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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/17—Monocytes; Macrophages
• • A—HUMAN NECESSITIES
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  • A61K40/41—Vertebrate antigens
  • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
• • 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
  • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

• the present invention relates in general to sub-populations of human monocytes useful in the treatment of retinal and optic nerve degeneration with or without inflammatory components and in the treatment of inflammatory diseases, disorders or conditions of the eye.
• AMD Age-related macular degeneration.
• AMD Age-related macular degeneration.
• VEGF vascular endothelial growth factor
• Uveitis is thought to be caused by autoimmune disorders such as rheumatoid arthritis or ankylosing spondylitis, infection, or exposure to toxins. However, in many cases the cause is unknown.
• the most common form of uveitis is anterior uveitis, which involves inflammation in the front part of the eye. The inflammation may be associated with autoimmune diseases, but most cases occur in healthy people.
• Posterior uveitis is a potentially blinding ocular inflammatory condition that affects the choroid of the eye and the neural retina.
• Retinitis pigmentosa a form of retinal dystrophy
• RP Retinitis pigmentosa
• nyctalopia night vision difficulties
• RP is caused by abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) of the retina leading to progressive sight loss.
• RPE retinal pigment epithelium
• Diabetic retinopathy is retinopathy caused by complications of diabetes, which can eventually lead to blindness. Diabetes causes a number of metabolic and physiologic abnormalities in the retina, but which of these abnormalities contribute to recognized features of diabetic retinopathy (DR) is less clear. Many of the molecular and physiologic abnormalities that have been found to develop in the retina in diabetes are consistent with inflammation. Moreover, a number of anti-inflammatory therapies have been found to significantly inhibit development of different aspects of DR in animal models (Tang et al., 2011).
• Glaucoma is a slow-progressing optic neuropathy with a high incidence in the elderly population (approximately 1%). Until recently, it was associated with high intraocular pressure (IOP) and therefore attempts have been focused on slowing down the disease progression by anti-hypertensive drugs. Over the years, it became apparent that glaucoma is a family of diseases and not all are associated with pressure. Moreover, it became clear that even when the disease is associated with pressure, the latter may be reduced to normal and even below normal values and degeneration may continue.
• IOP intraocular pressure
• Glaucoma results in neurodegeneration especially of the retinal ganglion cells (RGCs) leading to progressive loss of visual field up to loss of vision.
• Etiology Acute and/or chronic neuronal loss in the adult CNS results in the irreversible loss of function due to the very poor ability of mature nerve cells to proliferate and compensate for the lost neurons. Thus attenuating or reducing neuronal loss is essential for preservation of function. In most of the neurodegenerative diseases the etiology is not clear, hence they are incurable. Nevertheless, there are some primary and secondary risk factors, which are the target for therapeutic intervention aiming at inhibiting or attenuating progress of neuronal loss, collectively termed as neuroprotective therapy. Some of the risk factors are disease- specific but others, like excitatory amino acids, free radicals and nitric oxide, are common to all the neurodegenerative disorders. These factors are essential self-components in the healthy CNS, but with their accumulation in excess amounts in the degenerative tissue, they become cytotoxic leading to the spread of damage beyond the initial cause of neuron death.
• Glutamate is one of the most common mediators of toxicity in acute and chronic degenerative disorders (Pitt et al., 2000). Glutamate is a primary excitatory neurotransmitter in the human CNS. L-glutamate is present at a majority of synapses and is capable of displaying dual activity: it plays a pivotal role in normal functioning as an essential neurotransmitter, but becomes toxic when its physiological levels are exceeded.
• Regenerative medicine and protective autoimmunity Regenerative medicine is an emerging field that aims to repair, replace, and/or regenerate damaged tissues and organs by stimulating previously irreparable organs into healing themselves including tissue engineering, biomaterials, and cellular therapy.
• Cell renewal a common healing process in peripheral tissues, is limited in the adult neural retina as it is in the rest of the CNS.
• RPCs retinal progenitor cells
• CB retinal ciliary body
• This dormant progenitor cell niche was reported to be stimulated after retinal injury, although the underlying mechanisms are yet to be revealed. Unraveling the healing processes that operate in response to injury and finding ways to enhance them could lead to the development of new therapies for promoting neuroprotection and cell renewal, which is among the research goals in this field.
• Blood monocytes are heterogenic cellular population with different characteristics and activities. Utilizing blood monocytes for therapeutic purposes requires the identification of the cells with harmful functions and those that are beneficial.
• CD16 + enriched monocytes upon injection into the CSF of animals following spinal cord injury, attenuates spontaneous recovery as compared with animals that received total monocytic sub- population. It was also found that a monocytic sub-population devoid of CD16 expressing cells (CD16 ), are beneficial for recovery following spinal cord injury (PCT/IL2012/050522).
• This disclosure generally relates, in part, to a monocyte subpopulation of peripheral blood mononuclear cells (PBMCs) having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells and CD56 + cells, or a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + , for use in inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve, wherein the retina or optic nerve is damaged by a disease, disorder or condition of the eye.
• PBMCs peripheral blood mononuclear cells
• a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells and CD56 + cells or a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + , for use in treatment of an inflammatory disease, disorder or condition of the eye.
• compositions comprising a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells and CD56 + cells, or a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + , for inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve, wherein the retina or optic nerve is damaged by a disease, disorder or condition of the eye.
• the disclosure also generally relates to compositions comprising a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells and CD56 + cells, or a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + , for use in treatment of an inflammatory disease, disorder or condition of the eye.
