U.S. patent application number 10/582163 was filed with the patent office on 2011-05-19 for method and vaccine comprising copolymer 1 for treatment of psychiatric disorders.
Invention is credited to Michal Eisenbach-Schwartz, Jonathan Kipnis.
Application Number | 20110117115 10/582163 |
Document ID | / |
Family ID | 34386154 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110117115 |
Kind Code |
A1 |
Eisenbach-Schwartz; Michal ;
et al. |
May 19, 2011 |
Method and vaccine comprising copolymer 1 for treatment of
psychiatric disorders
Abstract
Copolymer 1, a Copolymer 1-related peptide, a Copolymer
1-related polypeptide, and T cells activated therewith are useful
in methods and compositions for treatment of psychiatric disorders,
diseases and conditions.
Inventors: |
Eisenbach-Schwartz; Michal;
(Rehovot, IL) ; Kipnis; Jonathan; (Modiin,
IL) |
Family ID: |
34386154 |
Appl. No.: |
10/582163 |
Filed: |
December 9, 2004 |
PCT Filed: |
December 9, 2004 |
PCT NO: |
PCT/IL04/01115 |
371 Date: |
March 5, 2007 |
Current U.S.
Class: |
424/185.1 ;
424/93.71; 514/17.5 |
Current CPC
Class: |
A61P 37/08 20180101;
C07K 14/705 20130101; A61P 37/04 20180101; A61P 25/18 20180101;
A61P 31/12 20180101; G01N 2500/00 20130101; A61P 1/16 20180101;
A61P 35/00 20180101; A61K 31/00 20130101; A61P 37/06 20180101; A61P
11/06 20180101; G01N 33/566 20130101; G01N 33/564 20130101; A61P
31/14 20180101 |
Class at
Publication: |
424/185.1 ;
514/17.5; 424/93.71 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 38/07 20060101 A61K038/07; A61K 38/02 20060101
A61K038/02; A61K 35/12 20060101 A61K035/12; A61P 25/18 20060101
A61P025/18; A61P 37/04 20060101 A61P037/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2003 |
JP |
2003-338331 |
Claims
1-38. (canceled)
39. A method for treatment of a psychiatric disorder, disease or
condition, which comprises administering to an individual in need
of such a treatment an effective amount of an agent selected from
the group consisting of (i) Copolymer 1, (ii) a Copolymer 1-related
peptide, (iii) a Copolymer 1-related polypeptide, and (iv) T cells
activated with (i), (ii) or (iii).
40. A method according to claim 39 wherein said individual is
immunized with a therapeutically effective amount of an agent
selected from the group consisting of (i) Copolymer 1, (ii) a
Copolymer 1-related peptide, (iii) a Copolymer 1-related
polypeptide, and (iv) T cells activated with (i), (ii) or
(iii).
41. The method according to claim 39 wherein said agent is
Copolymer 1.
42. The method according to claim 39 wherein said agent is a
Copolymer 1-related peptide or a Copolymer 1-related
polypeptide.
43. The method according to claim 39 wherein said agent is T cells
which have been activated by Copolymer 1.
44. A method according to claim 39 wherein said psychiatric
disorder, disease or condition is selected from the group
consisting of: (i) anxiety disorders; (ii) mood disorders; (iii)
schizophrenia and related disorders; (iv) drug use and dependence;
and (v) memory loss disorders.
45. A method according to claim 44 wherein said anxiety disorders
include phobic disorders, obsessive-compulsive disorder, stress,
post-traumatic stress disorder (PTSD), acute stress disorder and
generalized anxiety disorder.
46. A method according to claim 45 wherein said anxiety disorder is
post-traumatic stress disorder (PTSD) and said agent is Copolymer
1.
47. A method according to claim 44 wherein said mood disorders
include depression, dysthymic disorder, bipolar disorders and
cyclothymic disorder.
48. A method according to claim 44 wherein said psychiatric
disorder, disease or condition is schizophrenia and said agent is
Copolymer 1.
49. A method according to claim 44 wherein said schizophrenia
related disorders include brief psychotic disorder,
schizophreniform disorder, schizoaffective disorder and delusional
disorder.
50. A method according to claim 44 wherein said drug use and
dependence include alcoholism, cocaine dependence, amphetamine
dependence, hallucinogen dependence, and phencyclidine use.
51. A method according to claim 44 wherein said memory loss
disorder is cognitive impairment.
52. A method according to claim 41 wherein the Copolymer 1 is
administered in the form of a vaccine.
53. A method according to claim 52 wherein said vaccine comprises
Copolymer 1 without an adjuvant.
54. A method according to claim 52 wherein said vaccine comprises
Copolymer 1 emulsified in an adjuvant suitable for human clinical
use.
55. A method according to claim 54 wherein said adjuvant is
aluminum hydroxide, aluminum hydroxide gel or aluminum
hydroxyphosphate.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
for treatment of psychiatric disorders and, in particular, to
Copolymer 1 and related peptides and polypeptides and T cells
treated therewith for use in such compositions and methods.
[0002] Abbreviations: AMPH: D-amphetamine sulfate; ASR: acoustic
startle response; BDNF: brain-derived neurotrophic factor; CBC:
cut-off behavioral criteria; CFA: complete Freund's adjuvant; CNS:
central nervous system; COP-1: copolymer 1; EPM: Elevated
plus-maze; MBP: myelin basic protein; MK-801: (+)dizocilpine
maleate; MWM: Morris water maze; PNS: peripheral nervous system;
PPI: prepulse inhibition; PTSD: post-traumatic stress disorder;
SCID:severe combined immune deficiency; Teff: effector T cells;
Treg: regulatory T cells; WT: wild-type.
BACKGROUND OF THE INVENTION
[0003] Mental disorders are now known to be characterized not only
by behavioral abnormalities but also by somatic manifestations. In
a number of psychiatric disorders, a neurodegenerative component
has been identified. In schizophrenia, for example, there is loss
of hippocampal volume and death of hippocampal neurons (Lieberman
et al., 2001; Velakoulis et al., 1999), as well as anatomical and
molecular abnormalities of excitatory neurons in the dorsolateral
prefrontal cortex (McCullumsmith et al., 2002; Lewis et al., 1999).
The etiology and pathogenesis of schizophrenia are still obscure,
although there is general agreement that genetic predisposition is
a significant factor (Brzustowicz et al., 2002; Falkai et al.,
2003).
[0004] Symptoms of schizophrenia can be classified as positive,
negative, and cognitive, by using standard rating scales such as
the Positive and Negative Symptom Scale (Javitt et al., 1999).
Positive symptoms include hallucinations, agitation and paranoia;
negative symptoms reflect the loss of interpersonal drive and
normal interest in the environment; and cognitive symptoms include
conceptual disorganization and disorientation (Javitt et al.,
1999). Whereas the positive symptoms usually respond well to
dopamine-receptor antagonists (which however have significant and
devastating side effects such as induction of Parkinson's disease),
the negative symptoms and cognitive deficits typically persist,
resulting in chronic morbidity and poor long-term outcome (Rummel
et al., 2003).
[0005] Until quite recently, the body's principal adaptive
responses to stressful stimuli were attributed to the
hypothalamic-pituitary-adrenocortical axis (de Kloet, 2003). An
association between the immune system and the cognitive
performance, anxiety, and sensorimotor dysfunction seen in mental
disorders or acute psychological stress has generally been
considered unlikely or of little significance, despite a growing
body of evidence suggesting that such an association not only
exists, but might be an important consideration in the design of
therapy (Raison et al., 2001).
[0006] Contrary to long-held belief, the effect of the immune
system on the nervous system can also be beneficial. Recent studies
have shown that the injured CNS can benefit from the presence of a
well-controlled adaptive immunity (Schwartz et al., 1999a, 1999b;
Wekerle, 2002). T cells specifically reactive to proteins that
reside in the site of the insult promote post-injury neuronal
survival and restoration of function (Hauben et al., 2000; Mizrahi,
2002). It was further shown that this beneficial post-injury
response of autoimmune T cells to site-specific proteins is evoked
spontaneously (Yoles et al., 2001; Kipnis et al., 2002).
[0007] Immune abnormalities have been reported in patients with
schizophrenia, and there have been numerous attempts to find a
connection between schizophrenia and autoimmune disease. However,
studies over the last 60 years aimed at identifying schizophrenia
as an autoimmune disease have so far been unsuccessful (Amital and
Shoenfeld, 1993).
[0008] A link between brain maintenance and peripheral adaptive
immunity was suggested by recent findings that the ability to cope
with neurodegenerative conditions depends on CD4.sup.+T cells
(Moalem et al., 1999; Kipnis et al., 2001; Yoles et al., 2001). The
beneficial effect of CD4.sup.+ T cells in fighting off mediators of
degeneration was found to be specific to antigens residing in the
site of the lesion. Naturally occurring CD4.sup.+CD25.sup.+
regulatory T cells (Treg) normally suppress the ability to
spontaneously evoke this neuroprotective response, which is
amenable to boosting by weakening of Treg (Kipnis et al., 2002) or
by a well-controlled vaccination with self-antigens or with weak
agonists of self-antigens (Kipnis et al., 2000) such as
Copolymer-1.
[0009] Copolymer 1, also called Cop 1, is a random non-pathogenic
synthetic copolymer, a heterogeneous mix of polypeptides containing
the four amino acids L-glutamic acid (E), L-alanine (A), L-tyrosine
(Y) and L-lysine (K) in an approximate ratio of 1.5:4.8:1:3.6, but
with no uniform sequence. Although its mode of action remains
controversial, Cop 1 clearly helps retard the progression of human
multiple sclerosis (MS) and of the related autoimmune condition
studied in mice, experimental autoimmune encephalomyelitis (EAE).
One form of Cop 1, known as glatiramer acetate, has been approved
in several countries for the treatment of multiple sclerosis under
the trademark Copaxone.RTM. (Teva Pharmaceutical Industries Ltd.,
Petach Tikva, Israel).
[0010] Vaccination with Cop 1 or with Cop 1-activated T cells have
been shown by the present inventors to boost the protective
autoimmunity, after traumatic central nervous system (CNS) insult,
thereby reducing further injury-induced damage, and can further
protect CNS cells from glutamate toxicity. Reference is made to
Applicant's published International Applications Nos. WO 01/52878
and WO 01/93893, which disclose that Cop 1, Cop 1-related peptides
and polypeptides and T cells activated therewith prevent or inhibit
neuronal degeneration and promote nerve regeneration in the CNS or
peripheral nervous system (PNS), and protect CNS cells from
glutamate toxicity.
[0011] The main inventor, Prof Schwartz, and colleagues have shown
that Cop 1 acts as a low-affinity antigen that activates a wide
range of self-reacting T cells, resulting in neuroprotective
autoimmunity that is effective against both CNS white matter and
grey matter degeneration (Kipnis and Schwartz, 2002). The
neuroprotective effect of Cop 1 vaccination was demonstrated by the
inventors in animal models of acute and chronic neurological
disorders such as optic nerve injury (Kipnis et al., 2000), head
trauma (Kipnis et al., 2003), glaucoma (Schori et al., 2001),
amyotrophic lateral sclerosis (Angelov et al., 2003) and in the
applicant's patent applications WO 01/52878, WO 01/93893 and WO
03/047500.
