WO2005055920A2 - Compositions et methodes de traitement de troubles psychiatriques - Google Patents

Compositions et methodes de traitement de troubles psychiatriques Download PDF

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WO2005055920A2
WO2005055920A2 PCT/IL2004/001117 IL2004001117W WO2005055920A2 WO 2005055920 A2 WO2005055920 A2 WO 2005055920A2 IL 2004001117 W IL2004001117 W IL 2004001117W WO 2005055920 A2 WO2005055920 A2 WO 2005055920A2
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dopamine
disorder
treg
cells
agonist
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PCT/IL2004/001117
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WO2005055920A3 (fr
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Michal Eisenbach-Schwartz
Jonathan Kipnis
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Yeda Research And Development Co. Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to compositions and methods for treatment of psychiatric disorders and, in particular, to such a treatment with an agent that causes down-regulation of the suppressive activity of CD4 + CD25 + regulatory T (Treg) cells on CD4 + CD25 " effector T cells (Teff), modulation of the immune response and/or modulation of autoimmune response.
  • APCs antigen-presenting cells
  • APL altered peptide ligand
  • ASR acoustic startle response
  • BSA bovine serum albumin
  • CBC cut-off behavioral criteria
  • CNS central nervous system
  • CSPG chondroitin sulfate proteoglycans
  • CTLA-4 cytotoxic T-lymphocyte-associated antigen receptor 4
  • D- R a dopamine receptor
  • Dl-R dopamine receptor type 1
  • D2-R dopamine receptor type 2
  • DA dopamine
  • EAE experimental autoimmune encephalomyelitis
  • EPM Elevated plus-maze
  • ERK extracellular signal-regulated kinase
  • MAG myelin- associated glycoprotein
  • MBP myelin basic protein
  • MDC macrophage-derived chemokine
  • MOG myelin oligodendrocyte glycoprotein
  • mrIL-2 mouse recombinant interleukin-2
  • MWM myelin
  • Psychiatric or mental disorders The causes of psychiatric or mental disorders are complex. Genetic, biological, and environmental factors can contribute to their development. For many disorders, the causes have not yet been determined. For many of these disorders, it appears that they occur as a result of abnormalities in the levels of certain neurotransmitters in the brain, such as glutamate toxicity.
  • psychiatric or mental disorders are the: (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; and (iv) drug use and dependence such as alcoholism, opiate dependence, cocaine dependence, amphetamine dependence, hallucinogen dependence, phencyclidine use, etc.
  • anxiety disorders that include phobic disorders, obsessive-compulsive disorder, post-traumatic stress disorder (PTSD), acute stress disorder and generalized anxiety disorder
  • mood disorders that include depression, dysthymic disorder, bipolar disorders and cyclothymic disorder
  • schizophrenia and related disorders such as brief psychotic disorder, schizophreniform disorder, schizoaffectiv
  • Schizophrenia is a common and serious mental disorder which worldwide prevalence appears to be 1%, although pockets of higher and lower prevalence exist in some countries.
  • the cause of schizophrenia has not yet been determined, although research points to the interaction of genetic endowment and major environmental upheaval during development of the brain.
  • neurodevelopmental disruption may be the result of genetic and/or environmental stressors early in development, leading to subtle alterations in the brain.
  • environmental factors later in development can either exacerbate or ameliorate expression of genetic or neurodevelopmental defects.
  • the overarching message is that the onset and course of schizophrenia are most likely the result of an interaction between genetic and environmental influences.
  • Current research proposes that schizophrenia is caused by a genetic vulnerability coupled with environmental and psychosocial stressors, the so-called diathesis-stress model.
  • Family studies suggest that people have varying levels of inherited genetic vulnerability, from very low to very high, to schizophrenia. Whether or not the person develops schizophrenia is partly determined by this vulnerability.
  • the development of schizophrenia also depends on the amount and types of stresses the person experiences over time. Excessive levels of the neuro transmitter dopamine have long been implicated in schizophrenia, although it is unclear whether the excess is a primary cause of schizophrenia or a result of a more fundamental dysfunction.
  • the conventional or older antipsychotic medications e.g., chlorpromazine, haloperidol, fluphenazine, molindone
  • the more recently developed medications e.g., clozapine, risperidone, olanzapine, quetiapine, sertindole
  • the newer medications often called atypical because they have a different mechanism action than their predecessors, also appear in preliminary studies to be more effective against negative symptoms, display fewer side effects, and show promise for treating people for whom older medications are ineffective.
  • Their introduction has created more treatment options for people with schizophrenia and other serious mental illnesses.
  • Post-traumatic stress disorder is an anxiety disorder that can develop after exposure to a cosmic event or ordeal in which grave physical harm occurred or was threatened. Traumatic events that can trigger PTSD include violent personal assaults such as rape or mugging, natural or human-caused disasters, accidents, or military combat. PTSD can be extremely disabling. Research has demonstrated the effectiveness of cognitive-behavioral therapy, group therapy, and exposure therapy, in which the patient gradually and repeatedly relives the frightening experience under controlled conditions to help him or her work through the trauma. Studies have also shown that medications help ease associated symptoms of depression and anxiety and help promote sleep. Principles are attempting to determine which treatments work best for which type of trauma.
  • CD4 + CD25 + regulatory T cells The naturally occurring CD4 + CD25 + cells, which comprise about 10% of the total CD4 + population, are the so-called regulatory T cells (hereinafter designated "Treg"), originally called suppressor T cells. These cells express the transmembrane protein called CD25, the ⁇ chain of the IL-2 receptor (Sakaguchi et al., 1995).
  • Treg regulatory T cells
  • CD25 the transmembrane protein
  • CD25 the ⁇ chain of the IL-2 receptor
  • Treg effector T cells
  • This neuroprotective autoimmunity was shown by the inventors to be inhibited by naturally occurring CD4 + CD25 + cells, that suppressed an endogenous T-cell mediated neuroprotective mechanism to achieve maximal activation of autoimmunity and, therefore, to withstand injury to the CNS (Kipnis et al., 2002a).
  • Treg-imposed suppression is a multifactorial process, involving cell-to-cell contacts (Nakamura et al., 2001) and the activity of soluble factors [which presumably include IL-10 (Sundstedt et al., 2003) and TGF- ⁇ (Piccirillo et al.,
  • Treg can be inhibited by the addition of exogenous IL-2 (Thornton and Shevach, 1998), or blocking of the cytotoxic T-lymphocyte-associated antigen receptor 4 (CTLA-4) (Nakamura et al., 2001), or activation of the newly discovered glucocorticoid-induced TNF- ⁇ receptor (McHugh et al., 2002).
  • CTL-4 cytotoxic T-lymphocyte-associated antigen receptor 4
  • Some key adhesion molecules are more abundant on the surfaces of Treg than of Teff (Kohm et al., 2002).
  • the ability of Treg to enter tissues might help prevent autoimmune disease progression. In fighting off neurodegeneration or cancer, however, the presence of Treg is a liability.
  • nude mice replenished with splenocytes deprived of Treg are better able to withstand injurious conditions in the CNS than their matched wild-type controls or nude mice replenished with a population consisting of the full complement of spleen cells (Kipnis et al., 2002a; Schwartz and Kipnis, 2002).
  • Low-dose ⁇ -irradiation decreases the lymphocyte pool, causing homeostasis-driven proliferation of lymphocytes (Safwat, 2000; Ge et al., 2002). This proliferation restores the memory T cell compartment, producing more clones reactive to self-antigens; naive T cells are reconstituted by new arrivals from the thymus.
  • the cell proliferation is an outcome of cytokine stimulation and interaction between T-cell receptors, class II major histocompatibility complex proteins (MHC- II), and self-peptides.
  • MHC- II major histocompatibility complex proteins
  • the proliferating cells acquire a memory phenotype and become hypersensitive to antigen stimulation. Simulation of lymphopenic conditions in vitro inhibits the homeostasis-driven proliferation of cells in co- culture with Treg (Goldrath et al., 2000; Cho et al., 2000).
  • Treg enhances neuronal survival after CNS insults (Kipnis et al., 2002) and increases spontaneous anti-tumor autoimmunity (Sakaguchi et al., 2001a, 2001b). Therefore, to elicit the desired autoimmune response for anti-tumor therapy or protection of CNS neurons at risk of degeneration, the Treg-imposed suppression must be blocked (Kipnis et al., 2002a; Schwartz and Kipnis, 2002). How this is achieved physiologically is not known.
  • Dopamine (3,4-dihydroxyphenylethylamine or 3-hydroxytiramine) is a catecholamine formed in the body by the decarboxylation of dopa (3,4- dihydroxyphenylalanine) and acts as a neurotransmitter in the CNS. It is used in all areas of the brain and is particularly important for regulating the function of the basal ganglia. Parkinson's disease is a progressive degenerative disease caused principally by the degeneration of the dopaminergic cells in the substantia nigra pars compacta, with consequent loss of dopamine terminals in the striatum.
  • L-dopa levodopa
  • L- dopa is converted to dopamine in the blood and in the brain.
  • a medicine such as carbidopa, which blocks the conversion of L-dopa to dopamine in the blood. Therefore, more L-dopa is transported into the brain, where it is converted to dopamine.
  • dopamine agonists Due to the side effects of the treatment with L-dopa or with the combination L-dopa/carbidopa, dopamine agonists have been developed or are in development for the treatment of Parkinson's disease and other diseases or conditions in which dopamine is involved. Contrary to levodopa, that is converted to dopamine in the body, the dopamine agonists mimic the activity of dopamine by directly activating the dopamine receptor rather than replace it as levodopa does. The receptors for dopamine are primarily found in the striatum.
  • Dl through D5 There are at least five subtypes of dopamine receptors, called Dl through D5; the Dl and D5 subtypes belong to the dopamine receptor type 1 family and are referred to as "Dl- like” or “Dl-R” while the D2, D3, and D4 belong to the dopamine receptor type 2 family and are referred to as "D2-like” or “D2-R".
