Note: Descriptions are shown in the official language in which they were submitted.
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STEROL GLUCOSIDE TOXINS
FIELD OF THE INVENTION
The invention relates to the identification of sterol glucoside toxins, and
provides
methods for detecting and detoxifying the compounds, as well as therapeutic
methods for
treating subjects exposed to such toxins.
BACKGROUND OF THE INVENTION
Sterols are a diverse group of lipids, many of which are found in appreciable
quantities in animal and vegetal tissues. Sterols may include one or more of a
variety of
molecules belonging to C27-C30 crystalline alcohols, having a common general
structure
based on the cyclopentanoperhydrophenanthrene ring (also called sterane). In
the tissues of
vertebrates, the main sterol is the C27 alcohol cholesterol. There are a
variety of other
naturally-occurring animal sterols, such as lanosterol (a C30 compound) and 7-
dehydrocholesterol, which are illustrative of the structural similarities of
sterols. The
nomenaclature of sterols is based on the numbering of the carbons as
exemplified below for
cholesterol:
21 22 24 26
23 25
18
17
12 27
11 13 16
19 C D
14
1 9
2 10 8
A B
3 5 7
4
H 6
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Sterols are also found in plants. The denomination "phytosterol" has been used
for
sterols of vegetal origin. Chemically, plant sterols generally have the same
basic structure as
cholesterol, with differences occurring for example in the lateral chain on
carbon 17.
Cholesterol may itself be found in plants. Representative phytosterols are
compounds having
29 or 30 carbon atoms, such as campesterol, stigmasterol and beta-sitosterol
(stigmasta-5-en-
3beta-ol).
Steryl glycosides are sterol derivatives in which a carbohydrate unit is
linked to the
hydroxyl group of a sterol molecule. In plants, steryl glycosides have been
found in which the
sterol moiety is composed of various sterols: campesterol, stigmasterol,
sitosterol,
brassicasterol and dihydrositosterol. Similarly, the carbohydrate moiety may
be composed of
a variety of sugars, such as glucose, xylose or arabinose. Sterol glycosides
may be obtained
from biological sources such as plant tissues by a variety of methods (see for
example
Sugawara et al. Lipids 1999, 34, 1231; Ueno, et al. U.S. Patent No. 4,235,992
issued
November 25, 1980). An exemplary plant sterol glycoside is beta-sitostrol-beta-
D-glucoside
(5-cholesten-24b-ethyl-3b-ol-D-glucoside), for which the formula is give below
(also
showing the structure of the acylated compound):
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C H2OR
O
___" 'C :
HOQH
OH
R = H or C15H31CO
Acylated sterol glycosides may be formed in plants when a fatty acid is
acylated at the
primary alcohol group of the carbohydrate unit (such as glucose or galactose)
in the steryl
glycoside molecule (see Lepage, J Lipid Res 1964, 5, 587). For example, the 6'-
palmitoyl-
beta-D-glucoside of beta-sitosterol is reportedly present in potato tubers and
the 6'-linoleoyl-
beta-D-glucoside of beta-sitosterol is reportedly found in soybean extracts.
Acylated steryl
glucoside may be present at relatively high concentrations in a variety of
vegetable parts,
with the acylated form being generally more abundant that the non acylated
sterol glycoside
itself (Sugawara et al., Lipids 1999, 34, 1231).
Sterol glycosides also occur in bacteria. Helicobacter has for example been
described
as being particularly rich in cholesterol glucosides (Haque et at., J.
Bacteriol 1995, 177:
5334; Haque et al., April 1996, J Bacteriol;178(7):2065-70). A cholesterol
diglucoside has
been reported to occur in Acholeplasma axanthum (Mayberry et al., Biochim
Biophys Acta
1983, 752, 434).
Sterols and sterol glycosides have been reported to have a wide spectrum of
biological
activities in animals and humans (Pegel, et al., U.S. Patent No. 4,254,111
issued March 3,
1981; Pegel et al., U.S. Patent No. 4,260,603 issued April 7, 1981) and
techniques for
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transdermal administration of these compounds have been suggested (Walker, et
al. U.S.
Patent No. 5,128,324 issued July 7, 1992). It has been suggested that some
plant sterols, their
fatty acid esters and glucosides may be useful for treating cancers (Eugster,
et al., U.S. Patent
No. 5,270,041, December 14, 1993). There have been indications that sterols
and sterol
glycosides are generally non-toxic, or toxic only at relatively high doses
while being
beneficial at lower doses (Pegel, U.S. Patent No. 4,188,379 issued February
12, 1980). Some
phytosterols are thought to have therapeutic effects, such as anti-tumor
properties. Beta-
sitosterol is catogorized in the Merk Index, Tenth Edition, as an
antihyperlipoproteinemic. It
has been suggested that beta-sitosterol (BSS), and its glucoside (BSSG)
enhance the in vitro
proliferative response of T-cells (Bouic et al., Int J Immunopharmacol 1996
Dec;18(12):693-
700), may have other stimulatory effects as immunomodulators (Bouic et al.,
Int J Sports
Med 1999 May;20(4):258-62), and may therefore be therapeutically beneficial in
a wide
variety of diseases because of these immunostimulatory properties (Bouic and
Lamprecht,
Altern Med Rev 1999 Jun;4(3):170-7; Bouic et al., U.S. Patent No. 5,486,510,
January 23,
1996).
Cholesterol glucoside (5-cholesten-3b-ol-3b-D-glucoside) is reportedly made by
human cells in culture in conjunction with a heat shock response (Kunimoto et
al., Jan 2000,
Cell Stress Chaperones;5(1):3-7). Cholesteryl glucoside has also been reported
to occur in
Candida bogoriensis (Kastelic-Suhadolc, Biochim Biophys Acta 1980 Nov
7;620(2):322-5).
