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  • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system

Definitions

• the present invention relates to compositions comprising mononuclear phagocytes, and to methods for using mononuclear phagocytes , to promote axonal regeneration in mammals affected by injury or disease of the central nervous system, as well as to compositions and methods for enhancing the therapeutic capacity of mononuclear phagocytes to promote axonal regeneration.
• the invention relates to (a) pharmaceutical compositions comprising, and methods for administering, stimulated or non-stimulated allogeneic mononuclear phagocytes at or near a site of the mammalian central nervous system affected by injury or disease to promote axonal regeneration, (b) compositions and methods for stimulating mononuclear phagocytes so as to enhance their capacity to promote axonal regeneration, and (c) methods for screening tissues, cells, proteins, peptides and other biologically active agents for their ability to stimulate mononuclear phagocytes for promoting axonal regeneration.
• neurons of the mammalian central nervous system have a poor capacity for axonal regeneration.
• neurons of the mammalian peripheral nervous system have a substantially greater capacity for axonal regeneration. See Schwartz et al., 1989, FASEB J. 3.:2371-2378.
• axonal regeneration in the CNS and PNS has been attributed to the cellular environment of the neurons rather than to the neurons themselves.
• the Schwann cells that surround PNS neurons are modulated so as to become permissive or supportive for axonal regeneration.
• the astrocytes, oligodendrocytes and microglia that surround CNS neurons do not show such modulation and remain unsupportive or inhibitory for axonal regeneration. See Schwartz et al., 1987, CRC Crit. Rev. Biochem. 22 : 89-110.
• the present invention is directed to methods, and compositions, for use of allogeneic mononuclear phagocytes to promote axonal regeneration in the central nervous system of a mammal.
• the allogeneic mononuclear phagocytes are administered into the CNS at or near a site of injury or disease.
• administration of allogeneic mononuclear phagocytes into the CNS at or near a site of injury or disease is combined with administration to the mammal of at least one anti-inflammatory agent.
• administering is combined with administration to the CNS of one or more adjuvant factors, for instance acidic fibroblast growth factor (aFGF) , transforming growth factor- beta (TGF-/3) , interleukin 6 (IL-6) , nerve growth factor (NGF) , neurotrophic factor 3 (NT-3) , neurotrophic factor 4 (NT-4) , neurotrophic factor 5 (NT-5) and brain-derived neurotrophic factor (BDNF) .
• adjuvant factors for instance acidic fibroblast growth factor (aFGF) , transforming growth factor- beta (TGF-/3) , interleukin 6 (IL-6) , nerve growth factor (NGF) , neurotrophic factor 3 (NT-3) , neurotrophic factor 4 (NT-4) , neurotrophic factor 5 (NT-5) and brain-derived neurotrophic factor (BDNF) .
• aFGF acidic fibroblast growth factor
• TGF-/3 transforming growth factor- beta
• IL-6 interleukin 6
• Allogeneic mononuclear phagocytes useful for the methods and compositions of the invention include, but are not limited to, allogeneic onocytes, macrophages and dendritic cells, and autologous monocytes, macrophages and dendritic cells.
• the present invention further provides methods, and compositions, for stimulating allogeneic mononuclear phagocytes so as to enhance their capacity to promote axonal regeneration, and methods and compositions for use of stimulated allogeneic mononuclear phagocytes to promote axonal regeneration in the central nervous system of a mammal.
• the mononuclear phagocytes are stimulated by culturing them in medium to which has been added one or more of the following: -inter eron (IFN- ⁇ ) , ⁇ -interferon (IFN-7) , tumor necrosis factor ⁇ (TNF- ⁇ ) , interleukin 2 (IL-2) , interleukin 3 (IL-3) , interleukin 4 (IL-4) , interleukin 10 (IL-10) , monocyte chemotactic and activating factor (MCAF) , macrophage colony stimulating factor (M-CSF) , granulocyte- macrophage colony stimulating factor (GM-CSF) , colony stimulating factor 1 (CSF-1) , lipid A, the tripeptide fMet- Leu-Phe, muramyl dipeptide (MDP) , the ionophore A23187, or vitamin D3 binding protein.