  • the receptors are grouped in this manner because of the common properties of the receptor effects.
  • the different dopamine agonists may have affinity to both Dl and D2 families, albeit with different strength, or they may be specific to the Dl or the D2 family or to one of the receptors within one of the families.
  • dopamine agonists having varying activities at the different dopamine receptors are known, or being investigated, that exhibit subtly different effects.
  • Some of the dopamine agonists in use for treatment of Parkinson's disease include apomorphine (Dl and D2 agonist), the ergoline derivatives bromocriptine (D2 agonist), lisuride (Dl slightly partial agonist, D2 agonist), pergolide (Dl weak agonist, D2 and D3 strong agonist), and cabergoline (D2 agonist), and the non-ergoline derivatives ropinirole (D2 agonist) and pramipexole (D2 and D3 agonist).
  • dopamine agonists under investigation include the Dl agonists dihydrexidine (DHX, the first high affinity full Dl dopamine receptor agonist), SKF-38393, SKF-81297, and SKF-82958, and the D2 agonists quinpirole, LY 172555, PPHT and quinelorane. Besides their use in the treatment of Parkinson's disease, some dopamine agonists have been proposed for different indications. Bromocriptine and lisuride suppress prolactin secretion and can be used as prolactin inhibitor and in the treatment of prolactinomas.
  • Bromocriptine and cabergoline lower serum growth hormone levels in acromegaly patients and can be used for treatment of acromegaly.
  • US Patent No. 5,744,476 discloses the Dl-R agonist dihydrexidine either alone or together with levodopa or with a D2-R agonist, for raising extracellular brain acetylcholine levels to improve cognition in a human having senile or presenile dementia associated with neurodegeneration.
  • Dopamine antagonists have been developed for several indications, particularly D2 antagonists such as sulpride, spiperone, haloperidol, spiroperidol, clozapine, olanzapine and sertindole for use as antipsychotic agents.
  • the method includes down-regulating the suppressive effect of Treg on Teff, for example for treatment of cancer (but excluding treatment of a neurodegenerative condition, disorder or disease) by administering an agent selected from the group consisting of: (i) dopamine; (ii) a dopamine precursor; (iii) a Dl-R agonist; (iv) a D2-R antagonist; (v) a combination of (i) and (ii); and (vi) a combination of (i), (ii) or (iii) with (iv).
  • an agent selected from the group consisting of: (i) dopamine; (ii) a dopamine precursor; (iii) a Dl-R agonist; (iv) a D2-R antagonist; (v) a combination of (i) and (ii); and (vi) a combination of (i), (ii) or (iii) with (iv).
  • the method also includes up-regulating the suppressive effect of Treg on Teff, for example for treatment of cancer or for control of graft rejection, by administering: (i) a Dl-R antagonist; (ii) aD2-R agonist; and (iii) a combination of (i) and (ii).
  • Poly-YE Poly- YE or poly-Glu,Tyr is a non-pathogenic synthetic random copolymer composed of the two amino acids L-glutamic acid (Glu, E) and L-tyrosine (Tyr, Y) in different proportions, for example, the copolymer poly-Glu 50 Tyr 50 with an average length of 100 amino acids and a capacity to elicit strong immune response in certain mouse strains.
  • Poly-YE was described in WO 03/002140 of the present applicant for preventing or inhibiting neuronal degeneration or for promoting nerve regeneration in the CNS or PNS, or for protecting CNS or PNS cells from glutamate toxicity.
  • Modified CNS peptides and T cells activated thereby Activated T cells have been shown to enter the CNS parenchyma, irrespective of their antigen specificity, but only T cells capable of reacting with a CNS antigen seem to persist there.
  • T cells reactive to antigens of the CNS white matter, such as myelin basic protein (MBP) can induce the paralytic disease experimental autoimmune encephalomyelitis (EAE) and anti-MBP T cells may also be involved in the human disease multiple sclerosis.
  • EAE paralytic disease experimental autoimmune encephalomyelitis
  • anti-MBP T cell clones are present in the immune systems of healthy subjects.
  • Activated T cells which normally patrol the intact CNS, transiently accumulate at sites of CNS white matter lesions (Hirschberg et al., 1998).
  • the present inventors discovered recently that activated T cells that recognize an antigen of the nervous system (NS) of the patient confer neuroprotection, as described in PCT Publications WO 99/60021 and WO 03/002602. More specifically, T cells reactive to MBP were shown to be neuroprotective in rat models of partially crushed optic nerve (see also Moalem et al, 1999) and of spinal cord injury (see also Hauben et al, 2000b). Before this discovery, it had been thought that immune cells do not participate in NS repair. Furthermore, any immune activity in the context of CNS damage was traditionally considered detrimental for recovery.
  • NS-specific activated T cells could be used to protect nervous system tissue from secondary degeneration which may follow damage caused by injury or disease of the CNS or peripheral nervous system (PNS).
  • PNS peripheral nervous system
  • NS-specific activated T cells can be carried out also with a natural or synthetic NS-specific antigen such as myelin-associated glycoprotein (MAG), S-100, ⁇ -amyloid, Thy-1, P0, P2, a neurotransmitter receptor, and preferably human MBP, human proteolipid protein (PLP), human oligodendrocyte glycoprotein (MOG), and Nogo, or with a peptide derived from said antigen.
  • MAG myelin-associated glycoprotein
  • S-100 S-100
  • ⁇ -amyloid Thy-1
  • P0 P2
  • P2 a neurotransmitter receptor
  • peptides designated "altered peptides” or “altered peptide ligands” (APL), that are obtained by modification of a self-peptide derived from a CNS-specific antigen, which modification consists in the replacement of one or more amino acid residues of the self-peptide by different amino acid residues, said modified CNS peptide still being capable of recognizing the T-cell receptor recognized by the self-peptide but with less affinity (“modified CNS peptide”), particularly modified MBP-derived peptides, as described in WO 02/055010, and the modified Nogo and Nogo receptor-derived peptides as described in WO 03/002602.
  • modified CNS peptide particularly modified MBP-derived peptides, as described in WO 02/055010, and the modified Nogo and Nogo receptor-derived peptides as described in WO 03/002602.
  • Treg CD4 + CD25 + regulatory T
  • the present invention provides a method for treatment of an individual suffering from a psychiatric disorder which comprises administering to said individual in need of such a treatment an effective amount of an agent that causes down-regulation of the suppressive activity of CD4 + CD25 + regulatory T (Treg) cells on CD4 + CD25 ⁇ effector T cells (Teff), modulation of the immune response and/or modulation of autoimmune response, but excluding Copolymer 1, a Copolymer 1- related peptide and Copolymer 1 -related polypeptide, and further excluding a combination of dopamine, a dopamine precursor or an agonist of the dopamine receptor type 1 family (Dl-R agonist) with an antagonist of the dopamine receptor type 2 family (D2-R antagonist), when said individual is one suffering from schizophrenia.
  • an agent that causes down-regulation of the suppressive activity of CD4 + CD25 + regulatory T (Treg) cells on CD4 + CD25 ⁇ effector T cells (Teff), modulation of the immune response and/
  • the agent that causes down-regulation of the suppressive activity of Treg on Teff is selected from the group consisting of (i) dopamine or a pharmaceutically acceptable salt thereof; (ii) a dopamine precursor or a pharmaceutically acceptable salt thereof: (iii) an agonist of the dopamine receptor type 1 family (Dl-R agonist) or a pharmaceutically acceptable salt thereof: (iv) a combination of (i) and (ii); and (v) a combination of (i), (ii) or (iii) with an antagonist of the dopamine receptor type 2 family (D2-R antagonist) or a pharmaceutically acceptable salt thereof, provided that when said agent is a combination of (i), (ii) or (iii) with a dopamine D2-R antagonist, said individual is not one suffering from schizophrenia.
  • the agent that causes down-regulation of the Treg cells is low dose whole-body or total lymphoid organ ⁇ -irradiation that causes specific killing of Treg.
  • the agent is one that causes modulation of the autoimmune response and is, for example, a modified CNS-peptide obtained by modification of a self-peptide derived from a CNS-specific antigen, which modification consists in the replacement of one or more amino acid residues of the self-peptide by different amino acid residues, said modified CNS peptide still being capable of recognizing the T-cell receptor recognized by the self-peptide but with less affinity, or the agent is T cells that have been activated either by a CNS-specific antigen such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), Nogo, Nogo receptor, S-100, ⁇ -amyloid, Thy-1, P0, P2, and neurogen
  • the agent is the copolymer poly- YE or a poly- YE related peptide or polypeptide, that cause down-regulation of the suppressive activity of the Treg cells, modulation of the immune response and modulation of autoimmune response.
  • Poly-YE may be used as a vaccine for immunization of the individual in need.
  • the invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an agent as described above for treatment of psychiatric disorders.
  • the pharmaceutical composition may be a vaccine for immunization of the individual.
  • the invention still further provides the use of an agent as described above for the manufacture of a pharmaceutical composition for treatment of psychiatric disorders.
  • the invention yet further provides an article of manufacture comprising packaging material and a pharmaceutical composition contained within the packaging material, said pharmaceutical composition comprising an agent as defined above; and said packaging material includes a label that indicates that said agent is therapeutically effective for treating a psychiatric disorder.
  • psychiatric or mental disorders that can be treated according to the invention include, but are not limited to: (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
  • 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.
  • TMBP/RAG "7" mice exhibited the greater habituation of startle response (sharper slope) than WT, SCID and TMBP mice, however in these mice the startle amplitudes decreased rapidly during the repeated startle stimuli.
  • Figs. 3a-3c show that naturally occurring Treg suppress the ability to withstand psychological stress.
