Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR TREATING SECONDARY TISSUE DEGENERATION
ASSOCIATED WITH CENTRAL NERVOUS SYSTEM INJURY
This invention was made with government
support under Contract No. NS37336-O1 awarded by the
National Institutes of Health. The United States
Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
This invention relates to immunology and,
more specifically to treatment of secondary tissue
degeneration associated with central nervous system
(CNS) injury through neutralization of the chemokine
CXCL10.
The CNS consists of the brain and the spinal
cord, which form a continuous system containing nerve
cells, supporting cells, and nerve fibers. The brain
is a cognitive organ that has distinct regions and
layers, each associated with the reception and
processing of specific stimuli received from sense
organs. The human brain is divided into three main
areas that each have distinct functions: the forebrain
(prosencephalon), mid-brain (mesencephalon) and
hindbrain (rhombencephalon). The spinal cord is
arranged in segments, with higher segments controlling
movement and sensation in upper parts of the body and
lower segments controlling the lower parts of the body.
The consequences of CNS injury reflect the organization
of its component organs.
The types of disability associated with
spinal cord injury vary greatly depending on the type
and severity of the injury, the level of the cord at
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which the injury occurs, and the nerve fiber pathways
that are damaged. Severe injury to the spinal cord
causes paralysis and complete loss of sensation to the
parts of the body controlled by the spinal cord
segments below the point of injury. Spinal cord
injuries also can lead to many complications, including
pressure sores and increased susceptibility to
respiratory diseases. Brain injury can lead to a wide
spectrum of impairments in different areas, including
cognitive functioning, physical abilities,
communication, or social/behavioral functioning.
Damage to the central nervous system does not
stop immediately after the initial injury, but
continues in the hours and days following the initial
trauma. These delayed injury processes present windows
of opportunity for treatments aimed at reducing the
extent of disability resulting from brain and spinal
cord injury. In the absence of trauma or disease, most
types of immune cells only rarely enter the CNS.
However, when the brain or spinal cord are damaged by
trauma or disease, immune cells engulf the area,
eliminating debris and releasing a host of powerful
regulatory chemicals, both beneficial and harmful.
Influx of immune cells is associated with both acute
CNS injury and associated secondary degeneration.
The current standard of care treatment of
acute spinal cord injury is high-dose
methylprednisolone, a steroid that has to be
administered in the first three hours following injury
and whose overall efficacy has been questioned in
recent years. Symptomatic treatment of spinal cord
injury can be achieved with 4-aminopyridine (4-AP), a
blocker of potassium channels that prolongs the
duration of nerve action potentials and improves
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conduction in demyelinated axons. However, neither of
these agents prevents the complicated pathological
cascade associated with CNS trauma.
Thus, there exists a need to have additional
methods for treating central nervous system injury,
particularly spinal cord injury, and the secondary
tissue degeneration that results from trauma to the
CNS. The present invention satisfies this need and
provides related advantages as well.
SUMMARY OF THE INVENTION
The invention provides methods of reducing
the severity of secondary tissue degeneration
associated with central nervous system (CNS) injury in
a subject by administering to a subject having or at
risk of developing secondary tissue degeneration
associated with CNS injury an effective amount of a
neutralizing agent specific for interferon inducible
protein of 10 kDa (CXCL10). The methods of the
invention are useful for the treatment of both spinal
cord and brain injuries.
In additional aspects, the present invention
provides methods of reducing the severity of secondary
tissue degeneration associated with CNS injury in a
subject, comprising administering to a subject having
secondary tissue degeneration associated with CNS
injury an effective amount of a neutralizing agent
specific for interferon inducible protein of 10 kDa
(CXCL10).
In specific embodiments the subject is a
mammal. In other embodiments, the subject of the
method is a human. In yet additional embodiments the
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CNS injury is either a spinal cord injury, a brain
injury or both.
In yet further embodiments, the neutralizing
agent is an anti-CXCL10 antibody, fragment
corresponding to an anti-CXCL10 antibody, small
molecule, or polypeptide specific for CXCL10.
An additional aspect of the present invention
provides methods for reducing the severity of secondary
tissue degeneration associated with CNS injury in a
subject, comprising administering to a subject in need
thereof an effective amount of an antibody or fragment
thereof capable of binding specifically to interferon
inducible protein of 10 kDa (CXCL10).
In certain embodiments of the instant
invention, the CNS injury is created or induced by a
mechanical injury, bruising of the spinal cord, a
compression injury of the spinal cord, laceration of
the spinal cord, severance of the spinal cord, or a
demyelinating condition. In one embodiment, the
demyelinating condition is multiple sclerosis.
Also provided by the instant invention are
methods of reducing the severity of secondary tissue
degeneration associated with pathological CNS condition
in a subject, comprising administering to a subject in
need thereof an effective amount of an antibody or
fragment thereof capable of binding specifically to
interferon inducible protein of 10 kDa (CXCL10).
In particular embodiments, the pathological
condition is myelin loss. In other embodiments myelin
loss is due to acute disseminated encephalomyelitis,
post-infectious myelin loss, post-vaccinal myelin loss,
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acute necrotizing encephalomyelitis, or progressive
necrotizing myelopathy.
In further embodiments, the neutralizing
agent of the present invention is administered within
5 at least one, two, three, four, five, six, seven, or
eight hours of injury. In yet further embodiments, the
neutralizing agent is administered within 12 hours, 18
hours or within 24 hours of injury or diagnosis. In
other embodiments, the neutralizing agent is an
antibody or an antibody fragment. In yet additional
embodiments the neutralizing agent is administered
alone or in combination with additional agents such as
anti-inflammatories and administration occurs at least
once and in other embodiments, repeatedly for a number
of days. In one embodiment, the number of days is up
to 10 days, 15 days, 20 days, 25 days, or at least 30
days.
Also provided by the instant invention are
methods of reducing the severity of secondary tissue
degeneration associated with CNS injury in a subject,
comprising administering to a subject in need thereof
an effective amount of a polynucleotide agent capable
of reducing the amount of interferon inducible protein
of 10 kDa (CXCL10) in a cell.
In various embodiments, the polynucleotide
agent may be an antisense oligonucleotide or a
ribozyme, wherein said antisense oligonucleotide or
ribozyme specifically binds to a polynucleotide
encoding CXCL10.
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In yet additional aspects, the instant
invention provides methods for reducing the severity of
secondary tissue degeneration associated with
pathological CNS condition in a subject, comprising
administering to a subject in need thereof an effective
amount of a polynucleotide agent capable of reducing
the amount of interferon inducible protein of 10 kDa
(CXCL10) in a cell.
In certain embodiments, the agent may be an
antisense oligonucleotide or a ribozyme, wherein said
antisense oligonucleotide or ribozyme specifically
binds to a polynucleotide encoding CXCL10.
In still yet additional embodiments, the
neutralizing agent of the present invention may be
administered in a composition capable of enhancing
penetration of the blood-brain barrier, such as
liposomes.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows increased CXCL10 mRNA levels
after hemisection injury to the adult mouse spinal
cord.
Figure 2 shows behavioral deficit following
hemisection injury to the adult mouse spinal cord
progressively lessened in mice treated with anti-CXCL10
antibody as compared to untreated control mice as
measured by the following four kinematic parameters:
2(A) stride length; 2(B) toe spread; 2(C) stride width;
and 2(D) rear paw rotation.
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Figure 3 shows 3(A) gross pathology of a
spinal cord hemisection lesion from a dorsal view at
day 14 post-injury in an untreated mouse; 3(B) a
longitudinal section of a spinal cord dorsal
hemisection lesion at day 14 post-injury in an
untreated mouse; 3(C) gross pathology of a spinal cord
hemisection lesion from a dorsal view at day 14 post-
injury in an anti-CXCL10 antibody treated mouse; and
3(D) a longitudinal section of a spinal cord dorsal
hemisection lesion at day 14 post-injury in an
anti-CXCL10 antibody treated mouse. Anti-CXCL10
antibody mediated attenuation of the CD4+ T lymphocyte
response to traumatic spinal cord injury is associated
with tissue sparing.
Figure 4 shows CD4+ stained light microscopy
photographs demonstrating that 4 (A) anti-CXCL10
antibody treatment decreases the robust T-lymphocyte
recruitment following hemisection injury in treated
mice compared to 4(B) untreated mice.
Figures 5 shows that anti-CXCL10 antibody
mediated attenuation of the CD4+ T lymphocyte response
to traumatic spinal cord injury is associated with
tissue sparing.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a method of
treating and/or ameliorating CNS injury, including
spinal cord injury and brain injury, by neutralizing
interferon inducible protein of 10 kDA (CXCL10) with a
specific neutralizing agent. The invention also
provides a method of reversing the secondary tissue
degeneration associated with CNS injury or trauma as
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well as a method of facilitating nerve cell
regeneration and remyelination by neutralizing
interferon inducible protein of 10 kDA (CXCL10) with a
specific neutralizing agent.
Neutralization of CXCL10 represents a
significant treatment option for CNS injury because it
decreases the extent T-cell infiltration that is
responsible for the neurological degeneration
associated with tissue loss following CNS injury or
trauma. Neutralization of CXCL10 targets a cause
rather than merely the symptoms of secondary
degeneration associated with CNS injury or trauma.
The CNS contains nerve cells, or neurons,
that are characterized by long nerve fibers called
axons. Axons in the spinal cord carry signals downward
from the brain along descending pathways and upward
toward the brain along ascending pathways. Many axons
in these pathways are covered by sheaths of an
insulating substance called myelin, which gives them a
whitish appearance; therefore, the region in which they
lie is called "white matter." The nerve cells
themselves, with their tree-like branches called
dendrites that receive signals from other nerve cells,
make up "gray matter." Gray matter lies in a
butterfly-shaped region in the center of the spinal
cord. Both the brain and the spinal cord are enclosed
in three membranes (meninges): the pia mater, the
innermost layer; the arachnoid, a delicate middle
layer; and the dura mater, which is a tougher outer
layer.
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The spinal cord is organized into segments
along its length. Nerves from each segment connect to
specific regions of the body. The segments in the
neck, or cervical region, referred to as C1 through C8,
control signals to the neck, arms, and hands. Those in
the thoracic or upper back region, referred to as T1
through T12, relay signals to the torso and some parts
of the arms. Those in the upper lumbar or mid-back
region just below the ribs, referred to as Ll through
L5, control signals to the hips and legs. Finally, the
sacral segments, referred to as S1 through S5, lie just
below the lumbar segments in the mid-back and control
signals to the groin, toes, and some parts of the legs.
The effects of spinal cord injury at different segments
reflect this organization.
Several types of cells carry out CNS
functions. Large motor neurons have long axons that
control skeletal muscles in the neck, torso, and limbs.
Sensory neurons called dorsal root ganglion cells,
whose axons form the nerves that carry information from
the body into the spinal cord, are found immediately
outside the spinal cord. Spinal interneurons, which
lie completely within the spinal cord, help integrate
sensory information and generate coordinated signals
that control muscles. Glia, or supporting cells,
outnumber neurons in the brain and spinal cord and
perform a variety of functions. One type of glial
cell, the oligodendrocyte, creates the myelin sheaths
that insulate axons and improve the speed and
reliability of nerve signal transmission. Other glia
enclose the spinal cord like the rim and spokes of a
wheel, providing compartments for the ascending and
descending nerve fiber tracts. Astrocytes, large
star-shaped glial cells, regulate the composition of
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the fluids that surround nerve cells. Some of these
cells also form scar tissue after injury. Smaller
cells called microglia also become activated in
response to injury and help clean up waste products.
5 All of these glial cells produce substances that
support neuron survival and influence axon growth.