Sterol glucosides may be hydrolyzed in acid, such as in methanolic HC1
(Kastelic-
Suhadolc, Biochim Biophys Acta 1980 Nov 7;620(2):322-5). Enzymatic cleavage of
the beta-
glycosidic linkage may also be accomplished, for example by a beta-d-
glucosidase. A
therinostable beta-d-glucosidase from Thermoascus aurantiacus that hydrolysed
aryl and
alkyl beta-d-glucosides has for example recently been reported (Parry et al.,
1 January 2001,
Biochem J, 353 (Pt 1): 117-127). A steryl-beta-glucosidase (EC 3.2.1.104; CAS
Registration
No. 69494-88-8; cholesteryl-beta-D-glucoside glucohydrolase) has been
identified from
Sinapis alba seedlings that reportedly acts on glucosides of cholesterol and
sitosterol, but not
on some related sterols such as coprostanol, to hydrolyse the glucoside -
producing sterol and
D-glucose (Kalinowska and Wojciechowski, 1978, Phytochemistry 17: 1533-1537).
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Selective neuronal cell death is the common hallmark of various
neurodegenerative
disorders. At least two mechanisms of neuronal death have been identified
within the
mammalian central nervous system: necrosis and apoptosis. Necrosis is
generally
characterized as a passive form of `accidental' cell death that follows
physical damage and is
distinguished by membrane permeability changes leading to swelling of cell
organelles and
rupture of the plasma membrane (Simonian and Coyle, 1996). In contrast,
apoptosis is
generally characterized as an active form of programmed cell death involving
individual cells
that often remain surrounded by healthy neighbors. Apoptosis reportedly
requires ATP and
protein synthesis (Earnshaw, 1995) and has been characterized by cell
shrinkage, membrane
blebbing, and genomic fragmentation (Ellis et al., 1991; Nagata, 1997).
Both necrosis and apoptosis may be induced by stimulation of neurons by
glutamate
agonists acting through various glutamatergic excitatory amino acid (EAA)
receptor subtypes
(Choi, 1995). The actions of glutamate have been classified as either
"excitotoxicity" or
"excitotoxicity-independent". Excitotoxicity is thought to involve the over-
activation of
target EAA receptors leading to increased ionic flux. Two main types of
excitotoxicity have
been described: (1) chronic/slow excitotoxicity, which is thought to result
from defects in
energy metabolism leading to persistent receptor activation by ambient
glutamate (Zeevalk
and Nicklas, 1990); and, (2) acute/fast excitotoxicity, which is thought to
occur following
exposure to high levels of glutamate or glutamate agonists. For example, the
over-
stimulation of NMDA receptors by glutamate or NMDA may result in increased
calcium
flux, which in turn may lead to activation of cellular proteases and the
activation of other
potentially harmful molecules or pathways. It has been suggested that such
actions may
underlie the damage caused by ischaemia and hypoxia (Choi, 1995; Meldrum and
Garthwaite, 1990) or head trauma (Katayama et al., 1988).
Excitotoxicity-independent mechanisms of cell death have been shown to arise
due to
the accumulation of reactive oxygen species (ROS), elevation of calcium, and
the loss of
intracellular glutathione (GSH) (Tirosh et al., 2000). Each of these events
may induce
oxidative stress, described as an imbalance between oxidants (ROS) and
antioxidants (GSH,
GSH peroxidase, vitamins C and E, catalase, SOD, etc.) with the oxidants
becoming
dominant (Sies, 1991). Oxidative stress may trigger cellular necrosis (Wullner
et al., 1999) as
well as apoptosis (Zaman and Ratan, 1998; Hockenbery et al., 1993; Higuchi and
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Matsukawa, 1999; Nicole et al. 1998) and often arises due to factors leading
to GSH
depletion. For a number of reasons, neurons are thought to be particularly
susceptible to
oxidative stress, and oxidative stress-induced cell death has figured in a
number of
hypotheses concerning neurodegenerative diseases (see Evans, 1993; Simonian
and Coyle,
1996; Palmer, 1999; Russel et al., 1999) and aging (Verarucci et al., 1999).
Toxins present in the environment may play a role in human pathology. For
example,
agenized wheat flour was the most common source of processed flour in much of
the Western
world for the first fifty years of the 20th Century (see Shaw and Bains, 1998;
Campbell et al.,
1950) and was later found to contain methionine sulfoximine (MSO) in high
concentration.
MSO induced epileptic seizures in experimental animals ((Newell et al., 1947),
an action that
was not understood but thought to arise due to MSO acting to inhibit the
synthesis of both
GSH and glutamine (Meister and Tate, 1976). Subsequent studies have revealed
that MSO
also has neuro-excitotoxic actions, apparently via NMDA receptor activation
(Shaw et al.,
1999).
The etiology of various age-related neurological diseases remains largely
unknown.
Sporadic forms of Alzheimer's, Parkinson's, and Lou Gehrig's disease
(amyotrophic lateral
sclerosis, ALS) have been linked to environmental factors that cause neuronal
cell death by
either by excitotoxicity or by inducing oxidative stress. The experimental and
clinical
literature has been taken to support a potential role for excitotoxins in some
forms of
neurodegeneration, notably Lou Gehrig's disease and Alzheimer's disease. In
particular,
abnormalities in glutamate handling/transport have been linked to ALS
(Rothstein et al.,
1990, 1992, 1995) and domoic acid, a kainate receptor agonist, has been shown
to be causal
to memory losses much like those reported in Alzheimer's disease (Perl et al.,
1990).
Oxidative stress has also been linked to the same diseases, particularly
following GSH
depletion (see Bains and Shaw, 1997). Excitotoxicity and oxidative stress may
in fact be
innately linked in that neural excitation, particularly over-excitation which
occurs in
excitoxicity, may generate free radicals acting to increased oxidative stress
(Bindokas et al.,
1998).
The following abbreviations may be used in the present application: ALS,
amyotrophic lateral sclerosis; ALS-PDC, ALS-parkinsonism dementia complex;
AMPA, a-
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amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ATP, adenosine
triphosphate; BSSG,
(3-sitosterol-(3-D-glucoside; EAA, excitatory amino acid; G1uR, glutamate
receptor; GSH,
glutathione; LDH, lactate dehydrogenase; MSO, methionine sulfoximine; NMDA, N-
methyl-
D-aspartate; ROS, reactive oxygen species; SOD, superoxide dismutase.