  • (3a) A single 10-min exposure to the odor of a predator resulted in maladaptation in 70% of nude male BALB/c mice (see Fig. 1). The prevalence of maladaptation was somewhat decreased (50%) in nude mice that were replenished with normal splenocytes from WT BALB/c mice.
  • Figs. 4a-4c are micrographs showing that 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 WT mice and well-adapted animals from the group of nude mice replenished with splenocytes from WT mice devoid 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 fast blue and counterstained with eosin, or stained with anti-CD3 antibodies for the presence of autoimmune effector T cells and counterstained with hematoxylin.
  • Treg Activation of Treg for 96 hr, followed by addition of DA (10 _5 M) for 2 hr at the end of activation, significantly reduced the suppressive activity of Treg on Teff.
  • Figs. 6a-6d show that the dopamine (DA) effect on Treg is mediated via Dl- type receptor family.
  • Proliferation of Teff was assayed by incorporation of [ H]- thymidine into Teff cocultured with naturally occurring Treg. Recorded values are from one of three representative experiments and are expressed as means +/- SD of four replicates.
  • mRNA was extracted from freshly purified Teff and Treg.
  • the housekeeping gene ⁇ -actin was used for quantitative analysis. The results of one of five representative experiments are shown.
  • Figs. 8a-8d show the molecular mechanism underlying the effect of dopamine on Treg.
  • Treg were activated for 24 hr, then incubated for 2 hr with dopamine or SKF-38393 (control cells were activated but were not incubated with either dopamine or SKF-38393; note that different cell preparations were used for each treatment, and therefore the controls used for each treatment were not the same) and were stained 24 hr later for CTLA-4 on cell surfaces.
  • CTLA-4 expression was reduced after exposure to dopamine or to SKF- 38393. Representative results of one of five independent experiments with each treatment are shown. (8b) Production of IL-10.
  • Treg were activated for 24 hr with anti-CD3 and IL-2 in the presence of lethally irradiated splenocytes (APCs) and then for an additional 2 hr with dopamine.
  • Conditioned media were collected after 24, 48, or 72 hr of culture and were assayed for IL-10 using a sandwich ELISA. At any given time, significantly less IL-10 was detected in media conditioned by dopamine-treated Treg than in media conditioned by Treg not exposed to dopamine.
  • Statistical significance was verified using Student's t test analysis (**R ⁇ 0.01; *R ⁇ 0.05). The results shown are of one of three independent experiments, performed at each time point. (8c) Lack of IL-2 production by Treg.
  • Treg and Teff were activated separately for 48 hr with anti-CD3 and anti-CD28 (without mrIL-2) with or without dopamine. Conditioned media were collected after 48 hr and subjected to ELISA. Treg with or without dopamine did not secrete detectable levels of IL-2. Production of IL-2 by Teff was not affected by dopamine. (8d) Foxp3 expression in Treg. Treg were activated for 24 hr with anti-CD3 and anti- CD28 in the presence of IL-2, then exposed to dopamine for 2 hr, washed, and analyzed 30 min later for Foxp3 expression. No changes in Foxp3 were detected after 30 min of dopamine treatment of naive Treg. Figs.
  • 9a-9b show that ERK 1/2 phosphorylation inhibitors downregulate Treg-suppressive activity.
  • Treg were activated by incubation for 30 min with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and in the presence or absence of a tyrosine kinase inhibitor (genistein) and were then cocultured with Teff. The suppression of Teff by Treg was significantly reduced in the presence of genistein.
  • 9b Similarly, incubation of activated Treg with the specific MEK inhibitor PD98059, which inhibits the ERKl/2 signaling pathway, almost completely abolished their suppression of Treg. Figs.
  • lOa-lOc show the correlation between Treg activity and activation state of ERK 1/2.
  • 10a, 10b Western blot analyses of Treg lysates after activation for 20 min with anti-CD3 and anti-CD28, in the presence or absence of dopamine (10a) or SKF-38393 (10b). After activation, the amounts of phospho-ERKl/2 seen in Treg are larger than in Teff (Fig. 10a) but are reduced by dopamine (10a) or by SKF- 38393 (10b). Dopamine did not cause a significant change in phospho-ERKl/2 levels in Teff (10a, 10b).
  • 10c Quantitative analysis of phospho-bands using NIH Image version 1.62.
  • Figs, lla-lld show that dopamine alters the adhesive properties of Treg.
  • Treg and Teff were activated for 24 hr with anti-CD3 and anti-CD28 and were then incubated, with or without dopamine (10 "5 to 10 ⁇ 9 M), for 2 hr.
  • dopamine 10 "5 to 10 ⁇ 9 M
  • adhesion of Treg to the chondroitin sulfate proteoglycans (CSPG) matrix was significantly stronger than that of Teff.
  • Incubation with dopamine significantly reduced the adhesion of Treg in a concentration-dependent manner.
  • the effect of dopamine on Treg adhesion could be mimicked by SKF-38393.
  • FIG. 14 is a graph showing that administration of dopamine with the D2-R antagonist clozapine increases neuronal survival after glutamate-induced neuronal cell death.
  • Figs. 15A-15C show that low-dose total-body ⁇ -irradiation (TBI) increases neuronal survival after CNS mechanical injury. Young adult female rats of the Sprague-Dawley (SPD) and Lewis strains were subjected to low-dose TBI immediately after unilateral optic nerve crush injury. Two weeks later a fluorescent dye was applied, and after 5 more days retinas were excised and whole-mounted (see Materials and Methods).
  • TBI total-body ⁇ -irradiation
  • Figs. 17A-17G show that low-dose TBI induces lymphopenia and activates T cells.
  • Isolated Treg and Teff were each cultured for 48 h, then subjected to low-dose TBI (350 rad), which was followed by a further 48 h in culture.
  • T cells were then stained with propidium iodide (PI) and analyzed for dead cells by FACSort.
  • the numbers of dead cells did not differ significantly in Teff and Treg control populations. After ⁇ -irradiation, however, significantly more Treg died (68.4 ⁇ 5.6%) than Teff (23.3 ⁇ 2.5%).
  • PI propidium iodide
  • lymphocytes were prepared from the axial, mesenteric, and salivary lymph nodes, the spleens, and the peripheral blood of ⁇ -irradiated and control rats. The cells were stained for the ⁇ - chain of IL-2R (CD25), an activation marker (ergotype).
  • CD25 IL-2R
  • ergotype an activation marker
  • IFN- ⁇ was significantly increased 3 day after ⁇ -irradiation, whereas IL-12 was up- regulated on day 7 after induction of lymphopenia.
  • Figs. 18A-18C show that low-dose total-body or lymphoid organ ⁇ - irradiation increases resistance to glutamate-induced neuronal toxicity: Involvement of CD4 + CD25 + regulatory T cells.
  • C57B1/6J mice were subjected to TBI and BALB/c mice to TLI immediately after intravitreal injection of a toxic dose of glutamate. Neuronal survival was measured 1 week later by application of a fluorescent dye and subsequent counting of labeled retinal ganglion cells (RGCs) in whole-mounted retinas.
  • RRCs retinal ganglion cells
  • mice in one group were injected with Treg and mice in another group with Teff.
  • the bar graph shows the percentage increase in neuronal survival after TBI and treatments compared to those in nonirradiated controls. Injection of Treg wiped out this effect. Injection of Teff did not interfere with the beneficial effect.
  • Figs. 19A-19B show that incubation of activated Treg for 2 h with poly- YE prior to their co-culturing with Teff (TregYE) alleviated the Treg suppressive activity on Teff, as measured by the resulting proliferation of Teff, compared to that obtained with activated Treg not exposed to poly- YE (control).
  • Fig. 16B shows that the effect was even more significant in the co-cultures of Teff and TregYE to which poly- YE was added (TregYE+YE), as shown by the significantly higher Teff proliferation.
  • Figs. 20A-20D show that the cytokine phenotype of the Treg cells is changed in the presence of poly- YE and it becomes similar to the phenotype of Teff cells: there is up-regulation of IFN- ⁇ (20A), TGF- ⁇ 20(B) and IL-2 (20C) and down- regulation of IL-10 (20D).
  • FIG. 21 shows tracking of poly-YE-injected (left panels) and PBS-injected control mice (right panels) in the Morris water maze (MWM) after injection of the psychotomimetic drug MK-801.
  • Fig. 22 shows performance of a spatial memory task in the MWM after injection of the psychotomimetic drug MK-801, of poly-YE-injected (diamonds) and PBS-injected control mice (squares).
  • the present invention provides a method for treatment of an individual suffering from a psychiatric disorder which comprises administering to said individual in need of such a treatment an effective amount of an agent that causes down-regulation of the suppressive activity of CD4 + CD25 + regulatory T (Treg) cells on CD4 + CD25 ⁇ effector T cells (Teff), modulation of the immune response and/or modulation of autoimmune response, but excluding Copolymer 1, a Copolymer 1- related peptide and Copolymer 1 -related polypeptide, and further excluding a combination of dopamine, a dopamine precursor or an agonist of the dopamine receptor type 1 family (Dl-R agonist) with an antagonist of the dopamine receptor type 2 family (D2-R antagonist), when said individual is one suffering from schizophrenia.
  • an agent that causes down-regulation of the suppressive activity of CD4 + CD25 + regulatory T (Treg) cells on CD4 + CD25 ⁇ effector T cells (Teff), modulation of the immune response and/
  • the invention relates to a method for down-regulation of the suppressive activity of CD4 + CD25 + regulatory T cells (Treg) on CD4 + CD25 " effector T cells (Teff) in an individual suffering from a psychiatric disorder, which comprises administering to said individual in need an amount of an agent that causes said down-regulation of Treg, but excluding Copolymer 1, a Copolymer 1- related peptide and Copolymer 1 -related polypeptide, and further excluding a combination of dopamine, a dopamine precursor or an agonist of the dopamine receptor type 1 family (Dl-R agonist) with an antagonist of the dopamine receptor type 2 family (D2-R antagonist), when said individual is one suffering from schizophrenia.