The brain and spinal cord are confined within
bony cavities that protect them, but also render them
vulnerable to compression damage caused by swelling or
10 forceful injury. Cells of the CNS have a very high
rate of metabolism and rely upon blood glucose for
energy. Since the extent to which normal blood flow
exceeds the minimum required for healthy functioning,
is much smaller in the CNS than in other tissues, CNS
cells are particularly vulnerable to ischemia. Other
unique features of the CNS are the
"blood-brain-barrier" and the "blood-spinal cord-
barrier," which are formed by cells lining blood
vessels in the CNS and serve to protect nerve cells by
restricting entry of potentially harmful substances and
cells of the immune system. Trauma compromises these
barriers, contributing to further damage in the brain
and spinal cord.
As used herein, the terms "central nervous
system injury" and "CNS injury" are intended to refer
to an injury or condition of the spinal cord and/or
brain that is characterized by an inflammatory response
at a site of injury or lesion. Central nervous system
injury generally involves a primary mechanical insult,
but also encompasses lesions caused by non-traumatic
events such as pathological conditions. Injuries of
the CNS include contusions caused by bruising of the
spinal cord, and compression injuries caused by
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pressure on the spinal cord. Other types of spinal
cord injuries include lacerations, severance and
central cord syndrome, which affects the cervical
(neck) region of the cord and results from focused
damage to a group of nerve fibers called the
corticospinal tract that carries signal between the
brain and the spinal cord. A CNS injury or lesion can
affect one or more of the cell types or tissues of the
CNS, including neurons, glial cells and myelin.
Conditions of the CNS include demyelinating conditions
including Multiple Sclerosis (MS), which in which
demyelination occurs in the white matter of the brain
and spinal cord. Demyelination and local inflammation
are common to spinal cord injury and demyelinating
conditions of the CNS, including Multiple Sclerosis,
and influx of T-cells specific for myelin basic protein
(MBP) as a result of myelin destruction has been
demonstrated in both spinal cord injury and Multiple
Sclerosis.
Demyelinating diseases or conditions are an
important group of neurological disorders because of
the frequency with which they occur and the disability
that they cause. Demyelinating diseases have in common
a focal or patchy destruction of myelin sheaths that
can be accompanied by an inflammatory response. Myelin
loss also occurs in other conditions, for example, in
genetically determined defects in myelin metabolism,
and as a consequence of toxin exposure and infections
of oligodendrocytes or Schwann cells. Demyelinating
diseases of the CNS include, for example, MS, acute
disseminated encephalomyelitis (ADE) including
postinfectious and postvaccinal encephalomyelitis,
acute necrotizing hemorrhagic encephalomyelitis and
progressive (necrotizing) myelopathy.
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Injury to the CNS results in an influx of
inflammatory cells to the CNS parenchyma as described
by Dusart and Schwab, Eur. J. Neurosci., 6(5):712-724
(1994) and Schnell et al., Eur. J. Neurosci.,
11(10):3648-3458 (1999). The initial response is
dominated by neutrophils, which peak at 1 day
post-injury, and accompanies a breakdown of the Blood-
brain barrier and upregulation of cell adhesion
molecules on the vascular endothelium. Neutrophils
display phagocytic and bactericidal properties, and are
critical to the removal of microbial intruders and
debris (Clark, Dermatol. Clin., 11(4):647-66 (1994)).
Activated neutrophils produce a variety of
proinflammatory molecules including proteases,
cytokines, chemokines and free radicals as described by
Cassatella, Immunol. Today, 16(1):21-26 1994); Kunkel
et al., Semin. Cell Biol., 6(6):327-336 (1995); Conner
and Grisham, Nutrition 12(4):274-277 (1996); each of
which is incorporated herein by reference. The release
of these molecules contributes to the recruitment and
activation of other inflammatory cells, as well as
neuronal injury and glial cell activation as described
by Yong, Cytokines, astogliosis and neutrophils
following CNS trauma, in Cytokines and the CNS:
Development, Defense and Disease, CRC Press, Boca
Baton, Florida (1996). Hematogenous macrophages and
activated resident CNS microglia dominate the
inflammatory response beginning on the second day
post-injury, and are accompanied by infiltrating
lymphocytes. Chemoattractants for these cell types are
upregulated within hours of spinal cord injury, and
adhesion molecules necessary for the adherence of cells
to the cerebral endothelium are upregulated after
injury Bartholdi and Schwab, Eur. J. Neurosci.
9(7):1422-1438 1997); Hamada et al., J. Neurochem.
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66(4):1525-1531 (1996). The expression of CXCL10, a
CXC chemokine which attracts T cells, and MIP-la, a CC
chemokine which attracts macrophages, have been shown
to be upregulated within hours of spinal cord injury
(see Bartholdi, supra, 1997; and McTigue et al., J.
Neurosci. Res. 53(3):368-376 (1998).
The term "secondary tissue degeneration," as
used in herein in reference to CNS injury refers to the
secondary loss of adjacent cells and tissues that were
undamaged or marginally damaged by the initial trauma
or lesion to the CNS. The secondary tissue
degeneration can affect any of the cell types present
at the site of injury or lesion, including neurons and
filial cells as well as tissues, including the myelin
tissue surrounding the nerve fibers and the
vasculature. Secondary tissue degeneration generally
is associated with inflammation and CXCL10 mediated
recruitment of T cells having specificity for a wide
array of cell types and tissues. Secondary tissue
degeneration mediated by CXCL10 following CNS injury or
trauma also causes blood-brain-barrier disruption,
edema, demyelination, axonal damage and neuronal death,
which can be preceded by activation of
voltage-dependent or agonist-gated channels, ion leaks,
activation of calcium-dependent enzymes such as
proteases, lipases and nucleases, mitochondrial
dysfunction and energy depletion, culminating in cell
death (Yoles et al., Invest. Ophthalmol. Vis. Sci.
33(13):3586-3591 (1992); Zivin and Choi, Scientific
American 265(1):56-63 (1993); Hovda et al., Brain
Research 567(1):1-10 (1991); Yoshino et al., Brain
Research 561(1):106-119 (1991)), each of which is
incorporated herein by reference. CXCL10 mediated
neuronal cell death associated with secondary tissue
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degeneration following CNS injury or trauma can include
death by any mechanism, including necrosis, apoptosis,
paraptosis or any additional form of cell death, for
example, neurodegenerative cell death, such as has been
described in a transgenic mouse model of amyotrophic
lateral sclerosis (Canto and Gurney, American Journal
of Pathololoav 145:1271-1279 (1994), which is
incorporated herein by reference).
Inflammation contributes to secondary tissue
degeneration associated with a CNS injury (Dusart and
Schwab, Eur. J. Neurosci.6(5):712-724 (1994); Blight,
Central Nervous System Trauma 2(4):299-315 (1985);
Egerton et al., EMBO J. 11(10):3533-40 (1992); Popovich
et al., J Comp,. Neurol., 377(3):443-464 (1997), each of
which is incorporated herein by reference). A
successful host response to inflammation generally
requires the accumulation of specialized host cells at
the site of tissue damage. Inflammation in the injured
CNS is characterized by fluid accumulation, and the
influx of plasma proteins, neutrophils, T lymphocytes
and macrophages. This cellular accumulation is a
critical step in the normal inflammatory process and is
mediated by a family of secreted chemotactic cytokines
that have in common important structural features and a
role in pathogenic inflammation. CXCL10 belongs to
this protein family of over forty structurally and
functionally related proteins known as chemokines.
Chemokines are homologous 8 to 10 kDa heparin
binding proteins that possess a conserved structural
motif containing two cysteine pairs and are divided
into subfamilies based on the relative position of the
cysteine residues in the mature protein. There are at
least four subfamilies of chemokines, but only two, a-
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chemokines and (3-chemokines, have been well
characterized. In the cx-chemokines, the first two
cysteine residues are separated by a single amino acid
(CXC), whereas in the ~3-chemokines the first two
5 cysteine residues are adjacent to each other (CC). The
C-X-C chemokines include, for example, interleukin-8
(IL-8), human platelet derived factors, CXCL10 and Mig.
Members of the C-C chemokine subfamily include, for
example, macrophage chemoattractant and activating
10 factor (MCAF), macrophage inflammatory protein-la
(MIP-la), macrophage inflammatory protein-lei (MIP-1(3)
and regulated on activation, normal T-cell expressed
and secreted (RANTES).
Chemokines selectively attract leukocyte
15 subsets; some chemokines act specifically toward
eosinophils, ethers toward monocytes, dendritic cells,
or T cells (see Luster, A., New Engl. J. Med. 338: 436-
445 (1998), which is incorporated herein by reference).
In general, CC chemokines chemoattract monocytes,
eosinophils, basophils, and T cells; and signal through
the chemokine receptors CCR1 to CCR9. The CXC
chemokine family can be further divided into two
classes based on the presence or absence of an ELR
sequence (Glu-Leu-Arg) near the N terminal preceding
the CXC sequence. The ELR-containing CXC chemokines
including IL-8 chemoattract neutrophils, while the
non-ELR CXC chemokines including CXCL10 and Mig
chemoattract lymphocytes.
Chemokines induce cell migration and
activation in at least two ways: first, through direct
chemoattraction, and, second, by binding to specific G-
protein coupled cell-surface receptors on target cells.
More than 10 distinct chemokine receptors, each
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expressed on different subsets of leukocytes, have been
identified. Chemokine receptors are constitutively
expressed on some cells, whereas they are inducible on
others. CXCR3, the receptor recognized by CXCL10 is
expressed on activated T lymphocytes of the T helper
type 1 (Th 1) phenotype and Natural Killer (NK) Cells.
Significantly, a central mechanism in the pathology of
neuroinflammation associated with secondary tissue
degeneration is the migration of activated T cells to
the site of injury or lesion.
As used herein, the term "effective amount"
when used in reference to interferon inducible protein
of 10 kDa (CXCL10), is intended to mean an amount of a
neutralizing agent specific for CXCL10 sufficient to
reduce the severity of secondary tissue degeneration
associated with CNS injury.
As used herein, "reduction in severity" is
intended to refer to an arrest, decrease or reversal in
signs and symptoms, physiological indicators,
biochemical markers or metabolic indicators of
secondary tissue degeneration associated with a CNS
injury. In spinal cord injury, the destruction of
nerve fibers that carry motor signals from the brain to
the torso and limbs leads to muscle paralysis.
Physiological symptoms of secondary tissue degeneration
associated with CNS injury include, for example,
neurological impairments and neuroinflammation and
clinical symptoms vary greatly depending on the
severity of the injury, the segment of the spinal cord
at which the injury occurs, and which nerve fibers are
damaged. Destruction of sensory nerve fibers can lead
to loss of sensations such as touch, pressure, and
temperature; it sometimes also causes pain. Other
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serious consequences can include exaggerated reflexes;
loss of bladder and bowel control; sexual dysfunction;
lost or decreased breathing capacity; impaired cough
reflexes; and spasticity. Most people with spinal cord
injury regain some functions between a week and six
months after injury, but the likelihood of spontaneous
recovery diminishes after six months. Spinal cord
injuries can lead to many clinical complications,
including pressure sores, increased susceptibility to
respiratory diseases, and autonomic dysreflexia, a
potentially life-threatening increase in blood
pressure, sweating, and other autonomic reflexes in
reaction to bowel impaction or some other stimulus.
Physiological indicators of secondary tissue
degeneration associated with CNS injury include blood-
brain-barrier disruption, edema, demyelination, axonal
damage and neuronal death and tissue loss. Biochemical
markers 4f secondary tissue degeneration associated
with CNS injury are, for example, neuronal cell
markers, myelin, gamma globulin or the specific
molecules that give rise to oligoclonal banding.
Tissue loss in the CNS associated with secondary tissue
degeneration can be detected by a variety of clinical
methods well known in the art. A neuronal cell marker,
for example, NeuN can be utilized to visualize tissue
loss in the CNS (Wolf et al., J. Histochem. Cytochem.