• the disclosure also generally relates to methods for treatment of an inflammatory disease, disorder or condition of the eye, comprising administering to an individual in need an effective amount of a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid of, CD3 + cells, CD19 + cells and CD56 + cells, or a monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + cells.
• Figs. 1A-E show that syngeneic monocyte injection to the vitreous is neuroprotective after glutamate intoxication.
• Retinal Ganglion Cells RGCs
• RGCs Retinal Ganglion Cells
• C-E Representative micrographs of RGCs in retinas of noninjured (C) and injured mice (D-E) that were intravitreally injected with either PBS (D) or mouse monocytes (E), labeled with the RGC marker, Brn3a (red; scale bar 100 ⁇ ). Arrows point to Brn3a + RGCs. Note the loss of RGCs after the injury, as seen in the PBS treated group, and the sparing of RGCs in the monocyte (mouse mon) treated retina. Bar graphs throughout the figure show mean + SE of each group. *, P ⁇ 0.05; **, P ⁇ 0.01. GT - glutamate; mon - monocytes; RGC - retinal ganglion cell.
• Figs. 2A-H show micrographs depicting localization and characteristics of intravitreally-injected mouse monocytes. Representative micrographs of retinas from day 7 after glutamate intoxication, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected mouse monocytes (CX3CR1-GFP, green).
• CX3CR1-GFP mouse monocytes
• A TGFp
• B IL-10
• C CD38
• D IGF-1
• E BDNF
• F Arg-1 (Arginase-1
• G TNF-a
• H IL- ⁇ .
• Non-specific staining of cells was done with Hoechst stain (blue).
• the injected cells were found in the vitreous and in proximity to injured RGCs, as well as in the subretinal space. Arrows point to double labeled cells; insets show higher magnification of representative cells (scale bar 50 _m; inset scale
• GCL ganglion cell layer
• SRS subretinal space
• vit - vitreous vit - vitreous.
• Figs. 3A-C show that protective CNS -based autoimmune vaccination augments the immune response in the retina after optic nerve crush (ONC).
• One group of mice was vaccinated with MOG-derived altered peptide (45D) 1 week prior to ONC. Contralateral retinas from the vaccinated group were used as noninjured controls.
• Graphs show mean + SE of each group. *, P ⁇ 0.05; **, P ⁇ 0.01; P ⁇ 0.001.
• the present inventors have previously reported that after glutamate eye intoxication, monocyte-derived macrophages infiltrate the damaged retina of mice. Inhibition of this infiltration results in reduced survival of Retinal Ganglion Cells (RGCs) and diminished numbers of proliferating retinal progenitor cells (RPCs) in the ciliary body. Enhancement of the circulating monocyte pool leads to increased RGC survival and RPC renewal. The infiltrating monocyte-derived macrophages skews the milieu of the injured retina toward an antiinflammatory and neuroprotective one and down-regulates accumulation of other immune cells, thereby resolving local inflammation and enhancing tissue repair (London et al., 2011, incorporated by reference as if fully disclosed herein). Glutamate, at levels that exceed the physiological one, is one of the early factors that contribute to the process of self -perpetuating degeneration, irrespectively of the primary risk factor.
• the present inventors have also recently found that a sub-population of blood derived human monocytes that is defined by the absence, near absence, or low relative number of cells expressing CD3, CD19, CD56 and optionally CD16, is capable of homing from the cerebrospinal fluid (CSF) to the site of injury in the spinal cord, and promote there tissue restoration and improved functional recovery (PCT/IL2012/050522). It has further been recently found by the present inventors that the beneficial effect of the cells in healing the wounded spinal cord is obtained either by cells activated by co-incubation with a piece of skin administered into the spinal cord at the borders of the lesion, or by administering un-activated cells into the CSF of injured spinal cord.
• CSF cerebrospinal fluid
• CD X + Differentiation (CD) molecule X
• CD3 + a cell expressing on its surface a CD3 molecule
• the relative amount of the CD molecule expressed on the cell surface is referred to by adding "+”, e.g. CD3 ++ for high amounts of CD molecules, or the term "dim", showing a relative low level of CD molecules.
• a population of cells comprising a cell type defined by the expression of a certain CD, a population relatively enriched with these cells, or a population lacking such cells, is designated CD + , CD ++ or CD " , respectively.
• the human monocyte subpopulation of PBMCs has a low relative amount, or is substantially devoid, of CD3 + cells, CD19 + cells and CD56 + cells.
• the PBMCs of the present invention may have a low relative amount of CD3 + cells and be substantially devoid of CD19 + cells and CD56 + cells.
• CD16 + cells have been removed from the PBMC sub- population resulting in a monocyte subpopulation of PBMCs further substantially devoid of CD16 + cells, i.e. a monocyte subpopulation having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + cells.
• the PBMCs of the present invention may have a low relative amount of CD3 + cells and be substantially devoid of CD19 + cells, CD56 + cells and CD16 + cells.
• mouse CD115 + monocytes used in Example 4 herein below, is divided into two sub-populations; GR1 + and GR1 " , while the corresponding monocyte population in human is divided into three sub-populations defined based on their expression of the membrane markers CD14 and CD16 (Geissman et al. (2008) Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses. Immunology and Cell Biology (2008) 86, 398-408).