  • the agent that causes down-regulation of the Treg cells is low dose whole-body or total lymphoid organ ⁇ -irradiation.
  • the agent that causes down- regulation of the Treg cells is selected from the group consisting of (i) dopamine; (ii) a dopamine precursor: (iii) an agonist of the dopamine receptor type 1 family (Dl-R agonist): (iv) a combination of (i) and (ii); and (v) a combination of (i), (ii) or (iii) with an antagonist of the dopamine receptor type 2 family (D2-R antagonist), provided that when said agent is a combination of (i), (ii) or (iii) with an antagonist of the dopamine receptor type 2 family (D2-R antagonist), said individual is not one suffering from schizophrenia.
  • the terms "dopamine”, “dopamine precursor”, “Dl-R agonist” and “D2-R antagonist” are meant to include the compounds themselves as well as their pharmaceutically acceptable salts.
  • the agent is dopamine that can be administered parenterally by injection. According to the present invention, dopamine will not replace the dopamine in the brain but will down-regulate the suppressive effect of Treg cells on Teff cells in the periphery, thus allowing the Teff cells to exhibit the protective autoimmunity that is necessary to cope with the stressful condition.
  • dopamine can be used in combination with its precursor levodopa, optionally in further combination with carbidopa, even for the treatment of a Parkinson's patient in need of a treatment for a psychiatric disorder.
  • the agent is a dopamine Dl-R agonist.
  • the agent is a combination of dopamine with a dopamine D2-R antagonist.
  • the agent is a combination of dopamine Dl-R agonist with a dopamine D2-R antagonist.
  • the dopamine Dl-R agonist may be any such agonist known or to be developed in the future and includes, without being limited to, a Dl-R agonist selected from the group consisting of SKF-82958, SKF-38393, SKF-77434, SKF-81297, A-77636, fenoldopam and dihydrexidine.
  • the Dl-R agonist is SKF-38393 and its hydrochloride salt [(+/-)-l-Phenyl-2,3,4,5-tetrahydro-(lH)-3-benzazepine-7,8- diol.HCl].
  • the dopamine D2-R antagonist may be any such antagonist known or to be developed in the future and includes, without being limited to, a D2-R antagonist selected from the group consisting of amisulpride, eticlopride, raclopride, remoxipride, sulpride, tropapride, domperidone, iloperidone, risperidone, spiperone, haloperidol, spiroperidol, clozapine, olanzapine, sertindole, mazapertine succinate, zetidoline, CP-96345, LU111995, SDZ-HDC-912, and YM 09151-2.
  • the D2-R antagonist is clozapine.
  • the agent is a combination of dopamine with a dopamine D2-R antagonist, preferably dopamine and clozapine.
  • the agent is one that causes modulation of the autoimmune response such as, but not limited to, a modified CNS-peptide obtained by modification of a self-peptide derived from a CNS-specific antigen, which modification consists in the replacement of one or more amino acid residues of the self-peptide by different amino acid residues, said modified CNS peptide still being capable of recognizing the T-cell receptor recognized by the self-peptide but with less affinity.
  • the modified peptide may be derived by modification of a self-peptide derived from a CNS-specific antigen selected from the group consisting of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), Nogo, Nogo receptor, S- 100, ⁇ -amyloid, Thy-1, P0, P2, and neurotransmitter receptors.
  • the modified CNS peptide may be obtained by modification of an immunogenic epitope or a cryptic epitope of said CNS-specific antigen.
  • the modified peptide has from 9 to 20, preferably 9-18, 9-15 or 9-13, amino acid residues.
  • the CNS-specific antigen is MBP and the modified peptide is obtained by modification of a peptide selected from the group consisting of pll, p51-70, p87-99, p91-l 10, pl31-150, and pl51-170 of MBP.
  • the modified MBP peptide is obtained by modification of the self-peptide p87-99 of MBP VHFFKNIVTPRTP [SEQ ID NO:l] by replacing the lysine residue 91 with glycine (G91), to give a peptide of the sequence: VHFFGNIVTPRTP [SEQ ID NO:2], or with alanine (A91): VHFFANIVTPRTP [SEQ ID NO:3], or by replacing the proline residue 96 with alanine (A96): VHFFKNIVTARTP [SEQ ID NO:4].
  • the CNS-specific antigen is a mammalian Nogo or Nogo receptor molecule.
  • the modified peptide is obtained by modification of the peptide designated p472 containing the residues 623-640 of rat Nogo-A of the sequence SYDSIKLEPENPPPYEEA [SEQ ID NO: 5], in which the Lys 628 residue is replaced by Val or Ala or another similar residue, preferably the peptides: SYDSIVLEPENPPPYEEA [SEQ ID NO:6] SYDSIALEPENPPPYEEA [SEQIDNO:7]
  • the modified peptide is obtained by modification of the peptides derived from the Nogo receptor of the sequences: SGVPSNLPQR L A G R D [SEQ ID NO:8] orTRSHCRLGQAGSGSS [SEQ ID NO:9] in which an Arg residue is replaced by Val or Ala or another similar residue, preferably the peptides: SGVPSNLPQVLAGRD [SEQ ID NO 10] SGVPSNLPQRLAGVD [SEQ ID NO 11
  • the agent is the copolymer poly- YE or a poly- YE related peptide or polypeptide that causes down- regulation of the suppressive activity of the cells, modulation of the immune response and/or modulation of autoimmunity response.
  • Poly-YE as defined herein, is a random copolymer of Tyr and Glu that may contain the amino acids Glu and Tyr in any available ratio such as, for example, poly-(Glu, Tyr) 1 : 1 and poly(Glu, Tyr) 4: 1.
  • poly- YE related peptide or polypeptide refers to random copolymers of Tyr and Glu with different ratios of Glu and Tyr and/or different molecular weight and to random peptides containing several residues of Tyr and Glu.
  • Examples of psychiatric or mental disorders that can be treated according to the invention include, but are not limited to: (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 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.
  • 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.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in a 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.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • the pharmaceutical composition comprises a combination of agents, for example dopamine and levodopa, optionally with carbidopa
  • each of the agents may be presented in a separate container, and the resulting article of manufacture will contain a leaflet with instructions to the physician and to the patient about the order and dosage of administration of the agents.
  • 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.
  • parenteral e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes.
  • parenteral e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes.
  • mucosal e.g., oral, intranasal, buccal
  • the therapeutic effect depends at times on the condition or disease to be treated, on the individual's age and health condition, on other physical parameters (e.g., gender, weight, etc.) of the individual, as well as on various other factors, e.g., whether the individual is taking other drugs, etc., and thus suitable doses and protocols of administration will be decided by the physician taking all these factors into consideration.
  • All patents and patent applications cited in the specification are herewith incorporated by reference as if fully disclosed herein. The invention will now be illustrated by the following non-limiting examples and accompanying figures.
  • T-cell dependent Protection against neurodegenerative conditions in the CNS is T-cell dependent.
  • 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).
  • PTSD post-traumatic stress disorder
  • 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. Behavior on the EPM was recorded using Etho Vision 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. 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'.
  • ASR Acoustic startle response
  • 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, CA). 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).
  • mice or rats 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).
  • mice 7 days after the trauma indeed points to PTSD rather than to an acute stress reaction.
  • Antibodies and reagents Mouse recombinant IL-2 (mrIL-2) and anti mouse ⁇ -CD3 (clone 145-2C11) were purchased from R&D Systems (Minneapolis, MN).
  • Pharmingen Becton-Dickinson, Franklin Lakes, NJ.
  • 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.
  • ACK hypotonic buffer
  • ix Preparation of lymphocytes.
  • Mouse donor lymph nodes axillary, inguinal, superficial cervical, mandibular, and mesenteric
  • the lymphocytes were washed with ACK buffer to lyse red blood cells.
  • x Purification of murine CD4 + CD25 + /CD4 + CD25 ' T cells. Lymph nodes (axillary, inguinal, superficial cervical, mandibular, and mesenteric) and spleens were harvested and mashed. T cells were purified (enriched by negative selection) on T-cell columns (R&D Systems).
  • the enriched T cells were incubated with anti- CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and negatively selected CD4 + T cells were incubated with PE-conjugated anti-CD25 (30 ⁇ g/10 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 + CD25 + cells. The negative fraction consisted of CD4 + CD25 " T cells.
  • Example 1 Adaptation to acute psychological stress is T-cell dependent 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., 1999b; Cohen et al., 2000).
  • mice depleted of mature T cells and compared them to WT mice with properly functioning immune systems.
  • BALB/c strain was used to compare the stress response of the WT to that of mice with the same genetic background but suffering from SCID.
  • T cell required to withstand mental trauma are autoimmune T cells reactive to CNS-specific myelin-associated proteins
  • T cells are needed for the functional maintenance of higher brain functions; such dependencies can hardly be demonstrated in resting stage but are evident under stress conditions.
  • cells that can home to site of stress should be the cells that contributed to the observed effect.
  • homing and functioning of T cells means recognition and therefore specificity, we made an assumption that perhaps the relevant T cells are T cells that recognize abundant and highly distributed self-antigens in the brain.
  • transgenic mice expressing a T cell receptor for a 1-11 peptide of MBP To test this possibility we made use of transgenic mice expressing a T cell receptor for a 1-11 peptide of MBP.
  • mice bearing a transgene on a WT background (TMBP; these mice bear about 2% of endogenous T cells, which have a suppressive activity, namely regulatory T cells) or on a background of immune-deficient mice (RAGl "7" ), therefore these animals lack the endogenous population of regulatory T cells.