SUMMARY OF THE INVENTION
In one aspect, the present invention discloses the neuronal excitotoxicicity
of sterol
glycosides. In alternative embodiments, sterol glycosides that are
characterized by neuronal
excitotoxicity are (3-sitosterol- f3-D-glucoside (BSSG) and cholesterol
glucoside.
In one aspect of the invention, BSSG is identified as a toxin present in the
seed of the
cycad palm (Cycas circinalis), historically a staple of the diet of the
Chamorro people of
Guam. Cycad seed consumption has been linked to ALS-parkinsonism dementia
complex
(ALS-PDC), an endemic neurological disorder of Guam (Kurland, 1988).
Accordingly, in
various embodiments, the present invention provides methods of treating foods
to reduce the
concentration of sterol glycosides such as BSSG or cholesterol glucoside in
foods. In some
embodiments, the foods to be treated may for example include plant materials.
An alternative aspect of the present invention is the demonstration that mice
fed cycad
flour containing BSSG have severe behavioral abnormalities of motor and
cognitive function,
as well as significant levels of neurodegeneration in the cortex, hippo
campus, spinal cord,
substantia nigra and other CNS regions measured post mortem. Accordingly, in
one aspect
the present invention provides an animal model for studying neurodegenerative
disease, in
which a non-human mammal is fed an excitatory neurotixic sterol glycoside such
as BSSG or
cholesterol glucoside.
In one aspect, the present invention demonstrates that BSSG may mediate
neuronal
glutamate release followed by NMDA receptor activation. Accordingly, in one
aspect the
present invention provides in vitro assays for modulators of cytotoxic action,
such as assays
for identifying compounds that interfere with cytotoxic neuronal glutamate
release mediated
by BSSG or cholesterol glucoside. Lactate dehydrogenase assays may for example
be used to
assay cell death in vitro in conjunction with administration of BSSG and
putative inhibitors
of cytotoxicity.
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In an alternative aspect, the invention provides
kits for detecting BSSG or cholesterol glucoside, for example
to detect BSSG or cholesterol glucoside in foods or in body
fluids. Such kits may for example include immunoassays. In
one aspect, the invention accordingly provides antibodies, or
other ligands, that bind to the toxins of the invention.
In further alternative aspects, the invention
provides methods for treating subjects exposed to the toxins
of the invention. For example, such methods may include
vaccination with an antigenic composition effective to raise
antibodies to the toxins, or treatment of body fluids with an
adsorbent, such as an immunoadsorbent, to remove toxins of
the invention.
In yet another aspect, the invention provides an
animal model of a neurodegenerative disease comprising a non-
human mammal fed a sufficient amount of a neurotoxic sterol
glycoside to produce symptoms of the neurodegenerative
disease.
In yet a further aspect, the invention provides a
method for identifying neurological degeneration in a non-
human animal, the method comprising the steps of:
administering a neurotoxic sterol glycoside to the animal;
and identifying neurological degeneration in the animal.
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In one aspect, the present invention provides use, for examining
neurodegeneration in a non-human animal, of an animal model of neurotoxic
sterol
glycoside-induced neurodegeneration, said animal model comprising a non-human
mammal having neurodegeneration caused by administration of a synthetic
neurotoxic sterol glycoside in an amount sufficient to cause the
neurodegeneration in
the non-human mammal.
In another aspect, the present invention provides a method for
examining neurodegeneration in a non-human animal, the method comprising the
step of: administering a synthetic neurotoxic sterol glycoside to the animal,
wherein
the neurotoxic sterol glycoside causes the neurodegeneration in the animal,
and
wherein the neurodegeneration in the animal is examined by observing
behavioral
abnormalities in the mammal or is examined post-mortem in a central nervous
system tissue of the animal.
In still another aspect, the present invention provides a method for
producing an animal model of neurodegeneration, said method comprising the
steps
of: administering a synthetic neurotoxic sterol glycoside to a non-human
animal; and
allowing the animal to develop neurodegeneration, wherein the neurotoxic
sterol
glycoside causes the neurodegeneration, and wherein the neurodegeneration in
the
animal is examined by observing behavioral abnormalities in the animal or is
examined post mortem in a central nervous system tissue of the animal.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Shows neuro-excitotoxic action of cycad extract (7 x washed cycad
chips)
demonstrated by in vitro indices of cycad-induced neural activity and toxicity
on rat cortical slices. A.
Cortical wedge recording of adult rat neocortex. Drugs were administered to
the medium bathing
each wedge by gravity flow and neural activity differentially recorded as a
field potential. MK801
(MK) blocked the NMDA and cycad-induced depolarizations as did AP5 (not
shown); NBQX
blocked only the AMPA response, but had no effect on the cycad response (not
shown). B. Cortical
slice assays for LDH release following exposure to various compounds. Cycad
fractions in the same
concentration as applied to induce depolarization gave greater LDH release
than that evoked by
NMDA. The effects of both were attenuated by APS. Mg2+ diminished LDH release
while freeze-
thawing slices maximized cell death. *P<0.05, Student's t test.Drug
concentrations: NMDA (N),
20 M; AMPA (A), 10 M; cycad: 1: 50 dilution of crude extract of washed cycad
in Krebs-Heinseleit
buffer (Cyc).
Figure 2. Actions of isolated BSSG fractions on rat cortical slices. A. Field
potential
recording of isolated cycad sterol glucoside fraction D-2 (15 M) compared to
NMDA (20
M), other plant sterol glucosides (ouabain or emicymarin, 50 M), the (3-
sitosterol aglycone
(10 M), or D-2 plus AP5 (10 M). Arrows indicate onset of drug application.
B. LDH
release following exposure to the same BSSG D-2 fraction (75 M) compared to
NMDA (50
M), the sitosterol aglycone, R-SS (75 M); various compounds in the presence
of AP5 (20
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M). The action of cholesterol glucoside was qualitatively similar to (3-
sitosterol-(3-D-
glucoside (not shown). Statistics as in Fig. 1.
