu Lys Glu Lys Val Asn Ala Ala Arg
310 315 320
3O TGT CCT TTG CCT CTC TTC ACG TGT CTC CAC GTC AAA CCT GAT GTG TCA 1125
Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro Asp Val Ser
325 330 335
GAG CTG ATG TTT GCC GAT TGG GTG GAA TTT AGT CCA TAT GAG ATT GGC 1173
Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr Glu Ile Gly
340 345 350 355
ATG GCA AAA TAT GGT ACC TTT ATG GCT CCT GAC CTA TTT GGA AGC AAG 1221
Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu Phe Gly Ser Lys
360 365 370
TTT TTT ATG GGA ACA GTT GTA AAA AAA TAT GAA GAA AAC CCC TTG CAT 1269
Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn Pro Leu His
375 380 385
TTC TTG ATG GGT GTC TGG GGC AGT GCC TTT TCT ATA CTG TTC AAC AGA 1317
Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu Phe Asn Arg
390 395 400
GTT TTG GGA GTT TCT GGC TCA CAG AAT AAA GGC TCT ACA ATG GAA GAG 1365
Val Leu Gly Val Ser Gly Ser Gln Asn Lys Gly Ser Thr Met Glu Glu
405 410 415
GAA TTA GAA AAT ATT ACA GCA AAG CAC ATC GTG AGT AAT GAC AGC TCC 1913
Glu Leu Glu Asn Ile Thr Ala Lys His Ile Val Ser Asn Asp Ser Ser
420 925 430 435
GAC AGT GAT GAT GAG GCT CAA GGA CCC AAA GGC ACC GAG AAT GAA GAA 1461
Asp Ser Asp Asp Glu Ala Gln Gly Pro Lys Gly Thr Glu Asn Glu Glu
440 445 450
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CA 02388659 2002-05-31
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GCT GAA AAA GAG TAC CAA AGC GAC AAC CAA GCA AGT TGG GTC CAT CGG 1509
Ala Glu Lys Glu Tyr Gln Ser Asp Asn Gln Ala Ser Trp Val His Arg
455 460 465
ATG CTA ATG GCC TTG GTG AGC GAC TCG GCT TTA TTC AAT ACC CGA GAA 1557
Met Leu Met Ala Leu Val Ser Asp Ser Ala Leu Phe Asn Thr Arg Glu
470 475 480
GGA CGT GCC GGA AAG GTG CAT AAC TTC ATG CTG GGC TTG AAT CTC AAC 1605
Gly Arg Ala Gly Lys Val His Asn Phe Met Leu Gly Leu Asn Leu Asn
485 490 995
ACA TCA TAT CCA CTG TCT CCC CTG AGA GAC TTC AGC TCT CAG GAT TCC 1653
Thr Ser Tyr Pro Leu Ser Pro Leu Arg Asp Phe Ser Ser Gln Asp Ser
500 505 510 515
TTC GAT GAC GAG CTC GAC GCA GCG GTA GCA GAT CCA GAT GAA TTT GAA 1701
Phe Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp Glu Phe Glu
520 525 530
CGA ATA TAT GAA CCA CTG GAT GTC AAA AGT AAG AAG ATT CAT GTG GTA 1749
Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Ile His Val Val
535 540 545
GAT AGT GGG CTC ACA TTT AAC CTG CCA TAT CCC TTG ATT CTT CGA CCT 1797
Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile Leu Arg Pro
550 555 560
3 O CAG AGA GGT GTG GAT CTT ATC ATC TCC TTT GAC TTT TCT GCA AGG CCG 1845
Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser Ala Arg Pro
565 570 575
AGT GAC ACC AGT CCC CCT TTC AAG GAA CTT CTG CTT GCA GAG AAG TGG 1893
3 5 Ser Asp Thr Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala Glu Lys Trp
580 585 590 595
GCG AAA ATG AAC AAG CTT CCC TTT CCA AAG ATC GAT CCT TAT GTG TTT 1941
Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp Pro Tyr Val Phe
4 0 600 605 610
GAT CGG GAA GGA TTA AAG GAA TGC TAT GTT TTT AAA CCT AAG AAT CCT 1989
Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys Pro Lys Asn Pro
615 620 625