  • TMBP WT background
  • RAGl "7" immune-deficient mice
  • TMBP mice showed a prevalence of PTSD similar to WT animals (40%), whereas TMBp/Ragl-/- mice were completely well-adapted to stress and no PTSD-like behavior was seen in these animals (Fig. 2a), These results suggested that perhaps the better ability of the TMBp/Ragl "7" mice to adapt to the stress relative to TMBP mice was a reflection of the higher availability of autoimmune T cells in the former due to the absence of regulatory T cells. It is noted that SCID mice on this background (C57B1/6J) showed a higher prevalence than their WT counterpart, similar to what was obtained with BALB/c/OLA SCID mice (Fig. 1). TMBp/Ragl "7” mice spent less time than other groups of mice in closed arms (Fig. 2b), whereas the startle response of TMBp/Ragl "7” mice was significantly higher than that of other groups of mice (Fig. 2c), however they underwent habituation (Fig. 2d).
  • Example 3 Depletion of CD4 CD25* T cells increases the ability to cope with acute psychological stress
  • TMBp/Ragl "7" and TMBP mice though to a lesser extent, develop with age autoimmune disease due to the uncontrolled levels of autoimmune T cells. Nevertheless, these experiments suggested that normal animals might benefit for fighting off stressful conditions, from a transient reduction in the activity of regulatory T cells.
  • Naturally occurring Treg comprise approximately 10% of the CD4 + 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., 2002a).
  • nude mice replenished with splenocytes obtained from WT mice depleted of Treg depleted of Treg
  • nude mice replenished with a normal splenocyte population i.e., including Treg.
  • Significant differences between the two groups were observed both in the ASR (Fig. 3b) and in the time spent in the closed arms of the EPM (Fig. 3c).
  • T-cell accumulation in the brains of stressed mice correlates with behavioral adaptation
  • 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., 2000a).
  • 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 immunocytochemical 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. 4ai and 4aiii) and well-adapted mice (Fig. 4bi and 4biii) compared to the WT (Fig. 4ci).
  • Staining with anti-CD3 antibodies revealed large numbers of T cells in these brain regions of well-adapted mice (Fig. 4aii and 4aiv) and hardly any in maladapted (Figs. 4bii and 4biv) or normal WT mice (Fig.
  • T cells Once activated, such T cells ensure shaping of resident innate immune cells (i.e. microglia) to defense mode that could be tolerated by the delicate CNS tissue (Schwartz et al., 2003).
  • T cells that can endow mice with adaptation to stress are shown in the present invention to be directed against the brain-specific antigen MBP.
  • mice which differ from SCID animals only in a monospecific population of autoimmune 1-11 MBP peptide specific T cells, did not develop any signs of anxiety following exposure to stress, which implies that if MBP reactive T cells are not the cells that mediate the spontaneous effect in wild type animals (T cells specific to other brain-restricted proteins might have similar effect), at least these are the cells that have the potential of doing such an antipsychotic effect.
  • MBP reactive T cells are not the cells that mediate the spontaneous effect in wild type animals (T cells specific to other brain-restricted proteins might have similar effect)
  • T cells specific to other brain-restricted proteins might have similar effect
  • Therapeutic strategies based on re-charging peripheral immunity by vaccination with antigens that cross-react with a wide range of self- reacting T cells or by weakening regulatory T cells might provide ways for ensuring boosting peripheral immunity for CNS homeostasis under mental stress.
  • Complete deprivation of animal from Treg will predispose it for spontaneous development of autoimmune diseases. Therefore, we would not suggest to utilize this method for development of anti-psychotic drugs for human use.
  • partial and transient alleviation of suppression imposed by Treg might be of a powerful safe therapy for fighting off conditions leading to PTSD.
  • the present invention was undertaken in an attempt to identify physiological compounds potentially capable of controlling the Treg activity after CNS injury.
  • stressor pain-related physiological compounds are increased after CNS injury (Rothblat and Schneider, 1998; Thiffault et al., 2000)
  • one or more of them might transmit an early signal to Treg, with consequent reduction of the trafficking or suppressive activity, or both, of the latter.
  • likely candidate compounds might be key neurotransmitters such as dopamine, norepinephrine, serotonin, and substance P, all of which have been shown to participate in interactions between the brain and the immune system (Edgar et al., 2002).
  • dopamine was the only one that reduced the activity of Treg, and it did so via an ERK extracellular signal-regulated kinase- dependent pathway.
  • Dopamine affected both the suppressive and the trafficking activities of Treg, via dopamine type 1 (Dl-R and D5-R) receptors, found here to be preferentially expressed by Treg.
  • Dl-R and D5-R dopamine type 1 receptors
  • Mouse recombinant IL-2, anti mouse ⁇ -CD3 anti-mouse CTLA-4, and purified rabbit anti-mouse ERK2 antibody (R&D Systems, Minneapolis, Minnesota); rat anti-mouse phycoerythrin (PE)-conjugated CD25 antibody (PharMingen, Becton-Dickinson, Franklin Lakes, NJ); FITC-conjugated anti-CD4 antibody (Serotec, Oxford, UK); anti Dl-R (Calbiochem, Darmstadt, Germany); 3-hydroxytyramine (dopamine), norepinephrine, SKF-38393, SCH- 23390, quinpirole, clozapine, genistein, and PD98059 (Sigma-Aldrich, Rehovot, Israel); phosphatidyl serine detection kit (IQ Products, Houston, TX).
  • the neurotracer dye FluoroGold (5% solution in saline; Fluorochrome, Denver, CO) was applied (1 ⁇ l, at a rate of 0.5 ⁇ l/min in each hemisphere) using a Hamilton syringe, and the skin over the wound was sutured.
  • 51 Cr-labeled T-cells were left untreated or were preincubated (30 min, 37°C) with dopamine or the specified agonist or antagonist (10 "5M ).
  • the cells (10 5 cells in 100 ⁇ l of RPMI medium containing 0.1% BSA) were then added to the CSPG-coated wells, incubated (30 min, 37°C), and washed.
  • Adherent cells were lysed, and the resulting supernatants were removed and counted in a gamma counter. Results were expressed as the mean percentage of the total population before adhesion of bound T-cells from quadruplicate wells for each experimental group.
  • T-cells across polycarbonate filters (pore size, 5 ⁇ m; diameter, 6.5 mm) toward SDF-1 and monocyte-derived chemokine (MDC/CCL22) were assayed in 24-well Transwell chambers (Costar, Coming, Coming, NY).
  • T-lymphocytes (1.67 x 10 6 cells/ml) were suspended in RPMI medium/0.1% BSA, and 150 ⁇ l of the cell suspension was added to the upper chamber after incubation with or without dopamine (90 min, 37°C).
  • Chemokines were added to the lower chamber at concentrations of l ⁇ g/ml SDF-1 (CytoLab, Israel) and 0.25 ⁇ g/ml MDC (R & D Systems).
  • T-cells that migrated to the lower chambers were collected and stained with anti-CD4 and anti-CD25 antibodies.
  • the numbers of migrating T-cells were measured by flow cytometer acquisition for a fixed time (60 sec). To calculate specific migration, the number of cells in each subpopulation in the absence of chemokine was subtracted from the number in the corresponding cell subpopulation that migrated in the presence of chemokines.
  • the number of migrating CD4 + CD25 + T-cells was calculated as a percentage of the total T-cell population before migration. For migration of purified population, we used a similar protocol. (xxi) Activation of CD4 + CD25 + regulatory T cells.
  • Purified regulatory T cells (Treg; 0.5xl0 6 /ml) were activated in RPMI medium supplemented with L- glutamine (2 mM), 2-mercaptoethanol (5 ⁇ l0 "5 M), sodium pyruvate (1 mM), penicillin (100 IU/ml), streptomycin (100 ⁇ 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 (1.5xl0 6 /ml) were added to the culture.
  • T-cells were then washed and incubated with the secondary antibody (PE-labeled goat anti-rabbit IgG) for 30 min at room temperature, then washed, and analyzed by fluorescence and confocal microscopy.
  • the secondary antibody PE-labeled goat anti-rabbit IgG
  • Western blotting Cells were stimulated for 20 min with anti-CD3 and anti-CD28 antibodies in the presence or absence of dopamine or the Dl-R agonist
  • SKF-38393 Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (50mM Tris, pH 8, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 500 mM NaCl, and 10 mM MgCl 2 ). Supernatants were collected, and 5x sample buffer (containing 25 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 0.5M ⁇ -mercaptoethanol) was added before boiling. Activated ERK1/2 was detected by probing blots with a monoclonal antibody. Total ERK protein was detected by using a polyclonal rabbit antibody.
  • radioimmunoprecipitation assay lysis buffer 50mM Tris, pH 8, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 500 mM NaCl, and 10 mM MgCl 2 .
  • 5x sample buffer containing 25 mM Tri
  • IL-10 sense 5' ACCTGGTAGAAGTGATGCCCCAGGCA-3' (SEQ ID NO: 27); IL-10: sense 5' ACCTGGTAGAAGTGATGCCCCAGGCA-3' (SEQ ID NO: 27);
  • EXAMPLE 5 Dopamine reduces the suppression imposed by Treg Coculturing of Teff with Treg isolated from naive mice results in suppression of Teff proliferation.
  • the suppressive potency depends on the Treg/Teff ratio and the state of Treg activation; the suppression is significantly increased, for example, if the Treg are activated before being added to Teff (Thornton and Shevach, 1998). Inhibition of Teff proliferation, assayed by [ 3 H]-thymidine incorporation, can therefore be taken as a measure of the suppressive effect of Treg.
  • DA Dopamine
  • Treg regulatory T cells
  • Dl-R Dl receptor
  • a receptor-mediated pathway we used specific agonists and antagonists of dopamine receptors. Incubation of Treg with 10 "5 M SKF-38393, an agonist of the type 1 family of dopamine receptors (consisting of Dl-R and D5-R), reproduced the dopamine effect (Fig. 6b).