[0012] Reference is made to copending international application of
the same applicant entitled "Compositions and methods for treatment
of psychiatric disorders", filed on the same date at the Israel
Patent Office/Receiving Office, in which the present invention is
explicitly excluded.
[0013] Citation of any document herein is not intended as an
admission that such document is pertinent prior art, or considered
material to the patentability of any claim of the present
application. Any statement as to content or a date of any document
is based on the information available to applicant at the time of
filing and does not constitute an admission as to the correctness
of such a statement.
SUMMARY OF THE INVENTION
[0014] It has now been found, in accordance with the present
invention, that vaccination with Copolymer 1 can lessen behavioral
abnormalities and improve cognitive function in mice suffering from
dopamine imbalance induced by MK-801 or amphetamine.
[0015] In one aspect, the present invention relates to a method for
treatment of a individual suffering from a psychiatric disorder,
disease or condition which comprises administering to said
individual in need of such a treatment an effective amount of an
agent selected from the group consisting of (i) Copolymer 1, (ii) a
Copolymer 1-related peptide, (iii) a Copolymer 1-related
polypeptide, and (iv) T cells treated with (i), (ii) or (iii).
[0016] In another aspect, the present invention relates to a
pharmaceutical composition, preferably a vaccine, for treatment of
psychiatric disorders, diseases or conditions which comprises a
pharmaceutically acceptable carrier and an agent selected from the
group consisting of (i) Copolymer 1, (ii) a Copolymer 1-related
peptide, (iii) a Copolymer 1-related polypeptide, and (iv) T cells
treated with (i), (ii) or (iii).
[0017] In a further aspect, the present invention relates to the
use of an agent selected from the group consisting of (i) Copolymer
1, (ii) a Copolymer 1-related peptide, (iii) a Copolymer 1-related
polypeptide, and (iv) T cells treated with (i), (ii) or (iii), for
the preparation of a pharmaceutical composition, preferably a
vaccine, for treatment of psychiatric disorders, diseases or
conditions.
[0018] In still another aspect, the present invention provides an
article of manufacture comprising packaging material and a
pharmaceutical composition contained within the packaging material,
said pharmaceutical composition comprising an agent selected from
the group consisting of Copolymer 1, a Copolymer 1-related peptide,
and a Copolymer 1-related polypeptide; and said packaging material
includes a label that indicates that said agent is therapeutically
effective for treating a psychiatric disorder, disease or
condition.
[0019] The psychiatric disorders, diseases or conditions that may
be treated according to the invention include: (i) anxiety
disorders, that include phobic disorders, obsessive-compulsive
disorder, post-traumatic stress disorder (PTSD), acute stress
disorder and generalized anxiety disorder; (ii) mood disorders,
that include depression, dysthymic disorder, bipolar disorders and
cyclothymic disorder; (iii) schizophrenia and related disorders
such as brief psychotic disorder, schizophreniform disorder,
schizoaffective disorder and delusional disorder; (iv) drug use and
dependence such as alcoholism, opiate dependence, cocaine
dependence, amphetamine dependence, hallucinogen dependence, and
phencyclidine use; and (v) memory loss disorders such as amnesia or
memory loss associated with Alzheimer's type dementia or with
non-Alzheimer's type dementia, e.g. multi-infarct dementia or
memory loss associated with Parkinson's disease, Huntington's
disease, Creutzfeld-Jakob disease, head trauma, HIV infection,
hypo-thyroidism and vitamin B12 deficiency.
[0020] In preferred embodiments, the psychiatric disorder is
schizophrenia, an anxiety disorder such as stress or post-traumatic
stress disorder, or a mood disorder such as depression or a bipolar
disorder.
[0021] In the most preferred embodiment, the individual suffering
from a psychiatric disorder, disease or condition is immunized with
Copolymer 1. According to the invention, Copolymer 1 will also
treat cognitive disfunction caused by certain psychiatric disorders
such as schizophrenia, bipolar disorder, age-related dementia and
HIV dementia.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1a-1e show restoration of impaired prepulse inhibition
(PPI) of the acoustic startle response (ASR) in C57BL/6J mice by
Cop-1 immunization. C57BL/6J mice were immunized with Cop-1/CFA or
with PBS/CFA. Naive mice were used as controls. One week later, the
immunized mice were injected with MK-801 (0.1 mg/kg, i.p.; 1b, 1c)
or with D-amphetamine sulfate (AMPH) (2.5 mg/kg i.p.; 1 d and 1 c).
The PPI of the ASR in control mice served as a baseline (1a).
Vehicle-immunized mice injected with MK-801 showed significantly
disrupted PPI (1b). In mice immunized with Cop-1/CFA, PPI was
monotonically increased as a function of prepulse intensity (1c; F
(1,9)=14.05, P<0.005). Vehicle-immunized mice injected with AMPH
also showed significantly disrupted PPI (1d). In mice immunized
with Cop-1/CFA, PPI was monotonically increased as a function of
prepulse intensity (4e; F (1,10)=8.6, P<0.015).
[0023] FIGS. 2a-2f show that cognitive activity is affected by the
integrity of the immune system. BALB/c/OLA wild-type (WT), nude,
and SCID mice were monitored while attempting a spatial learning
memory task in the Morris water maze (MWM) behavioral test. (2a-2c)
Comparison of WT and SCID mice. During the acquisition (2a),
extinction (2b), and reversal (2c) phases of the task, SCID mice
took significantly longer than WT mice to acquire the spatial
learning needed (3-way ANOVA, repeated measures: groups, df (1,20),
F=23.0, P<0.0001; trials, df (3,60), F=10.995, P<0.00001;
days, df (1,60), F=4.6, P<0.006, for the acquisition phase; and
groups, df (1,20), F=7.9, P<0.01; trials, df-(3,60), F=10.77,
P<0.00001; days, df (1,20), F=34.4, P<0.001, for the reversal
phase). The presented results are from one of two experiments
performed, with 10 mice per group in each experiment. (2d-2f)
Comparison of nude mice with nude mice that were replenished with T
cells 3 weeks before being tested on the MWM. During the
acquisition (2d), extinction (2e), and reversal (2f) phases of the
task, nonreplenished nude mice took significantly longer to acquire
spatial learning than nude mice replenished with T cells from naive
wild-type mice (3-way ANOVA, repeated measures: groups, df (1,18),
F=32.3, P<0.00001; trials, df (3,54), F=10.1, P<0.00001;
days, df (3,54), F=20.56, P<0.00001, for the acquisition phase;
and groups, df (1,18), F=58.6, P<0.00001; trials, df (3,54),
F=12.6, P<0.00001; days, df (1,18), F=19.2, P<0.0004, for the
reversal phase). The presented results are from one of two
experiments performed, with 10 mice per group in each
experiment.
[0024] FIGS. 3a-3i show the effect of Cop-1 vaccination on the
performance by C57BL/6J mice of a spatial learning/memory task in
the MWM after injection of a psychotomimetic drug. Acquisition of
the spatial learning task in the MWM took significantly longer in
mice injected with MK-801 (0.1 mg/kg i.p.) or AMPH (2.5 mg/kg i.p.)
than in naive (PBS-injected) C57BL/6J mice in the acquisition (3a)
and the reversal (3b) phases. Immunization with Cop-1/CFA resulted
in decreased escape latencies in acquisition and reversal phases
after injection of [MK-801 (3c, 3d) or amphetamine (3e, 3f). MK-801
(3c, 3d); 3-way ANOVA, repeated measures: groups, df (1,9), F=56.6,
P<0.0001; trials, df (3,27), F=54.0, P<0.00001; days, df
(3,27), F=15.6, P<0.00001, for the acquisition phase; and
groups, df (1,9), F=42.7, P<0.0001; trials, df (3,27), F=24.4,
P<0.00001; days, df (1,9), F=7.9, P<0.02, for the reversal
phase; or AMPH (3e,. 3f); 3-way ANOVA, repeated measures: groups,
df (1,10), F=9.8, P<0.01; trials, df (3,30), F=29.9,
P<0.00001; days, df (1,30), F=21.3, P<0.00001, for the
acquisition phase; and groups, df (1,10), F=53.7, P<0.00003;
trials, df (3,30), F=16.1, P<0.00002; days, df (1,10), F=5.0,
P<0.05 for the reversal phase]. The performance of mice
immunized with Cop-1 did not differ significantly from normal
behavior. (3h) The decrease in time spent in a training quadrant,
obtained in control mice, was abolished upon injection of MK-801
(3g); however, mice immunized with Cop-1 behaved similarly to WT
mice (3i).
[0025] FIG. 4 illustrates tracking of Cop-1-immunized and control
mice in the Morris water maze after injection of MK-801 and shows
that Cop-1 counteracted MK-801-induced impairment of learning and
memory. The figure depicts the swimming strategies of C57BL/6J mice
that were immunized with Cop-1/CFA or PBS/CFA, and injected 1 week
later with MK-801. Naive mice served as controls for performance in
the MWM. On the second day after administration of the drug,
performance was tested in four consecutive trials at 5-min
intervals. The Cop-1-vaccinated mice, like the naive mice, learned
to swim away from the wall to search for the platform in the inner
50% of the pool and to use the platform as a refuge when they found
it. A significantly less efficient strategy was used by
MK-801-injected mice immunized with vehicle.
[0026] FIGS. 5a-5f show strain dependence and T-cell dependence on
the ability to withstand psychological stress. (FIGS. 5a, 5d) Mouse
strains differ in their ability to adapt to psychological stress. A
single 10-min exposure to the odor of a predator caused behavioral
changes in 36.8% of male C57BL/6J mice but in only 10.5% of male
BALB/c mice (x2=3.7, P<0.05). (FIGS. 5b, 5e) In the BALB/c
strain, maladaptation was significantly more prevalent in SCID mice
(61.9%) than in the wild type (17.2%; x2=10.6, P<0.001). (FIGS.
5c, 5f). Maladaptation was similarly more prevalent in nude mice
(devoid of mature T cells only) than in the wild type (70% and
17.2%, respectively; x2 test: P<0.0002), verifying that the
observed differences were attributable to the absence of mature T
cells.
[0027] FIG. 6 shows the ability of mice immunized with Cop-1 to
withstand psychological stress in comparison with PBS-treated mice
(control), after a single 10-min exposure to the odor of a
predator.
[0028] FIGS. 7a-7c show that naturally occurring
CD4+CD25+regulatory T cells suppress the ability to withstand
psychological stress. (FIG. 7a) A single 10-min exposure to the
odor of a predator resulted in maladaptation in 70% of nude male
BALB/c mice (see FIG. 6). The prevalence of maladaptation was
somewhat decreased (50%) in nude mice that were replenished with
normal splenocytes from wild-type BALB/c mice. In nude mice that
were replenished with a splenocyte population depleted of Treg, the
prevalence of maladaptation (20%) was significantly lower than that
of control (non-replenished) nude mice (x2=6.7, P<0.009). (FIG.
7b) The startle response (mean.+-.SD) of nude mice replenished with
splenocytes depleted of Treg was significantly weaker than that of
nude mice replenished with a normal splenocyte population
(P<0.03) or than that of control nude mice (F(df=2,37)=9.2,
P<0.0006). (FIG. 7c) Nude mice replenished with splenocytes
depleted of Treg spent significantly less time exploring the closed
arms of the elevated plus-maze than did nude mice replenished with
a normal splenocyte population (P<0.02) or than control nude
mice (F(df=2,37)=8.7, P<0.0008).