• PBMC peripheral blood mononuclear cell
• [a certain cell] is used herein interchangeably with the phrase "near absence of and preferably includes a population of cells lacking a certain cell type, or alternatively comprising a relative amount of a certain cell type not exceeding about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total number of cells in said population of cells.
• the phrase "a population of cells having a low relative amount of... [a certain cell]” refers to a population of cells being present at a low relative amount of cells in comparison with the relative amount of the same kind of cells in a freshly isolated and unprocessed human PBMC population, but at a higher relative amount than the population being "substantially devoid" of this cell population.
• the relative amount of each one of CD19 + cells and CD56 + cells is not exceeding about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total number of cells in the PBMC population.
• the relative amount of CD16 + cells in the monocyte subpopulation substantially devoid of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + cells is not exceeding about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total number of cells.
• a normal human PBMC cell population comprises inter alia more than 70% CD3 + cells and about 10-15% monocytes, so for a "population of cells having a low relative amount of CD3 + cells", the relative amount of CD3 + cells was reduced from more than 70% to about 10%, 15%, 20% or 25% of the total number of cells in the PBMC population.
• the PBMC population of the present invention will have near absence of CD3 + cells, i.e. their relative amount being about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total number of cells in the PBMC population.
• the monocyte subpopulations having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells and CD56 + and optionally also substantially devoid of CD16 + cells are substantially enriched in CD14 + cells.
• a population of cells substantially enriched in refers to a population of cells comprising a relative amount of a certain cell type exceeding about 60%, 70%, 80%, 90%, 95% or 99% of the total number of cells in said population of cells.
• the monocyte subpopulation of PBMCs having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells and CD56 + cells comprises at least about 60% CD14 + cells, and the monocyte subpopulation substantially devoid of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + cells comprises at least about 80% CD14 + cells.
• the monocyte subpopulation as defined herein is also referred to herein as a CD14 ++ , CD 16 " population.
• the monocyte subpopulation of the present invention is useful for inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve, wherein the retina or optic nerve is damaged by a disease, disorder or condition of the eye.
• the disease, disorder or condition that cause neuronal degeneration in the eye and is exacerbated by glutamate toxicity is selected from a retinal degeneration disorder selected from age-related macular degeneration or retinitis pigmentosa; anterior ischemic optic neuropathy; glaucoma or uveitis.
• a retinal degeneration disorder selected from age-related macular degeneration or retinitis pigmentosa; anterior ischemic optic neuropathy; glaucoma or uveitis.
• These diseases cause damage to the nerve tissue and in certain embodiments, administration of the monocyte subpopulation of the present invention promote nerve regeneration.
• the invention also contemplates the treatment of chronic or acute retinopathy, retinopathy as a result of systemic disease such as diabetes or hypertension, radiation retinopathy and solar retinopathy.
• the monocyte subpopulation used in the present invention will be effective in treating inflammatory diseases of the eye, such as scleritis (inflammation of the sclera); keratitis (inflammation of the cornea); corneal ulcer (loss of the surface epithelial layer of the eye's cornea); snow blindness (a painful condition caused by exposure of unprotected eyes to bright light); Thygeson's superficial punctate keratopathy; corneal neovascularization; Fuchs' dystrophy (cloudy morning vision); keratoconus (change in the thickness and shape of the cornea); keratoconjunctivitis sicca (dry eyes); or ulceris (inflammation of the iris).
• inflammatory diseases of the eye such as scleritis (inflammation of the sclera); keratitis (inflammation of the cornea); corneal ulcer (loss of the surface epithelial layer of the eye's cornea); snow blindness (a painful condition caused by exposure of unprotected eyes
• the PBMCs are useful for inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve, wherein the retina or optic nerve is damaged by an inflammatory disease, disorder or condition of the eye.
• the monocyte subpopulation of PBMCs used in any one of the aspects of the present invention comprises human PBMCs.
• the monocyte subpopulation of human PBMCs may be isolated from autologous
• PBMCs i.e. blood is collected from a patient in need of treatment of an eye disease
• a PBMC monocyte subpopulation according to the present invention is prepared as defined herein below, and then administered back to the patient.
• the monocyte subpopulation of human PBMCs may be isolated from allogeneic PBMCs, i.e. blood is collected from a genetically similar, but not identical, donor, a PBMC monocyte subpopulation according the present invention is prepared as defined herein below and optionally stored in a cell-bank before being administered to the patient.
• the monocyte subpopulation of PBMCs is formulated for injection, for example for injection into the eye.
• compositions comprising a monocyte subpopulation of PBMCs as defined herein above, for inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve, wherein the retina or optic nerve is damaged by a disease, disorder or condition of the eye, or for treatment of an inflammatory disease, disorder or condition of the eye.
• the composition may comprise cells of the monocyte subpopulation of PBMCs suspended in a pharmaceutically acceptable carrier adapted for injection, for example for administration into the eye.
• compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
• the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the cells are administered.
• the carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.
• a binder such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate
• a disintegrating agent such as alginic acid, maize starch and the like
• a lubricant or surfactant such as magnesium
• compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the administration of the cells or the composition into the eye may be via intra- vitreal injection or sub-retinal injection.
• PBMC monocyte subpopulation upon homing to the boundaries of an injury in the spinal cord, mitigates the injury and improves functional recovery, whether the cells have previously been activated by co-culture with a piece of skin, i.e. they are skin-activated cells, or whether they are non-activated (PCT/IL2012/050522).
• the cells of the monocyte subpopulation of PBMCs of the present invention injected into the eye may be skin-activated cells.