  • Treg No signs of apoptosis were detectable in Treg, which, after being incubated with dopamine, were stained with propidium iodide and analyzed for apoptotic cells (sub-Gl) by flow cytometry (Fig. 6c).
  • Fig. 6c To further verify the absence of apoptotic death in Treg, after incubating Treg with dopamine, we stained them for phosphatidylserine with annexin V. Again, we could not detect any signs of apoptosis in Treg beyond the background levels seen in the absence of dopamine (Fig. 6d).
  • the reduction in Treg activity after their encounter with dopamine or a related agonist evidently results not from the death of Treg but rather from alteration of their behavior.
  • EXAMPLE 7 Expression of dopamine receptors by Teff and Treg Because dopamine reduced the suppressive activity of Treg on Teff but did not alter the susceptibility of Teff to suppression by Treg, we examined the possibility that Teff and Treg express different subtypes or different amounts of the relevant dopamine receptors. This was done by assaying the expression of the dopamine type 1 receptors, Dl-R and D5-R, in Treg and Teff. PCR assays showed that Treg expressed significantly more Dl-R and D5-R transcripts (4-fold and 14- fold, respectively) than Teff (Fig. 7a,b).
  • IL-10 Another molecule that participates in the suppressive activity of Treg is IL-10 (Maloy et al., 2003). It was therefore of interest to measure the production of IL-10 by Treg after their exposure to dopamine. Media collected after incubation of Treg with dopamine (10 '5 M) for 24, 48, and 72 hr showed a significant decrease in the amount of IL-10 at all time points examined (Fig. 8b). Dopamine did not, however, alter the anergic state of Treg; production of IL-2 was not detected in Treg that had been incubated in the presence of dopamine, as verified by ELISA for a secreted cytokine in media conditioned for 48 hr by activated Treg (Fig. 8c).
  • Teff as expected, secreted IL-2, the level of which was not affected by dopamine (Fig. 8c). It should be noted that activation of both T-cell populations was performed in the absence of mrIL-2. A gene encoding the Foxp3 protein was recently found to be associated with Treg (Ramsdell, 2003). We therefore examined whether the dopamine-induced reduction of Treg activity alters the expression of this gene. mRNA isolated from Treg that were activated for 24 hr, exposed for 2 hr to dopamine, and maintained in culture for an additional 30 min or 24 hr was analyzed for Foxp3 expression. Foxp3, as expected, was detected in Treg, but no significant change in its expression was observed after Treg were exposed to dopamine for 30 min (Fig. 8d) or 24 hr (data not shown).
  • EXAMPLE 9 ERK1/2 is deactivated by dopamine in Treg
  • Dl-type, but not D2-type, receptors taken together with the recent report that the ERK pathway can be activated by Dl-R dependent signaling (Takeuchi and Fukunaga, 2003), led us to suspect that the downregulatory effect of dopamine on the suppressive activity of Treg might be exerted via the ERK pathway.
  • Treg activity is affected by PD98059, a specific MEK inhibitor that blocks the ERK 1/2 signaling pathway (Sharp et al., 1997).
  • PD98059 significantly reduced the suppressive activity of Treg relative to that of control-activated Treg (Fig. 9b).
  • the above findings prompted us to examine the state of ERK phosphorylation in activated Treg in the presence or absence of dopamine.
  • Treg were activated with anti-CD3 and anti-CD28 for 20 min in the presence or absence of dopamine (10 "5 M), and Western blot analysis of phospho-ERKl/2 expression in lysates of Treg and Teff was performed. Significantly more phosphorylated ERK 1/2 was detected in activated Treg than in activated Teff.
  • phospho-ERKl/2 was found to be downregulated in Treg that had been activated in the presence of dopamine (Fig. 10a).
  • ERK 1/2 phosphorylation in Treg was also reduced by the specific Dl-type receptor agonist SKF (Fig. 10b). Results of the quantitative analysis of the phospho-bands are shown in Figure 10c.
  • EXAMPLE 10 Dopamine alters the adhesive and migratory properties of Treg
  • One of the main features of T-cells is their ability to migrate to tissues in need of rescue or repair [such as a diseased or damaged CNS (Hickey, 1999)]. We therefore considered the possibility that dopamine reduces not only the suppressive activity but also the migratory ability of Treg. Because T-cell migration and adhesion have been linked to ERK activation (Tanimura et al., 2003), this assumption appeared even more feasible in light of the above observation that dopamine reduced ERK activation in Treg.
  • Treg we incubated Treg with dopamine for 2 hr and then examined their adhesion to CSPG, extracellular matrix proteins often associated with injured tissues (Jones et al., 2003).
  • the ability of Treg to adhere to CSPG was significantly greater than that of Teff (Fig. 11a) and was significantly decreased, in a concentration-dependent manner (10 "9 to 10 " 5 M), by dopamine (Fig. 11a).
  • the dopamine effect on Treg could be mimicked by the Dl-typespecific agonist SKF-38393 and inhibited by the Dl-type antagonist SCH-23390. Dopamine had only a slight, nonsignificant effect on the adhesion of Teff to CSPG (Fig. 11a).
  • Treg The ability of Treg to adhere to fibronectin was greater than that of Teff (Fig. lib). Exposure to dopamine resulted in no effect on adhesion of Treg to fibronectin and a slight increase in the adhesion of Teff (Fig. lib). To verify that the effect of dopamine on adhesion of Treg exerted through the ERK 1/2 pathway, we incubated Treg with the ERK 1/2 signaling pathway inhibitor PD98059 before performing the adhesion assay. PD98059 significantly reduced the ability of Treg to adhere to CSPG (Fig. lie).
  • EXAMPLE 11 Exogenous dopamine increases the ability to fight off neurodegeneration
  • BALB/c activated Treg into mice
  • Treg and Teff respond differentially to dopamine prompted us to examine the effect of dopamine on the ability to withstand neurotoxic conditions in vivo.
  • mice We subjected two groups of mice to a severe optic nerve crush injury and immediately thereafter gave injections of dopamine (0.4 mg/kg) to the mice in one group and injections of PBS to those in the other group. Two weeks later, their retinas were excised and neuronal survival was assessed. Significantly more viable neurons (1110 ⁇ 56/mm 2 ; mean ⁇ SD) were found in the retinas of dopamine- injected mice than in the retinas of vehicle-treated mice (789 + 23) (Fig. 13a).
  • We tested the beneficial effect of systemic dopamine in an additional model of neuronal degeneration induced by glutamate, a common player in many neurodegenerative conditions Keratayama et al., 1990; Xiong et al., 2003).
  • a single systemic injection of dopamine (0.4 mg/kg) or its Dl-type agonist given immediately after intraocular injection of a toxic dose of glutamate increased neuronal survival by 18 + 2.5 or 19 + 3.2%, respectively, relative to that in glutamate-injected controls treated with PBS (Table 1).
  • Injection of the same agonist to scid mice resulted in no effect, thus supporting the assumption that systemic dopamine benefit CNS neurons via the peripheral immune system.
  • the systemic injection of Dl-type antagonist was done in an attempt to find out whether dopamine is involved, at least in part, in the spontaneous ability to withstand the insult.
  • mice were given systemic injections of the indicated drugs. Neuronal survival was determined 10 d later (see Materials and Methods). The results are expressed by changes (in percentage) in neuronal survival in treated mice relative to untreated mice. Each value represents a mean ⁇ SEM of a group at least five animals, and each experiment was performed at least twice, independently. Asterisks (***/? ⁇ 0.001; **p ⁇ 0.0 ⁇ ) indicate statistical significance of the presented data from a single experiment using a Student's t test statistical analysis. NT, Not tested; ns, no statistical significance.
  • EXAMPLE 12 Exposure of Treg to dopamine in vitro reduces their suppressive activity in vivo The above results suggested that systemic dopamine can benefit injured CNS tissue in a T-cell-dependent pathway. To show this could be a consequence of a direct effect of dopamine on Treg activity in vivo, we examined whether dopamine can reduce the suppressive activity of Treg in an in vivo model of neuronal survival. Systemic injection of Treg after glutamate intoxication resulted in a 25% increase in neuronal death. Incubation of Treg with dopamine before their systemic injection into mice abolished their suppressive effect, indicated by the lack of change in the number of surviving neurons.
  • FIG. 13b shows representative micrographs of fields from retinas excised from mice that were exposed to intravitreally injected glutamate and then injected with either Treg or Teff.
  • EXAMPLE 13 Dopamine with the D2-R antagonist clozapine increase neuronal survival after glutamate-induced neuronal cell death
  • Mice were injected intraocularly with a toxic dose of glutamate followed by an immediate injection i.v of the D2-R family antagonist clozapine (5 mg/kg) or with clozapine in combination with dopamine. Retinas were excised 7 days afterwards and survived neurons were counted. The results are depicted in Fig. 14.
  • Mice injected with clozapine alone showed a significant increase in neuronal survival compared to vehicle-injected mice.
  • mice injected with clozapine in combination with dopamine showed even higher neuronal survival.
  • the above scenario exemplifies how the brain might control an autoimmune response, with dopamine as a short-lived physiological molecule enabling Treg to sense the need for autoimmunity.
  • the site of dopamine interaction with Treg has yet to be discovered.
  • Teff autoimmune T cells
  • Treg adhesive and migratory abilities of Treg were reduced by dopamine via the ERK pathway.
  • Treg might exert their suppressive activity on Teff (autoimmune T-cells) either in the lymphoid organs or at the site of the neural tissue degeneration.
  • Teff autoimmune T-cells
  • Mediation of the suppressive activity of Treg has been attributed partially to IL-10 and CTLA-4, whereas their migration and adhesion have been attributed to the specific repertoire of chemokine receptors and adhesion molecules that they express (Sebastiani et al., 2001).
  • Treg Reduction of the suppressive activity of Treg was correlated with a decrease in their IL-10 production (Zhang et al., 2004) and CTLA- 4 expression, which might participate in the cytokine-mediated and cell-cell- mediated suppression by Treg, respectively.