Figure 3: [3H]-glutamate release in rat cortical slices. A. [3H]-glutamate
release with
isolated BSSG D-1-2 fraction (25 M) compared to NMDA (50 M) with or without
AP5 (20
M). For this experiment, calcium concentration was either 0 (L) or 2mM (H).
Note the
calcium dependence for both NMDA and BSSG. B. [3H]-glutamate release by D-2
BSSG
fraction. Concentrations as in B.
Figure 4: Behavioral test results in the mouse model of neurodegenerative
disease. A.
Leg extension: the mouse is held by its tail, and in a normal mouse, both of
its legs flex out (a
score of 2 is recorded). If one or both of the legs do not flex out a score of
1 or 0 is given
accordingly. B. Gait Length: the mouse walks through a tunnel with paint on
its backpaws.
Distance between subsequent paw prints is recorded as the gait length (stride
length). C.
Rotarod: the mouse is placed in a rotating cylinder, at increasing speeds. The
time to fall of
the cylinder and number of spins (rotations with out falling off) are
recorded. D. Wire Hang:
the mouse is placed up side down on a wire mesh and time to fall into a padded
box is
recorded. E. Water Maze: the mouse is placed in a small swimming pool of water
and swims
to find a hidden platform located near the middle of the pool. Time to find
the platform and
percentage of time in each quadrant of the pool is recorded. F. Radial Arm
Maze: the mouse
is placed in a 8 arm maze, in which 4 of the arms are baited with food. Errors
are recorded as
entries into unbaited tubes and re-entry in to tubes already visited.
Figure 5: Caspase-3 labeling of human cortical astrocytes in tissue culture.
Fetal
human telencephalic astrocytes were grown to confluency and then exposed to
NMDA,
BSSG, cholesterol glucoside (CG), or hydrogen peroxide (H202) for various
periods. The
peak of caspase-3 positive labeling was seen at 24 hrs. after exposure to the
various
compounds. Note significant caspase-3 positive labeling in all experimental
conditions vs.
control (DMEM), including for the sterol glucosides BSSG and CG. At longer
time points,
the overall numbers of cells in these conditions declined.
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Figure 6: Cumulative motor and cognitive deficits of seven cycad-fed animals
(cyc 1
through cyc 7). Averages of the values recorded for each of the cycad animals
on each of the
behavioural tests described were expressed as a percentage in relation to the
cumulative
control values. Values were computed for motor, cognitive, or combined motor
and
cognitive functions as follows: Control values for any measure were averaged
across all
control animals and set to 100%. For each cycad animal, the individual
response for each
separate measure was described as % response/ 100; total cumulative response
for cycad
mouse is the sum average of all such separate measures/100. Left: Motor
deficits. Centre:
Cognitive deficits. Right: Combined motor and cognitive deficits. S.E.M.
indicates variance
across individual behavioural measures in each of the cycad-fed mice.
DETAILED DESCRIPTION OF THE INVENTION
Kits and assays of the invention may include a variety of techniques for
detecting
toxins. For example, antibodies to BSSG or cholesterol glucoside may be used
in kits or
assays. Antibodies, or other ligands, that bind to the toxins of the invention
may for example
be used to prepare kits such as immunoassay agglutination kits designed to
detect the toxins
in biological specimens, such as blood or feces. Affinity purified antibodies
against toxins of
the invention may for example be used to passively coat small particles, such
as polystyrene
particles, that form visible aggregates when they are mixed with a sample
containing the
toxins. Many alternative immunoassay procedures for detecting toxins of the
invention in
body fluids may be adapted from methods known in the immunoassay art. Such
techniques
may include radio-immunoassay techniques; and, enzyme-immunoassay techniques
such as
competitive, double antibody solid phase ("DASP") and sandwich procedures.
Various solid
phase immunoassays may be performed using various solid supports, including
finely divided
cellulose, solid beads or discs, polystyrene tubes and microtiter plates.
Enzyme
immunoassays may be adapted to include color formation as an indicator of a
result.
Procedures for raising polyclonal antibodies are also well known. Typically,
such
antibodies can be raised by administering an antigenic formulation of the
toxin of the present
invention subcutaneously to an antibody producing animal, such as New Zealand
white
rabbits. The antigens may for example be injected at a total volume of 100
microlitres per site
at six different sites. Each injected material may contain adjuvants. The
rabbits are then bled
two weeks after the first injection and periodically boosted with the same
antigen three times
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every six weeks. A sample of serum is then collected 10 days after each boost.
Polyclonal
antibodies are then recovered from the serum by affinity chromatography using
the
corresponding antigen to capture the antibody. This and other procedures for
raising
polyclonal antibodies are disclosed in E. Harlow, et. al., editors,
Antibodies: A Laboratory
Manual (1988). United States Patent No. 5,753,260 issued to
Alving, et al. May 19, 1998 discloses immunoreactive compositions
and methods for immunizing animals to produce antibodies
against sterols, which may be used to produce anti-BSSG or anti-cholesterol
glucoside
antibodies. Antigenic compositions for raising such antibodies may for example
include
liposomes containing phosphatidylcholine, toxin (such as BSSG or cholesterol
glucoside),
and adjuvant such as lipid A in molar ratios of approximately 2:5:0.02.
Delivery vehicles
other than liposomes would also be suitable, including microcapsules,
microspheres,
lipospheres, polymers, and slow release devices could serve instead of
liposomes.
Monoclonal antibody production to toxins of the present invention may
similarly be
effected by known techniques involving first obtaining immune cells
(lymphocytes) from the
spleen of a mammal (e.g., mouse) which has been previously immunized with the
antigen of
interest either in vivo or in vitro. The antibody-secreting lymphocytes are
then fused with
(mouse) myeloma cells or transformed cells, which are capable of replicating
indefinitely in
cell culture, thereby producing an immortal, immunoglobulin-secreting cell
line. Fusion with
mammalian myeloma cells or other fusion partners capable of replicating
indefinitely in cell
culture is effected by standard and well-known techniques, for example, by
using
polyethylene glycol ("PEG") or other fusing agents (see Milstein and Kohler,
Eur. J. Immunol. 6:511 (1976)). The resulting fused cells,
or hybridomas, are cultured, and the resulting colonies screened for the
production of the
desired monoclonal antibodies. Colonies producing such antibodies are cloned
and grown
either in vivo or in vitro to produce large quantities of antibody. A
description of the
theoretical basis and practical methodology of fusing such cells is set forth
in Kohler and
Milstein, Nature 256:495 (1975).