• the cells of the monocyte subpopulation of PBMCs of the present invention injected into the eye may be non-activated cells.
• the monocyte subpopulation of PBMCs as defined herein and the composition comprising them are useful for treatment of a neurodegenerative diseases, disorders or conditions of the eye.
• the treatment comprises promotion of neural tissue restoration including, for example, preventing or inhibiting neuronal degeneration, promotion of neuronal survival, axonal regeneration and/or sprouting, neurogenesis in a damaged retina or optic nerve tissue, and/or promotion of functional recovery. It has been shown herein that at least partially, the beneficial effect on the nerve cells results from the modulation of the inflammatory response by the specific monocyte subpopulation of PBMCs as defined herein.
• the present invention further provides a method for inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve, wherein the retina or optic nerve is damaged by a disease, disorder or condition of the eye, or for treatment of an inflammatory disease, disorder or condition of the eye, said method comprising administering to an individual in need an effective amount of a monocyte subpopulation of PBMCs as defined herein.
• PCT/IL2012/050522 of the same applicant provides a method for isolation from blood of the monocyte subpopulation of human PMBC substantially devoid of CD3 + cells, CD19 + cells and CD56 + cells described herein, comprising the steps: (i) isolating mononuclear cells from blood; and (ii) removing the CD3 + cells, CD19 + cells and CD56 + cells from the mononuclear cells of (i) by contacting said mononuclear cells with anti-CD3 + antibodies, anti- CD ⁇ "1" antibodies and anti-CD56 + antibodies, each one of which is linked to micro beads, thereby binding said cells to said micro beads, and removing the micro beads, thereby obtaining a monocyte subpopulation of human peripheral blood mononuclear cells (PMBC) from blood having a low relative amount of CD3 + cells and substantially devoid of CD19 + cells and CD56 + cells.
• PMBC peripheral blood mononuclear cells
• PCT/IL2012/050522 further provides a method for isolation from blood of the PMBC monocyte subpopulation substantially devoid of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + cells, comprising the steps: (iii) isolating mononuclear cells from blood; and (iv) removing the CD3 + cells, CD19 + cells, CD56 + cells and CD16 + cells from the mononuclear cells of (iii) by contacting said mononuclear cells with anti-CD3 + antibodies, anti-CD19 + antibodies, anti-CD56 + antibodies and anti-CD16 + antibodies, each one of which is linked to microbeads, thereby binding said cells to said microbeads, and removing the microbeads, thereby obtaining a monocyte subpopulation of human PMBC from blood having a low amount of CD3 + cells and substantially devoid of CD19 + cells, CD56 + cells and CD16 + .
• step (iv), i.e. the removal of CD3 + cells, CD19 + cells, CD56 + cells and CD16 + comprises the sub-steps: (v) removing the CD3 + cells, CD19 + cells and CD56 + cells from the mononuclear cells of (iii) thereby obtaining a monocyte subpopulation of human PMBCs from blood substantially devoid of CD3 + cells CD19 + cells and CD56 + cells; and (vi) removing the CD16 + cells from the PMBC population of (v), thereby obtaining a monocyte subpopulation of human PMBC from blood substantially devoid of CD3 + cells CD19 + cells, CD56 + cells and CD16 + cells.
• the step (iv) is performed in a single step.
• the antibodies that capture the cells expressing the desired antigen on their surface may be biotinylated and then linked to microbeads comprising avidin or streptavidin or equivalent biotin-binding proteins, or the antibodies may be supplied to the cells when already bound to micro beads.
• the micro beads may be magnetic or non-magnetic and the cells, when bound to the micro beads, may be removed from the solution in which the cells are suspended, by centrifugation, if the micro beads are not magnetic, or by exposure to the magnetic field of a magnet, if they are.
• the antibodies are linked to magnetic micro beads when brought in contact with the cells and the cells are removed by removing the micro beads to which the cells are bound by pulling them from the solution with a magnet.
• the above disclosed method may be used for isolating the monocyte subpopulations of human peripheral blood mononuclear cells (PMBC) from blood having a low relative amount, or substantially devoid, of CD3 + cells, CD19 + cells, CD56 + cells and/or CD16 + cells, which are used herein for inhibition of neuronal degeneration, protection of neurons from glutamate toxicity or promotion of nerve regeneration in the retina or optic nerve damaged by a disease, disorder or condition of the eye or for treatment of an inflammatory disease, disorder or condition of the eye.
• PMBC peripheral blood mononuclear cells
• CDl lcGFP/+ transgenic mice (B6.FVB-Tg Itgax-DTR/GFP 57Lan/J; Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, et al. 2002.
• CDl lc+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity. 17:211-220) carrying a transgene encoding a GFP reporter under control of the murine CDl lc promoter are used.
• FoxP3GFP mice were a generous gift from Dr. Alexander Rudensky, Memorial Sloan-Kettering Cancer Center.
• Transgenic R6/2 mice overexpressing the human gene encoding huntingtin were obtained from the Jackson Laboratory.
• BM chimeras [Cx3crl GFPI+ > WT] BM chimeras are prepared by subjecting WT recipient mice to lethal whole-body irradiation (950 rad) while shielding the head, as previously described (Rolls, A., R. Shechter, A. London, Y. Segev, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, and M. Schwartz. 2008. Two faces of chondroitin sulfate proteoglycan in spinal cord repair: a role in microglia/macrophage activation. PLoS Med. 5:el71.; Shechter et al.,2009).