[0029] FIGS. 8a-8c are micrographs showing that the
immunohistochemistry of T cells in the brain is correlated with
adaptation to psychological stress. Maladapted animals from the
group of nude mice replenished with a normal splenocyte population
from wild-type mice and well-adapted animals from the group of nude
mice replenished with splenocytes from wild-type mice depleted of
Treg were killed and their brains were removed, perfused, embedded
in paraffin, and sliced for histology. Brain slices from the
hippocampal area and fimbria of the hippocampus were stained for
myelinated axons with Luxol and counterstained with eosin, or
stained with anti-CD3 antibodies for the presence of T cells and
counterstained with hematoxylin. (FIG. 8a) Brain slices from the
maladapted mice showed no staining for T cells (ii and iv). (FIG.
8b) Brain slices from the well-adapted mice showed T-cell
reactivity in hippocampal areas (ii and iv) corresponding to myelin
reactivity (Luxol-positive areas; i and iii). (FIG. 8c) Wild-type
mice that were not exposed to stress showed, as expected, no T-cell
reactivity in brain slices. The micrographs show representative
results of at least 6 brain slices from each mouse, and from at
least 3 mice in each group.
[0030] FIG. 9 shows that Cop-1 alleviates the suppressive activity
mediated by Treg (CD4.sup.+CD25.sup.+). T cell proliferation was
assayed by incorporation of [.sup.3H]-thymidine into effector T
cells (Teff) co-cultured with Treg. Recorded values are from one
representative experiment out of three and are expressed as
means.+-.SD of 4 replicates.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention relates to a method for treatment of a
psychiatric disorder, disease or condition which comprises
administering to an individual in need of such a treatment an
effective amount of an agent selected from the group consisting of
(i) Copolymer 1, (ii) a Copolymer 1-related peptide, (iii) a
Copolymer 1-related polypeptide, and (iv) T cells treated with (i),
(ii) or (iii).
[0032] In the present invention, we examined whether adaptive
immunity plays a role in higher brain functions, both under normal
conditions and in the abnormal situation generated by
neurotransmitter imbalance. It is shown herein that systemic immune
deficiency resulted in cognitive impairment which could be reversed
by replenishment with T cells. Moreover, vaccination with Cop-1, a
synthetic low-affinity agonist of a wide-range of self-reactive T
cell clones, presumably by boosting the number of T cells capable
of crossreacting with relevant CNS antigens, prevented drug-induced
psychosis and reduced cognitive impaiment.
[0033] The results herein also show that another brain function
critically affected by the integrity of the peripheral adaptive
immune system is cognition. When tested in the MWM (on a task that
generates stress), immune-deficient mice showed impaired cognitive
function, which was prevented by restoration of immune system
integrity. The observed impairment of cognitive activity, which
occurred here when the immune system was intact but the brain was
suffering from an imbalance in neurotransmitters, suggests that the
peripheral immune system can contain the small fluctuations in
neurotransmitter levels that occur daily and enable normal
cognitive performance, but is unable to cope with a pathological
imbalance that causes cognitive impairment. In the latter
situation, therefore, the relevant T cells need to be boosted.
Boosting was achieved in the present invention by vaccination with
Cop-1, which significantly prevented the cognitive impairment
induced by psychotomimetic drugs. Both behavioral and cognitive
abnormalities were observed in our mouse model within 15 min of
administration of MK-801 or AMPA and were counteracted by the
effects of the Cop-1 vaccination given 1 week earlier. Due to the
rapid onset of symptoms that occurs in the experimental paradigm
used here, the mice had to be vaccinated before psychosis was
induced. It should be noted, however, that the vaccination
according to the present invention encompasses not only preventive
use, but also as a remedial therapy for chronic patients, in whom
intervention at any stage of the disease should be beneficial.
[0034] The rapidity of the Cop-1-reactive T cells in counteracting
the psychotic effects of the drugs suggests that T cells already
elicited by the immunization were patrolling the healthy brain. It
was recently shown that Cop-1 vaccination in healthy animals indeed
leads to increased accumulation of T cells in the CNS and local
production of BDNF (Kipnis et al., 2000). It is also possible that,
as a result of the early immunization, a significant number of
Cop-1 -reactive T cells circulate in the blood, and that they home
to the relevant site only when it is under stress, caused for
example by a pathological alteration in the brain level of dopamine
(by injection of AMPH) or of glutamate (by injection of MK-801).
Regardless of whether the effector T cells circulate in the blood
or reside in the brain, the rapidity of the T cell response to
neurotransmitter imbalance testifies to the importance of well
functioning adaptive immunity in the daily maintenance of the brain
(Kipnis and Schwartz, 2002). These results might also explain why,
in the elderly population, who are known to suffer a
disproportionate reduction in immunity and increased metabolic and
neurotransmitter imbalance in the brain, the incidence of dementia
is increased (Wick et al., 2003).
[0035] Psychological trauma, like physical insults to the CNS, can
cause widespread, long-term changes in neurological and
neurohormonal functioning, which seem to be related to
morphological changes (Markowitsch et al., 1998). It seems
reasonable to assume that exacerbation of both the behavioral and
the cognitive manifestations of schizophrenia over time can be
correlated with the neurodegeneration occurring in certain regions
of the brain (Deutsch et al., 2001). The present finding that
Cop-1-reactive T cells in mice mediated the prevention of
MK-801-induced or amphetamine-induced psychoses, which mimic in
part symptoms of schizophrenia in humans, coupled with the
increasing recognition that neurodegeneration plays a role in
schizophrenia and other mental diseases, suggests that the
development of immune-based neuroprotection might provide a global
remedy that addresses both positive and negative symptoms of
schizophrenia and possibly also of other psychiatric conditions,
including age-related and HIV-related dementias.
[0036] It is shown herein that a cross-talk between the brain and
the adaptive immune system (T cells) affects the consequences of a
single instance of psychological trauma. Complete T cell deficiency
was found here to correlate with maladaptation to psychological
stress, whereas removal of only a subpopulation of T cells, the
naturally occurring regulatory T cells (Treg), improved the ability
to adapt to the stress. This indicates that in normal animals
subjected to traumatic mental stress, the T cell-mediated response
cannot reach its full therapeutic potential, as it is suppressed by
the presence of the naturally occurring regulatory T cells.
Down-regulation of Treg by Cop-1, as shown herein, is beneficial
and can improve an individual's ability to withstand and cope with
stressful conditions. Our present results indeed show that boosting
of a T cell response to the self-like antigen Cop-1 significantly
reduced cognitive impairment and eased psychosis in animal models
with schizophrenia-associated symptoms. Both behavioral and
cognitive abnormalities were observed in our mouse model within 15
minutes of administration of the psychotomimetic drugs MK-801 and
AMPH, and were counteracted by the effects of the Cop-1 vaccination
given one week earlier. The rapidity of the Cop-1-reactive T cells
in counteracting the psychotic effects of the drugs suggests that T
cells has already been elicited by the immunization, and were
patrolling the healthy brain in a non-activated state, though on
alert and ready for action if needed. Alternatively, it is possible
that as a result of the immunization a significant number of
Cop-1-reactive T cells, as yet unactivated, were circulating in the
blood, ready to home to the site of the threat when needed there. A
pathological alteration in the brain level of dopamine (by
injection of amphetamine) or of glutamate (by injection of MK-801)
might activate these T cells, allowing rapid protection against the
devastating effects of the neurotransmitter imbalance. The observed
effect of Cop-1 on the alleviation of psychosis induced by
psychomimetic drugs indicates that it is a suitable candidate of
choice for the development of a new generation of anti-psychotic
drugs.
[0037] Glutamatergic imbalance is a feature common to
neurodegenerative and mental disorders. After injury to the CNS, T
cells directed to CNS-related self-antigens participate in CNS
maintenance, which includes protection against glutamate toxicity.
This neuroprotective effect can be boosted by agonists of
self-antigens, such as Cop-1. According to the present invention,
it is shown that a similar T cell-dependent mechanism is protective
against neurotransmitter imbalance in the brain, leading to
behavioral and cognitive malfunction. Vaccination with Cop-1
protected mice from psychotic behavior and cognitive impairment
induced by MK-801 or amphetamine (simulating symptoms of
schizophrenia). It is also shown herein that Cop-1-reactive T
cells, upon encountering relevant self-antigens (MBP), produced
brain-derived neurotrophic factor (BDNF), a neurotrophin known to
be protective in schizophrenic brain, to be linked to bipolar
disorder development and connected with nerve activity, memory
function and mood.
[0038] As used herein, the terms "Cop-1" and "Copolymer 1" are used
interchangeably. For the purpose of the present invention,
"Copolymer 1-related peptide or Copolymer 1-related polypeptide" is
intended to include any peptide or polypeptide, including a random
copolymer that cross-reacts functionally with myelin basic protein
(MBP) and is able to compete with MBP on the MHC class II in the
antigen presentation.
[0039] The composition or vaccine of the invention may comprise as
active agent a random copolymer comprising a suitable quantity of a
positively charged amino acid such as lysine or arginine, in
combination with a negatively charged amino acid (preferably in a
lesser quantity) such as glutamic acid or aspartic acid, optionally
in combination with a non-charged neutral amino acid such as
alanine or glycine, serving as a filler, and optionally with an
amino acid adapted to confer on the copolymer immunogenic
properties, such as an aromatic amino acid like tyrosine or
tryptophan. Such compositions may include any of those copolymers
disclosed in WO 00/05250, the entire contents of which being hereby
incorporated herein by reference.
[0040] More specifically, the composition for use in the present
invention comprises at least one copolymer selected from the group
consisting of random copolymers comprising one amino acid selected
from each of at least three of the following groups: (a) lysine and
arginine; (b) glutamic acid and aspartic acid; (c) alanine and
glycine; and (d) tyrosine and tryptophan.
[0041] The copolymers for use in the present invention can be
composed of L- or D-amino acids or mixtures thereof. As is known by
those of skill in the art, L-amino acids occur in most natural
proteins. However, D-amino acids are commercially available and can
be substituted for some or all of the amino acids used to make the
terpolymers and other copolymers used in the present invention. The
present invention contemplates the use of copolymers containing
both D- and L-amino acids, as well as copolymers consisting
essentially of either L- or D-amino acids.
[0042] In a more preferred embodiment, the pharmaceutical
composition or vaccine of the invention comprises Copolymer 1, a
mixture of random polypeptides consisting essentially of the amino
acids L-glutamic acid (E), L-alanine (A), L-tyrosine (Y) and
L-lysine (K) in an approximate ratio of 1.5:4.8:1:3.6, having a net
overall positive electrical charge and of a molecular weight from
about 2 KDa to about 40 KDa. In one preferred embodiment, the Cop 1
has average molecular weight of about 2 KDa to about 20 KDa, more
preferably of about 4,7 KDa to about 13 KDa, still more preferably
of about 4 KDa to about 8.6 KDa, of about 5 KDa to 9 KDa, or of
about 6.25 KDa to 8.4 KDa. In another preferred embodiment, the Cop
1 has average molecular weight of about 13 KDa to about 20 KDa,
more preferably of about 13 KDa to about 16 KDa or of about 15 KDa
to about 16 KDa. Other average molecular weights for Cop 1, lower
than 40 KDa, are also encompassed by the present invention.