  • Treg express relatively large amounts of the CD44 receptor (needed for their adhesion to CSPG) and the chemokine receptor CCR-4 (needed for their migratory ability).
  • the exposure of Treg to dopamine resulted in a decrease in both their adhesion to CSPG and their migration toward MDC, in correlation with their diminished expression of CD44 and CCR-4, respectively.
  • Treg exist in a state of anergy, neither proliferating in response to mitogenic stimuli nor producing IL-2.
  • dopamine downregulated the suppressive activity of Treg, it did not reverse the anergic state of these T-cells with respect to proliferation or IL-2 production, supporting the contention that dopamine induces changes in the activity rather than in the phenotype of Treg.
  • the in vivo relevance of the effect of dopamine on the suppressive activity of Treg was demonstrated in the experimental paradigms of mouse optic nerve mechanical crush injury and glutamate intoxication in the mouse eye. Significantly more neurons survived consequences of optic nerve crush or neurotoxic insult in mice given injections of dopamine or its Dl-type agonist.
  • the loss of Treg activity in vivo might reflect the effect of dopamine both on homing of Treg to the damaged site and on their suppression.
  • the potential ability of endogenous dopamine to operate spontaneously in vivo was demonstrated by the decrease in neuronal survival in mice given injections of the Dl-type antagonist SCH-23390 immediately after glutamate intoxication.
  • the weak (11%) effect of SCH-23390 on neuronal survival appears to be attributable, at least in part, to the nature of the experimental model (Kipnis et al., 2002).
  • the 11% decrease observed in the wild-type mice represents>30% of the maximal possible T-cell-dependent effect (the difference between nude and wild type).
  • dopamine is a member of a family of physiological compounds capable of controlling Treg activity after a CNS insult.
  • Previous studies have documented the effect of dopamine on T-cell adhesion (Levite et al., 2001), on activation (Hani et al., 2001), and on T-lymphocyte suppression of IgG production by peripheral blood mononuclear cells (Kirtland et al., 1980). No attempt was made in any of those studies to attribute the dopamine effect to subpopulations of CD4 + T-cells.
  • Our results suggest that dopamine has a direct and preferential effect on Treg in initiating the immune response.
  • Rat anti-mouse PE- conjugated CD25 antibody (PC61) was purchased from Pharmingen (Becton- Dickinson, Franklin Lakes, NJ). (xxix) Crush injury of the optic nerve in rats and mice. The optic nerve was cmshed as previously described in detail (Yoles and Schwartz, 1998). Using a binocular operating microscope, we anesthetized the animals and exposed their right optic nerves. In rats, we used calibrated cross-action forceps to inflict a moderate or severe c sh injury on the optic nerve, 1-2 mm from the eye. The severity of the injury determines the number of directly injured neurons.
  • the neurotracer dye FluoroGold (5% solution in saline; Fluorochrome, Denver, CO) was injected into the anesthetized mouse (1 ⁇ l, at a rate of 0.5 ⁇ l/min in each hemisphere) using a Hamilton syringe, at a depth of 2 mm from the exposed brain surface, 2.92 mm posterior to the bregma and 0.5 mm lateral to the midline.
  • a Hamilton syringe 5% solution in saline; Fluorochrome, Denver, CO
  • One week after cmsh injury the mice were killed and their retinas were detached and prepared as flattened whole mounts in 4% paraformaldehyde solution. Labeled cells from 4 to 6 selected fields of identical size (0.7 mm 2 ) were counted (Fisher et al., 2001).
  • splenocytes were washed with hypotonic buffer (ACK) to lyse red blood cells.
  • ACK hypotonic buffer
  • CD4 + CD25 + T cells were purified according to a previously described procedure (Thornton and Shevach, 1998). Briefly, splenocytes and lymph node cells were passed through mouse CD3 + columns (MTCC-25; R&D Systems, Minneapolis, MN) according to the manufacturer's instmctions. Purified T cells (CD3 + ) were incubated with anti CD8 antibody conjugated to magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
  • LNF- ⁇ product 405 bp
  • 5'-ATGAGTGCTACACGCCGCGTCTTGG-3' SEQ ID NO: 30
  • 3'-GAGTTCATTGACTTTGTGCTGG-5' SEQ ID NO: 31
  • IL-12 product 386 bp
  • 5'-AGATGACATCACCTGGACCT-3' SEQ ID NO: 32
  • 3'-CTTTGGTTCAGTGTGACCTTC-5' SEQ ID NO: 33
  • IL-2R product 423 bp
  • 5'-TGCCACGTTCAAAGCCCTCTCCTA-3' SEQ ID NO: 34
  • 3'-TGCGTCCACCTTATCTCCCCACAC-5' SEQ ID NO: 35
  • L19 product 194 bp
  • mice that were subjected to a single low dose of ⁇ -irradiation recovered better from mechanical injury (optic nerve axotomy or spinal cord contusion) or biochemical insult (local glutamate toxicity) than matched controls not subjected to irradiation.
  • Replenishment of ⁇ -irradiated mice with activated Treg wiped out their advantage in terms of ability to withstand neuronal toxicity.
  • EXAMPLE 14 Low-dose total body or total lymphoid organ ⁇ -irradiation in rats protects CNS neurons from injurious conditions.
  • TBI low-dose total body irradiation
  • SPD rats received a single dose (350 rad) of TBI immediately after a cmsh injury to the left optic nerve.
  • Neuronal survival (presented here as the mean number of surviving RGCs/mm ⁇ SEM; P values were calculated by Student's t-test), assessed 2 weeks after the injury, was significantly higher in irradiated rats (72.8 ⁇ 8.6) than in nonirradiated controls (49.1 ⁇ 6; P ⁇ 0.01; Fig. 15A).
  • EXAMPLE 15 Low-dose total body irradiation improves spontaneous recovery from spinal cord injury
  • rats to severe spinal contusion, as previously described (Hauben et al., 2000a).
  • rats were matched by weight and age, so that the primary loss due to the mechanical insult would be of equal severity in all rats (Hauben et al., 2000a). Any differences in recovery would then reflect the change in secondary loss as a result of TBI.
  • EXAMPLE 16 Low-dose ⁇ -irradiation leads to immune activation through homeostasis-driven proliferation of adaptive immunity.
  • Treg suppress the spontaneous ability to manifest a protective autoimmunity
  • Teff and Treg differ in their sensitivity to ⁇ -irradiation.
  • Each of these two cell populations was purified and activated for 48 h with anti-CD3 and anti-CD28 antibodies.
  • Each was then subjected to ⁇ -irradiation (350 rad) and left for an additional 48 h in the stimulation medium. At the end of the incubation period the cells were washed and stained with propidium iodide (PI) for detection of dead cells.
  • PI propidium iodide
  • TBI induced a significant increase in the numbers of CD4 + T cells expressing the CD25 marker (activated T cells) in the spleen, lymph nodes, and blood relative to nonirradiated CD4 + T cells (Fig. 17C). This finding was further substantiated by the observed increase in CD25 mRNA 3 days and 7 days after irradiation (Fig. 17D). An increase in CD25-expressing cells could result from activation of Teff or an increase in the incidence of Treg. Since, as shown above, Treg are more sensitive than Teff to the ⁇ -irradiation, the later possibility is very unlikely.
  • T cells were further verified the irradiation-induced activation of T cells by comparing the expression of the pro inflammatory cytokines IFN- ⁇ and IL-12 in T cells purified from splenocytes and lymph nodes of irradiated rats with that expressed by T cells isolated from nonirradiated rats. Expression of IFN- ⁇ was found to be increased 3 days after TBI and decreased 1 week later, whereas IL-12 expression was still significantly increased 1 week after TBI (Figs. 17E, 17F).
  • EXAMPLE 17 Neuroprotection mediated by lymphopenia-induced activation of T cells in mice is diminished by injection of naturally occurring Treg. Since glutamate is among the most common mediators of neuronal degeneration in the CNS, we also examined the beneficial effect of TBI in a model of glutamate toxicity. After intravitreal injection of a toxic dose of glutamate (400 nmol), significantly more RGCs survived in mice that were subjected, immediately after the glutamate injection, to TBI (350 rad) than in nonirradiated mice (mean number of RGCs ⁇ SD mm 2 ; 2709 ⁇ 51 compared to 1692 ⁇ 47; P ⁇ 0.001).
  • TBI beneficial effect of TBI decreased as the time interval between irradiation and the glutamate injection increased, suggesting that synchrony between proliferation and antigen presentation, both induced by the insult, is needed for the increase in the subpopulation of T cells specific to the insult-associated antigen.
  • the irradiated mice could still withstand the effects of glutamate toxicity injected 6 days after TBI (2366 ⁇ 94; P ⁇ 0.01) and as late as 14 days after TBI (2037 ⁇ 70; P ⁇ 0.05; Fig. 18A).
  • Poly-YE was described in WO 03/002140 of the present applicants as a neuroprotective agent capable of preventing or inhibiting neuronal degeneration in the CNS or PNS, for promoting nerve regeneration in the CNS or PNS, or for protecting CNS or PNS cells from glutamate toxicity. It was therefore of interest to examine in established models as described above whether poly- YE will be beneficial for treatment of mental disorders.
  • mice were injected with MK-801 or vehicle (PBS) 15 min before being subjected to behavioral tests.
  • mrIL-2 Mouse recombinant IL-2
  • anti-CD3 anti-CD3; clone 145-2C11
  • PC61 Rat anti-mouse PE-conjugated CD25 antibody (PC61) was purchased from Pharmingen (Becton-Dickinson, Franklin Lakes, NJ, USA).
  • PC61 Rat anti-mouse PE-conjugated CD25 antibody
  • 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.
  • ACK hypotonic buffer
  • Preparation of splenocytes was carried out as in Materials and Methods, Section III(xxxv).