GAT GTG GAG AAG GAT TGC CCA ACC ATT ATC CAC TTT GTT CTG GCC AAC 2037
Asp Val Glu Lys Asp Cys Pro Thr Ile Ile His Phe Val Leu Ala Asn
630 635 640
ATC AAC TTC AGA AAG TAC AAG GCC CCA GGT GTT CTA AGG GAA ACC AAA 2085
Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Leu Arg Glu Thr Lys
645 650 655
GAA GAG AAA GAA ATT GCT GAC TTT GAC ATT TTT GAT GAC CCC GAA TCG 2133
Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp Asp Pro Glu Ser
660 665 670 675
CCA TTT TCA ACC TTC AAC TTT CAG TAT CCC AAT CAA GCA TTC AAA AGG 2181
Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn Gln Ala Phe Lys Arg
680 685 690
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CA 02388659 2002-05-31
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CTT CAC GAT TTG ATG TAC TTC AAC ACA CTG AAC AAC ATT GAT GTG ATA 2229
Leu His Asp Leu Met Tyr Phe Asn Thr Leu Asn Asn Ile Asp Val Ile
695 700 705
AAG GAT GCC ATT GTT GAG AGC ATT GAA TAC AGA AGA CAG AAC CCA TCT 2277
Lys Asp Ala Ile Val Glu Ser Ile Glu Tyr Arg Arg Gln Asn Pro Ser
710 715 720
CGT TGC TCT GTT TCC CTC AGT AAT GTT GAA GCA AGA AAA TTC TTC AAT 2325
Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala Arg Lys Phe Phe Asn
725 730 735
AAG GAG TTT CTA AGT AAA CCC ACT GTG TAA TTTCTGTGCT GGGATGATCA 2375
Lys Glu Phe Leu Ser Lys Pro Thr Val
740 745
AGCCATTTGA ATTCCATGAC AATTTGAGTT CAGAAGACAT TAGAGGTCAT CTTACTATGC 2935
AGAAGAGACT GGCTGCTACT CAAAGTTGTG GAGATTTAGC CATGTGTTAG GTGAAAATGA 2495
TGTTGATTAT GTAATACTTA GCAACAGTTTCTGACAGTATGAATTTTTTG ACATTAGCAT2555
AGAGCTATAT ACTGTATTTT AAACATTCCTCACATTTTTTACCTGTACTT TTTATATAAA2615
TATGACATGT CTTTTCTTTT GAAAATATTTAATAGTTTAACTCAGTAAAG GAGACTTCCC2675
ATTGTGTGTG AATGTTATTC TGAACTAGATTTGTTCATGCCATGTTACAA CACTATTTTT2735
ATTTAAATGT TTATATTTAC ACATACGAAATAAATACTTTGCTGTACAAA TT 2787
(2) INFORMATION FOR SEQ 9:
ID NO.:
(i) SEQUENCE CHARACTERISTICSv
(A) LENGTH: 748
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: ID NO.:
SEQ 4:
Met Ser Phe Ile Asp Pro Tyr Gln His Ile Ile Val Glu His Gln Tyr
1 5 10 15
Ser His Lys Phe Thr Val Val Val Leu Arg Ala Thr Lys Val Thr Lys
20 25 30
Gly Thr Phe Gly Asp Met Leu Asp Thr Pro Asp Pro Tyr Val Glu Leu
35 40 45
Phe Ile Ser Thr Thr Pro Asp Ser Arg Lys Arg Thr Arg His Phe Asn
55 60
50 Asn Asp Ile Asn Pro Val Trp Asn Glu Thr Phe Glu Phe Ile Leu Asp
65 70 75 80
Pro Asn Gln Glu Asn Val Leu Glu Ile Thr Leu Met Asp Ala Asn Tyr
85 90 95
Val Met Asp Glu Thr Leu Gly Thr Ala Thr Phe Pro Val Ser Ser Met
100 105 110
Lys Val Gly Glu Lys Lys Glu Val Pro Phe Ile Phe Asn Gln Val Thr
115 120 125
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CA 02388659 2002-05-31
83
Glu Met Ile Leu Glu Met Ser Leu Glu Val Cys Ser Cys Pro Asp Leu
130 135 140
Arg Phe Ser Met Ala Leu Cys Asp Gln Glu Lys Thr Phe Arg Gln Gln
145 150 155 160
Arg Lys Glu Asn Ile Lys Glu Asn Met Lys Lys Leu Leu Gly Pro Lys
165 170 175
Lys Ser Glu Gly Leu Tyr Ser Thr Arg Asp Val Pro Val Val Ala Ile
180 185 190
Leu Gly Ser Gly Gly Gly Phe Arg Ala Met Val Gly Phe Ser Gly Val
195 200 205
Met Lys Ala Leu Tyr Glu Ser Gly Ile Leu Asp Cys Ala Thr Tyr Ile