Copolymer 1 of said molecular weight ranges can be prepared by
methods known in the art, for example by the processes described in
U.S. Pat. No. 5,800,808, the entire contents of which are hereby
incorporated by reference in the entirety. The Copolymer 1 may be a
polypeptide comprising from about 15 to about 100, preferably from
about 40 to about 80, amino acids in length. In one preferred
embodiment, the Cop 1 is in the form of its acetate salt known
under the generic name glatiramer acetate, that has been approved
in several countries for the treatment of multiple sclerosis (MS)
under the trade name, Copaxone.RTM. (a trademark of Teva
Pharmaceuticals Ltd., Petach Tikva, Israel). The activity of
Copolymer 1 for the vaccine disclosed herein is expected to remain
if one or more of the following substitutions is made: aspartic
acid for glutamic acid, glycine for alanine, arginine for lysine,
and tryptophan for tyrosine.
[0043] In another embodiment of the invention, the Cop 1-related
peptide or polypeptide is a copolymer of three different amino
acids each from a different one of three groups of the groups (a)
to (d). These copolymers are herein referred to as terpolymers.
[0044] In one embodiment, the Cop 1-related peptide or polypeptide
is a terpolymer containing tyrosine, alanine, and lysine,
hereinafter designated YAK, in which the average molar fraction of
the amino acids can vary: tyrosine can be present in a mole
fraction of about 0.05-0.250; alanine in a mole fraction of about
0.3 -0.6; and lysine in a mole fraction of about 0.1-0.5. More
preferably, the molar ratios of tyrosine, alanine and lysine are
about 0.10:0.54:0.35, respectively. It is possible to substitute
arginine for lysine, glycine for alanine, and/or tryptophan for
tyrosine.
[0045] In another embodiment, the Cop 1-related peptide or
polypeptide is a terpolymer containing tyrosine, glutamic acid, and
lysine, hereinafter designated YEK, in which the average molar
fraction of the amino acids can vary: glutamic acid can be present
in a mole fraction of about 0.005-0.300, tyrosine can be present in
a mole fraction of about 0.005-0.250, and lysine can be present in
a mole fraction of about 0.3-0.7. More preferably, the molar ratios
of glutamic acid, tyrosine, and lysine are about 0.26:0.16:0.58,
respectively. It is possible to substitute aspartic acid for
glutamic acid, arginine for lysine, and/or tryptophan for
tyrosine.
[0046] In another preferred embodiment, the Cop 1-related peptide
or polypeptide is a terpolymer containing lysine, glutamic acid,
and alanine, hereinafter designated
[0047] KEA, in which the average molar fraction of the amino acids
can vary: glutamic acid can be present in a mole fraction of about
0.005-0.300, alanine in a mole fraction of about 0.005-0.600, and
lysine can be present in a mole fraction of about 0.2-0.7. More
preferably, the molar ratios of glutamic acid, alanine and lysine
are about 0.15:0.48:0.36, respectively. It is possible to
substitute aspartic acid for glutamic acid, glycine for alanine,
and/or arginine for lysine.
[0048] In a preferred embodiment, the Cop 1-related peptide or
polypeptide is a terpolymer containing tyrosine, glutamic acid, and
alanine, hereinafter designated YEA, in which the average molar
fraction of the amino acids can vary: tyrosine can be present in a
mole fraction of about 0.005-0.250, glutamic acid in a mole
fraction of about 0.005-0.300, and alanine in a mole fraction of
about 0.005-0.800. More preferably, the molar ratios of glutamic
acid, alanine, and tyrosine are about 0.21: 0.65:0.14,
respectively. It is possible to substitute tryptophan for tyrosine,
aspartic acid for glutamic acid, and/or glycine for alanine.
[0049] The average molecular weight of the terpolymers YAK, YEK,
KEA and YEA can vary between about 2 KDa to 40 KDa, preferably
between about 3 KDa to 35 KDa, more preferably between about 5 KDa
to 25 KDa.
[0050] Copolymer 1 and related peptides and polypeptides may be
prepared by methods known in the art, for example, under
condensation conditions using the desired molar ratio of amino
acids in solution, or by solid phase synthetic procedures.
Condensation conditions include the proper temperature, pH, and
solvent conditions for condensing the carboxyl group of one amino
acid with the amino group of another amino acid to form a peptide
bond. Condensing agents, for example dicyclohexylcarbodiimide, can
be used to facilitate the formation of the peptide bond. Blocking
groups can be used to protect functional groups, such as the side
chain moieties and some of the amino or carboxyl groups against
undesired side reactions.
[0051] For example, the copolymers can be prepared by the process
disclosed in U.S. Pat. No. 3,849,550, wherein the
N-carboxyanhydrides of tyrosine, alanine, .gamma.-benzyl glutamate
and N .epsilon.-trifluoroacetyl-lysine are polymerized at ambient
temperatures (20.degree. C.-26.degree. C.) in anhydrous dioxane
with diethylamine as an initiator. The .gamma.-carboxyl group of
the glutamic acid can be deblocked by hydrogen bromide in glacial
acetic acid. The trifluoroacetyl groups are removed from lysine by
1M piperidine. One of skill in the art readily understands that the
process can be adjusted to make peptides and polypeptides
containing the desired amino acids, that is, three of the four
amino acids in Copolymer 1, by selectively eliminating the
reactions that relate to any one of glutamic acid, alanine,
tyrosine, or lysine.
[0052] The molecular weight of the copolymers can be adjusted
during polypeptide synthesis or after the copolymers have been
made. To adjust the molecular weight during polypeptide synthesis,
the synthetic conditions or the amounts of amino acids are adjusted
so that synthesis stops when the polypeptide reaches the
approximate length which is desired. After synthesis, polypeptides
with the desired molecular weight can be obtained by any available
size selection procedure, such as chromatography of the
polypeptides on a molecular weight sizing column or gel, and
collection of the molecular weight ranges desired. The copolymers
can also be partially hydrolyzed to remove high molecular weight
species, for example, by acid or enzymatic hydrolysis, and then
purified to remove the acid or enzymes.
[0053] In one embodiment, the copolymers with a desired molecular
weight may be prepared by a process, which includes reacting a
protected polypeptide with hydrobromic acid to form a
trifluoroacetyl-polypeptide having the desired molecular weight
profile. The reaction is performed for a time and at a temperature
which is predetermined by one or more test reactions. During the
test reaction, the time and temperature are varied and the
molecular weight range of a given batch of test polypeptides is
determined. The test conditions which provide the optimal molecular
weight range for that batch of polypeptides are used for the batch.
Thus, a trifluoroacetyl-polypeptide having the desired molecular
weight profile can be produced by a process, which includes
reacting the protected polypeptide with hydrobromic acid for a time
and at a temperature predetermined by test reaction. The
trifluoroacetyl-polypeptide with the desired molecular weight
profile is then further treated with an aqueous piperidine solution
to form a low toxicity polypeptide having the desired molecular
weight.
[0054] In a preferred embodiment, a test sample of protected
polypeptide from a given batch is reacted with hydrobromic acid for
about 10-50 hours at a temperature of about 20-28.degree. C. The
best conditions for that batch are determined by running several
test reactions. For example, in one embodiment, the protected
polypeptide is reacted with hydrobromic acid for about 17 hours at
a temperature of about 26.degree. C.
[0055] As binding motifs of Cop 1 to MS-associated HLA-DR molecules
are known (Fridkis-Hareli et al, 1999), polypeptides derived from
Cop 1 having a defined sequence can readily be prepared and tested
for binding to the peptide binding groove of the HLA-DR molecules
as described in the Fridkis-Hareli et al (1999) publication.
Examples of such peptides are those disclosed in WO 00/05249 and WO
00/05250, the entire contents of which are hereby incorporated
herein by reference, and include the peptides of SEQ ID NOs. 1-32
hereinbelow.
TABLE-US-00001 TABLE 1 SEQ Peptide ID NO. Sequence 1
AAAYAAAAAAKAAAA 2 AEKYAAAAAAKAAAA 3 AKEYAAAAAAKAAAA 4
AKKYAAAAAAKAAAA 5 AEAYAAAAAAKAAAA 6 KEAYAAAAAAKAAAA 7
AEEYAAAAAAKAAAA 8 AAEYAAAAAAKAAAA 9 EKAYAAAAAAKAAAA 10
AAKYEAAAAAKAAAA 11 AAKYAEAAAAKAAAA 12 EAAYAAAAAAKAAAA 13
EKKYAAAAAAKAAAA 14 EAKYAAAAAAKAAAA 15 AEKYAAAAAAAAAAA 16
AKEYAAAAAAAAAAA 17 AKKYEAAAAAAAAAA 18 AKKYAEAAAAAAAAA 19
AEAYKAAAAAAAAAA 20 KEAYAAAAAAAAAAA 21 AEEYKAAAAAAAAAA 22
AAEYKAAAAAAAAAA 23 EKAYAAAAAAAAAAA 24 AAKYEAAAAAAAAAA 25
AAKYAEAAAAAAAAA 26 EKKYAAAAAAAAAAA 27 EAKYAAAAAAAAAAA 28
AEYAKAAAAAAAAAA 29 AEKAYAAAAAAAAAA 30 EKYAAAAAAAAAAAA 31
AYKAEAAAAAAAAAA 32 AKYAEAAAAAAAAAA
[0056] Such peptides and other similar peptides derived from Cop 1
would be expected to have similar activity as Cop 1. Such peptides,
and other similar peptides, are also considered to be within the
definition of Cop 1-related peptides or polypeptides and their use
is considered to be part of the present invention.
[0057] The definition of "Cop 1-related peptide or polypeptide"
according to the invention is meant to encompass other synthetic
amino acid copolymers such as the random four-amino acid copolymers
described by Fridkis-Hareli et al., 2002 (as candidates for
treatment of multiple sclerosis), namely copolymers (14-, 35- and
50-mers) containing the amino acids phenylalanine, glutamic acid,
alanine and lysine (poly FEAK), or tyrosine, phenylalanine, alanine
and lysine (poly YFAK), and any other similar copolymer to be
discovered that can be considered a universal antigen similar to
Cop 1.
[0058] In another embodiment, the present invention relates to the
treatment of a psychiatric disease, disorder or condition which
comprises administering to an individual in need T cells that have
been activated preferably in the presence of Cop 1, or by a Cop
1-related peptide or polypeptide. Such T cells are preferably
autologous, most preferably of the CD4 and/or CD8 phenotypes, but
they may also be allogeneic T cells from related donors, e.g.,
siblings, parents, children, or HLA-matched or partially matched,
semi-allogeneic or fully allogeneic donors. T cells for this
purpose are described in U.S. Ser. No. 09/756,301 and U.S. Ser. No.
09/765,644, corresponding to WO 01/93893, each and all of them
hereby incorporated by reference in its entirety as if fully
disclosed herein.