• mice are reconstituted with 5 x 10 6 BM cells according to a previously described protocol (Shechter et al., 2009). Chimeric mice are subjected to glutamate intoxication or EAU induction protocol 8-12 wk after BM transplantation.
• MC-21 administration.
• MC-21 (an antibody to CCR2; Mack, M., J. Cihak, C. Simonis, B. Luckow, A.E. Proudfoot, J. Plachy, H. Briihl, M. Frink, H.J. Anders, V. Dahlhauer, et al. 2001. Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice. J. Immunol. 166:4697-4704) is injected intraperitoneally starting immediately after the injury and throughout the experimental period. In the case of EAU induction, it was injected before or after the peak of EAU, every other day for a total of 5 injections (8 ⁇ g per injection).
• CD115+ monocytes are isolated as previously reported (Varol, C, L. Landsman, D.K. Fogg, L. Greenshtein, B. Gildor, R. Margalit, V. Kalchenko, F. Geissmann, and S. Jung. 2007. Monocytes give rise to mucosal, but not splenic, conventional dendritic cells. J. Exp. Med. 204: 171-180).
• BM cells are harvested from the femora and tibiae of mice and enriched for mononuclear cells on a Ficoll density gradient.
• the CD 115+ BM monocyte population is isolated through MACS enrichment using biotinylated anti-CD 115 antibodies and streptavidin-coupled magnetic beads (MiltenyiBiotec), according to the manufacturer's protocols. After this procedure, monocytes (WT, (3 ⁇ 4cri GFP/+ ,IL-10 deficient, or MHC-II deficient) are injected i.v. (4-5 x 10 6 cells per mouse) on day 1 after injury.
• BrdU regimen BrdU (Sigma- Aldrich) is dissolved in PBS and injected intravitreally
• mice are injected with 1 ⁇ 5% Fluoro-Gold (Fluorochrome) solution in saline on day 3 after injury to both superior colliculi at the following coordinates: 2.92 mm posterior to the bregma, 0.5 mm lateral to the midline, and at a depth of 2 mm from the skull. After 72 h, the mice are killed, their eyes are enucleated, and each retina is flattened as whole mount in 4%paraformaldehyde (PFA) in PBS.
• PFA paraformaldehyde
• the selected fields were located at approximately the same distance from the optic disk (0.3 mm) to overcome the variation in RGC density as a function of distance from the optic disk. Fields were counted under the fluorescence microscope (magnification x800) by observers blinded to the treatment received by the mouse. The average number of RGCs per field in each retina was calculated. For more details, see Schori et al., 2001 (Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: implications for glaucoma. Proc. Natl. Acad. Sci. USA. 98:3398-3403)and Schwartz and Kipnis, 2007 (Model of acute injury to study neuroprotection. Methods Mol. Biol. 399:41-53).
• the following fluorochrome-labeled mAbs were purchased from BD, BioLegend, eBioscience or AbDSerotec, and used according to the manufacturers' protocols: PE-conjugated anti-CDl lb, CD206 (MMR), and IL-4Ra antibodies; PerCP-cy5.5-conjugated anti-Ly6C and CDl lb antibodies; allophycocyanin (APC)-conjugated anti-CD115, TCRp, FoxP3 and CD204 antibodies, Alexa 647-conjugated anti-Dectin-1 antibody; and Pacific Blue/Brilliant Violet-conjugated anti-TCRp, CD4 and CD45.2 antibodies.
• FoxP3 staining was performed using the Foxp3 staining buffer set (eBioscience), according to the manufacturer's protocol. Cells were analyzed on a FACS LSRII cytometer using FACSDiva software (both from BD). Analysis was performed with FlowJo software (Tree Star, Inc.). In each experiment, relevant negative and positive control groups were used to determine the populations of interest and to exclude the rest.
• the excitation and emission for the red filter set are 510-550 nm and 590 nm (long pass), respectively.
• the green filter set is460-490 nm for excitation and 510-550 nm for emission. Fluorescence exposure time is50 ms. Images are acquired using the Camware camera-controlling software program (PCO). Image analysis is performed using ImageJ 1.43 software (W. Rasband, National Institutes of Health, Bethesda,
• Example 1 Isolation of human mononuclear cells that express high level of CX 3 CRI and low level of CCR2 from PBMC
• CD14 + CD16 represent 80% to 90% of blood monocytes, and express high levels of the chemokine receptor CCR2 and low levels of the chemokine receptor CX 3 CRI (the receptor of Fractalkine).
• human CD16+ monocytes express high levels of CX 3 CRI and low levels of CCR2 (Cros et al., 2010).
• gene expression analyses indicated similarities between human CD14 dim CD16 + and murine patrolling Grl ⁇ TM monocytes.
• the CD14 dim CD16 + cells are bona fide monocytes involved in the innate local surveillance of tissues and the pathogenesis of autoimmune diseases.
• CCR2+ depletion First the mononuclear cells were Fc -receptor-blocked by treatment with FcR blocking reagent (2.5 ⁇ 1/10 6 cells) (130-059-901, MiltenyiBiotec) for 15 minutes at room temperature. Then, without washing, the monoclonal anti-human CCR2-biotin reagent (FAB 151B, R&D Systems) was added (10 ⁇ 1/10 6 cells) and incubated for 35 minutes at 2° - 8° C.