• a biologically active protein or peptide may be used in its native or recombin
• the present invention further provides an assay for screening or identifying additional tissues, cells and biologically active agents that are suitable for stimulating mononuclear phagocytes to enhance their capacity to promote axonal regeneration.
• mononuclear phagocytes are first cultured together with the tissue or cells to be tested, or in medium that has been conditioned by the tissue or cells to be tested or in medium to which has been added the biologically active agent to be tested.
• the cultured mononuclear phagocytes are then assayed for nitric oxide production, or both nitric oxide production and phagocytic activity.
• Mononuclear phagocytes with increased production of nitric oxide, or increased nitric oxide production and increased phagocytic activity have an enhanced capacity to promote axonal regeneration.
• FIGURE 1 illustrates axonal regeneration in transected optic nerves of rats as detected by retrograde transport of fluorescent dye to retinal ganglion cells (RGCs) .
• RGCs retinal ganglion cells
• FIGURE 2 illustrates axonal regeneration in transected optic nerves of rats as a function of the number and type of monocytes applied to the site of injury shortly after transection. See text. Section 6, for experimental details.
• 2 ⁇ l DCCM-1 medium were applied to the site of injury containing optic nerve-stimulated monocytes (OS) or sciatic nerve-stimulated monocytes (SS) at a total dose of 2.5 x 10 3 cells; 5 x 10 3 cells; 10 4 cells; or 10 5 cells.
• OS optic nerve-stimulated monocytes
• SS sciatic nerve-stimulated monocytes
• FIGURE 3 presents representative photomicrographs showing retrograde labeling of retinal ganglion cells in rats subjected to optic nerve transection followed by administration of (A) 5 x 10 3 sciatic nerve-stimulated monocytes or (B) control medium. See text, Section 6, for experimental details.
• FIGURE 4 presents representative photomicrographs showing anterograde labeling of optic nerve fibers in rats subjected to optic nerve transection followed by administration of sciatic nerve-stimulated monocytes (A-D) or control medium (E) . See text, Section 6, for experimental details.
• FIGURE 4A is a low magnification view showing the point at which HRP was applied (H) , the site of transection (ST) and the surrounding dura mater (DU) .
• the bracketed region, distal to the site of transection, is shown at higher magnification in FIGURES 4B, 4C and 4D, in which growth conelike structures (gc) are shown at the tips of the fibers.
• FIGURE 5 illustrates axonal regeneration in transected optic nerves of rats after application to the site of injury of monocytes cultured with sciatic nerve for 2-17 hours. See text, Section 6, for experimental details.
• 2 ⁇ l of DCCM-1 medium were applied to the site of injury containing 5 x 10 3 non-stimulated monocytes (NS) or 5 x 10 3 monocytes cultured with rat sciatic nerve for 2 hours (2h) , 12 hours (12h) or 17 hours (17h).
• FIGURE 6 illustrates axonal regeneration in transected optic nerves after administration, at the site of injury, of rat monocytes stimulated with mouse sciatic nerve or rat sciatic nerve. See text, Section 6, for experimental details.
• 2 ⁇ l DCCM-1 medium were applied to the site of injury containing 5 x 10 3 monocytes cultured for 24 hours with either mouse sciatic nerve (MOUSE) or rat sciatic nerve (RAT) .
• MOUSE mouse sciatic nerve
• RAT rat sciatic nerve
• FIGURE 7 illustrates the phagocytic activity of rat monocytes cultured for 2 hours with rat sciatic nerve. See text, Section 6, for experimental details. 2.5 x 10 5 rat monocytes were cultured in 1 ml DCCM-1 medium alone (CONTROL) or in 1 ml DCCM-1 medium with 2 segments of rat sciatic nerve (2SS) or with 4 segments of rat sciatic nerve (4SS) . After 2 hours, the monocytes were exposed to fluorescent beads and cell-associated fluorescence was measured by flow cytometry.