210 215 220
2 0 Ala Gly Leu Ser Gly Ser Thr Trp Tyr Met Ser Thr Leu Tyr Ser His
225 230 235 240
Pro Asp Phe Pro Glu Lys Gly Pro Glu Glu Ile Asn Glu Glu Leu Met
245 250 255
Lys Asn Val Ser His Asn Pro Leu Leu Leu Leu Thr Pro Gln Lys Val
260 265 270
Lys Arg Tyr Val Glu Ser Leu Trp Lys Lys Lys Ser Ser Gly Gln Pro
275 280 285
Val Thr Phe Thr Asp Ile Phe Gly Met Leu Ile Gly Glu Thr Leu Ile
290 295 300
Gln Asn Arg Met Ser Met Thr Leu Ser Ser Leu Lys Glu Lys Val Asn
305 310 315 320
Ala Ala Arg Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro
325 330 335
Asp Val Ser Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr
340 345 350
Glu Ile Gly Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu Phe
355 360 365
Gly Ser Lys Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn
370 375 380
Pro Leu His Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu
385 390 395 400
Phe Asn Arg Val Leu Gly Val Ser Gly Ser Gln Asn Lys Gly Ser Thr
405 410 415
Met Glu Glu Glu Leu Glu Asn Ile Thr Ala Lys His Ile Val Ser Asn
420 425 430
Asp Ser Ser Asp Ser Asp Asp Glu Ala Gln Gly Pro Lys Gly Thr Glu
435 940 445
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CA 02388659 2002-05-31
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Asn Glu Glu Ala Glu Lys Glu Tyr Gln Ser Asp Asn Gln Ala Ser Trp
450 455 460
Val His Arg Met Leu Met Ala Leu Val Ser Asp Ser Ala Leu Phe Asn
465 470 475 980
Thr Arg Glu Gly Arg Ala Gly Lys Val His Asn Phe Met Leu Gly Leu
485 490 495
Asn Leu Asn Thr Ser Tyr Pro Leu Ser Pro Leu Arg Asp Phe Ser Ser
500 505 510
Gln Asp Ser Phe Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp
515 520 525
Glu Phe Glu Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Tle
530 535 540
His Val Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile
545 550 555 560
Leu Arg Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser
565 570 575
Ala Arg Pro Ser Asp Thr Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala
580 585 590
Glu Lys Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp Pro
595 600 605
Tyr Val Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys Pro
610 615 620
Lys Asn Pro Asp Val Glu Lys Asp Cys Pro Thr Ile Ile His Phe Val
625 630 635 640
Leu Ala Asn Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Leu Arg
645 650 655
Glu Thr Lys Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp Asp
660 665 670
Pro Glu Ser Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn Gln Ala
675 680 685
Phe Lys Arg Leu His Asp Leu Met Tyr Phe Asn Thr Leu Asn Asn Ile
690 695 700
Asp Val Ile Lys Asp Ala Ile Val Glu Ser Ile Glu Tyr Arg Arg Gln
705 710 715 720
Asn Pro Ser Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala Arg Lys
725 730 735
Phe Phe Asn Lys Glu Phe Leu Ser Lys Pro Thr Val
740 745
(2) INFORMATION FOR SEQ ID NO.: 5:
~ I; ~ ,I': I 61
CA 02388659 2002-05-31
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS:
5 (D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 5:
10 Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu
1 5 10 15
Tyr Arg Asn Gly Lys