[0059] The dosage of Cop 1 to be administered will be determined by
the physician according to the age of the patient and stage of the
disease and may be chosen from a range of 1-80 mg, preferably 20
mg, although any other suitable dosage is encompassed by the
invention. The treatment should be preferably carried out by
administration of repeated doses at suitable time intervals,
preferably every 1, 4 or 6 weeks, but any other suitable interval
between the immunizations is envisaged by the invention according
to the psychiatric disease to be treated, the age and condition of
the patient.
[0060] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients. The
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the composition and not deleterious
to the recipient thereof.
[0061] For the purposes of the present invention, the composition
comprising Copolymer 1 or a Copolymer 1-related peptide or
polypeptide is administered in a regimen that confers protective
autoimmunity (also sometimes referred to as a vaccine for
neuroprotective vaccination). Such a vaccine, if desired, may
contain Copolymer 1 emulsified in an adjuvant suitable for human
clinical use.
[0062] Thus, according to the present agent, the active agent may
be administered without any adjuvant or it may be emulsified in an
adjuvant suitable for human clinical use. The adjuvant is selected
from aluminum hydroxide, aluminum hydroxide gel, and aluminum
hydroxyphosphate, or any other adjuvant that is found to be
suitable for human clinical use. In a preferred embodiment, the
vaccine adjuvant is amorphous aluminum hydroxyphosphate having an
acidic isoelectric point and an Al:P ratio of 1:1 (herein referred
to as Alum-phos). It is clear that this is given by way of example
only, and that the vaccine can be varied both with respect to the
constituents and relative proportions of the constituents. Methods
of administration include, but are not limited to, parenteral,
e.g., intravenous, intraperitoneal, intramuscular, subcutaneous,
mucosal (e.g., oral, intranasal, buccal, vaginal, rectal,
intraocular), intrathecal, topical and intradermal routes.
Administration can be systemic or local.
[0063] According to the present invention, Cop 1 or a Cop 1-related
peptide or polypeptide may be used as a sole therapy or in
combination with one or more drugs for the treatment of the
psychiatric disorder, disease or condition. When administered
together with another drug or drugs suitable for treatment of the
psychiatric disorder, disease or condition, the additional drug or
drugs is/are administered at the same day of vaccination, and daily
or at any other interval thereafter, according to the
manufacturer's instructions, with no association to the vaccine
regimen.
[0064] The invention will now be illustrated by the following
non-limiting examples.
EXAMPLES
Materials and Methods
[0065] (i) Animals. Inbred adult male wild-type and nu/nu BALB/c
and C57B1/6J mice, adult female Lewis rats and BALB/c/OLA mice, as
well as BALB/c/OLA mice with severe combined immune deficiency
(SCID) (due to RAG1/2 knockout) and nude mice (deficient in mature
T cells), all 8-12 weeks old, were supplied by the Animal Breeding
Center of The Weizmann Institute of Science (Rehovot, Israel). The
animals were housed in a light- and temperature-controlled room and
matched for age in each experiment. Animals were handled according
to the regulations formulated by IACUC (Institutional Animal Care
and Use Committee).
[0066] (ii) Antigens. Copolymer 1 (Cop-1) was purchased from Teva
Pharmaceuticals Ltd. (Petach Tikva, Israel).
[0067] (iii) Immunization. Each animal was injected with a total of
100 .mu.g of Cop-1 emulsified in an equal volume of complete
Freund's adjuvant (CFA) containing 5 mg/ml of Mycobacteria H37 RA
(Difco). The emulsion, in a total volume of 0.1 mL, was injected
into the flank 1 week before the mouse was first injected with a
psychotomimetic drug. Control mice were injected with an equal
volume of phosphate-buffered saline (PBS) emulsified in CFA.
[0068] (iv) T-cell lines. Ten days after Cop-1 immunization, T-cell
lines were generated from draining lymph node cells obtained from
Lewis rats immunized with Cop-1 emulsified in CFA. The lymph nodes
were surgically removed, dissociated, washed, and then activated
with the antigen (10 .mu.g/ml) in stimulation medium as previously
reported (Kipnis et al., 2000). The T-cell lines were expanded by
repeated stimulation and propagation.
[0069] (v) Enzyme-linked immunosorbent assay. T cell-reactive Cop-1
cells were grown for 1 week in a propagation medium, then washed
with PBS and re-suspended in stimulation medium. The cultured T
cells (0.5.times.10.sup.6 cells/mL) were then incubated, in the
presence of irradiated thymocytes (10.sup.7 cells/mL), with
concanavalin A (Con A; 1.25 .mu.g/mL), myelin basic protein (MBP;
10 .mu.g/ml), Cop-1 (10 .mu.g/mL), ovalbumin (OVA; 10 .mu.g/mL), or
no antigen, in stimulation medium. After 48 h the cells were
centrifuged and their supernatants were collected and sampled.
Concentrations of brain-derived neurotrophic factor (BDNF) in the
samples were determined with a sensitive sandwich ELISA. In brief,
96-well flat-bottomed plates were coated with a chicken anti-human
BDNF antibody (Promega, Madison, WI) in 0.025 M NaHCO.sub.3 and
0.025 M Na.sub.2CO.sub.3 (pH 8.2). Recombinant human BDNF (used as
a standard; Research Diagnostics, Flanders, N.J.) was used in
serial dilutions in blocking solution containing 3% bovine serum
albumin, 0.05% polyoxyethylene-sorbitan monolaurate (Tween-20), and
1% fetal calf serum in PBS (pH 8.2). Bound BDNF was detected by
incubating the plates with a mouse anti-human BDNF antibody
(Research Diagnostics) and then with peroxidase-conjugated goat
anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.) in
blocking solution. The plates were developed using a
3,3',5,5'-tetramethyl-benzidine liquid substrate system
(Sigma-Aldrich). The reaction was stopped by adding 1M
H.sub.3PO.sub.4, and the optical density was determined at 450 nm.
Results for each experiment were calculated as the amount of
secreted BDNF per 1 mL of sample, after subtraction of background
levels of the irradiated thymocytes incubated with the stimulation
medium.
[0070] (vi) Drug solutions. Fresh solutions of dizocilpine maleate
(MK-801; Sigma-Aldrich) were prepared in physiological saline (0.9%
NaCl in sterile distilled water) for each batch of mice.
Physiological saline was also used as a vehicle for D-amphetamine
sulfate (AMPH; Sigma). MK-801 (0.1 mg/kg) or AMPH (2.5 mg/kg) or
saline was injected i.p. in a total volume of 5 ml per kg of body
weight. Mice were injected with MK-801, AMPH, or vehicle 15 min
before being subjected to behavioral tests.
[0071] (vii) Morris water maze (MWM) behavioral test. Spatial
learning/memory was assessed by performance on a
hippocampal-dependent visuospatial learning task in the MWM. Mice
were given four trials per day, for four consecutive days, to find
a hidden platform located 1.5 cm below the water surface in a pool
1.4 m in diameter. Within the testing room only distal
visuo-spatial cues were available to the mice for location of the
submerged platform. The escape latency, i.e., the time required by
the mouse to find and climb onto the platform, was recorded for up
to 60 s. Each mouse was allowed to remain on the platform for 30 s,
and was then moved from the maze to its home cage. If the mouse did
not find the platform within 120 s, it was manually placed on the
platform and returned to its home cage after 30 s. The inter-trial
interval was 30 s. On day 5, the platform was removed from the
pool, and each mouse was tested by a probe trial for 60 s. On days
6-7 the platform was placed at the opposite location, and the mouse
was retrained in four sessions. Data were recorded by using an
EthoVision automated tracking system (Noldus Information
Technology, Wageningen, The Netherlands).
[0072] (viii) Prepulse inhibition (PPI). All sessions for testing
of PPI consisted of startle trials (pulse-alone), prepulse trials
(prepulse plus pulse), and no-stimulus trials (no-stim). The
pulse-alone trial consisted of a 40-ms, 120-dB pulse of broadband
noise. Acoustic PPI was measured by prepulse plus pulse trials
consisting of a 20-ms prepulse, 100 ms delay, and then a 40-ms,
120-dB startle pulse. The onset-to-onset interval was 120 ms.
Acoustic prepulse intensities were 4, 8, 13, and 16 dB above the
65-dB background noise (i.e., 69, 73, 78, and 81 dB).
[0073] The no-stim trial consisted of background noise only. The
acoustic section of the test session began and ended with five
presentations of the pulse-alone trial; in between, each acoustic
or no-stim trial type was presented 10 times in a pseudorandom
order. The average time between trials was 15 s (range: 12-30 s).
After the mice were placed in the startle chambers, a 65-dB
background noise was presented for a 5-min period of acclimation,
and then throughout the test session.
[0074] PPI was calculated as a percentage score for each acoustic
prepulse trial type: % PPI=100-{[(startle response for
prepulse+pulse)/(startle response for pulse-alone)].times.100}. The
magnitude of the acoustic startle response was calculated as the
average response to all of the pulse-alone trials, excluding the
first and last blocks of five pulse-alone trials each. For brevity,
the main effects of prepulse intensity (which were always
significant) are not discussed here. Data from the nostim trials
are not included in the examples because the values obtained were
negligible relative to values from trials containing startle
stimuli.
[0075] (ix) Induction of psychological stress. Mice in the
experimental groups were exposed for 10 minutes to thoroughly
soiled cat litter (used by a cat for 2 days and sifted for faeces).
Control (WT) mice were exposed for the same time to unused
litter.
Behavioral Testing:
[0076] (x) Elevated plus-maze (EPM). The maze we used was a black
opaque perspex platform with four arms in the shape of a plus,
elevated 78 cm above the ground, as described by File (Griebel et
al., 1995). Each arm was 24 cm long and 7.5 cm wide. One pair of
opposite arms was "closed", i.e., the arms were enclosed by
20.5-cm-high perspex walls on both sides and on the outer edges of
the platform, and the other pair was "open", surrounded only by a
3-mm-high perspex lip, which served as a tactile guide for animals
in the open areas. The apparatus was illuminated by dim red
lighting that provided 40-60 lux in both the open and the closed
arms. Mice were placed one at a time in the central platform,
facing towards different arms on different days in randomized
order. Between each test session the maze was cleaned with an
aqueous solution of 5% ethanol and dried thoroughly.
[0077] Behavior on the EPM was recorded using EthoVision programs
(Noldus) that recorded the location of the mouse over the 5-minute
test period. To ensure that the software provided accurate
monitoring of the various parameters selected for analysis,
videotaped replay of the behavior of randomly chosen mice was
scrutinized by an experienced observer.
[0078] Five behavioral parameters were assessed: (1) time spent in
the open arms; (2) time spent in the closed arms; (3) number of
entries into open arms; (4) number of entries into closed arms; (5)
total number of entries into all arms. Mice were recorded as having
entered an open or closed arm only when all four paws had passed
over the dividing line between open and closed arms. The number of
entries into any arm of the maze (total arm entries) was defined as
`exploration activity`.
[0079] (xi) Acoustic startle response (ASR). Pairs of mice were
tested in startle chambers. The ASR and pre-pulse inhibition were
measured using two ventilated startle chambers (SR-LAB system, San
Diego Instruments, San Diego, Calif.). Each chamber consists of a
Plexiglas cylinder resting on a platform inside a ventilated
sound-attenuated chamber. A high-frequency loudspeaker inside the
chamber produces both a continuous broad-band background noise of
68 dB and different acoustic stimuli. Movement inside the tube is
detected by a piezoelectric accelerometer located below the frame.