44:1167-1171 (1996), which is incorporated herein by
reference). In addition, evoked potentials (EP) can be
used to measure how quickly nerve impulses travel along
the nerve fibers in various parts of the nervous system
and computer-assisted tomography (CT) can be used to
scan the CNS to detect areas of tissue loss caused by
cell death or demyelination of nerve fibers. Magnetic
resonance imaging (MRI) also can be used to scan the
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CNS, but without the use of x-rays. More sensitive
than the CT scan, MRI can detect areas of CNS tissue
loss that may not be seen by the CT scanner. Moreover,
lumbar puncture or spinal tap procedures can be used to
draw out cerebrospinal fluid. The fluid can be
examined for increased levels of gamma globulin and
oligoclonal banding. These and other methods well
known in the art can be used to determine the severity
of secondary tissue degeneration associated with CNS
injury by measuring tissue loss, including tissue loss
caused by neuronal, glial cell death and demyelination
of nerve fibers.
As used herein, the term "neutralizing agent
specific for interferon inducible protein of 10 kDa
(CXCL10)" is intended to refer to an agent effecting a
decrease in the extent, amount or rate of CXCL10
expression or effecting a decrease in the activity of
CXCL10. Neutralizing agents useful for practicing the
claimed invention include, for example, binding
molecules such as antibodies against CXCL10. A
neutralizing agent can be any molecule that binds
CXCL10 with sufficient affinity to decrease CXCL10
activity. Additionally, a neutralizing agent can be
any molecule binds to a regulatory molecule or gene
region so as to inhibit or promote the function of the
regulatory protein or gene region and effect a decrease
in the extent or amount or rate of CXCL10 expression or
activity. For example, a fragment or peptidomimetic of
the CXCR3 receptor that binds CXCL10 with sufficient
affinity to decrease CXCL10 activity, is useful for
practicing the claimed methods. In addition, examples
of neutralizing agents which effect a decrease in
CXCL10 expression can include antisense nucleic acids
and transcriptional inhibitors.
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The invention provides a method of reducing
the severity of secondary tissue degeneration
associated with CNS injury. The method comprises
administering to a subject having secondary tissue
degeneration associated with CNS injury an effective
amount of a neutralizing agent specific for interferon
inducible protein of 10 kDa (CXCL10).
As described herein, CXCL10 is involved in
mounting a host defense against inflammation of the
nervous system. Specifically, CXCL10 coordinates the
trafficking of Thl T lymphocytes into the CNS in
response to a CNS injury, CNS inflammation,
pathological aberration such as an infectious agent or
autoagressive immune cells. T lymphocytes can be
subdivided into two major categories known as CD4+
cells and CD8+ cells. CD4 and CD8 are surface proteins
that facilitate interactions between T cell receptors
for antigen and antigen itself which is presented to T
cells by antigen-presenting cells. Antigens recognized
by T cells are contained within clefts of major
histocompatibility complex (MHC) proteins expressed on
the surface of antigen-presenting cells. CD4 cells
recognize peptide fragments presented by class II
histocompatibility alleles, CD8 cells recognize
fragments presented by class I histocompatibility
alleles. The DR2 allele, over-represented in multiple
sclerosis, is a MHC class II allele, pointing to a role
for CD4+ cells in lesion formation in the CNS.
CD4+ cells can be further subdivided into Thl
and Th2 subtypes. Thl cells are responsible for
delayed type hypersensitivity responses and secrete
numerous cytokines including interleukin-2 (IL-2), a
stimulator of T cell proliferation, and interferon, an
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activator of macrophages, and lymphotoxin, a protein
which has the capacity to damage oligodendrocytes, the
myelin-forming cells of the CNS. Interferon, in
combination with IL-2 activates macrophages which
5 directly strip myelin from nerve fibers as well as
secrete tumor necrosis factor-a (TNF-a), a cytokine
that damages the myelin-producing oligodendrocytes.
As described herein, neutralization of
CXCL10, which is expressed following a CNS injury
10 within and around the injury site or lesion (Figure 1),
reduces the secondary tissue degeneration that
manifests itself through tissue loss at the injury site
and surrounding tissue (Figure 3). As such,
neutralizing CXCL10 activity can lead to a reduction in
15 the severity of secondary tissue degeneration
associated with CNS injury. A CXCL10 neutralizing
agent including an antibody, antisense nucleic acid or
other compound identified by the methods described
below is useful for treating or reducing the severity
20 of secondary tissue degeneration associated with CNS
ink ury .
Administration of a CXCL10 neutralizing agent
targets a variety of distinct CXCL10 mediated
destructive events associated with secondary tissue
degeneration including, for example, activation of
voltage-dependent or agonist-gated channels, ion leaks,
activation of calcium-dependent enzymes such as
proteases, lipases and nucleases, inflammation,
mitochondrial dysfunction and energy depletion,
culminating in cell death. Administration of a CXCL10-
specific neutralizing agent provides an enhanced method
of reducing the severity of secondary degeneration that
represents a therapeutic improvement by targeting and
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21
treating each of these distinct CXCL10 mediated events
rather than a single aspect of the secondary tissue
degeneration.
In addition, to the unexpected finding that
CXCL 10 has such a dramatic effect on secondary tissue
degeneration, is the astounding finding that, not only
can damage be halted, but CXCL10 neutralizing agents
also facilitate myelin regrowth thereby inducing nerve
regeneration and remyelination. Therefore, the
invention also provides a method of reversing secondary
tissue degeneration associated with CNS injury or
trauma as well as a method of facilitating nerve cell
regeneration and remyelination by neutralizing
interferon inducible protein of 10 kDA (CXCL10) with a
specific neutralizing agent.
Mechanical injury to the adult mammalian
spinal cord results in an increase in vascular
permeability, and a widespread activation and
recruitment of inflammatory cells (Dusart, I. and M. E.
Schwab (1994), "Secondary cell death and the
inflammatory reaction after dorsal hemisection of the
rat spinal cord," Eur J Neurosci 6(5): 712-24)
(Schnell, L., S. Fearn, et al. (1999), "Acute
inflammatory responses to mechanical lesions in the
CNS: differences between brain and spinal cord," Eur J
Neurosci 11(10): 3648-58). The control and
consequences of this robust inflammatory response to
spinal cord injury are largely unknown. A greater
understanding of neuroimmune interactions can be seen
in the field of multiple sclerosis research, where it
has recently been demonstrated that attenuation of the
T lymphocyte response to demyelinating pathology in the
MHV model of multiple sclerosis resulted in diminished
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histopathogenesis and behavioral impairment (Liu, M.
T., H. S. Keirstead, et al. (2001), "Neutralization of
the chemokine cxc110 reduces inflammatory cell invasion
and demyelination and improves neurological function in
a viral model of multiple sclerosis," J Immunol
167(7):4091-7). Given the predominance of T
lymphocytes within sites of spinal cord injury
(McTigue, D. M., M. Tani, et al. (1998), "Selective
chemokine mRNA accumulation in the rat spinal cord
after contusion injury," J Neurosci Res 53(3):368-76),
and recent studies suggesting that they play a central
role in the anatomical and functional outcome of spinal
cord trauma (Hauben, E., O. Butovsky, et al. (2000),
"Passive or active immunization with myelin basic
protein promotes recovery from spinal cord contusion,"
J Neurosci 20(17):6421-30) (Hauben, E., U. Nevo, et al.
(2000), "Autoimmune T cells as potential
neuroprotective therapy for spinal cord injury," Lancet
355(9200):286-7), the consequences of inhibiting T
lymphocyte recruitment to sites of spinal cord injury
were further investigated. The availability of
functionally blocking antibodies to the T lymphocyte
chemoattractant CXCL10 (Liu, M. T., H. S. Keirstead, et
al. (2001), "Neutralization of the chemokine cxc110
reduces inflammatory cell invasion and demyelination
and improves neurological function in a viral model of
multiple sclerosis," J Immunol 167(7):4091-7) provided
an opportunity to test the role of this chemokine in
recruiting T lymphocytes to sites of spinal cord
trauma, and the contribution of T lymphocytes to
posttraumatic histopathogenesis and behavioral
impairment. The data herein clearly demonstrate that
CXCL10 is upregulated after injury to the adult
mammalian spinal cord (Figure 1), and that
antibody-mediated neutralization of CXCL10 in injured
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animals reduced the dramatic T lymphocyte infiltration
that normally occurs after spinal cord trauma (Figure
5). These data corroborate the inventors' recent
demonstration that administration of anti-CXCL10
antisera to mice with established viral-induced
demyelination resulted in a significant reduction in
CD4+ T lymphocyte infiltration to the CNS, as well as
diminished expression of the TH1-associated
proinflammatory cytokine IFN-g (Liu, M. T., H. S.
Keirstead, et al. (2001), "Neutralization of the
chemokine cxc110 reduces inflammatory cell invasion and
demyelination and improves neurological function in a
viral model of multiple sclerosis," J Immunol
167(7):4091-7). The attenuation of T lymphocyte
infiltration to sites of spinal cord injury following
anti-CXCL10 treatment present strong evidence that
CXCL10 in particular is a key T lymphocyte
chemoattractant during spinal cord injury, consistent
with the expression of CXCR3 receptors on the surface
of these cells (Rollins, B. J. (1997), "Chemokines,"
Blood 90(3):909-28) (Luster, A. D., J. C. Unkeless, et
al. (1985), "Gamma-interferon transcriptionally
regulates an early-response gene containing homology to
platelet proteins," Nature 315(6021):672-6).
Importantly and unexpectedly, the data
disclosed herein indicate that anti-CXCL10 treatment
alone was sufficient to decrease posttraumatic tissue
degeneration and locomotor deficits following injury.
Morphometric analyses indicated that
hemisection-injured mice that received anti-CXCL10
treatment had a 68% reduction in tissue loss around the
injury site compared to untreated hemisection-injured
mice (Figure 3). Neuronal cell counts around the
injury site indicated that the reduction in tissue loss
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in hemisection-injured mice that received anti-CXCL10
treatment was associated with the presence of 438% more
neurons around the injury site than untreated
hemisection-injured mice. These data indicate that
anti-CXCL10 treatment significantly decreased
posttraumatic tissue loss following dorsal hemisection
inj ury .
Behavioral analyses indicated that
hemisection-injured mice that received anti-CXCL10
treatment showed a statistically significant
progressive improvement in all kinematic parameters
tested during the recovery period (Figure 2). By 14
days post-injury, the behavioral scores for all 4
kinematic parameters were not significantly different
from uninjured control mice. This recovery was likely
contributed to by reorganization of the sensorimotor
pathways caudal to the lesion rather than regeneration
of severed fibers. Biochemical and synaptic
reorganization has been demonstrated following spinal
cord injury, and can contribute to functional
improvement (Edgerton, V. R., R. D. Leon, et al. (2001)
"Retraining the injured spinal cord," J Physiol 533(Pt
1):15-22). Although little data exist pertaining to
synaptogenesis in the injured spinal cord, quantitative
electron micrographic studies using the well-documented
model of the denervated dentate gyrus indicate that
substantial numbers of new synapses are formed on
denervated neurons by 10 days post-injury (Steward, O.,
S. L. Vinsant, et al. (1988), "The process of
reinnervation in the dentate gyrus of adult rats: an
ultrastructural study of changes in presynaptic
terminals as a result of sprouting," J Comp Neurol
267(2):203-10). Although the mechanism of behavioral
recovery in the current study is unknown, the data
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disclosed herein clearly indicate that anti-CXCL10
treatment reduces neurological impairment following
spinal cord injury.