  • xlv Purification of CD 4 CD 25 and CD4 CD25 " 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 + T cells were incubated with PE- conjugated anti-CD25 (30 ⁇ g/10 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 + CD25 + cells. The negative fraction consisted of CD4 + CD25 ⁇ T cells.
  • Spatial memory was assessed by performance on the Morris water maze task, a hippocampal-dependent visuo-spatial learning task. Mice were given four trials per day, for 4 consecutive days, to find the 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.
  • the platform was removed from the pool, and each mouse was tested by a probe trial for 60 s.
  • the platform was placed at the opposite location, and the mouse was retrained in four sessions. Data were recorded using an EthoVision automated tracking system (Noldus).
  • EXAMPLE 18 Poly-YE alleviates the suppressive activity mediated by Treg Naive Teff cells (50x10 3 cells/well) were co-cultured with decreasing numbers (50, 25, 12.5 and 6.5xl0 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 poly- YE (control) or, after 24 h, activated Treg cells were incubated for 2 h with poly- YE (20 ⁇ g/ml in PBS) before co-culturing them with Teff (TregYE).
  • Figs. 19A- 19B show that incubation of the activated Treg for 2 h with poly- YE prior to their co-culturing with Teff (TregYE) alleviated the Treg suppressive activity on Teff, as measured by the resulting proliferation of Teff, compared to that obtained with activated Treg not exposed to poly- E (control). Fig.
  • Teff and TregYE show that the effect was even more significant in the co-cultures of Teff and TregYE to which poly- YE was added (TregYE+YE) as shown by the significantly higher Teff proliferation.
  • the proliferation of Teff also increased with decreasing concentrations of activated Treg.
  • T cell proliferation was assayed by incorporation of [ H]-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.
  • EXAMPLE 19 Poly-YE causes changes in the cytokine phenotype of Treg cells
  • Treg cells were incubated with mrIL-2 and anti-CD3 for 72 hours, washed and further incubated for 48 hours using fresh medium and poly- YE (20 ⁇ g/ml in PBS).
  • Conditioned media were collected 24 hours after incubation with poly- YE and analyzed for cytokines using commercial kits for INF- ⁇ , IL-10, TGF- ⁇ and IL-2, according to the manufacturer's instmctions (all kits from R&D Systems, Biotest Ltd., Kfar Saba, Israel). Figs.
  • cytokine phenotype of the T regulatory cells is changed in the presence of poly- YE and it becomes similar to the phenotype of T effector cells, namely, there is up regulation of IFN- ⁇ (Fig. 20A ), TGF- ⁇ (20B) and IL-2 (Fig. 20C) and down regulation of IL-10 (Fig. 20D).
  • the change of the cytokine secretion profile of the Treg cells after incubation with poly- YE reflects the changes induced by poly- YE in the biological activity of the Treg cells.
  • the inhibitory cytokine IL-10 secretion is significantly reduced while secretion of the pro-inflammatory cytokine INF- ⁇ is elevated.
  • the appearance of IL-2 secretion goes in line with the observation of increased proliferation of the Treg cells in the presence of poly-YE.
  • EXAMPLE 20 Poly-YE immunization is protective against cognitive impairment induced by psychotomimetic agents Since poly-Glu,Tyr was shown in Example 18 above to alleviate the suppressive activity of the Treg cells, we have then tested its effect in an animal model of psychotic behavior that simulates schizophrenia.
  • Dizocilpine maleate (+)MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) receptor channel) act as psychotomimetic agents, inducing - via neurotransmitter imbalance - psychotic symptoms in healthy individuals and exacerbating psychotic symptoms in schizophrenic patients.
  • NMDA N-methyl-D-aspartate
  • MK-801 also induces cognitive deficits in the mice. Numerous authors have reported an MK-801 -induced learning deficit in acquisition of spatial memory (Whishaw and Auer, 1989; Ahlander et al., 1999) and non-spatial memory tasks (Griesbach et al., 1998). We therefore examined the effect of poly- YE immunization on the ability to prevent or reverse the cognitive deficit induced by MK-801.
  • MK-801 One week before administration of MK-801, each mouse was either immunized with poly-YE (25 ⁇ g/mouse subcutaneously) or with vehicle (PBS), and then injected intraperitoneally (i.p.) with MK-801 (0.1 mg/kg) 15 min before the mouse was tested.
  • FIG. 21 depicts representative tracks of MK-801 -injected poly-YE-immunized mice and of MK-801 -injected PBS- injected control mice when tested in the MWM at the first day (trails 1 and 4).
  • the swimming strategies of the poly-YE-immunized mice (left panels) and the PBS-treated controls (right panels) differed: the poly-YE- immunized mice employed more methodical swimming strategies than the controls.
  • mice 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.
  • PBS-treated mice encountered the hidden platform they behaved in an abnormal and maladaptive way.
  • mice 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.
  • NMDA receptors mediate lasting increases in anxiety-like behavior produced by the stress of predator exposure-implications for anxiety associated with posttraumatic stress disorder.
  • Dhabhar FS Enhancing versus suppressive effects of stress hormones on skin immune function. Proc. Natl. Acad. Sci. USA 96, 1059-64 (1999). Dhabhar FS, BS McEwen, Acute stress enhances while chronic stress suppresses cell-mediated immunity in vivo: a potential role for leukocyte trafficking. Brain Behav. Immun. 11, 286-306 (1997). Dummer, W, Niethammer AG, Baccala R, Lawson BR, Wagner N, Reisfeld RA, Theofilopoulos, T cell homeostatic proliferation elicits effective antitumor autoimmunity. J Clin Invest 110, 185-192 (2002).
  • Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation.
  • Kipnis J Mizrahi T, Yoles E, Ben-Nun A, Schwartz M, Ben-Nur A. Myelin specific Thl cells are necessary for post-traumatic protective autoimmunity. J Neuroimmunol. 130(l-2):78-85 (2002b). Kipnis J, Cardon M, Avidan H, Lewitus GM, Mordechay S, Rolls A, et al. Dopamine, through the extracellular signal-regulated kinase pathway, downregulates CD4+CD25+ regulatory T-cell activity: implications for neurodegeneration. J Neurosci; 24: 6133-43 (2004).
  • McEwen BS Protective and damaging effects of stress mediators: the good and bad sides of the response to stress. Metabolism 51, 2-4 (Jun, 2002). McHugh RS, Whitters MJ, Piccirillo CA, Young DA, Shevach EM, Collins M, Byrne MC, CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid- induced TNF receptor. Immunity 16:311-323 (2002). Mizrahi, T., Hauben, E. & Schwartz, M.
  • the tissue-specific self-pathogen is the protective self-antigen: the case of uveitis. J Immunol 169, 5971-5977 (2002). Moalem, G., Leibowitz-Amit R, Yoles E, Mor F, Cohen IR, Schwartz M.. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med 5, 49-55 (1999). Moalem, G., Yoles, E., Leibowitz-Amit, R., Muller-Gilor, S., Mor, F., Cohen, I.R., and Schwartz, M. Autoimmune T cells retard the loss of function in injured rat optic nerves.
  • CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor betal production and responsiveness. J Exp Med 196:237-246 (2002). Pozzi L, Hakansson K, Usiello A, Borgkvist A, Lindskog M, Greengard P,
  • Fisone G Opposite regulation by typical and atypical antipsychotics of ERKl/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum. J Neurochem 86:451- 459 (2003). Ramsdell F. Foxp3 and natural regulatory T cells: key to a cell lineage? Immunity 19:165-168 (2003). Ricci A, Mariotta S, Greco S, Bisetti A. Expression of dopamine receptors in immune organs and circulating immune cells. Clin Exp Hypertens 19:59-71 (1997). Robertson, G.S. & Jian, M.
  • Dl and D2 dopamine receptors differentially increase Fos-like immunoreactivity in accumbal projections to the ventral pallidum and midbrain.
  • Tanimura S Asato K, Fujishiro SH, Kohno M Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: downregulation of matrix metalloproteinase-3/-9/-14 and CD44.
  • Thiffault C Langston JW, Di Monte DA.
  • Thornton, A.M. & Shevach, E.M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production.

Abstract

L'invention concerne un agent sélectionné parmi (i) la dopamine, (ii) un précurseur de la dopamine, (iii) un agoniste D1-R de la dopamine, (iv) un mélange de dopamine et d'un précurseur de dopamine, (v) un mélange de dopamine, d'un précurseur de dopamine ou d'un agoniste D1-R de dopamine avec un antagoniste D2-R de dopamine, (vi) un peptide du système nerveux central (SNC) modifié, (vii) des lymphocytes qui ont été activés par un antigène spécifique de SNC ou par un peptide SNC modifié, et (viii) poly-YE, un peptide ou un polypeptide lié à poly-YE. Ledit agent peut provoquer une régulation vers le bas de l'activité suppressive de lymphocytes régulateurs CD4+CD25+ sur des lymphocytes effecteurs CD4+CD25- (Teff), la modulation de la réponse immunitaire et/ou la modulation de la réponse auto-immune, et il est utilisé dans le traitement de troubles psychiatriques.
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US7399740B2 (en) 2001-06-28 2008-07-15 Yeda Research And Development Co. Ltd. Poly-Glu,Tyr for neuroprotective therapy
WO2009081395A1 (fr) * 2007-12-21 2009-07-02 Ben Gurion University Of The Negev Research And Development Authority Procédé de traitement de maladies neurodégénératives
WO2009128070A1 (fr) * 2008-04-15 2009-10-22 Yeda Research And Development Co. Ltd Agents et procédés pour le traitement de troubles de l'anxiété
US11464756B1 (en) 2017-05-19 2022-10-11 Jerry Darm Mecuna pruriens, L-DOPA and 5-HTP based dietary supplements, pharmaceutical formulations and uses thereof

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