The amplitude of the ASR of the whole body to an acoustic pulse was
defined as the average of 100 accelerometer readings, 100 ms each,
collected from pulse onset. These readings (signals) were digitized
and stored in a computer. Sound levels within each test chamber are
routinely measured using a sound-level meter (Radio Shack, San
Diego Instruments) to ensure consistent presentation. An SR-LAB
calibration unit was used routinely to ensure consistency of the
stabilimeter sensitivity between test chambers and over time
(Swerdlow and Geyer, 1998). The mice were placed inside the tube,
and the startle session started with a 5-minute period of
acclimatization to the background noise level of 68 dB, which was
maintained throughout the session.
[0080] (xii) Study design for determination of "cut-off behavioral
criteria" (CBC).
[0081] The studies were designed in two steps:
[0082] Step I--Prior to attempting to distinguish the
differentially affected sub-groups, we routinely perform a
preliminary assessment of the overall response of the exposed
population intended to ascertain the accuracy of our
zero-hypothesis, i.e. to demonstrate that exposure to the stressor
did in fact have significant overall behavioral effects on the
exposed animals as a group compared to controls, in each of our
studies. The data are also ascertained to demonstrate a range of
varying degrees of behavioral changes.
[0083] Behavioral changes, such as extremely compromised
exploratory behavior on the plus maze and markedly increased
startle reaction that does not undergo any adaptation reflect
anxiety-like behaviors, i.e. fearfulness and hypervigilance. In
keeping with the work of Blanchard and Blanchard (Blanchard et al.,
1990; Blanchard et al., 1993; Blanchard et al., 1998), Adamec
(Adamec et al., 1998; Adamec et al., 1999a) and Cohen (Cohen et
al., 1996; Cohen et al., 2000; Cohen et al., 2003) the observed
behaviors at this time-point are considered to reflect relatively
long-term and persistent changes. Since it has as yet not been
possible to design an animal model for the intrusive cluster of
symptoms, changes such as these, which persist over the space of a
week or more, are considered to represent a fair representation of
PTSD-like symptoms in terms of animal models.
[0084] Step II--The CBC applied to exposed animals:
[0085] Having established that the stressor had an effect on the
animals and that not all animals responded to it in the same
manner, we focus only on animals that demonstrate extremes of
behavioral change on the one hand or virtually no change on the
other.
[0086] In order to maximize the clarity of which animals to define
as "affected", and so to minimize the chance of including "false
positives", we define the behavioral cut-off criteria to represent
the most extreme degree of behavioral disturbance in each of two
consecutive behavioral paradigms. In order to be defined as
"affected", the individual animal has to have conformed to both
sets of criteria, consecutively. Conversely, in order to be
considered to have responded hardly at all, animals must conform to
equally extreme criteria for "near normal" behaviors. The validity
of the criteria are re-affirmed in each study by ascertaining that
the vast majority of unexposed control animals conform to the
latter and none, or almost none, to the former. The CBCs determined
as above, were as follows: (a) Maladapted: 1) Five minutes spent in
closed arms and zero (0) entries into the open-arms; 2) Mean
amplitude of the startle response (at 110 Db)>800 units and
nonhabituation of the acoustic startle response; (b) Well-Adapted:
1) 0-1 minutes spent in closed arms and .gtoreq.8 open-arm entries;
2) Mean amplitude of the startle response (at 110 Db)<600 units
and normal habituation of the acoustic startle response.
[0087] (xiii) Supplemental Information. The behavioral outcome in
mice exposed to the odor of a predator has been used as a model for
PTSD (Adamec et al., 1999b; Cohen et al., 2003). Brief, escapable
exposure of mice or rats to a cat or cat odor increases the
defensive behaviors observed in a visible burrow system for many
hours after removal of the threat (Rodgers et al., 1990). The
long-lasting behavior abnormality is being viewed as maladaptation
to the predator stress (i.e. PTSD). The stressor and the time scale
used in the present study might justify view the results as
relevance to PTSD according to the following criteria (Yehuda and
Antelman, 1993): (a) The stressor is strong and transient and
provides a more natural setting than that offered by other types of
stressors, such as electrical shocks to the tail (Adamec et al.,
1997). (b) The observed reduction in time spent by the stressed
mice in the open arms of the EPM is reminiscent of the avoidance
behavior seen in patients with PTSD. Stressed mice did not show
fewer total entries in the EPM than unstressed mice. This finding
is consistent with anxiety rather than with nonspecific impairment
of locomotion. The DSM-IV defines this symptom as the persistent
avoidance of reminders of the trauma and the numbing of
responsiveness.
[0088] Since the traumatic event in these mice took place in an
open space, it is in line with this definition. (c) Seven days in
the life of a mouse that normally lives for 3 years is roughly
equivalent to 6 months in a human life span of 72 years (Adamec et
al., 1997). It thus seems that our assessment of mice 7 days after
the trauma indeed points to PTSD rather than to an acute stress
reaction.
[0089] (xiv) Antibodies and reagents. Mouse recombinant IL-2
(mrIL-2) and anti mouse .zeta.-CD3 (anti-CD3; clone 145-2C11) were
purchased from R&D Systems (Minneapolis, Minn.). Rat anti-mouse
phycoerythrin (PE)-conjugated CD25 antibody (PC61) was purchased
from Pharmingen (Becton-Dickinson, Franklin Lakes, N.J.).
[0090] (xv) Histology and immunohistochemistry of paraffin-embedded
brain sections. Paraffin-embedded brain tissues from maladapted
nude mice replenished with a normal population of wild-type
splenocytes, or from well-adapted nude mice replenished with wild
type splenocytes depleted of Treg cells, were cut into 4
.mu.m-thick coronal sections, deparaffinized with xylene, and
dehydrated with a graded series of ethanol solutions. The sections
were then stained with Luxol fast blue (Sigma-Aldrich, Israel) and
counterstained with Fast Red (Sigma, Israel). For
immunohistochemical analyses, deparaffinized and dehydrated
sections were immersed (30 min) in methanol containing 3%
H.sub.2O.sub.2 and 1% of concentrated HC1 to block endogenous
peroxidase activity, treated (1 h) with phosphate-buffered saline
(PBS), pH 7.4, containing 20% normal rabbit serum and 0.3% Triton
X-100, and incubated overnight at room temperature with anti-CD3
antibodies (Serotec, Oxford, UK; diluted 1:50 in PBS containing 2%
normal rabbit serum). The sections were washed with PBS and
incubated for 30 min, first with biotinylated anti-rabbit IgG and
then with avidin-biotinylated peroxidase complex (Vector
Laboratories, Burlingame, Calif.). Peroxidase activity in a
solution of 3,3'-diaminobenzidine was visualized by light
microscopy.
[0091] (xvi) Preparation of splenocytes. Donor splenocytes from
rats (aged up to 10 weeks) were obtained by rupturing the spleen
and following conventional procedures. The splenocytes were washed
with hypotonic buffer (ACK) to lyse red blood cells.
[0092] (xvii) Preparation of lymphocytes. Donor mice lymph nodes
(axillary, inguinal, superficial cervical, mandibular, and
mesenteric) were ruptured through mesh. The lymphocytes were washed
with hypotonic buffer (ACK) to lyse red blood cells.
[0093] (xviii) Purification of CD4.sup.+ CD25.sup.+ and CD4.sup.+
CD25.sup.- T cells. Lymph nodes were harvested and mashed. T cells
were enriched by negative selection and purified on CD3-cell
columns (MTCC-25; R&D Systems). The enriched T cells were
incubated with anti-CD8 microbeads (Miltenyi Biotec, Bergisch
Gladbach, Germany), and negatively selected CD4.sup.+ T cells were
incubated with PE-conjugated anti-CD25 (30 .mu.g/10.sup.8 cells) in
PBS/2% fetal calf serum. They were then washed and incubated with
anti-PE microbeads (Miltenyi Biotec) and subjected to magnetic
separation with AutoMACS (Miltenyi Biotec). The retained cells were
eluted from the column as purified CD4.sup.+ CD25.sup.+ cells. The
negative fraction consisted of CD4.sup.+CD25.sup.- T cells. Cell
purity was checked by FACSort (Becton-Dickinson) and typically
ranged from 88% to 95%. Purified cells were cultured in 24-well
plates (1 ml).
[0094] (xix) Activation of CD4.sup.+CD25.sup.+ regulatory T cells.
Purified regulatory T cells (Treg; 0.5.times.10.sup.6/ml) were
activated in RPMI medium supplemented with L-glutamine (2 mM),
2-mercaptoethanol (5.times.10.sup.-5 M), sodium pyruvate (1 mM),
penicillin (100 IU/ml), streptomycin (100 .mu.g/ml), non-essential
amino acids (1 ml/100 ml), and autologous serum 2% (vol/vol) in the
presence of mrIL-2 (5 ng/ml) and soluble anti-CD3 antibodies (1
ng/ml). Irradiated (2500 rad) splenocytes (1.5.times.10.sup.6/ml)
were added to the culture. Cells were activated for 24 or 96
hr.
[0095] (xx) Inhibition assay (co-culturing of Teff with Treg).
Naive effector T cells (Teff; 50.times.10.sup.3/well) were
co-cultured with decreasing numbers of activated Treg for 72 h in
96-well flat-bottomed plates in the presence of irradiated
splenocytes (10.sup.6/ml) supplemented with anti-CD3 antibodies.
[.sup.3H]-thymidine (1 .mu.Ci) was added for the last 16 hr of
culture. After the cells were harvested, their analyzed
[.sup.3H]-thymidine content was analyzed by the use of a gamma
counter.
Example 1
[0096] Vaccination with Cop-1 has an Anti-Psychotic Effect and
Protects Against Sensorimotor Dysfunction induced by
Psychotomimetic Agents
[0097] Dizocilpine maleate ((+)MK-801, an antagonist of the
N-methyl-D-aspartate (NMDA) receptor channel) and AMPH act as
psychotomimetic agents, inducing--via neurotransmitter
imbalance--psychotic symptoms in healthy individuals and
exacerbating psychotic symptoms in schizophrenic patients (Lahti et
al., 2001). We therefore used these two compounds in an animal
model to induce psychotic behavior that simulates behavioral and
cellular abnormalities associated with schizophrenia (Tenn et al.,
2003).
[0098] The neurotransmitter imbalance induced by MK-801 or AMPH
also causes sensorimotor dysfunction, another characteristic
feature of patients with schizophrenia. Because (as shown below) T
cell-based therapy counteracted the effect of these drugs on
cognition, we assumed that other functions impaired by these drugs
would be similarly affected. Sensorimotor gating can be assessed
experimentally by drug-induced PPI of the acoustic startle
response. One week before administration of MK-801 or AMPH,
C57BL/6J mice were either inoculated with Cop-1/CFA or with
PBS/CFA. The psychotomimetic drugs were injected 15 min before
measurement of the PPI (Van den Buuse et al., 2003). As expected,
PPI in untreated normal mice increased with increasing prepulse
intensity (FIG. 1a) whereas, in mice injected with MK-801
(C57BL/6J) or AMPH (FIG. 1d) the PPI response was abnormal.