A detrimental role for the immune response
5 following extravasation to the injured CNS parenchyma
is indicated by the etiology of multiple sclerosis and
disease states seen in animal models of multiple
sclerosis, in which pro-inflammatory cells are
considered central to the development of disease
10 (Olsson, T. (1995), "Cytokine-producing cells in
experimental autoimmune encephalomyelitis and multiple
sclerosis," Neurology 45(6 Suppl 6):511-5)
(Schulze-Koops, H., P. E. Lipsky, et al. (1995),
"Elevated Th1- or Th0-like cytokine mRNA in peripheral
15 circulation of patients with rheumatoid arthritis.
Modulation by treatment with anti- ICAM-1 correlates
with clinical benefit," J Immunol 155(10):5029-37). A
detrimental role for the immune response in traumatic
spinal cord injury is suggested by the temporal
20 association of chemokine expression and immune cell
influx with secondary degeneration (Dusart, I. and M.
E. Schwab (1994), "Secondary cell death and the
inflammatory reaction after dorsal hemisection of the
rat spinal cord," Eur J Neurosci 6(5):712-24) (Blight,
25 A. R. (1985), "Delayed demyelination and macrophage
invasion: a candidate for secondary cell damage in
spinal cord injury," Cent New Syst Trauma
2 (4) :299-315) (Popovich, P. G. , P. 4~Iei, et al . (1997) ,
"Cellular inflammatory response after spinal cord
injury in Sprague-Dawley and Lewis rats," J Comp Neurol
377 (3) :443-64) .
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Popovich and colleagues have convincingly
demonstrated that clodronate-mediated depletion of
hematogenous macrophages leads to a decrease in
posttraumatic tissue loss and an improvement in
overground locomotion following spinal cord injury.
These findings led the authors to conclude that
infiltrating immune cells are effectors of acute
secondary degeneration, and suggest that cell-specific
immunodepletion may prove therapeutic for spinal cord
injury (Popovich, P. G., P. Wei, et al. (1997),
"Cellular inflammatory response after spinal cord
injury in Sprague-Dawley and Lewis rats," J Comp Neurol
377(3):443-64). Indeed, activated mononuclear
phagocytes release neurotoxins after traumatic CNS
injury (Giulian, D., M. Corpuz, et al. (1993),
"Reactive mononuclear phagocytes release neurotoxins
after ischemic and traumatic injury to the central
nervous system," J Neurosci Res 36(6):681-93), induce
NMDA receptor-mediated neurotoxicity (Paini, D., K.
Frei, et al. (1991), "Murine brain macrophages induce
NMDA receptor mediated neurotoxicity in vitro by
secreting glutamate," Neurosci Lett 133:159-162), and
activated leukocytes release a wide variety of lytic
enzymes as well as reactive oxygen and nitrogen
intermediates (Martiney, J. A., C. Cuff, et al. (1998),
"Cytokine-induced inflammation in the central nervous
system revisited," Neurochem Res 23(3):349-59).
These findings are interesting in light of
the present demonstration that anti-CXCL10 treatment
leads to a decrease in the infiltration of both T
lymphocytes and hematogenous macrophages to sites of
spinal cord injury (Figure 5). As hematogenous
macrophages do not express the CXCR3 receptor for
CXCL10 it is likely that their depletion is a
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27
downstream effect of T lymphocyte depletion; this is
supported by the demonstration that activated CD4+ T
lymphocytes secrete RANTES, a chemoattractant for
hematogenous macrophages.
Given the complexity of immune cell
regulation within sites of CNS disease/trauma and the
downstream consequences of immune cell activation
within such sites, targeting the recruitment of immune
cell populations to sites of injury is a can elucidate
the role of particular populations in the secondary
degenerative cascade that follows a CNS insult. The
results described herein provide new insight into the
functional significance of CXCL10 expression during CNS
injury and further provide a basis for methods of
treating CNS injuries with a neutralizing agent
specific for CXCL10, which is shown herein to be a key
chemoattractant during spinal cord injury. Therapies
that target CXCL10 as provided by the invention
represent a viable treatment strategy for reducing
posttraumatic histopathogenesis and behavioral
impairment following CNS injury.
A CXCL10 neutralizing agent that binds CXCL10
with sufficient affinity can reduce activity of CXCL10
related to immune cell recruitment, tissue loss or
demyelination. A CXCL10-specific neutralizing agent
can be a macromolecule, such as polypeptide, nucleic
acid, carbohydrate or lipid. A CXCL10-specific
neutralizing agent can also be a derivative, analogue
or mimetic compound as well as a small organic compound
as long as CXCL10 activity is reduced in the presence
of the neutralizing agent. The size of a neutralizing
agent is not important so long as the molecule exhibits
or can be made to exhibit selective neutralizing
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activity towards CXCL10. For example, a neutralizing
agent can be as little as between about one and six,
and as large as tens or hundreds of monomer building
blocks which constitute a macromolecule or chemical
binding molecule. Similarly, an organic compound can
be a simple or complex structure so long as it binds
CXCL10 with sufficient affinity to reduce activity.
Neutralizing agents specific for CXCL10 can
include, for example, antibodies and other receptor or
ligand binding polypeptides of the immune system. Such
other molecules of the immune system include for
example, T cell receptors (TCR) including CD4 cell
receptors. A neutralizing agent for CXCL10 also can be
a molecule that inhibits the interaction of CXCL10 and
a chemokine receptor, for example, the CXCR3 receptor.
Additionally, cell surface receptors such as integrins,
growth factor receptors and chemokine receptors, as
well as any other receptors or fragments thereof that
bind CXCL10, or can be made to bind with sufficient
affinity to reduce activity are also neutralizing
agents useful for practicing the methods of the
invention. Additionally, receptors, growth factors,
cytokines or chemokines, for example, which inhibit the
expression of CXCL10 or their receptors are also
neutralizing agents useful for practicing the methods
of the invention. Furthermore, DNA binding
polypeptides such as transcription factors and DNA
replication factors are likewise included within the
definition of the term binding molecule so long as they
have selective binding activity for CXCL10, regulatory
molecules that control the expression or activity of
CXCL10, or gene regions that control the expression of
CXCL10. Finally, polypeptides, nucleic acids and
chemical compounds such as those selected from random
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and combinational libraries are also included within
the definition of the term so long as such a molecule
binds CXCL10 with sufficient affinity to decrease
activity.
Various approaches can be used for
identifying neutralizing agents selective for CXLC10.
For example, one approach is to use the information
available regarding the structure and function of
CXCL10 to generate binding molecule populations from
molecules known to function as chemokine binding
molecules or known to exhibit or be capable of
exhibiting binding affinity specific for CXCL10, such
as fragments or mimetics of the CXCR3 receptor found on
CD4+ T cells and NK cells. A neutralizing agent
specific for CXCL10 can be an antibody and other
receptor of the immune repertoire. The normal function
of such immune receptors is to bind essentially an
infinite number of different antigens and ligands.
Therefore, generating a diverse population of binding
molecules from an immune repertoire, for example, can
be useful for identifying a neutralizing agent specific
for CXCL10.
A neutralizing agent specific for CXCL10 can
further be identified from a large population of
unknown molecules by methods well known in the art.
Such a population can be a random library of peptides
or small molecule compounds. The population can be
generated to contain a sufficient diversity of sequence
or structure so as to contain a molecule which will
bind to the CXCL10 protein or their respective nucleic
acids. Those skilled in the art will know what size
and diversity is necessary or sufficient for the
intended purpose. A population of sufficient size and
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complexity can be generated so as to have a high
probability of containing a CXCL10 neutralizing agent
that binds CXCL10 with sufficient affinity to decrease
activity. Numerous other types of library molecule
5 populations exist and are described further below.
Any molecule that binds to CXCL10, to a
CXCL10 receptor, to a gene region that controls CXCL10
expression, or to a regulatory molecule that modulates
CXCL10 activity or expression as well as to any
10 regulatory molecule that modulates CXCL10 receptor
expression is a CXCL10-specific neutralizing agent
useful for practicing the invention. For example, a
CXCL10-specific neutralizing agent can be a regulatory
molecule affects CXCL10 expression by reducing or
15 inhibiting the action of a transcription factor that
controls or upregulates transcription of CXCL10. In
addition, a regulatory molecule that binds with
sufficient affinity to a molecule involved in the
activation of CXCL10 to reduce CXCL10 activation is a
20 neutralizing agent useful for practicing the methods of
the invention.
A neutralizing agent can bind to CXCL10 with
sufficient affinity to decrease their activity is
useful for practicing the claimed methods of reducing
25 the severity of secondary tissue degeneration
associated with CNS injury and in a subject affected
with secondary tissue degeneration associated with CNS
injury. In addition, a CXCL10-specific neutralizing
agent can decrease CXCL10 activity or expression by
30 binding to a CXCL10 receptor, to a regulatory molecule
that modulates the activity or expression of CXCL10, or
to a gene region that controls CXCL10 expression. For
example, a neutralizing agent useful for practicing the
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claimed invention can be an antibody against a
regulator molecule that modulates CXCL10 expression or
activity. Furthermore, as described herein, an anti-
CXCL10 antibody is a useful neutralizing agent for
practicing the methods of the invention. In addition,
since CXCL10 is a secreted protein known to bind to a
specific T-cell receptor, a neutralizing agent useful
for practicing the invention can be any binding
polypeptide, receptor or fragment thereof of the
immunoglobulin superfamily of receptors.
Alternatively, it may be desired to use populations of
random peptide populations to identify further
neutralizing agents specific for CXCL10. Those skilled
in the art will know or can determine what type of
approach and what type of neutralizing agent is
appropriate for practicing the methods of the invention
for reducing the severity of secondary tissue
degeneration associated with CNS injury in a subject
affected with secondary tissue degeneration associated
with CNS injury.
A moderate sized population for
identification of a CXCL10-specific neutralizing agent
can consist of hundreds and thousands of different
binding molecules within the population whereas a large
sized binding molecule population will consist of tens
of thousands and millions of different binding molecule
species. More specifically, large and diverse
populations of binding molecules for the identification
of a neutralizing agent will contain any of about 104,
105, 106, 10', 108, 109, 101°, or more, different binding
molecule species. One skilled in the art will know the
approximate diversity of the population of binding
molecules sufficient to identify a neutralizing agent
specific for CXCL10.
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Recombinant libraries of binding molecules
can be used to identify a neutralizing agent specific
for IP-10 since large and diverse populations can be
rapidly generated and screened with CXCL10.
Recombinant libraries of expressed polypeptides useful
for identifying a neutralizing agent specific for
CXCL10 can be engineered in a large number of different
ways known in the art. Recombinant library methods
similarly allow for the production of a large number of
binding molecule populations from naturally occurring
repertoires. Whether recombinant or otherwise,
essentially any source of binding molecule population
can be used so long as the source provides a sufficient
size and diversity of different binding molecules to
identify a neutralizing agent specific for CXCL10. If
desired, a population of binding molecules useful for
identifying a neutralizing agent specific for CXCL10
can be a selectively immobilized to a solid support as
described by Watkins et al., Anal. Biochem. 256 (92):
169-177 (1998), which is incorporated herein by
reference.
A phage expression library in which lysogenic
phage cause the release of bacterially expressed
binding molecule polypeptides is a specific example of
a recombinant library that can be used to identify a
neutralizing agent specific for CXCL10. In another
type of phage expression library, large numbers of
potential binding molecules can be expressed as fusion
polypeptides on the periplasmic surface of bacterial
cells. Libraries in yeast and higher eukaryotic cells
exist as well and are similarly applicable in the
methods of the invention. Those skilled in the art
will know or can determine what type of library is
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useful for identifying a neutralizing agent specific
for CXCL10.