• FIGURE 8 illustrates the phagocytic activity of rat monocytes cultured for 24 hours with rat sciatic nerve. See text, Section 6, for experimental details. 2.5 x 10 5 rat monocytes were cultured in 1 ml DCCM-1 medium alone (CONTROL) or in 1 ml DCCM-1 medium with 1 segment of rat sciatic nerve (1SS) or with 4 segments of rat sciatic nerve (4SS) . After 16-24 hours, the monocytes were exposed to fluorescent beads and cell-associated fluorescence was measured by flow cytometry.
• FIGURE 9 illustrates the phagocytic activity of rat monocytes cultured for 2 hours with rat optic nerve. See text, Section 6, for experimental details. 2.5 x 10 5 rat monocytes were cultured in 1 ml DCCM-1 medium alone (CONTROL) or in 1 ml DCCM-1 medium with 4 segments of rat optic nerve (40S) . After 2 hours, the monocytes were exposed to fluorescent beads and cell-associated fluorescence was measured by flow cytometry.
• FIGURE 10 illustrates the phagocytic activity of rat monocytes cultured for 24 hours with rat optic nerve. See text. Section 6, for experimental details.
• rat monocytes 2.5 x 10 5 rat monocytes were cultured in 1 ml DCCM-1 medium alone (CONTROL) or in 1 ml DCCM-1 medium with 4 segments of rat optic nerve (40S) . After 24 hours, the monocytes were exposed to fluorescent beads and cell-associated fluorescence was measured by flow cytometry.
• FIGURE 11 illustrates the phagocytic activity of rat monocytes cultured overnight with rat sciatic nerve in the presence of medium conditioned by rat optic nerve.
• 5 x 10 5 rat monocytes were cultured in 1 ml DCCM-1 medium with 6 segments of rat sciatic nerve with no further additions (0) or with the addition of optic nerve-conditioned medium at a total protein concentration of 0.1 ⁇ g/ l (0.1), 1.0 ⁇ g/ l (i), or 10 ⁇ g/ml (10). After 24 hours, the monocytes were exposed to fluorescent beads and cell-associated fluorescence was measured by flow cytometry.
• FIGURE 12 illustrates nitric oxide production by rat monocytes cultured for 24, 48, 72 or 96 hours with rat sciatic nerve or with rat optic nerve. See text, Section 6, for experimental details. 10 6 rat monocytes were cultured in 1 ml DCCM-1 medium alone (CONTROL) , or in the same medium with 1 segment of rat sciatic nerve (1SS) , with 1 segment of rat optic nerve (10S) , or with four segments or rat optic nerve (40S) . After 24, 48, 72 or 96 hours, the media were collected and the levels of nitric oxide were measured.
• FIGURE 13 illustrates nitric oxide production by rat monocytes cultured for 72 hours with medium conditioned by rat sciatic nerve or rat optic nerve.
• rat monocytes were cultured in 1 ml DCCM-1 medium with no further additions or with the addition of sciatic nerve-conditioned medium or optic nerve- conditioned medium at a total protein concentration of 10, 100, 200 or 300 ⁇ g/ml. After 72 hours, the media were collected and the levels of nitric oxide were measured.
• FIGURE 14 illustrates axonal regeneration in transected optic nerves of rats following administration of optic nerve- stimulated monocytes combined with anti-inflammatory therapy. See text, Section 6, for experimental details.
• 2 ⁇ l DCCM-1 medium were applied to the site of injury containing no cells or 5 x 10 3 sciatic nerve- stimulated rat monocytes.
• some of the rats received an intraperitoneal injection of 0.8 mg dexa ethasone, producing the following treatment groups: no therapy (CONTROL) , dexamethasone only (DEX) , monocytes only (SS) , and both dexamethasone and monocytes (DEX/SS) .
• CONTROL no therapy
• DEX dexamethasone only
• SS monocytes only
• DEX/SS both dexamethasone and monocytes
• FIGURE 15 illustrates recovery of voluntary motor function following administration of stimulated rat monocytes to rats that have undergone complete spinal cord transection. See text. Section 7, for experimental details.