• FcR blocking reagent 2.5 ⁇ 1/10 6 cells
• FcR blocking reagent 130-059-901, MiltenyiBiotec
• the cells were washed with cold MACSTM buffer (ImM EDTA, 2% FCS in PBS) and streptavidin microbeads (130-048-101, MiltenyiBiotec) were added (20 ⁇ 1/10' cells) for 20 minutes at 2° - 8° C.
• the cells were washed and resuspended with 0.5 ml of MACSTM buffer.
• the depletion of the CCR2+ cells was done with LD column (130-042-901, MiltenyiBiotec) according to the manufacturers' protocols.
• FACSTM Fluorescence-activated cell sorting
• Fluor® 700 conjugated anti CD16 (BLG-302026), PE conjugated anti CX 3 CR1 (MBL- D070-5) and PerCP conjugated anti CCR2 (BLG-335303).
• Example 2 Inhibition of neuronal degeneration in the retina.
• retinal degeneration induction of EAU retinal intoxication with glutamate, a common mediator of toxicity under neurodegenerative conditions; optic nerve crush; elevated IOP model; and transgenic mice that best mimic the pathology of pigment epithelium degeneration.
• EAU induction This is a model for human posterior uveitis. Since uveitis often results in the deterioration of the retina, it is considered a model of CNS neurodegeneration.
• Mice are injected subcutaneously with 500 ⁇ g peptide derived from human interphotoreceptor retinoid binding protein (IRBP), residues 1-20 (22) (AnaSpec, GL Biochem (Shanghai) Ltd., or the peptide synthesis unit at the Weizmann Institute), as a 1: 1 emulsion with Complete Freund's Adjuvant (CFA) containing Mycobacterium tuberculosis strain H37.RA at a concentration of 2.5 mg/ml (1.25 mg/ml final). Mice are co-injected with 1 ⁇ g Bordella pertussis toxin (Sigma) intraperitoneally .
• IRBP interphotoreceptor retinoid binding protein
• CFA Complete Freund's Adjuvant
• This regimen induces disease in 80-100% of animals by 14 days after immunization.
• the peak of disease occurs between d22 and d29 after immunization.
• Surviving RGCs are assessed 7 d after glutamate intoxication, or before, in the course of, and after the peak of the EUA disease, by immunostaining for Brn3a, a specific RGC marker (Nadal-Nicolas, F.M., M. Jimenez-Lopez, P. Sobrado-Calvo, L. Nieto-Lopez, I. Canovas-Martinez, M. Salinas-Navarro, M. Vidal-Sanz, and M. Agudo. 2009.
• Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest. Ophthalmol. Vis. Sci. 50:3860-3868), and by retrograde labeling of RGCs with Fluoro-Gold, a commonly used method which labels the cell bodies of anatomically intact axons (Schori et al., 2002, supra; Schwartz and Kipnis, 2007, supra).
• Intra- vitreal glutamate toxicity model Elevation of glutamate has been reported in many CNS disorders. In its role as an excitotoxic compound, glutamate is one of the most common mediators of toxicity in acute and chronic (including optic nerve degeneration in glaucoma) degenerative disorders (Doble, 1999). This is therefore a general model for neurodegeneration of central nervous system nerves. When the glutamate is injected into the eye, this model is particularly representative of retinal ganglion neurodegeneration as it occurs in various diseases of the eye, such as retinal degeneration disorders, e.g. age-related macular degeneration or retinitis pigmentosa; anterior ischemic optic neuropathy, glaucoma or uveitis.
• retinal degeneration disorders e.g. age-related macular degeneration or retinitis pigmentosa
• anterior ischemic optic neuropathy glaucoma or uveitis.
• mice are anesthetized, treated with local anesthesia (Localin; Dr. Fischer) applied directly to the eye, and injected intravitreally with a total volume of 1 ⁇ saline containing 400 nmol 1-glutamate (Sigma-Aldrich), as previously described (Yoles E, Friedmann I, Barouch R, Shani Y, Schwartz M. Self-Protective mechanism awakened by glutamate in retinal ganglion cells. J Neurotrauma. 2001, Mar;18(3):339-4; Schori, H., E. Yoles, L.A. Wheeler, T. Raveh, A. Kimchi, and M. Schwartz. 2002. Immune-related mechanisms participating in resistance and susceptibility to glutamate toxicity. Eur. J. Neurosci. 16:557-564).
• N-methyl-N-nitrosourea (MNU) toxicity model A single systemic administration of MNU causes retinal degeneration in various animal species. The retinal degeneration is highly reproducible, and the photoreceptor cell loss occurs within seven days after MNU administration via apoptosis resembling human retinitis pigmentosa (Tsubura A, Yoshizawa K, Kuwata M, Uehara N. Animal models for retinitis pigmentosa induced by MNU; disease progression, mechanisms and therapeutic trials. Histol Histopathol. 2010 Jul;25(7):933-44. Review)
• mice Optic nerve crush in the mice.
• a calibrated optic nerve crush induces progressive ganglion cell death due to retrograde signaling of axonal injury (Levkovitch-Verbin H, Harris- Cerruti C, Groner Y, Wheeler LA, Schwartz M, Yoles E. RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4169-74).
• mice are anesthetized and subjected to severe crush injury in the intraorbital portion of the optic nerve, 1-2 mm from the eyeball. With the aid of a binocular operating microscope, the conjunctiva is incised and the optic nerve exposed. Using cross-action calibrated forceps and taking special care not to interfere with the blood supply, the nerve is crushed for 2 s.