In addition to utilizing whole antibodies, the kits and processes of the
present
invention encompass use of binding portions of such antibodies that recognize
toxins of the
invention. Such binding portions include Fab fragments, F(ab')2 fragments, and
Fv fragments.
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These antibody fragments can be made by conventional procedures, such as
proteolytic
fragmentation procedures, as described in Goding, Monoclonal Antibodies:
Principles and
Practice, pp. 98-118, New York:Academic Press (1983).
In one aspect of the invention, ligands (such as antibodies) to the toxins of
the
invention may be administered to a patient in need of such treatment to
ameliorate the effect
of the toxins on the patient. In an alternative aspect of the invention, an
animal, such as a
human, may be vaccinated with an antigenic composition effective to raise
antibodies against
sterol glucoside toxins of the invention, such as BSSG or cholesterol
glucoside, to retard or
reduce the severity of toxicity caused by ingestion of toxins of the
invention.
In alternative embodiments, the invention provides methods of detoxification
of
compositions containing toxins, such as foodstuffs, in which sterol glucoside
toxins of the
invention may be hydrolyzed, for example in acid, such as in methanolic HCl
(Kastelic-
Suhadolc, Biochim Biophys Acta 1980 Nov 7;620(2):322-5). Detoxification by
enzymatic
cleavage of the beta-glycosidic linkage may also be accomplished, for example
by a beta-d-
glucosidase. A thermostable beta-d-glucosidase from Thermoascus aurantiacus
that
hydrolyses aryl and alkyl beta-d-glucosides has for example recently been
reported (Parry et
al., 1 January 2001, Biochem J, 353(Pt 1): 117-127). A steryl-beta-glucosidase
(EC
3.2.1.104; CAS Registration No. 69494-88-8; cholesteryl-beta-D-glucoside
glucohydrolase)
has been identified from Sinapis alba seedlings that reportedly acts on
glucosides of
cholesterol and sitosterol (Kalinowska and Wojciechowski, 1978, Phytochemistry
17: 1533-
1537).
In one aspect, the invention provides methods for treatment of materials, such
as body
fluids, with an adsorbent, such as an immunoadsorbent, to remove toxins of the
invention. In
such methods, an antibody in an insoluble form may be used to bind the toxin
antigen to
remove it from a mixture of substances. For example, solid-phase
immunoadsorbent gels may
be used, in which purified antibodies, for example from the serum of immunized
animals, is
coupled to cyanogen bromide-activated 4% agarose gels. In alternative
embodiments,
sephadex, derivatives of cellulose, or other polymers can be used as the
matrix as an
alternative to agarose.
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Alternative aspects of the invention are illustrated in the following
Examples, which
are merely illustrative of some embodiments and do not necessarily reflect the
full scope of
the invention.
Examples
Animals
In vitro experiments were performed on adult (>70do) male Sprague-Dawley
colony
rats maintained on a light-dark cycle (12 hr: 12 hr). In vivo experiments were
conduced using
CD-1 colony reared 5-7 mo. old male mice.
Chemicals
MSO was obtained from Sigma-Aldrich Canada Ltd. (Mississauga, Ontario). AMPA,
NMDA, AP5, and NBQX were obtained from Precision Biochemicals Inc. (Vancouver,
British Columbia). [3H] CGP 39653 and [3H] glutamate were purchased from NEW
Mendel
Scientific Co.(Guelph, Ontario). LDH kits and DNAase were obtained from Sigma
(St.
Louis). TUNEL kits were purchased from Intergen (ApopTag).(Oxford). Other
chemicals
were of analytical grade available from BDH Inc. (Vancouver, British
Columbia).
Cycad Extracts and Purification of BSSG
Initial experiments were performed with crude cycad flour extracts made by
extensively grinding chips of cycad in a small volume of distilled water.
These cycad chips
had been extensively soaked over a period of 7 days. This cycad extract was
diluted by
various factors in Krebs-Henseleit buffer for use in bath application to field
potential or LDH
assays. Based on early experiments (e.g., see Fig. 2), cycad fractions were
extensively
screened for potency based on the size of the evoked field potential response
or on amount of
LDH released. From each stage, the most potent batch was selected and further
separated by
column chromatography. The fractions ultimately yielded several variants of a
sterol
glucoside, (3-sitosterol-(3-D glucoside (BSSG) with a range of molecular
weights ranging
from 574-576). These fractions have been given fraction identification codes
indicating stage
in the isolation procedure and are described in the following as D-2, D-1-1,
and D-2.
Electrophysiology: Field Potential Recordings
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Cortical `wedges' were prepared as described previously (Shaw et al., 1996).
In brief,
animals were anesthetized with C02, decapitated, and a cortical block rapidly
removed and
placed in cold Krebs-Henseleit buffer containing (in mM): NaCl 124, KC1 3.3,
NaHCO3 25,
glucose 10, KH2PO4 1.2, CaC12 2.4, and MgSO4 1.2, bubbled with 5% C02/ 95% 02,
pH 7.4.
The cortical block was sectioned into 500 M thick coronal slices using a
Vibratome
(Campden Instruments) and the slices cut into pie shaped wedges in which the
white matter
formed the narrow edge of the wedge. Each wedge was placed on a net across a
grease gap
between two fluid filled chambers. The cortical side of the wedge was bathed
(at room
temperature, approx. 25 C) in buffer lacking Mgt+; the callosal portion was
bathed in buffer
containing Mg 2+ to minimize neural activity. Field potentials were
differentially recorded
between the two chambers using two Ag/AgC1 electrodes. Recordings from up to 6
wedges,
each in individual chambers, could be made simultaneously for each experiment.