In addition to the methods described above,
which utilize purified polypeptide to screen libraries
of compounds for those which specifically bind CXCL10,
a neutralizing agent specific for CXCL10 can be
identified by using purified polypeptide to produce
antibodies. For example, antibodies which are specific
for CXCL10 can be used as neutralizing agents of the
invention and can be generated using methods that are
well known in the art. Neutralizing agents useful for
practicing the methods of the invention include both
polyclonal and monoclonal antibodies against CXCL10 or
any molecule that modulates CXCL10 expression or
activity, as well as antigen binding fragments of such
antibodies including Fab, F(ab')2, Fd and Fv fragments
and the like. In addition, neutralizing agents useful
for practicing the methods of the invention encompass
non-naturally occurring antibodies, including, for
example, single chain antibodies, chimeric antibodies,
bifunctional antibodies, complementarity determining
region-grafted (CDR-grafted) antibodies and humanized
antibodies, as well as antigen-binding fragments
thereof .
Methods of preparing and isolating
antibodies, including polyclonal and monoclonal
antibodies, using peptide immunogens, are well known to
those skilled in the art and are described, for
example, in Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1988),
which is incorporated herein by reference. Non-
naturally occurring antibodies can be constructed using
solid phase peptide synthesis, can be produced
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recombinantly or can be obtained, for example, by
screening combinatorial libraries consisting of
variable heavy chains and variable light chains as
described by Huse et al., Science 246:1275-1281 (1989),
which is incorporated herein by reference. These and
other methods of making, for example, chimeric,
humanized, CDR-grafted, single chain, and bifunctional
antibodies are well known to those skilled in the art
(Hoogenboom et al., U.S. Patent No. 5,564,332, issued
October 15, 1996; Winter and Harris, Immunol. Todav
14:243-246 (1993); Ward et al., Nature 341:544-546
(1989); Harlow and Lane, supra, 1988); Hilyard et al.,
Protein Engineering: A practical approach (IRL Press
1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford
University Press 1995); each of which is incorporated
herein by reference).
A CXCL10-specific antibody neutralizing agent
can be raised using as an immunogen a substantially
purified CXCL10 protein, which can be prepared from
natural sources or produced recombinantly, or a peptide
portion of a CXCL10 protein including synthetic
peptides. A non-immunogenic peptide portion of a
CXCL10 protein can be made immunogenic by coupling the
hapten to a carrier molecule such bovine serum albumin
(BSA) or keyhole limpet hemocyanin (KLH), or by
expressing the peptide portion as a fusion protein.
Various other carrier molecules and methods for
coupling a hapten to a carrier molecule are well known
in the art (see Harlow and Lane, supra, 1988; see,
also, Hermanson, Bioconjuaate Techniques, Academic
Press, 1996, which is incorporated herein by
reference). As described above, an antibody
neutralizing agent specific for CXCL10 can also be
raised against a regulatory molecule that modulates
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CXCL10 expression or activity rather than against
CXCL10 directly.
A neutralizing agent specific for CXCL10,
such as an antibody can be labeled so as to be
5 detectable using methods well known in the art
(Hermanson, su ra, 1996; Harlow and Lane, su ra,1988;
chap. 9). For example, a neutralizing agent specific
for CXCL10 can be linked to a radioisotope or
therapeutic agent by methods well known in the art. A
10 neutralizing agent that directly binds CXCL10 linked to
a radioisotope or other moiety capable of visualization
can be useful to diagnose or stage the progression of a
clinical stage of secondary tissue degeneration
associated with CNS injury that is characterized by the
15 organ or tissue-specific presence or absence of CXCL10.
Methods for raising polyclonal antibodies,
for example, in a rabbit, goat, mouse or other mammal,
are well known in the art (Harlow and Lane, supra,
1988). The production of anti-peptide antibodies
20 commonly involves the use of host animals such as
rabbits, mice, guinea pigs, or rats. If a large amount
of serum is needed, larger animals such as sheep,
goats, horses, pigs, or donkeys can be used. Animals
are usually chosen based on the amount of antiserum
25 required and suitable animals include rabbits, mice,
rats, guinea pigs, and hamsters. These animals yield a
maximum of 25 mL, 100-200 uL and 1-2 mL of serum per
single bleed (Harlow and Lane, supra, 1988). Rabbits
are very useful for the production of polyclonal
30 antisera, since they can be safely and repeatedly bled
and produce high volumes of antiserum. Two injections
two to four weeks apart with 15-50~Zg of antigen in a
suitable adjuvant such as, for example, Freund's
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Complete Adjuvant can be followed by blood collection
and analysis of the antiserum.
In addition, monoclonal antibodies can be
obtained using methods that are well known and routine
in the art (Harlow and Lane, supra, 1988). A peptide
portion of a protein such as CXCL10 for use as an
immunogen can be determined by methods well known in
the art. Spleen cells from a CXCL10 immunized mouse
can be fused to an appropriate myeloma cell line to
produce hybridoma cells. Cloned hybridoma cell lines
can be screened using a labeled CXCL10 protein to
identify clones that secrete anti-CXCL10. Hybridomas
expressing anti-CXCL10 monoclonal antibodies having a
desirable specificity and affinity can be isolated and
utilized as a continuous source of the antibody
neutralizing agent.
Neutralizing agents specific for CXCL10 can
be used to reduce the severity of secondary tissue
degeneration associated with CNS injury in a mammal,
including in a human subject. Humanized antibodies can
be constructed by conferring essentially any antigen
binding specificity onto a human antibody framework.
Methods of constructing humanized antibodies are useful
to prepare an antibody neutralizing agent appropriate
for practicing the methods of the invention and
avoiding host immune responses against the antibody
neutralizing agent when used therapeutically.
The antibody neutralizing agents described
above can be used to generate therapeutic human
neutralizing agents by methods well known in the art
such as complementary determining region (CDR)-grafting
and optimization of framework and CDR residues. For
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example, humanization of an antibody neutralizing agent
can be accomplished by CDR-grafting as described in
Fiorentini at al., Immunotechnoloay 3(1): 45-59 (1997),
which is incorporated herein be reference. Briefly,
CDR-grafting involves recombinantly splicing CDRs from
a nonhuman antibody neutralizing agent into a human
framework region to confer binding activity onto the
resultant grafted antibody, or variable region binding
fragment thereof. Once the CDR-grafted antibody, or
variable region binding fragment is made, binding
affinity comparable to the nonhuman antibody
neutralizing agent can be reacquired by subsequent
rounds of affinity maturation strategies known in the
art. Humanization of antibody neutralizing agents in
the form of rabbit polyclonal antibodies can be
accomplished by similar methods as described in Rader
et al., J. Biol. Chem. 275(18): 13668-13676 (2000),
which is incorporated herein be reference.
Humanization of a nonhuman CXCL10 antibody
neutralizing agent can also be achieved by simultaneous
optimization of framework and CDR residues, which
permits the rapid identification of co-operatively
interacting framework and CDR residues, as described in
Wu et al., J. Mol. Biol. 294(1): 151-162 (1999), which
is incorporated herein by reference. Briefly, a
combinatorial library that examines a number of
potentially important framework positions is expressed
concomitantly with focused CDR libraries consisting of
variants containing random single amino acid mutations
in the third CDR of the heavy and light chains. By
this method, multiple Fab variants containing as few as
one nonhuman framework residue and displaying up to
approximately 500-fold higher affinity than the initial
chimeric Fab can be identified. Screening of
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combinatorial framework-CDR libraries permits
identification of monoclonal antibodies with structures
optimized for function, including instances in which
the antigen induces conformational changes in the
monoclonal antibody. The enhanced humanized variants
contain fewer nonhuman framework residues than
antibodies humanized by sequential in vitro
humanization and affinity maturation strategies known
in the art.
As described above, antibody neutralizing
agents of the invention include, for example,
polyclonal antibodies, monoclonal antibodies as well as
recombinant versions and functional fragments thereof.
Recombinant versions of antibody neutralizing agents
include a wide variety of constructions ranging from
simple expression and co-assembly of encoding heavy and
light chain cDNAs to speciality constructs termed
designer antibodies. Recombinant methodologies,
combined with the extensive characterization of
polypeptides within the immunoglobulin superfamily, and
particularly antibodies, provides the ability to design
and construct a vast number of different types, styles
and specificities of binding molecules derived from
immunoglobulin variable and constant region binding
domains. Specific examples include chimeric
antibodies, where the constant region of one antibody
is substituted with that of another antibody, and
humanized antibodies, described above, where the
complementarity determining regions (CDR) from one
antibody are substituted with those from another
antibody.
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Other recombinant versions of antibody
neutralizing agents include, for example, functional
antibody variants where the variable region binding
domain or functional fragments responsible for
maintaining antigen binding is fused to an F~ receptor
binding domain from the antibody constant region. Such
variants are essentially truncated forms of antibodies
that remove regions non-essential for antigen and F~
receptor binding. Truncated variants can be have
single valency, for example, or alternatively be
constructed with multiple valencies depending on the
application and need of the user. Additionally,
linkers or spacers can be inserted between the antigen
and F~ receptor binding domains to optimize binding
activity as well as contain additional functional
domains fused or attached to effect biological
functions other than CXCL10 neutralization. Those
skilled in the art will know how to construct
recombinant antibody neutralizing agents specific for
CXCL10 in light of the art knowledge regarding antibody
engineering and given the guidance and. teachings
herein. A description of recombinant antibodies,
functional fragments and variants and antibody-like
molecules can be found, for example, in Antibodv
Engineer,ing, 2nd Edition, (Carl A.K. Borrebaeck, Ed.)
Oxford University Press, New York,(1995).
Additional functional variants of antibodies
that can be used as antibody neutralizing agents
include antibody-like molecules other than antigen
binding-F~ receptor binding domain fusions. For
example, antibodies, functional fragments and fusions
thereof containing a F~ receptor binding domain can be
produced to be bispecific in that one variable region
binding domain exhibits binding activity for one
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antigen and the other variable region binding domain
exhibits binding activity for a second antigen. Such
bispecific antibody neutralizing agents can be
advantageous in the methods of the invention because a
5 single bispecific antibody will contain two different
target antigen binding species. Therefore, a single
molecular entity can be administered to achieve
neutralization of CXCL10.
An antibody neutralizing agent specific for
10 CXCL10 can also be an immunoadhesion or bispecific
immunoadhesion. Immunoadhesions are antibody-like
molecules that combine the binding domain of a non-
antibody polypeptide with the effector functions of an
antibody of an antibody constant domain. The binding
15 domain of the non-antibody polypeptide can be, for
example, a ligand or a cell surface receptor having
ligand binding activity. Immunoadhesions for use as
CXCL10 neutralizing agents can contain at least the F~
receptor binding effector functions of the antibody
20 constant domain. Specific examples of ligands and cell
surface receptors that can be used for the antigen
binding domain of an immunoadhesion neutralizing agent
include, for example, a T cell or NK cell receptor such
as the CXCR3 receptor that recognizes CXCL10. Other
25 ligands and ligand receptors known in the art can
similarly be used for the antigen binding domain of an
immunoadhesion neutralizing agent specific for CXCL10.
In addition, multivalent and multispecific
immunoadhesions can be constructed for use as CXCL10
30 neutralizing agents. The construction of bispecific
antibodies, immunoadhesions, bispecific immunoadhesions
and other heteromultimeric polypeptides which can be
used as CXCL10-specific neutralizing agents is the
subject matter of, for example, U.S. Patent Numbers
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41
5,807,706 and 5,428,130, which are incorporated herein
by reference.
In one embodiment of the invention, the
polynucleotides encoding CXCL10, regulatory molecules
that modulate the expression or activity of CXCL10, or
any fragment thereof, or antisense molecules, can be
used as neutralizing agents for therapeutic purposes.
In one aspect, antisense molecules to the CXCL10
encoding nucleic acids can be used to block the
transcription or translation of the mRNA.
Specifically, cells can be transformed with sequences
complementary to CXCL10 nucleic acids. Such methods
are well known in the art, and sense or antisense
oligonucleotides or larger fragments, can be designed
from various locations along the coding or control
regions of sequences encoding CXCL10. Thus, antisense
molecules can be used to neutralize CXCL10 activity, or
to achieve regulation of gene function.