• IOP Intraocular pressure
• Ischemic injury is produced as previously described (Da, T., and A.S. Verkman. 2004. Aquaporin-4 gene disruption in mice protects against impaired retinal function and cell death after ischemia.
• IOP intraocular pressure
• Saline isotonic salt solution
• mice are treated by a single injection of autologous monocytes at different time points after damage onset. Two routes of administration are examined per each animal model: (i) Intra-vitreal injection and (ii) Sub-retinal injection.
• the endpoint of the study is evaluation of neuronal survival in the retina using morphological criteria and by counting the cellular element that survives in cress sections of the retina.
• Subretinal injection of the monocyte sub-population is performed for example according to Warfinge et al., 2011; however any technique for injecting monocytes sub-retinally may be used. Animals are placed under general anesthesia, and vitreoretinal surgery is performed as previously described (K.Warfvinge, J. F. Kiilgaard, E. B. Lavik et al., "Retinal progenitor cell xenografts to the pig retina: morphologic integration and cytochemical differentiation," Archives of Ophthalmology, vol. 123, no. 10, pp. 1385-1393, 2005).
• the pigs are pre-anesthetized with intramuscular injections consisting of midazolam, zolazepam, tiletamine, xylazine, ketamine, and methadone. They are then intubated, artificially ventilated, and anesthetized with isoflurane/oxygen.
• the operative pupil is dilated with topical phenylephrine, tropicamide, and atropine.
• the surgical field is prepared and draped in the usual sterile fashion before commencement of surgery.
• a localized 3-port pars plana vitrectomy is performed and green argon laser burns applied in a grid pattern to the area centralis of the retina.
• a retinotomy is prepared and the monocytes delivered via an appropriate needle to the subretinal space in the region corresponding to the laser burns. Correct graft placement is ascertained by direct visualization at the time of injection. Chloramphenicol is given prophylactically at the end of surgery to avoid infection.
• Example 3 Treatment of age-related macular degeneration with monocyte subpopulation.
• Albino rats are exposed to 12 hours of 3000-luxcyclic light for 1, 3, or 6 months.
• Fundus examination, fundus photography, fluorescein and indocyanine green angiography, and optical coherence tomography are performed prior to euthanization.
• Light-exposed animals are euthanized after 1, 3, or 6 months for histopathological evaluation.
• Retinas are examined for the presence of 4-hydroxy-2-nonenal- and nitrotyrosine-modified proteins by immunofluorescence staining (Daniel M. Albert et al., 2010. Development of choroidal neovascularization in rats with advanced intense cyclic light-induced retinal degeneration.
• AMD and Alzheimer's disease are both chronic neurodegenerative disorders that affect a substantial proportion of elderly persons. Characteristic of these disorders is the irreversible loss of function, for which there is no cure.
• the degeneration occurring in AMD and Alzheimer's disease may, to some extent, have a common pathogenesis (Klaver et al., 1999. Is age-related maculopathy associated with Alzheimer's disease? The Rotterdam study. Am J Epidemiol; 150:963-8).
• the pathogeneses of the two diseases show some striking similarities.
• Alzheimer's disease an early pathologic hallmark in Alzheimer's disease is the presence of extracellular senile plaques (Selkoe, 1991. The molecular pathology of Alzheimer's disease. Neuron 6, 487-98). These plaques are composed of many components, including small peptides generated by proteolytic cleavage of a family of transmembrane polypeptides known as amyloid precursor proteins. Two peptides that are widely regarded as major contributors to the pathology of Alzheimer's disease are known as amyloid- ⁇ ( ⁇ ) peptides.
• amyloid deposits and drusen include proteins such as vitronectin, amyloid P, apolipoprotein E, and even the ⁇ peptides and amyloid oligomers that are associated with amyloid plaques in Alzheimer's disease (Luibl et al., 2006. Drusen deposits associated with aging and age-related macular degeneration contain nonfibrillar amyloid oligomers. J Clin Invest 116, 378-85; Mullins et al., 2000. Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. Faseb J 14, 835-46; Yoshida et al., 2005. The potential role of amyloid beta in the pathogenesis of age-related macular degeneration. J Clin Invest 115, 2793-800).
• the ⁇ peptides present in Alzheimer's disease activate microglial cells to produce potentially neurotoxic substances such as reactive oxygen and nitrogen species, proinflammatory cytokines, complement proteins, and other inflammatory mediators that bring about neurodegenerative changes.
• the inflammatory response that has been associated with Alzheimer's disease often involves CDl lb + activated microglia, representing the innate arm of the immune system in the CNS.
• CDl lb + microglia were reported to be associated with age- related normal human brain (Streit, 2004. Microglia and Alzheimer's disease pathogenesis. J Neurosci Res 77, 1-8), and it is possible that such microglia are the ones that contribute both to age-related cognitive loss and to impaired neurogenesis (Monje et al., 2003.
• Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760-5). CDl lb have also been found in patients with Alzheimer's disease (Akiyama &McGeer, 1990. Brain microglia constitutively express beta-2 integrins. J Neuroimmunol 30, 81-93). Moreover, inflammatory mediators are present in amyloid deposits as well as in drusen, suggesting a possible common role for the inflammatory pathway in AMD and Alzheimer's disease (Hageman et al., 2001. Molecular composition of drusen as related to substructural phenotype. Mol Vis 5, 28). A role for local inflammation in drusen biogenesis suggests that it is analogous to the process that occurs in Alzheimer's disease, where accumulation of extracellular plaques and deposits elicits a local chronic inflammatory response that exacerbates the effects of primary pathogenic stimuli
• the efficacy of the monocyte subpopulation as defined above on AMD treatment may be assessed using a model utilizing exposure of neurons to aggregated ⁇ .