The
wedges were continuously perfused on the cortical side with oxygenated, Mg2+-
free buffer
using a gravity feed system. Using this system, drugs could be rapidly
substituted for control
media to examine response characteristics. Wedges typically survived for up to
8 hrs.
Responses were recorded on LabViewTM after amplification and A/D conversion
and the
traces were charted in ExcelTM for WindowsTM. Statistical analysis of peak
response
amplitude was performed by one-way ANOVA using Bonferroni's post test with
GraphPad
PrismTM
[3H]-Glutamate Release Studies
Brain slices were taken from cortical blocks in which all subcortical tissue
had been removed.
400 M slices were cut using a modified slice cutter (Van Huizen et al., 1989).
Slices were rinsed
twice for 5 min in Mg +2 containing Krebs-Henseleit buffer pH 7.4. Incubation
media consisted of
100 M cold glutamate, 20 M AP5 and 10 M DNQX, the latter NMDA or AMPA
antagonists,
respectively. 10 nM of [3H]-glutamate was added to the mixture and incubated
for lhr at 37 C under
in oxygenated atmosphere (02/CO2=95/5%). Experimental treatments were
performed in 500 M
Mg+2 free buffer placed in tissue culture wells containing different
concentrations of MSO or isolated
BSSG fractions of cycad flour. Slices were removed at the end of incubation
period and the
supernatant removed for scintillation counting. The supernatant fractions were
placed in scintillation
vials containing NEN Formula 989 for a minimum of 12 hrs before being counted
in a Beckman
LS6000 scintillation counter. Results were normalized to the dpm counts of
respective controls.
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LDH Assays
Cortical slices were prepared as described above in the glutamate release
experiments and
placed in tissue culture wells containing Krebs-Heinsleight buffer
supplemented with 0.0004% H202
and lmg/mL glucose. Extensive previous studies have demonstrated that this
medium supports
cellular activity for prolonged periods (Van Huizen et al., 1989; Shaw et al.,
1996). (Note that
hydrogen peroxide, added as the source of molecular oxygen, was not
deleterious at this low
concentration (see Van Huizen et al., 1989). In our preliminary experiments,
hydrogen peroxide did
not affect LDH release up to a 1mM concentration (0.0034%)). All slices were
washed twice with
buffer for 20 min each at room temperature before incubation in media
containing the test
compounds for lhr at 37 C. Test compounds included MSO, NMDA, kainate,
different
concentrations of cycad extract or different fractions or concentrations of
isolated BSSG. MSO,
NMDA, and cycad/BSSG were each tested alone or in combination with AP5, and
compared to
control slices maintained in buffer alone. For additional comparison and to
establish the limits of the
method, some slices were freeze-thawed to kill all the cells. Alternatively,
some slices were
incubated in buffer containing 1.2 mM Mg2+ in order to diminish spontaneous
neural activity. At the
end of the lhr incubation period, 3 samples (100 l of buffer, each sample)
were taken from each
well. LDH assays were performed on these samples using a LDH diagnostic kit
(Sigma) following
the manufacturer's protocol with some modifications. In brief, 0.5 ml of
pyruvate solution was
mixed with 0.5 mg pre-weighed NADH. 100 l of slice medium (free of slices)
was added to the
mixture and incubated for 30 min at 37 C. 0.5 ml of Sigma coloring reagent (2,
4-
dinitrophenylhydrazine in HCI, 2mg/ml) was added to develop the color and the
mixture was
incubated for 20 min at room temperature. 5 ml of 0.4 N NaOH were added to
each tube. After 5
min, optical density was read at 440nm. Standard curves were prepared for each
assay using
different concentrations of pyruvate solution (0-960 units). LDH activity (in
International Units) was
calculated from the standard curve and normalized by total protein content of
each slice as
determined by the Lowry protein assay (Peterson, 1979). One International Unit
represents the
amount of enzyme required to convert 1 mol of substrate/minute at room
temperature.
In situ labeling of DNA Fragmentation/Apo tosis
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-digoxigenin (DIG)
nickend
labeling (TUNEL) was carried out using an Intergen ApopTag Plus peroxidase kit
using the
manufacturer's protocol adapted from Gavrieli et al. (1992) with some
modifications. More specific
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antibody labels for apoptosis, eg. caspase 3 also showed cell death in the
same regions. Briefly, 20
M thick coronal sections were cut on a cryostat then fixed in 1 %
paraformaldehyde at room
temperature for 2 days. The endogenous peroxidase was quenched by 3% hydrogen
peroxide in
phosphate buffered solution (PBS). After rinsing with PBS, the sections were
then exposed to
11 L/cm2 working strength of TdT enzyme for lhr at 37 C. After washing in PBS,
15 L/em2 of anti-
digoxigenin-peroxidase was applied for 30 min in a humidified chamber at room
temperature. Colour
was developed by adding 125 l DAB substrate working solution for 6 min.
Slides were counter-
stained with methyl green for 25 minutes at room temperature. Positive
apoptosis controls were
generated by pre-incubating sections with DNAase (Sigma). These methods have
been successfully
used to indicate apoptotic neurons when used in other preparations (Simpson et
al., 2000).
Data for LDH and glutamate release experiements were analyzed for significance
by one way
ANOVA using Dunnett's and Bonferroni's post tests with GraphPad PrismTM
Tissue culture studies of BSSG toxicity
We have raised cortical astrocytes on coverslips in culture, exposing them to
kainic acid,
BSSH, or cholesterol glucoside. Cell loss was measured directly by cell
density measurements;
apoptosis was measured by staining cells for caspase-3 labeling. Each of these
compounds generated
a time-dependent apoptotic cell loss. These data are shown in Fig. 5.
Results
MSO and cycad mechanisms of action in CNS
MSO, crude cycad extract, and BSSG isolated from cycad seed flour were tested
for
neural action and neuro-excitotoxicity in a series of bioassays. Figure 1 a
shows the neural
response to MSO measured as field potential in the cortical wedge preparation
from adult rat.