Expression vectors derived from retroviruses,
adenovirus, adeno-associated virus (AAV), herpes or
vaccinia viruses, or from various bacterial plasmids
can be used for delivery of antisense nucleotide
sequences. The viral vector selected should be able to
infect the CNS cells and be safe to the host and cause
minimal cell transformation. Retroviral vectors and
adenoviruses offer an efficient, useful, and presently
the best-characterized means of introducing and
expressing foreign nucleotide sequences efficiently in
mammalian cells. These vectors are well known in the
art and have very broad host and cell type ranges,
express genes stably and efficiently. Methods well
known to those skilled in the art can be used to
construct such recombinant vectors and are described in
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Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Press, Plainview,
New York (1989), and Ausubel et al., Current Protocols
in Molecular Biology, John Wiley & Sons, New York
(1999); each of which is incorporated herein by
reference. Even in the absence of integration into the
DNA, such vectors can continue to transcribe RNA
molecules until they are disabled by endogenous
nucleases. Transient expression can last for a month
or more with a non-replicating vector and even longer
if appropriate replication elements are part of the
vector system.
Ribozymes, enzymatic RNA molecules, can also
be used to catalyze the specific cleavage of CXCL10
mRNA or the mRNA of any regulatory molecule that
modulates the expression or activity of CXCL10. The
mechanism of ribozyme action involves sequence-specific
hybridization of the ribozyme molecule to complementary
target the mRNA, followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within any potential
RNA target are identified by scanning the RNA for
ribozyme cleavage sites which include the following
sequences: GUA, GUU, and GUC. Once identified, short
RNA sequences of between 15 and 20 ribonucleotides
corresponding to the region of the target gene
containing the cleavage site can be evaluated for
secondary structural features which can render the
oligonucleotide inoperable. The suitability of
candidate targets can also be evaluated by testing
accessibility to hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Antisense molecules and ribozymes of the invention can
be prepared by any method known in the art for the
synthesis of nucleic acid molecules.
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The neutralizing agents useful for practicing
the methods of the invention can be formulated and
administered by those skilled in the art in a manner
and in an amount appropriate for the severity of the
tissue degeneration to be treated; the rate or amount
of tissue loss; the weight, gender, age and health of
the subject; the biochemical nature, bioactivity,
bioavailability and side effects of the particular
compound; and in a manner compatible with concurrent
treatment regimens. An appropriate amount and
formulation for decreasing the severity of secondary
tissue degeneration associated with CNS injury in
humans can be extrapolated from credible animal models
known in the art of the particular disorder. It is
understood, that the dosage of a neutralizing agent
specific for CXCL10 has to be adjusted based on the
binding affinity of the neutralizing agent for CXCL10,
such that a lower dose of a neutralizing agent
exhibiting significantly higher binding affinity can be
administered compared to the dosage necessary for a
neutralizing agent with lower binding affinity. Thus,
appropriate dosage will vary with the particular
treatment and with the duration of desired treatment;
,however, it is anticipated that dosages between about
10 micrograms and about 1 milligram per kilogram of
body weight per day will be used for therapeutic
treatment. A therapeutically effective amount is
typically an amount of a neutralizing agent that, when
administered in a physiologically acceptable
composition, is sufficient to achieve a plasma
concentration of from about 0.1 ug/ml to about 100
pg/ml, from about 1.0 pg/ml to about 50 pg/ml, or at
least about 2 ~Zg/ml and usually 5 to 10 ug/ml.
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It is contemplated that the method of the
invention of reducing the severity of secondary tissue
degeneration associated with CNS injury in a subject,
comprising administering to a subject having secondary
tissue degeneration associated with CNS injury an
effective amount of a neutralizing agent specific for
interferon inducible protein of 10 kDa (CXCL10) can
further comprise a time-course regimen consisting of
repeated treatments starting at the time of injury or
trauma. Since secondary tissue degeneration associated
with CNS injury is a progressive process that ensues
over the days or early weeks post injury,
administration of a CXCL10 neutralizing agent can be
initiated immediately or as soon as feasible following
injury and repeated daily for an appropriate length of
time. An appropriate length of time can be determined
on a variety of factors known to those skilled in the
art and can be, for example, 1, 2, 3, 5, 6, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 20, 25 or more days post
injury. Treatments also can be administered every
second or third day as well as two, three or more times
per day of treatment. For example, treatments can be
continued for the amount of time that CXCL10 levels are
upregulated above their base level following CNS injury
or trauma (see Lee et al. Neurochemistry International
36:417-425 (2000), which is incorporated herein by
reference). Thus, a time course regimen is useful for
practicing the claimed methods because a neutralizing
agent specific for interferon inducible protein of 10
kDa (CXCL10) can be administered starting immediately
following the CNS injury, for example, within about 10
minutes, about 20 minutes, about 30 minutes, about 40
minutes, about 50 minutes or within about 1 hour, about
2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 10
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hours, about 12 or more hours and repeated throughout
the time period that has been determined to correlate
with upregulation of CXCL10 following a CNS injury.
The total amount of neutralizing agent can be
5 administered as a single dose or by infusion over a
relatively short period of time, or can be administered
in multiple doses administered over a more prolonged
period of time. Such considerations will depend on a
variety of case-specific factors such as, for example,
10 whether the disease category is characterized by acute
episodes or gradual tissue deterioration. For example,
for a subject affected with chronic tissue
deterioration the neutralizing agent can be
administered in a slow-release matrice, which can be
15 implanted for systemic delivery or at the site of the
target tissue. Contemplated matrices useful for
controlled release of therapeutic compounds are well
known in the art, and include materials such as
DepoFoamTM, biopolymers, micropumps, and the like.
20 The neutralizing agents of the invention can
be administered to the subject by any number of routes
known in the art including, for example, systemically,
such as intravenously or intraarterially. A CXCL10-
specific neutralizing agent can be provided in the form
25 of isolated and substantially purified polypetides and
polypeptide fragments in pharmaceutically acceptable
formulations using formulation methods known to those
of ordinary skill in the art. These formulations can
be administered by standard routes, including for
30 example, topical, transdermal, intraperitoneal,
intracranial, intracerebroventricular, intracerebral,
intravaginal, intrauterine, oral, rectal or parenteral
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(e.g., intravenous, intraspinal, subcutaneous or
intramuscular) routes. Intraspinal and intravenous
administration of a CXCL10-specific neutralizing agent
are particularly suitable routes for practicing the
methods of the invention. Intraspinal administration
can be performed so as to target the delivery of the
neutralizing agent to the site of injury or trauma. In
addition, a CXCL10-specific neutralizing agent variant
can be incorporated into biodegradable polymers
allowing for sustained release of the compound useful
for reducing the severity of secondary tissue
degeneration associated with CNS injury. Biodegradable
polymers and their use are described, for example, in
Brem et al., J. Neurosura. 74:441-446 (1991), which is
incorporated herein by reference.
A CXCL10-specific neutralizing agent can be
administered as a solution or suspension together with
a pharmaceutically acceptable medium. Such a
pharmaceutically acceptable medium can be, for example,
sterile aqueous solvents such as sodium phosphate
buffer, phosphate buffered saline, normal saline or
Ringer's solution or other physiologically buffered
saline, or other solvent or vehicle such as a glycol,
glycerol, an oil such as olive oil or an injectable
organic ester. A pharmaceutically acceptable medium
can additionally contain physiologically acceptable
compounds that act, for example, stabilize the
neutralizing agent, increase its solubility, or
increase its absorption. Such physiologically
acceptable compounds include, for example,
carbohydrates such as glucose, sucrose or dextrans;
antioxidants such as ascorbic acid or glutathione;
receptor mediated permeabilizers, which can be used to
increase permeability of the blood-brain barrier;
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chelating agents such as EDTA, which disrupts microbial
membranes; divalent metal ions such as calcium or
magnesium; low molecular weight proteins; lipids or
liposomes; or other stabilizers or excipients. Those
skilled in the art understand that the choice of a
pharmaceutically acceptable carrier depends on the
route of administration of the compound containing the
neutralizing agent and on its particular physical and
chemical characteristics.
Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile
injection solutions such as the pharmaceutically
acceptable mediums described above. The solutions can
additionally contain, for example, buffers,
bacteriostats and solutes which render the formulation
isotonic with the blood of the intended recipient.
Other formulations include, for example, aqueous and
non-aqueous sterile suspensions which can include
suspending agents and thickening agents. The
formulations can be presented in unit-dose or
multi-dose containers, for example, sealed ampules and
vials, and can be stored in a lyophilized condition
requiring, for example, the addition of the sterile
liquid carrier, immediately prior to use.
Extemporaneous injection solutions and suspensions can
be prepared from sterile powders, granules and tablets
of the kind previously described.
For applications directed to brain injury
that a CXCL10 neutralizing agent can be administered in
a formulation that can cross the blood-brain barrier,
for example, a formulation that increases the
lipophilicity of the CXCL10 neutralizing agent. For
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example, the neutralizing agent can be incorporated
into liposomes (Gregoriadis, Liposome Technology, Vols.
I to III, 2nd ed. (CRC Press, Boca Raton FL (1993)).
Liposomes, which consist of phospholipids or other
lipids, are nontoxic, physiologically acceptable and
metabolizable carriers that are relatively simple to
make and administer.
A neutralizing agent specific for CXCL10 can
also be prepared as nanoparticles. Adsorbing peptide
compounds onto the surface of nanoparticles has proven
effective in delivering peptide drugs to the brain (see
Kreuter et al., Brain Res. 674:171-174 (1995)).
Exemplary nanoparticles are colloidal polymer particles
of poly-butylcyanoacrylate with a neutralizing agent
specific for~CXCL10 adsorbed onto the surface and then
coated with polysorbate 80.
Image-guided ultrasound delivery of a CXCL10
neutralizing agent through the blood-brain barrier to
selected locations in the brain can be utilized as
described in U.S. Patent No. 5,752,515. Briefly, to
deliver a CXCL10 neutralizing agent past the blood-
brain barrier a selected location in the brain is
targeted and ultrasound used to induce a change
detectable by imaging in the CNS (CNS) tissues and/or
fluids at that location. At least a portion of the
brain in the vicinity of the selected location is
imaged, for example, via magnetic resonance imaging
(MRI), to confirm the location of the change. A
CXCL10-specific neutralizing in the patient's
bloodstream can delivered to the confirmed location by
applying ultrasound to effect opening of the
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blood-brain barrier at that location and, thereby, to
induce uptake of the neutralizing agent.
In addition, polypeptides called receptor
mediated permeabilizers (RMP) can be used to increase
the permeability of the blood-brain barrier to
molecules such as therapeutic agents or diagnostic
agents as described in U.S. Patent Nos. 5,268,164;
5,506,206; and 5,686,416. These receptor mediated
permeabilizers can be intravenously co-administered to
a host with molecules whose desired destination is the
cerebrospinal fluid compartment of the brain. The
permeabilizer polypeptides or conformational analogues
thereof allow therapeutic agents to penetrate the
blood-brain barrier and arrive at their target
destination.
In accordance with another embodiment of the
present invention, there are provided therapeutic
systems, preferably in kit form, containing a CXCL 10
neutralizing agent and instructions for its use in
methods of reducing the severity of secondary tissue
degeneration associated with central nervous system
injury. In one embodiment, for example, the
therapeutic agent is an anti-CXCL10 antibody.
Invention kits are useful for reversing the severity of
secondary tissue degeneration associated with central
nervous system injury.
A suitable kit includes at least one CXCL10
neutralizing agent, as a separately packaged chemical
reagents) in an amount sufficient for at least one
therapeutic application. Instructions for use of the
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packaged kit are also typically included. Those of
skill in the art can readily incorporate a CXCL10
neutralizing agent into kit form in combination with
appropriate buffers and solutions for the practice of
5 the invention methods as described herein.