Vaccination with Cop-1/CFA, but not with PBS/CFA, prevented the
abnormal behavior induced by MK-801 (FIGS. 1b, 1c). Similar results
were obtained with AMPH with (FIG. 1d) and without Cop-1
immunization (FIG. 1e). Thus, sensorimotor dysfunction induced by
administration of each of the psychotomimetic agents was prevented
or partially restored by vaccination with Cop-1/CFA.
[0099] Due to the rapid onset of symptoms that occurs in the
experimental paradigm used here, the mice had to be vaccinated
before psychosis was induced. Further experiments with a wider
window for therapeutic intervention have indeed shown that
vaccination with Cop-1 after exposure of rats to psychotomimetic
drugs was beneficial (not shown).
Example 2
Cognitive Functions are Impaired in the Absence of T Cells
[0100] To establish whether learning and memory processes are
dependent on the integrity of the immune system, we compared the
spatial learning/memory of wild-type and SCID BALB/c/OLA mice, by
using the MWM, a hippocampal-dependent visuo-spatial
learning/memory task. The SCID mice manifested significant
impairment of spatial memory compared with their wild-type
counterparts (FIGS. 2a-2c). During the acquisition (FIG. 2a),
extinction (FIG. 2b), and reversal (FIG. 2c) phases of the MWM
task, mice devoid of adaptive immunity showed significantly
increased latency in finding the hidden platform compared with
wild-type mice (FIGS. 2a-2c). Unlike the wild-type, the
immune-deficient (SCID) mice failed to recall data from the
previous day's training trial (FIGS. 2a, 2c). Moreover, the SCID
mice started out at a lower level of performance than the
wild-type, indicating that their general skills in carrying out the
task were impaired, at least to some extent (FIG. 2a). The two
groups did not differ in their performance of the visual platform
task, or in a test in which distal cues were removed and the mice
were tested once a day for 4 days, or in their swimming strategies,
distance, or speed (data not shown).
[0101] It should be emphasized that, in the above set of
experiments, we used SCID mice (deficient in both T cell and B cell
responses) rather than nude mice (deficient only in mature T cells)
to exclude differences that might be attributable to absence of fur
(in nude mice) rather than to the mere differences in immune system
activity. The immune deficiency of SCID mice, however, is a result
of knockout of RAG1/2 genes. Because RAG1 is expressed in brains of
normal mice (Kim et al., 2003) (although their functions there are
still unknown), it was necessary to further substantiate our
conclusion that the observed difference between the SCID and the
wild-type mice was attributable to T cell immunity. We therefore
compared nude mice replenished with a normal T cell population with
nonreplenished nude mice (FIG. 2). We replenished nude mice with T
cells from matched wild-type mice and tested them 3 weeks later on
the MWM task (FIGS. 2d-2f). During the acquisition and the reversal
phases, nude mice replenished with T cells showed significantly
shorter latency in finding the hidden platform than did
nonreplenished nude mice. In the extinction phase, the replenished
mice needed to spend significantly less time than the
nonreplenished mice in the training quadrant for the trials.
Moreover, the nonreplenished mice were significantly less able to
recall data from the previous day's training trial and showed
significantly slower rates of learning than those of their
replenished counterparts (FIGS. 2d-2f).
Example 3
Cop-1 Vaccination is Protective Against Cognitive Impairment
Induced by Psychotomimetic Agents
[0102] The above results encouraged us to examine the possibility
that, in mice with impaired cognitive functions caused for example
by neurotransmitter imbalance, boosting of the relevant T cells
(e.g., by T cell-based vaccination) might have a therapeutic
effect.
[0103] As mentioned above, MK-801 and AMPH induce in humans
psychotic symptoms. In mice, such symptoms evidently simulate the
cognitive impairment and behavioral abnormalities associated with
schizophrenia (Tenn et al., 2003). A number of authors have
reported an MK-801-induced deficit in acquisition of spatial memory
(Whishaw et al., 1989; Ahlander et al., 1999) and nonspatial memory
tasks (Griesbach et al., 1998).
[0104] To examine whether T cell-based vaccination can overcome
behavioral abnormalities and cognitive impairment resulting from
neurotransmitter imbalance, we immunized mice with Cop-1. This
synthetic antigen apparently acts as a weak agonist of a wide range
of self-reactive T cells, thereby stimulating a response that is
mediated by T cells that crossreact with CNS antigens and is needed
to fight off neurodegenerative conditions (Kipnis et al., 2000). It
should be emphasized that, unlike myelin-associated and other
CNS-associated antigens, antigens (such as ovalbumin) that do not
crossreact with CNS-specific proteins fail to accumulate in the
healthy brain and have no protective effect (Kipnis et al., 2000;
Moalem et al., 2000).
[0105] One week before receiving the psychotomimetic drug, C57BL/6J
mice were immunized with Cop-1 emulsified in CFA or with PBS
emulsified in CFA. Relative to behavior in naive normal mice,
performance of a task requiring spatial learning and memory in the
MWM was significantly impaired in mice injected with MK-801 or AMPH
(FIGS. 3a, 3b), as indicated by their significantly higher escape
latencies. During the acquisition (FIGS. 3c, 3e) and the reversal
(FIGS. 3d, 30 phases of the MWM task, PBS/CFA injected mice that
had received either of the psychotomimetic drugs took significantly
longer than the corresponding Cop-1/CFA vaccinated mice to acquire
the spatial navigation task, if they were able to acquire it at
all. In the extinction phase, naive normal mice showed a decrease
over successive trials in the time spent in the training quadrant
(FIG. 3g). Injection of MK-801 weakened this characteristic feature
in mice immunized with PBS/CFA (FIG. 3h), but not in mice immunized
with Cop-1/CFA (FIG. 3i). Similar results were obtained in
amphetamine-injected mice (data not shown).
[0106] The Cop-1/CFA-vaccinated mice injected with each of the
psychotomimetic drugs learned to swim to the hidden platform and
make use of it as a refuge by climbing onto it and remaining there,
as indicated by decreasing latencies in successive trials. In
contrast, when the corresponding PBS/CFA-injected mice encountered
the hidden platform, they behaved in an abnormal and maladaptive
way. Even when placed directly on the hidden platform after a trial
in which they had failed to locate it, these mice quickly walked or
jumped off and continued swimming in a haphazard and disorganized
manner. In all of these tasks, the behavior of normal naive mice
and of normal CFA/PBS-injected mice was identical (data not
shown).
[0107] FIG. 4 depicts the behavior of naive normal mice and of
MK-801-treated mice immunized with Cop-1/CFA or PBS/CFA, and shows
that Cop-1/CFA immunized mice, unlike the PBS/CFA immunized mice,
adopted methodical swimming strategies similar to those seen in
normal mice. Thus, Cop-1/CFA-vaccinated mice injected with a
psychotomimetic drug learned to swim away from the wall to search
for the platform in the inner part of the pool and to use the
platform as a refuge when they found it. In contrast, the behavior
of the PBS/CFA-treated mice injected with a psychotomimetic drug
showed severe disturbances, including hyperactivity, swimming over
the platform, and aimless swimming in circles. Their subsequent
performance in the elevated-plus-maze task indicated, however, that
the observed differences in spatial learning ability in the MWM
between these two groups of mice was not caused by a difference in
anxiety. Furthermore, in the social behavior test, the mice that
were vaccinated with Cop-1 also showed better communicative
behavior than the controls (data not shown).
Example 4
Strain Dependence and T-cell Dependence on the Ability to Withstand
Psychological Stress
[0108] Protection against neurodegenerative conditions in the CNS
is T-cell dependent. Psychological trauma, like physical CNS
insult, can cause widespread long-term changes in neurological and
neurohormonal functioning, and appears to be related to structural
changes (Markowitsch et al., 1998; Myhrer, 1998). There is evidence
that mental/emotional state directly affects the immune system
status (de Groot et al., 2002; McEwen, 2002; Dhabhar and McEwen,
1999). It was therefore of interest to examine the effect of CD4+
(adaptive immunity) T cells on the ability to withstand
psychological trauma.
[0109] Here we examined whether T cells also play a role in the
ability of mice to withstand psychological stress (caused, for
example, by predator odor) associated with behavioral changes
reminiscent of post-traumatic stress disorder (PTSD). Previous
studies have shown that exposure of rats or mice to a predator
(cat) or odor of a predator (thoroughly soiled cat litter) for 10
minutes causes major stress in these animals (Adamec et al., 1997,
1999a, 1999b; Cohen et al., 1996, 2000, 2003). This stress model
was used herein.
[0110] Measurement of behavioral adaptation (acoustic startle
response and avoidance behavior) in mice after their exposure to
predator odor revealed that maladaptation was significantly more
prevalent (x.sup.2=10.6, P<0.001) in immune-deficient mice (62%)
than in their wild-type counterparts (17%). The prevalence of
maladaptation in the normal mice was reduced upon removal of
naturally occurring Treg cells, which normally suppress
autoimmunity. The ability to cope with stress was correlated with
recruitment of T cells in the brain. These findings suggest that a
well-controlled T cell-dependent dialog between the brain and the
immune system is needed for mens sana in corpore sano.
[0111] We first exposed naive adult mice of two strains (C57B1/6J
and BALB/c) to the odor of a cat, as previously described (Cohen et
al., 2003). Seven days later we assessed their behavioral responses
to two sequentially administered behavioral challenges, the
elevated plus-maze (EPM) and the acoustic startle response (ASR),
which together provide a framework for selected cutoff behavioral
criteria (CBC). By classifying the tested mice as either
"maladapted" or "well adapted", we could determine the prevalence
of the more severely affected animals.
[0112] The two strains (C57B1/6J and BALB/c) differed in their
overall adaptation to the imposed psychological trauma (FIGS.
5a-5c). The incidence of maladaptation in C57B1/6J mice was 36.8%,
whereas in BALB/c mice it was only 10.5% (x.sup.2=3.7, P<0.05;
FIG. 5a). The differences were significantly manifested in the ASR
(FIG. 5b), but not in the time spent in the closed arms of the EPM
(FIG. 5c). This finding is in line with previous observations in
connection with the strain-related ability of mice to withstand
glutamate toxicity and optic nerve injury (Kipnis et al.,
2001).
[0113] The results prompted us to investigate whether the adaptive
immune system (represented by CD4+ T cells) affects the behavioral
consequences of traumatic mental stress and the adaptation to such
stress. We therefore examined the ability to adapt to the
psychological stress in the absence of well-functioning immune
system. Because of the relatively low incidence of maladaptation in
the BALB/c mice (FIG. 5a) we used this strain to compare the
response to stress in the wild-type (WT) to that in mice with
severe combined immune deficiency (SCID) with the same genetic
background. Significantly more mice showed symptoms of
maladaptation in the SCID mice than in the WT (61.9% compared to
17.2%; x.sup.2=10.6, P<0.001; FIG. 5d. The same comparison
between nude mice (devoid of mature T cells only) and the WT data
yielded similar results (70% compared to 17.2%; x.sup.2=13.9,
P<0.0002; FIG. 5d, verifying that the observed differences were
due to the absence of mature T cells. Differences between the WT
mice and both the SCID and the nude mice were significantly
manifested in the ASR (FIG. 5e) as well as in the time spent in the
closed arms of the EPM (FIG. 5f).