Bath application of MSO led to a relatively rapid depolarizing field
potentials over a range of
concentrations beginning at approx. 50 M. The responses to glutamate receptor
agonists
NMDA and AMPA are also shown in the traces of Fig. la for comparison. MSO
responses,
like those of NMDA, could be blocked by the co-application of NMDA receptor
antagonists
AP5, kynurenate, or MK 801 (the latter not shown here). MSO responses were not
blocked
by application of AMPA antagonists NBQX or other AMPA antagonists. Figure lb
shows
LDH assays for rat cortical slices following exposure to MSO and other
excitotoxins. Both
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NMDA and MSO increased cell death as measured by LDH release, and both
treatments
were blocked by the addition of AP5.
The actions of cycad flour extracts on the cortical wedge preparation are
shown in
Fig. 2. Cycad extracts gave depolarizing field potentials that could be
blocked by MK801
(Fig. 2a) or AP5 (data not shown), but not NBQX (data not shown). LDH assays
confirmed
that cycad extract (3-sitosterol-p-D-glucoside fractions gave increased LDH
release that could
induce cell death, an effect that was blocked by AP5 (Fig. 2b). Assays were
also performed
for comparison using other plant sterol glucosides (ouabain and emicymarin),
and for
synthetic cholesterol glucoside. The (3-sitosterol agylcone and cholesterol
were screened for
comparison to the glucosides. Ouabain and emicymarin gave small
hyperpolarizing
responses and gave little LDH release (not shown). The aglycone sterols were
without effect.
The action of cholesterol glucoside was qualitatively similar to (3-sitosterol-
(3-D-glucoside
(not shown).
Figure 3 shows results from the cortical wedge preparation (Fig. 3a) and in
LDH
assays (Fig. 3b) using isolated BSSG. The isolated BSSG fractions gave similar
field
potential responses that were blocked by NMDA antagonists. Cell death in LDH
assays was
also blocked by NMDA antagonists.
To test whether the actions of MSO and BSSG might act indirectly by releasing
glutamate from intracellular compartments, we examined radiolabeled glutamate
release from
rat cortical slices. Preloaded [3H]-glutamate release was significantly
increased in the
presence of MSO and BSSG fractions D-1-2 and D-2 (Figs. 4abc) in a calcium
dependent
manner, and these effects could be blocked by AP5.
Cycad-fed animals showed significant and progressive deficits in both motor
and
cognitive function. Post-sacrifice histological examinations of the brains of
cycad-fed
animals revealed the presence of significant levels of apoptosis in
hippocampal formation,
cortex, and spinal cord compared to control mice. Rats fed MSO also showed
evidence of
apoptosis in CNS.
Synthesis of R-sitosterol- 3-D-gllucoside and related analogues.
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The above experiments have demonstrated in vitro and in vivo neurotoxicity of
cycad and have
suggested that the toxic component in cycad is f3-sitosterol-(3-D-glucoside
and related sterol
glucosides. To test the hypothesis that P-sitosterol-(3-D-glucoside is the
active toxin requires either
more of this compound extracted from cycad or the synthesis of the molecule de
novo. As cycad is
limited in supply, not to mention tedious to process, we have attempted to
provide a synthetic pathway
to acquire sufficient (3-sitosterol-(3-D-glucoside for future experiments. We
have now accomplished
this using the high yield methods set out below (provided by Drs. D.E.
Williams and L. Lermer,
UBC).
a. Preparation of D-glucose-pentaacetate
Glucose (20 g, 0.111 mols) and NaOAC (9.10 g, 0.111 mols) is added to a dry 1L
RBF under N2. Acetic anhydride (125.7 ml,1.332 mol, l2eq) is added using a
syringe. The
solution is rapidly stirred and then warmed using a Bunsen burner until the
solution becomes
clear and colorless. The solution is allowed to cool to room temperature,
forming a white
precipitate. The solution is stirred for 2 hrs, taken up in EtOAc, washed with
H2O 3 x, 5%
NaHCO3 3 x, and 1 x with brine. The organic layer is dried with MgSO4,
filtered and
concentrated under reduced pressure, to afford D-glucose-pentaacetate, a white
powder. The
powder is re-crystallized in EthOH (approx. 400 ml) to afford 35.96 g of a
white powder.
The re-crystalized D-glucose-pentaacetate gives 42.02 g (65% yield). The
mother liquor is
concentrated and re-crystallized to afford a second crop of 6.60 g of D-
glucose-pentaacetate
as a white powder. The remaining mother liquor is concentrated to afford 18.82
grams of a
white solid. An over all yield of 92.5 % is obtained.
b. Preparation of 2,3 ,4,6-Tetra-O-acetyl-D- lg ucop rranose
Hydrazine acetate 5.1 g (55.39 mmol, 1.20 eq) is placed in a dry 500 ml RBF
under nitrogen.
Dry DMF (dried over CaSO4 and distilled at 5 mmHg) is added to the flask using
a syringe.
D-glucose-pentaacetate 17.96 g (46.00 mmol) is placed in a dry 250 ml RBF
under nitrogen
and dissolved in 100 ml dry DMF. The D-glucose-pentaacetate is added to the
reaction flask
via a cannula. After the addition of D-glucose-pentaacetate, the solution has
a slightly yellow
colour. Solid hydrazine remained suspended. After 3 hrs the suspension has
dissolved and
the solution remains clear pale yellow in colour. The solution is taken up in
EtOAc, washed
3 x with H2O and 1 x with brine, dried over MgSO4, filtered and concentrated
under reduced
pressure. 2,3,4,6-Tetra-O-acetyl-D-glucopyranose as clear colourless viscous
oil is obtained.
This oil is used without further purification in the next step.
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c. Preparation of 2,3 ,4,6-Tetra-o-acetEl-aD- lucos 1 trichloroacetimidate
The viscous 2,3,4,6-Tetra-O-acetyl-D-glueopyranose oil is dissolved in dry
CH2C12
150 ml in a 500 ml RBF under nitrogen and cooled to - 40 C (cooling bath of
CH3CN/C02).