In current treatment regimes for CNS injury,
more than one compound is often administered to an
individual for management of the same or different
aspects of the disease. Similarly, in the methods of
10 the invention involving decreasing the rate of
secondary tissue degeneration, a neutralizing agent
specific for CXCL10 can advantageously be formulated
with a second therapeutic compound such as an anti-
inflammatory compound, immunosuppressive compound or
15 any other compound that manages the same or different
aspects of the disease. Such compounds include, for
example, methylprednisolone acetate, CM-1 ganglioside
and 4-aminopyridine (4-AP). Contemplated methods of
reducing the severity of secondary tissue degeneration
20 associated with CNS injury include administering a
neutralizing agent specific for CXCL10 alone, in
combination with, or in sequence with, such other
compounds as well as in combination with other
therapies, for example, rehabilitation and neural
25 prostheses. Alternatively, combination therapies can
consist of fusion proteins, where the neutralizing
agent specific for CXCL10 is linked to a heterologous
protein, such as a therapeutic protein.
The following examples are intended to
30 illustrate but not limit the present invention. It is
understood that modifications which do not
substantially affect the activity of the various
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~1
embodiments of this invention are also included within
the definition of the invention provided herein.
Accordingly, the following examples are intended to
illustrate but not limit the present invention.
Unless described separately below, the
experimental procedures corresponding to the Examples
set forth below are as follows:
Antibody administration. Age-matched adult
female C57/BL6 mice were used for all studies. Mice
received intraperitoneal injections of 100mg of
monoclonal CXCL 10 antibody (suspended in 200m1 of
sterile PBS) 1 day prior to injury, and every other day
thereafter until sacrifice. The monoclonal antibodies
used in these studies are specific for CXCL 10 and
block CXCL 10-induced T cell chemotaxis.
Spinal cord injury. Spinal cord dorsal
hemisection lesions were performed under Avertin
anesthesia (0.6m1/20g) administered via intraperitoneal
injection. A midline incision was made over the
spinous processes of T4-L2 and the paravertebral
muscles were separated from the vertebrae. A complete
dorsal laminectomy of the T9 vertebrae was performed
using fine scissors and rongeurs. The column was
stabilized with a clamp attached to a micromanipulator.
A microlesion knife was then be used to produce a
dorsal hemisection injury. Muscle layers were then
sutured and the superficial tissue and skin closed with
4-0 silk. Body temperature was maintained on a heating
pad. Postoperative care included bladder voiding and
hydration with lactated Ringer's solution.
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Kinematic Analysis. All mice were acclimated
to a linear running arena for 4 days prior to the onset
of functional testing. Kinematic analysis was
conducted one day prior to injury, and every day
thereafter until sacrifice at approximately midday, and
scored independently by two observers blinded to the
treatment group. Animals were videotaped using a
Hitachi 8mm Video Camcorder VM-E555LA from underneath a
plexiglass surface bearing defined lcm grid lines. The
videos were then downloaded using an MPEG-2 compression
devise, and analyzed using FMV 2.0 software, which
allowed the video to be viewed frame by frame for
kinematic assessment. Four kinematic parameters were
assayed; stride length, stride width, paw rotation and
toe spread. Rear paw stride length was defined as the
point from which the start of a step with the rear paw
through to the end of a step with the same paw
(measurements taken on individual sides and for three
consecutive steps in mms which were subsequently
averaged). Stride width was defined as the width in
mms of the hind limb strides (distance from the left
outermost lateral hind paw digit to the right outermost
contralateral hind paw digit). Toe spread was defined
as the width in mms in the hind paw from the most
lateral point of the lateral digit to the most medial
point of the medial digit (both right and left hind paw
respectively). Paw rotation was defined as the angle
between the longitudinal axis of the rear paws and the
midline axis of the body in degrees. The SPSS 4.0
T-test was used to determine significant differences
between treated and untreated groups.
RNase protection assay. Mice were killed at
the following post-injury time points by C02 fixation
and decapitation: 6hr (n=3), l2hr (n=3), l8hr (n=3),
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24hr (n=3), 3days (n=3), 7days (n=3), and l4days (n=2).
Two non-injured mice were used as controls. The spinal
cord was removed in two pieces and immediately frozen.
Total RNA was extracted with Trizol reagent (Gibco) and
ethanol precipitated. The pellets were dissolved in
50u1 RNase-free water and the RNA concentration was
determined. The multiprobe set mCK-5 was used to
detect CXCL 10 mRNA transcripts. This included a probe
for L32 which was used to as a control for RNA loading.
Analysis was performed on l0ug protein. Fragments were
separated by polyacrylamide gel electrophoresis, and
visualized by film autoradiography. Autoradiographs
were scanned and imaging software was used to quantify
the bands.
Mononuclear cell isolation and flow
cytometry. A single cell suspension was obtained from
spinal cords of mice treated with either anti-CXCL10,
or control antibody at days 3 and 14 post-injury. FACS
analysis was performed as previously described (Lane,
Liu et al., "A central role for CD4(+) T cells and
RANTES in virus-induced central nervous sytem
inflammation and demyelination," J Virol, (2000)
74(3):1415-24). Briefly, brains were removed and a
single cell suspension was obtained by grinding the
tissue. All techniques were performed within sterile
tissue culture plates on ice; the plates contained
Dulbecco modified Eagle medium supplemented with 10°s
fetal bovine serum. Cell suspensions were transferred
to 15 ml conical tubes and Percoll (Pharmacia, Uppsala,
Sweden) was added for a final concentration of 30%.
One milliliter of 70°s Percoll was underlaid and the
cells were spun at 1300 x g for 30 min at 4oC. Cells
were removed from the interface and washed twice.
Fluorescein isothiocyanate-conjugated (FITC) rat
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anti-mouse CD4 and (Phycoerythrin-conjugated PE) rat
anti-mouse CD8 (Pharmingen, San piego, CA) were used
to detect infiltrating CD4+ and CD8+ T cells
respectively. As a control isotype-matched FITC and
PE antibody was used. Cells were incubated with
antibodies for 1 hour at 40°C, washed, fixed in to
paraformaldehyde, and analyzed on a FACStar (Becton
Dickinson, Mountain View, Calif.). Percent of cells
infiltrating into the CNS were determined on Cell quest
flow cytometric software. Positive cells were
determined by subtracting experimental sample
fluorescence from background and control samples.
Total numbers of CD4 and CD8 T cells were determined by
multiplying the percentage of positive cells within the
gated population by the number of cells isolated from
the spinal cord. Data is presented as the mean + SEM.
H&E Stamina. Longitudinal frozen spinal
cord sections were cut and fixed in acetic alcohol.
The sections were stained in Harris' Hematoxylin,
washed with tap water, and counterstained in 1°s eosin.
Slides were dehydrated in ascending alcohols and
mounted with permount (Fisher Scientific).
Immunohistochemistry. Animals were
transcardially perfused with 4°s paraformaldehyde 24
hrs, 3 days, and 14 days post-injury. The spinal cords
were removed and sunk in 25% sucrose overnight. The
tissue was then embedded in OCT and l2um thick
longitudinal cryosections were cut and placed onto
slides. Sections were blocked in 10% normal goat serum
(NGS; diluted with PBS) for lhr at room temperature.
Primary antisera (rat anti-mouse CD4 monoclonal ab,
1:200 dilution in 10% NGS, PharMingen; rat anti-mouse
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CDllb, Serotec) were applied to sections overnight at
4°C. Sections were rinsed three times with PBS and
goat anti-rat IgG biotinylated antisera (1:200 dilution
in 10~ NGS, Vector Laboratories) were applied and
5 slides were incubated lhr at room temperature.
Sections were rinsed three times in PBS and incubated
in a methanol: 30o hydrogen peroxide solution (100:1)
for l0min. The sections were washed three times in PBS
and incubated in ABC solution (Vector Laboratories) for
10 30min at room temperature. DAB substrate solution
(Vector Laboratories) was used to visualize the binding
of the antibodies. Sections were dehydrated in
ascending alcohols. Permount (Fisher Scientific) was
used for mounting. No immunoreactivity was seen when
15 the primary antibody was omitted. The same procedure
was used to stain for neurons with the following
modifications: The sections were blocked overnight and
the primary antisera used were mouse anti-NeuN
monoclonal (Chemicon, 1:100 dilution). The secondary
20 antibody used was biotinylated rat adsorbed horse
anti-mouse at (Vector Laboratories, 1:200 dilution).
Neurons and CD4 positive T cells within one millimeter
of each side of the injury were counted at a lOX
magnification. Only clearly labeled cells were
25 counted.
EXAMPLE I
Upreaulation of CXCL10 Levels and T Cell Numbers
following CNS In'~ury
This example describes upregulation of CXCL10
30 levels and T cell numbers following CNS injury.
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To determine whether CXCL10 mRNA levels were
increased after hemisection injury, hemisection
injuries of adult mouse spinal cords were performed on
adult C57B16 female mice using a pointed scalpel blade
at T9, following removal of the dorsal half of the T9
vertebrae. Total RNA was extracted from hemisectioned
spinal cords using TRIzol~ reagent (Life Technologies
(GIBCO-BRL) Rockville, MD) at 6 hrs, 12 hrs, 18 hrs, 24
hrs, 3 days, 7 days and 14 days post injury, and the
level of IP-10 mRNA transcripts was determined for each
time point by ribonuclease protection assay (RPA) using
the CK-1 chemokine probe set previously described in
Lane et al., J. Virol. 74(3):415-424 (2000), which is
incorporated herein by reference. The abundance of
mRNA transcripts was determined by scanning
autoradiographs to determine the density of individual
bands as it related to internal L32 controls using NIH
1.61 image software.
As shown in Figure 1, CXCL10 mRNA levels
increased by 6 hours post-injury and then gradually
declined, remaining above basal levels even after 14
days post-injury. CXCL10 mRNA was undetectable in
uninjured spinal cord tissue.
Spinal cords also were dissected 3 and 14
days post-injury such that the injury site, which was
marked at surgery, was centrally located within the
dissected tissue. Longitudinal sections in which the
central canal was clearly visible were selected and
digitalized. For cell counts, the number of CD4
immunopositive cells within the total tissue area
extending one millimeter either side of the injury site
was determined using stereology. Only immunolabeled
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cells with a clearly discernable Hoechst stained
nucleus were scored.
Quantitative analysis of CD4 immunostained
longitudinal tissue sections indicated that CD4+ T
cells were present at an increased density within the
region extending 1 mm either side of the injury site at
3 days post-injury in hemisection injured spinal cord,
as compared to uninjured spinal cord. The above
studies indicate that IP-10 levels and T cell numbers
are upregulated after CNS injury.
EXAMPLE II
Neutralization of CXCL10 Levels Diminished T Cell
Accumulation within and around the In'Ly Site
following CNS Iniury
To determine the effect of anti-IP-10
treatment following dorsal hemisection injury,
hemisection-injured adult mice received 0.5 ml
intraperitoneal injections of anti-IP-10 antibodies
(approximately 0.5mg/ml) every other day starting at 1
day prior to injury and continuing until 7 days
post-injury. The mice were sacrificed at 3 days or 14
days post-injury and the spinal cords were dissected
such that the injury site was centrally located within
the dissected tissue. Longitudinal sections in which
the central canal was clearly visible were selected and
digitalized. For cell counts, the number of CD4
immunopositive cells within the total tissue area
extending one millimeter either side of the injury site
was determined using stereology. As described in
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Example I above, only immunolabeled cells with a
clearly discernable Hoechst stained nucleus were
scored.