• the solid line presents the BBB loco otor scores (mean + SEM) for 8 out of 12 animals that showed motor recovery after spinal cord transection and treatment with stimulated monocytes, and the broken line presents the BBB locomotor scores of control animals following spinal cord transection.
• FIGURE 15B shows serial BBB locomotor scores for an individual animal subjected to spinal cord transection and treated with 4 x 10 5 stimulated monocytes plus aFGF.
• FIGURE 16 presents low-power micrographs of transected spinal cord in rats treated with stimulated monocytes (A) or control medium (B) , processed for immunohistochemical detection of GFAP (a) or neurofilament antigens (b) . See text. Section 7, for experimental details. Each picture is a montage of approximately 100 frames, each photographed at lOx magnification.
• FIGURE 17 presents high-power micrographs of transected spinal cord in rats treated with stimulated monocytes (A) or control medium (B) , processed for immunohistochemical detection of neurofilament antigens (a) or GAP-43 (b) . See text, Section 7, for experimental details. Bar: 2.5 ⁇ m.
• the present invention provides methods and compositions for use of allogeneic mononuclear phagocytes to promote axonal regeneration following injury or disease of the central nervous system (CNS) . Allogeneic mononuclear phagocytes are introduced at or near the site of CNS injury or disease.
• CNS central nervous system
• the term "mononuclear phagocytes" is intended to comprise, without limitation, monocytes obtained from central or peripheral blood, macrophages obtained from any site, including any tissue or cavity, macrophages derived by culturing macrophage precursors obtained from bone marrow or blood, dendritic cells obtained from any site, including spleen, lymph node, skin and lymphatic fluid, and dendritic cells derived from culturing dendritic cell precursors obtained from bone marrow or blood. Allogeneic mononuclear phagocytes can be obtained from the circulation or from any tissue in which they reside.
• Peripheral blood is an easily accessible ready source of allogeneic monocytes and is used as a source according to a preferred embodiment of the invention. Especially preferred is the use of autologous monocytes purified from the peripheral blood of a subject to whom the therapeutic preparation is intended to be administered.
• Allogeneic mononuclear phagocytes from other sources are well known in the art and include, without limitation, macrophages obtained from serosal cavities such as the peritoneal or pleural cavity, alveolar macrophages, and macrophages associated with other tissues (e.g. liver, spleen, thymu ⁇ ) , where they may be known by various terms such as Kupffer cells (in the liver) and microglial cells (in the CNS) . Allogeneic mononuclear phagocytes further include dendritic cells, which likewise may be known by various terms, such as Langerhans cells (in the skin), veiled cells (in lymphatic fluid) and interdigitating cells (in lymph nodes) .
• dendritic cells which likewise may be known by various terms, such as Langerhans cells (in the skin), veiled cells (in lymphatic fluid) and interdigitating cells (in lymph nodes) .
• mononuclear phagocytes can be derived by culture from allogeneic brain-derived mixed glial cells or from allogeneic precursor cells, which may be obtained from bone-marrow or blood.
• the allogeneic mononuclear phagocytes are not microglia and are not derived by culture from brain-derived mixed glial cells.
• cells other than mononuclear phagocytes are depleted from the cell population to be administered.
• Enrichment techniques include, without limitation, elutriation; centrifugation through material of suitable density, such as a Percoll gradient (Colotta et al., 1983, J. Immunol. 132 : 936-944) ; selective adhesion on suitable surfaces followed by removal at reduced temperature or at reduced concentrations of divalent cations (Rosen and Gordon, 1987, J. Exp. Med.
• 166: 1685-1701 f mechanical removal, or removal in the presence of lidocaine; and techniques for isolating dendritic cells from blood (O'Doherty et al. , 1993, J. Exp. Med. 178: 1067-1078) , bone marrow (Inaba et al. , 1992, J. Exp. Med. 176: 1693-1702) and lymphoid tissue (Macatonia et al., J. Exp. Med. 6_9: 1255-1264) .
• at least 50%, more preferably at least 70%, still more preferably at least 80%, and yet more preferably at least 90% of the cells are mononuclear phagocytes.