C13CCN 46.11 ml (459.9 mmol, 10 eq) is added to the reaction flask drop wise
using a
syringe, followed by the addition of DBU (0.68 ml, 0.1 eq). After two hrs the
solution is
allowed to warm to room temperature. The solvent is removed under reduce
pressure and the
oil is loaded using a minimum of CH2C12 on a 8 cm diameter silica column. The
column is
eluted with 3:1 pet. ether:EtOAC followed by 2:1 pet.Ether: EtOAc. Three
fractions are
collected. The first fraction RF=0.63 contains 16.3 94 g of 2,3,4,6-Tetra-o-
acetyl-a-D-glucosyl
trichloroacetimidate (a-TAG-I) as a viscous oil. A second oil fraction is
collected containing
a mixture of a-TAG-I and small impurity of (3-TAG-I 1.644 grams and a third
fraction
containing a 4.060 grams of (3-TAG-I as a white powder. A 71% yield of 2,3,4,6-
Tetra-o-
acetyl-a-D-glucosyl trichloroacetimidate is obtained and a total overall yield
of 87% is
achieved.
d. Coupling - preparation of 2,3,4,6-Tetra-o-acetyl-(3-D-glucosyl-(3-
sitosterol
2,3,4,6-Tetra-o-acetyl-a-D-glucosyl trichloroacetimidate (a-TAG-I) (1.771 g,
0.54
mmol, 1.5 eq) is pumped on over night and dissolved in dry CH2C12. The solvent
is then
removed under low pressure. Dry a-TAG-I in a 10 ml flask is re-dissolved in 5
ml of
CH2C12. The a-TAG-I is transferred into the 25 ml RBF reaction flask
containing activated
3A molecular sieves via a cannula. The solution is stirred for 1 hr over the
sieves to take up
any residual water. (3-sitosterol (0.0994 g, 0.24 mmol) is dissolved in 5m 1
dry CH2C12 and
transferred in the reaction flask via a cannula. The flask containing
sitosterol is rinsed with 1
ml CH2C12. The rinse is added to the reaction flask. The reaction is cooled to
-23 C (cooling
bath of CC14/ C02) and some precipitation occurs. A syringe is used to inject
a 0.99 ml
volume of a stock solution of 0.1 ml BF3*Et20 in 8 ml CH2C12 drop wise over 20
min to the
reaction flask. The solution remains clear and colorless. After 2 hrs no
precipitation remains
in the reaction flask. After 4 hours a fine white precipitate is observed. The
reaction is
completed by TLC using (9:1 CHC13: MeOH). A second addition of TAG-I (0.1180
g, 0.239
mmol, 1 eq) dissolved in 5 inl CH2C12 is added drop wise to the flask. After 1
hr, no starting
material is present. The solvent is evaporated under reduced pressure and the
oil is loaded on
a silica column. The column is eluted with 10:1 pet. ether:EtOAc (10
collection tubes )
followed by 3:1 pet. ether:EtOAc (15:tube) followed by 1:1 3:1 pet. ether
:EtOAc. Two
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WO 02/37122 PCT/CA01/01567
fractions are obtained. The first fraction contains 0.0219 g- (3-sitosteryl
acetate. The second
fraction contains 0.1392 g (78 %) of 2,3,4,6-Tetra-o-acetyl-(3-D-glucosyl-(3-
sitosterol. (Note:
The sterol and the product have very similar RF's using Pet. Ether: EthOAc
solvent to elute.
The reaction may be followed using a 9:1 solution of CHC13: MeOH to elute the
TLCs).
e. Deacetylation - preparation of (3-Sitosterl-(3-D- lucopyranoside
The 2,3,4,6-Tetra-o-acetyl-(3-D-glucosyl-(3-sitosterol 0.1392g is dissolved in
80 ml
warm MeOH. The solution is allowed to cool back to room temperature. Some
precipitation
is observed. Et3N (12 ml) is added followed by 2 ml of H2O. After three hrs
the precipitate
has re-dissolved, and the clear, colorless solution is stirred overnight. A
fine white
precipitate is observed in the reaction flask the next morning. TLC using 15%
McOH/CHC13
indicates that the reaction is complete. The solvent is removed under reduced
pressure to
afford a white solid. The solid is re-dissolved in a minimum amount of 15%
McOH/CHC13.
The solution is loaded on a silica column eluted with 15% MeOH/CHC13. A single
white
powder 0.1079 g (100% yield) is obtained. These methods result in an overall
yield of 78%
for the combination of the coupling and deacetylation reactions. The white
solid is re-
crystallized in Ethanol to afford 0.0452 g (42%) of a white powder. The mother
liquor is
concentrated and a second crop is obtained of 0.0165 g (15%) of a white
powder. The
mother liquor is concentrated and the solid is re-crystallized using a
H20/MeOH mixed
solvent system to afford 0.0044g (4%). The remaining mother liquor is
concentrated to
afford 0.0180 g (17%). The overall yield from the 0.0994 g of sitosterol is
0.0661g (48%) of
re-crystallized (3-sitosteryl-(3-D-glucopyranoside and 0.018g (13%) of
remaining non-
recrystallized (3-sitosteryl-(3-D-glucopyranoside product. Note that the
0.0219 g of (3-
sitosteryl acetate from the coupling reaction can be recycled back to
sitosterol and then
carried through to the desired product in order to increase yields.
Wedge recording and LDH assays using synthetic (3-sitosterol-(3-D-glucoside
created
by these methods have been shown qualitative similarity between the synthetic
and natural D-
2 fractions
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Conclusion
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 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. Given the overlap in the occurrence
of particular
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64005-1200
sterols in plants, animals and other organisms, the present application refers
to all such
compounds collectively as sterols. Numeric ranges are inclusive of the numbers
defining the
range. In the specification, the word "comprising" is used as an open-ended
term,
substantially equivalent to the phrase "including, but not limited to", and
the word
.5 "comprises" has a corresponding meaning. Citation of references herein
shall not be
construed as an admission that such references are prior art to the present
invention.
The invention includes all embodiments and variations substantially as
hereinbefore described and with reference to the examples and drawings.
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