• Example 4 Intravitreal injection of mouse monocytes - effect on retinal ganglion survival after glutamate intoxication and characterization of the injected cells.
• mice were anesthetized and intravitreally injected with 400nmol L- glutamate (in ⁇ saline) into the right eye.
• L- glutamate in ⁇ saline
• cells were purified for intravitreal injection: CD115 + Bone Marrow Monocytic Cells (BMMCs) from mouse - bone marrow cells were harvested from the femurs and tibiae of donor mice (that carry a GFP reporter under the cx ⁇ crl myeloid promoter), and enriched for mononuclear cells on a Ficoll density gradient.
• BMMCs Bone Marrow Monocytic Cells
• the CD115 + BM monocyte population was isolated through Magnetic Activated Cell Sorting (MACS) system using biotinylated anti-CD 115 antibodies and streptavidin-coupled magnetic beads (Miltenyi Biotec) for positive selection of the targeted population.
• MCS Magnetic Activated Cell Sorting
• Cells were injected intravitreally to the GT-intoxicated eye (through the same hole through which the GT was introduced the day before), 10 5 cells per mouse (in ⁇ PBS).
• a control group was injected with PBS.
• mice were perfused and eyes were collected into 2.5% PFA overnight and then the solution was replaced to 1% PFA and the eyes were immersed for an additional 3-4 days, and further processed for immunohistological staining with the RGC marker, Brn3a.
• RGC marker Brn3a
• the PBS- injected eyes served as negative control, and the non-injured, contralateral eyes from the same mice served as a positive control for RGC survival.
• the injected mouse cells were traced in the eye by staining for GFP, and their phenotype was characterized by staining for several immune markers (cytokines, growth factors, phagocytosis markers).
• FIG. 1A- C Mouse monocytes injected intravitreally had a protective effect on RGCs (Figs. 1A- C). The monocytes were detected in the vitreous of the injected eyes, as well as in proximity to the ganglion cell layer (where damaged RGCs are located). In addition, some cells could be detected in the subretinal space (Fig 2). [00103] Double immunostaining for GFP together with an array of immune markers revealed the expression of IL-10, TGF- ⁇ , IGF-1, BDNF, CD36 and Arg-l(Arginase-l), as well as TNFa and IL- ⁇ , among the CX 3 CR1-GFP + mouse monocytes (Fig 2). Notably, this was a qualitative analysis, and so the frequency at which the different types of markers are expressed among the injected cells has yet to be determined.
• Example 5 CNS-based autoimmune vaccination augments the infiltration of macrophages and T cells into the retina after optic nerve crush.
• [Cx3crl GFP/+ - WT] BM chimeras were prepared by subjecting WT recipient mice to lethal whole-body irradiation (950 rad) while shielding the head, to prevent any direct insult to the retina or infiltration of myeloid cells other than that induced by optic nerve crush (ONC).
• mice were reconstituted with 5xl0 6 bone marrow cells derived from Cx3crl GFPI+ transgenic mice by i.v. injection to the tail vein. In the resultant mice, only monocytes infiltrating from the blood carry the GFP label.
• mice One group of mice was subcutaneously injected with a ⁇ emulsion containing 100 ⁇ g MOG altered peptide (45D; sequence: ME VGW YRS PFDR V VHLYRNGK) in 2.5 mg/ml complete Freund's adjuvant (Difco).
• MOG altered peptide 45D; sequence: ME VGW YRS PFDR V VHLYRNGK
• mice (vaccinated and non-vaccinated) were anesthetized and subjected to severe crush of the right optic nerve under a binocular operating microscope, using cross-action forceps. The contralateral nerve was left undisturbed.
• mice were perfused and retinas were collected and processed to a single-cell suspension for flow cytometric analysis.
• ONC resulted in an increase in total leukocytes in the retina (CD45 + ), an increase which was enhanced in 45D-vaccinated injured mice (Fig. 3A).
• Macrophages and T cells were also increased in retinas of mice that had been vaccinated with 45D prior to ONC, compared to retinas from non-vaccinated, injured mice (Figs. 3B and C).
• Example 6 Assesment of the capacity of CNS-based autoimmune vaccination to confer neuroprotection to retinal ganglion cells after optic nerve crush.
• mice are subcutaneously injected with a ⁇ emulsion containing 10( g MOG altered peptide 45D (myelin oligodendrocyte glycoprotein (MOG) 3 5 55 altered at position 45 from serine to aspartic acid) in 2.5 mg/ml complete Freund's adjuvant (Difco).
• MOG altered peptide 45D myelin oligodendrocyte glycoprotein (MOG) 3 5 55 altered at position 45 from serine to aspartic acid
• mice vaccinated and non- vaccinated are anesthetized and subjected to severe crush of the right optic nerve under a binocular operating microscope, using cross-action forceps. The contralateral nerve is left undisturbed.
• mice are perfused and eyes are collected into fixative (2.5% PFA 1% PFA), and further processed for immunohistological staining with the RGC marker, Brn3a.
• RGC marker Brn3a
• Four sections are quantified per sample, taken from different depths of the eye. Cells counted in the ganglion cell layer of the retina are Brn3a + , IB-4 " and Hoechst "1" .
• the noninjured, contralateral eyes from the non-vaccinated mice serve as a positive control for RGC survival (100%).

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