Example 5
Behavioral Adaptation to Psychological Stress
[0114] FIG. 6 shows the ability of mice immunized with Cop-1 to
withstand psychological stress in comparison with PBS-treated mice
(control), after a single single 10-min exposure to the odor of a
predator. It can be seen that this single exposure caused
behavioral changes in 40.8% of male C57B1/6J control mice
(immunized with PBS emulsified in CFA) and in only 14.8% of Cop-1
immunized mice. Maladaptation, resembling PTSD symptoms. was more
prevalent in control mice than in Cop-1 immunized mice (P<0.05),
verifying that the observed differences were attributable to the
presence of protective Cop-1 reactive T cells.
Example 6
[0115] Production of BDNF by Cop-1-Reactive T Cells upon
Encountering Brain Proteins
[0116] Several research groups have reported that T cells reactive
to Cop-1 home to sites of pathology in the CNS (Kipnis et al.,
2000; Aharoni et al., 2002), and that activated Cop-1-reactive T
cells, being able to cross-react with various CNS-related
self-antigens, can produce neurotrophic factors such as BDNF
(Kipnis et al., 2000; Aharoni et al., 2002; Kerschensteiner et al.,
2003), which is known for its ability to confer neuroprotection to
injured CNS tissue. BDNF deficiency has been reported in patients
with schizophrenia (Weickert et al., 2003, Egan et al., 2003); it
is unclear, however, whether the deficiency is a cause or an
effect, and whether BDNF-based treatment will be beneficial.
[0117] Production of neurotrophic factors by T cells depends on the
state of activation of the T cells (Moalem et al., 2000).
Production of neurotrophic factors by Cop-1-reactive T cells
therefore evidently requires a local signal from resident
antigen-presenting cells that these T cells can recognize. We
therefore carried out an experiment in vitro to determine whether
Cop-1-reactive T cells, on encountering CNS myelin, can produce
BDNF.
[0118] Cop-1-reactive T cells were cultured for 48 h with Cop-1,
MBP, ovalbumin, or concanavalin A in stimulation medium. T cell
supernatants were collected and subjected to sandwich ELISA. Table
2 shows that the production of BDNF by Cop-1-reactive T cells was
increased when these T cells encountered not only their specific
antigen (Cop-1) but also the CNS-related self-antigen MBP. Compared
with unstimulated T cells, secretion of BDNF by Cop-1-reactive T
cells was significantly increased after stimulation of the T cells
with a specific antigen (Cop-1) or with a self-antigen (MBP) that
crossreacts with Cop-1. Values are mean values (pg/ml) +/- SE (from
three independent experiments) of the amounts of BDNF secreted by
Cop-1-specific T cells in response to stimulation by various
antigens.
TABLE-US-00002 TABLE 2 ELISA of BDNF secreted by Cop-1-reactive T
cells Antigen -- Cop-1 MBP OVA Con A pg/ml 400 +/- 1220 +/- 1500
+/- 440 +/- 1100 +/- 45 100 150 50 120
Example 7
[0119] Naturally Occurring CD4.sup.+CD25.sup.+ Regulatory T cells
(Treg) Suppress the Ability to Withstand Psychological Stress
[0120] The spontaneous ability to fight off the sequelae of a
mechanical (e.g. crush) injury or a biochemical insult (e.g. from
glutamate toxicity) to the CNS (Kipnis et al., 2002) is suppressed
by naturally occurring Treg, which comprise approximately 10% of
the CD4.sup.+T-cell population (Shevach, 2000). These cells were
shown to suppress ability to fight off degenerative conditions in
the CNS imposed for example by axonal injury (Kipnis et al.,
2002).
[0121] To address the possibility that these cells control the
spontaneous ability to fight off mental stress, we compared nude
BALB/c mice with splenocytes obtained from WT mice depleted of Treg
(devoid of Treg) to nude mice replenished with a normal splenocyte
population (i.e., including Treg). In nude mice replenished with
splenocytes free of Treg the prevalence of maladaptation was
significantly lower (20%) than in the mice replenished with a
normal T-cell population containing both
[0122] Treg and effector T cells (50%) (x.sup.2=4.0, P<0.046
(FIG. 7a). Significant differences between the two groups were
observed both in the ASR (FIG. 7b) and in the time spent in the
closed arms of the EPM (FIG. 7c).
Example 8
[0123] T-Cell Accumulation in The Brains of Stressed Mice
Correlates with Behavioral Adaptation
[0124] In mice and rats suffering from neurodegenerative
conditions, the beneficial effect of T cells is correlated with
accumulation of T cells at the site of the lesion (Butovsky et al.,
2001; Hauben et al., 2000). To determine the relationship between
the observed beneficial effect of the T-cell response and the
consequences of exposure to stressful psychological conditions we
examined whether it involved homing of T cells to the CNS. This was
done by comparing the immuno-cytochemical staining for T cells in
brain slices obtained from mice replenished with splenocytes
depleted of Treg with those from mice replenished with a whole
splenocyte population. Staining of brain slices with hematoxylin
and eosin revealed no structural alterations in the hippocampus or
amygdala (data not shown) between these two groups and the WT mice.
Luxol fast blue staining for myelin reactivity also showed no
differences between maladapted (FIGS. 8ai, 8aiii) and well-adapted
mice (FIGS. 8bi, 8biii) compared to the WT (FIG. 8ci). Staining
with anti-CD3 antibodies, however, revealed large numbers of T
cells in these brain regions of well-adapted mice (FIGS. 8aii,
8aiv) and hardly any in maladapted (FIGS. 8bii, 8biv) or normal WT
mice (FIG. 8cii), suggesting that the recruitment of T cells to the
brain is correlated with the resistance to mental stress. We wish,
however, to emphasize that accumulation of T cells altogether is
modest and by no means imply that PTSD can benefit from
inflammation, rather that a well-controlled adaptive immunity
assists in resisting consequences of mental stress, similarly to
physical stress, and through the same mechanism.
Example 9
Cop-1 Alleviates the Suppressive Activity Mediated by Treg
[0125] Naive Teff cells (50.times.10.sup.3 cells/well) were
co-cultured with decreasing numbers (50, 25, 12.5 and
6.5.times.10.sup.3 cells/well) of Treg cells that have been
activated for 24 h with anti-CD3 and mrIL-2. The activation of the
Treg cells was carried out in the absence of Cop-1 (control) or,
after 24 h, activated Treg cells were incubated for 2 h with Cop-1
(20 .mu.g/ml in PBS) before co-culturing them with Teff, and then
Cop-1 (20 .mu.g/ml) was added to the co-cultures of Teff and Treg
(Tregcop+cop) and the co-cultures were further incubated. FIG. 9
shows that incubation of the activated Treg (reg) for 2 h with
Cop-1 prior to their co-culturing with Teff (eff) and further
incubation of the co-culture with Cop-1 alleviated the suppression
of Teff compared to the control. The proliferation of Teff also
increased with decreasing concentrations of activated Treg. T cell
proliferation was assayed by incorporation of [.sup.3H]-thymidine
into effector T cells co-cultured with Treg. Recorded values are
from one representative experiment out of three and are expressed
as means.+-.SD of 4 replicates.
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Sequence CWU 1
1
32115PRTArtificial SequenceSynthetic peptide 1Ala Ala Ala Tyr Ala
Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10 15215PRTArtificial
SequenceSynthetic peptide 2Ala Glu Lys Tyr Ala Ala Ala Ala Ala Ala
Lys Ala Ala Ala Ala1 5 10 15315PRTArtificial SequenceSynthetic
peptide 3Ala Lys Glu Tyr Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala
Ala1 5 10 15415PRTArtificial SequenceSynthetic peptide 4Ala Lys Lys
Tyr Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10
15515PRTArtificial SequenceSynthetic peptide 5Ala Glu Ala Tyr Ala
Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10 15615PRTArtificial
SequenceSynthetic peptide 6Lys Glu Ala Tyr Ala Ala Ala Ala Ala Ala
Lys Ala Ala Ala Ala1 5 10 15715PRTArtificial SequenceSynthetic
peptide 7Ala Glu Glu Tyr Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala
Ala1 5 10 15815PRTArtificial SequenceSynthetic peptide 8Ala Ala Glu
Tyr Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10
15915PRTArtificial SequenceSynthetic peptide 9Glu Lys Ala Tyr Ala
Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10 151015PRTArtificial
SequenceSynthetic peptide 10Ala Ala Lys Tyr Glu Ala Ala Ala Ala Ala
Lys Ala Ala Ala Ala1 5 10 151115PRTArtificial SequenceSynthetic
peptide 11Ala Ala Lys Tyr Ala Glu Ala Ala Ala Ala Lys Ala Ala Ala
Ala1 5 10 151215PRTArtificial SequenceSynthetic peptide 12Glu Ala
Ala Tyr Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10
151315PRTArtificial SequenceSynthetic peptide 13Glu Lys Lys Tyr Ala
Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala1 5 10 151415PRTArtificial
SequenceSynthetic peptide 14Glu Ala Lys Tyr Ala Ala Ala Ala Ala Ala
Lys Ala Ala Ala Ala1 5 10 151515PRTArtificial SequenceSynthetic
peptide 15Ala Glu Lys Tyr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala1 5 10 151615PRTArtificial SequenceSynthetic peptide 16Ala Lys
Glu Tyr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10
151715PRTArtificial SequenceSynthetic peptide 17Ala Lys Lys Tyr Glu
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 151815PRTArtificial
SequeneceSynthetic peptide 18Ala Lys Lys Tyr Ala Glu Ala Ala Ala
Ala Ala Ala Ala Ala Ala1 5 10 151915PRTArtificial SequenceSynthetic
peptide 19Ala Glu Ala Tyr Lys Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala1 5 10 152015PRTArtificial SequenceSynthetic peptide 20Lys Glu
Ala Tyr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10
152115PRTArtificial SequenceSynthetic peptide 21Ala Glu Glu Tyr Lys
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 152215PRTArtificial
SequenceSynthetic peptide 22Ala Ala Glu Tyr Lys Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala1 5 10 152315PRTArtificial SequenceSynthetic
peptide 23Glu Lys Ala Tyr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala1 5 10 152415PRTArtificial SequenceSynthetic peptide 24Ala Ala
Lys Tyr Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10
152515PRTArtificial SequenceSynthetic peptide 25Ala Ala Lys Tyr Ala
Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 152615PRTArtificial
SequenceSynthetic peptide 26Glu Lys Lys Tyr Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala1 5 10 152715PRTArtificial SequenceSynthetic
peptide 27Glu Ala Lys Tyr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala1 5 10 152815PRTArtificial SequenceSynthetic peptide 28Ala Glu
Tyr Ala Lys Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10
152915PRTArtificial SequenceSynthetic peptide 29Ala Glu Lys Ala Tyr
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 153015PRTArtificial
SequenceSynthetic peptide 30Glu Lys Tyr Ala Ala Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala1 5 10 153115PRTArtificial SequenceSynthetic
peptide 31Ala Tyr Lys Ala Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala1 5 10 153215PRTArtificial SequenceSynthetic peptide 32Ala Lys
Tyr Ala Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala1 5 10 15
* * * * *