Quantitative analysis of CD4 immunostained
longitudinal tissue sections indicated that CD4+ T
cells were present at an increased density within the
region extending 1 mm either side of the injury site at
3 days post-injury in untreated hemisection injured
spinal cord, as compared to anti-CXCL10 treated
hemisection injured spinal cord. These studies
indicate that neutralization of IP-10 decreased T cell
recruitment following CNS injury.
To determine the effect anti-IP 10 treatment
on behavioral deficits associated with CNS injury,
anti-CXCL10 antibody treated hemisection-injured mice,
untreated hemisection-injured mice and uninjured
control mice were first acclimated for 4 days prior to
hemisection injury and subsequently subjected to daily
kinematic analyses from 1 to 13 days following
hemisection injury by two observers blinded to the
treatment group. Animals were videotaped from
underneath a 4'x4' plexiglass surface bearing defined
grid lines, and the recording analyzed using Adobe
Premiere video editing software (Adobe Systems, Inc.,
San Jose, CA).
Four kinematic parameters were assessed:
stride length, stride width, paw rotation and toe
spread. As shown in Figure 2, all hemisection-injured
mice treated with anti-IP-10 antibody had significantly
greater stride length, and significantly less stride
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59
width, toe spread and paw rotation, then untreated
hemisection-injured mice. Progressive behavioral
improvement was assessed by comparing, for each
kinematic parameter, the data points for all animals
over the first three days post-injury with the data
points for all animals over the last three days
post-injury. Untreated hemisection-injured mice showed
no change in kinematic parameters during the recovery
period (p>0.05). In contrast, treated
hemisection-injured mice showed a statistically
significant progressive improvement in all kinematic
parameters during the recovery period (p« 0.01).
Spinal cords were dissected 14 days
post-injury such that the injury site was centrally
located within the dissected tissue. Longitudinal
sections in which the central canal was clearly visible
were selected and digitalized. The total tissue area
extending one millimeter either side of the injury site
was measured using an MCID analysis system as described
in Zhang et al., J. Comp. Neurol. 371:485-495(1996),
which is incorporated herein by reference.
As shown in Figure 3, untreated
hemisection-injured mice had on average a 49.4%
reduction in total tissue area extending 1 mm either
side of the injury, as compared to uninjured control
spinal cords. In contrast, morphometric analyses of
anti-IP-10 antibody treated hemisection-injured mice
indicated a 20.90 reduction in total tissue area
extending lmm on either side of the injury compared to
uninjured control spinal cords, which represents a 68°s
reduction in tissue loss compared to untreated
hemisection-injured mice. The number of NeuN
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immunopositive neurons was determined within the total
tissue area extending one millimeter either side of the
injury site was determined using stereology. The NeuN
monoclonal antibody (MAB377, Chemicon, Temecula, CA),
5 which recognizes neuron-specific nuclear protein and
reacts with most neuronal cell types was used according
to Manufacturer's instructions. Only immunolabeled
cells with a clearly discernable Hoechst-stained
nucleus were scored. Quantitative analysis of NeuN
10 immunostained longitudinal tissue sections indicated
that NeuN+ neurons were present at an increased density
within the region extending 1 mm either side of the
injury site in anti-CXCL1010 treated
hemisection-injured mice, as compared to untreated
15 hemisection-injured mice.
These data indicate that anti-CXCL10
treatment decreased posttraumatic tissue loss following
hemisection injury.
EXAMPLE III
20 Effect of CXCL10 Antibodies on Lymphocyte Infiltration
CXCL10 mRNA levels were increased after
hemisection injury to the adult mouse spinal cord.
Total RNA was extracted from hemisectioned spinal cords
at 6 hrs (n=3), 12 hrs (n=3), 18 hrs (n=3), 24 hrs
25 (n=3), 3 days (n=3), 7 days (n=3) and 14 days (n=3)
after injury, and the level of CXCL10 mRNA transcripts
was determined for each time point by ribonuclease
protection assay (RPA) using the CK-1 chemokine probe
set (Lane, Liu et al . , ~~A central role for CD4 (+) T
30 cells and R,ANTES in virus-induced central nervous
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61
system inflammation and demyelination," J. Virol.,
(2000) 74 (3) :1415-24) . The abundance of mRNA
transcripts was determined by scanning autoradiographs
to determine the density of individual bands as it
related to internal L32 controls. CXCL10 mRNA levels
increased by 6 hours post-injury to an average level of
74.8 +/- 21.78 and then gradually declined, remaining
above basal levels even after 14 days past-injury with
an average level of 41.57 +/- 1.80 (Figure 1). CXCL 10
mRNA was undetectable in uninjured spinal cord tissue.
Treatment with anti-CXCL10 reduced lymphocyte
and activated macrophage infiltration into the CNS
following injury. In hemisectioned adult mice,
quantitative analyses of CD4 immunostained longitudinal
tissue sections indicated that the total number of CD4+
T lymphocytes within the region extending lmm either
side of the injury site at 3 days post-injury was 2050
+/- 102 (n=3; Figure 5). CD4+ T lymphocytes were not
detected in uninjured spinal cord tissue. These data
indicate that CXCL 10 levels and T lymphocyte numbers
are upregulated after traumatic injury and support
previous studies demonstrating an upregulation of
CXCL10 6 hours after contusion injury to adult rats,
and a peak of T lymphocyte infiltration within the
first week post-injury (McTigue, Tani et al.,"Selective
chemokine mRNA accumulation in the rat spinal cord
after contusion injury," J. Neurosci. Res., (1998)
53 (3) :368-76) .
In hemisectioned adult mice that received
anti-CXCL10 treatment, quantitative analyses of CD4
immunostained longitudinal tissue sections indicated
that the total number of CD4+ T lymphocytes within the
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62
region extending lmm either side of the injury site at
3 days post-injury was 622 +/- 202 (n=3), representing
a 70% decrease relative to untreated
hemisection-injured animals (Figure 5).
FACS analysis indicated that the number of
CD4+ lymphocytes, CD8+ lymphocytes, and F480/CD45+
macrophages were decreased at both 3 days and 14 days
after injury in anti-CXCL10 treated hemisection-injured
mice as compared to untreated hemisection-injured mice.
In particular, FACS data summarized in Table 1 below
demonstrated a significant reduction in CD4+ T
lymphocytes (66.4°s reduction), CD8+ T lymphocytes
(57.4% reduction) and activated macrophage/microglia
cells (35.5°s reduction) present within the spinal cords
of these animals at day 3 post-injury as compared with
T lymphocyte and activated macrophage/microglia levels
in untreated hemisection-injured mice. In addition,
FACS data demonstrated a significant reduction in CD4+
T lymphocytes (58% reduction), CD8+ T lymphocytes (640
reduction) and activated macrophage/microglia cells
(43.2% reduction) present within the spinal cords of
treated animals at day 14 post-injury as compared with
T lymphocyte and activated macrophage/microglia levels
in untreated hemisection-injured mice. (n=4; Table 1).
Table 1. Cellular Infiltration is Reduced in anti-
CXCL10 treated Mice.
Number infiltrating cells in
of
spinalcord
Treatment n Days CD4 CD8 F480/CD45
p.i.
Anti-CXCL10 4 3 3.7 103 2.6 103 2.0 105
x x x
Anti-CXCL10 4 14 4.2 10' 2.7 103 2.5 105
x x x
No treatment 4 3 1.1 10' 6.1 103 3.1 105
x x x
No treatment 4 14 1.0 104 7.5 10' 4.4 105
x x x
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EXAMPLE IV
Effect of CXCL10 Treatment on Tissue Sparing and
Function Deficit
In order to determine whether an attenuated
CD4+ T lymphocyte response to traumatic spinal cord
injury was associated with tissue sparing or a decrease
in functional deficit, hemisectioned adult mice
received intraperitoneal injections of anti-CXCL10
antibodies every other day from 1 day prior to injury
to 9 days post-injury and were sacrificed 14 days after
injury (n=11). Tissue sparing after hemisection injury
was significantly greater in mice treated with
anti-CXCL10 antibody as compared to untreated
hemisection-injured mice (n=8). Morphometric analyses
of longitudinal tissue sections in which the central
canal was clearly visible were conducted using the MCID
analysis system, as described in Zhang et al., (1996)
(Zhang, Fujiki et al., "Genetic influences on cellular
reactions to spinal cord injury: a wound-healing
response present in normal mice is impaired in mice
carrying a mutation (WldS) that causes delayed
Wallerian degeneration," J. Comp. Neurol., (1996)
371(3):485-95). Untreated hemisection-injured mice had
on average a 49.4% reduction in total tissue area
extending lmm either side of the injury, as compared to
uninjured control spinal cords (Figure 3). In
contrast, morphometric analyses of anti-CXCL10 antibody
treated hemisection-injured mice indicated a 20.9%
reduction in total tissue area extending lmm either
side of the injury compared to uninjured control spinal
cords, which represents a 68% reduction in tissue loss
compared to untreated hemisection-injured mice (Figure
3) .
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Consistent with greater tissue sparing,
treated hemisection-injured mice contained
significantly more neurons around the injury site at 14
days post-injury than untreated hemisection-injured
mice. Quantitative analyses of NeuN immunostained
longitudinal tissue sections from untreated
hemisection-injured mice indicated that the averaged
total number of NeuN+ neurons within the region
extending lmm either side of the injury site at 14 days
post-injury was 267 +/- 72. In contrast, quantitative
analysis of NeuN immunostained longitudinal tissue
sections from anti-CXCL10 antibody treated
hemisection-injured mice indicated that the averaged
total number of NeuN+ neurons within the region
extending lmm either side of the injury site at 14 days
post-injury was 1170 +/- 184, representing 438°s more
neurons than the untreated hemisection-injured animals.
These data indicate that anti-CXCL 10
treatment significantly decreased posttraumatic tissue
loss following dorsal hemisection injury.
EXAMPLE V
Effect of Anti-CXCL10 Antibodv Treatment on Behavioral
Deficit
Behavioral deficit following hemisection
injury progressively lessened in mice treated with
anti-CXCL10 antibody as compared to untreated control
mice. Treated and untreated hemisection-injured mice,
as well as uninjured control mice (n=8), were subject
to daily kinematic analyses from 1-13 days following
hemisection injury, by two observers blinded to the
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treatment group. Animals were videotaped from
underneath a 3'x 1' plexiglass surface bearing defined
1cm grid lines, and the recording analyzed using video
editing software. Four kinematic parameters were
5 assessed; rear paw stride length, rear paw stride
width, rear paw rotation and rear paw toe spread.
These analyses indicated that all hemisection-injured
mice treated with anti-CXCL10 antibody had
significantly greater rear paw stride length, and
10 significantly less rear paw stride width, rear paw toe
spread and rear paw rotation, then untreated
hemisection-injured control mice at 14 days post-injury
(Figure 2). Progressive behavioral improvement was
assessed by comparing the data points for all animals
15 over the first three days post-injury with the data
points for all animals over the last three days
post-injury, for each kinematic parameter. Untreated
hemisection-injured mice showed no change in kinematic
parameters during the recovery period (p>0.05). In
20 contrast, treated hemisection-injured mice showed a
statistically significant progressive improvement in
all kinematic parameters during the recovery period
(p<0.01). By 14 days post-injury, the behavioral
scores for all 4 kinematic parameters of treated
25 hemisection-injured mice were not significantly
different from uninjured control mice.
These data indicate that anti-CXCL10 antibody
treatment reduces neurological impairment following
spinal cord injury.
30 Throughout this application various
publications have been referenced within parentheses.
The disclosures of these publications in their
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entireties are hereby incorporated by reference in this
application in order to more fully describe the state
of the art to which this invention pertains.
Although the invention has been described
with reference to the disclosed embodiments, those
skilled in the art will readily appreciate that the
specific experiments detailed are only illustrative of
the invention. It should be understood that various
modifications can be made withQUt departing from the
spirit of the invention. Accordingly, the invention is
limited only by the following claims.