• a substantially purified preparation of mononuclear phagocytes e.g. a preparation in which at least 95% of the cells are mononuclear phagocytes.
• the mononuclear phagocytes may be used therapeutically at any desired time, according to the needs of the patient.
• the mononuclear phagocytes may, if desired, be cultured prior to administration in any suitable culture medium.
• the mononuclear phagocytes are cultured in a vessel made from sterile material to which these cells show limited or no adherence.
• the mononuclear phagocytes are cultured in sterile Teflon bags prior to administration.
• stimulated mononuclear phagocytes are mononuclear phagocytes with an enhanced capacity to promote axonal regeneration.
• the capacity of the mononuclear phagocytes to promote axonal regeneration is enhanced at least three-fold over non-stimulated mononuclear phagocytes, more preferably the capacity of the mononuclear phagocytes to promote axonal regeneration is enhanced at least 15-fold over non-stimulated mononuclear phagocytes.
• Stimulatory tissue, cells and biologically active agents are tissue, cells and biologically active agents that, when cultured together with mononuclear phagocytes, enhance the capacity of the mononuclear phagocytes to promote axonal regeneration.
• stimulatory tissue, cells or at least one stimulatory biologically active agent is added to the culture in order to enhance the capacity of the mononuclear phagocytes to promote axonal regeneration.
• one or more segments of a nerve most preferably a peripheral nerve such as the sciatic nerve, are added to the culture.
• a xenogeneic nerve is suitable for this purpose or, more preferably, an allogeneic or autologous nerve.
• a human nerve can be obtained from any available human tissue, such as a human cadaver or a surgical specimen (e.g. an amputated limb) .
• other stimulatory tissue or cells are added to the culture.
• Dermis is suitable for this purpose and can be obtained, from a living donor or a cadaver, by punch biopsy, by surgical resection, or by any other suitable technique. Especially preferred is skin obtained by punch biopsy, particularly skin obtained from a patient to whom the stimulated mononuclear phagocytes are intended to be administered. Synovial tissue, tendon sheath and liver are also suitable for this purpose, as are other regenerating tissues. Additional stimulatory tissues and cells can be identified according to the assay described below. If desired, the stimulatory tissue or cells are homogenized before addition to the culture. As will be evident to those skilled in the art, the stimulatory tissue or cell homogenate can be preserved, e.g. by cryopreservation, before use.
• At least one stimulatory biologically active agent is added to the culture in order to enhance the capacity of the mononuclear phagocytes to promote axonal regeneration.
• Neurotrophic factor 3 (NT3), nerve growth factor (NGF) , brain-derived neurotrophic factor, ⁇ - interferon (IFN- ⁇ ) , ⁇ -interferon (IFN- ⁇ ) , tumor necrosis factor ⁇ (TNF- ⁇ ) , interleukin 2 (IL-2) , interleukin 3 (IL-3) , interleukin 4 (IL-4) , interleukin 10 (IL-10) , monocyte chemotactic and activating factor (MCAF) , macrophage colony stimulating factor (M-CSF) , granulocyte-macrophage colony stimulating factor (GM-CSF) , colony stimulating factor 1 (CSF-1) , lipid A, fMet-Leu-Phe (fMLP) , muramyl dipeptide (MDP), the
• a biologically active protein or peptide may be used in its native or recombinant form, at a concentration (for each protein or peptide) of 1 to 5000 ng/ml, more preferably 10 to 5000 ng/ml, still more preferably 100 to 2500 ng/ml, most preferably about 1000 ng/ml.
• mononuclear phagocytes are stimulated by culturing them in medium to which IL-4 or IL-10 (and more preferably both IL-4 and IL-10) have been added.
• the mononuclear phagocytes are cultured together with stimulatory tissue, stimulatory cells, homogenate of stimulatory tissue or stimulatory cells, or at least one stimulatory biologically active agent for 24 hours. Shorter periods of culture, such as approximately 2 hours, are also effective, as are longer periods of culture, such as one or more weeks.
• stimulatory conditioned medium is prepared by incubating stimulatory tissue or cells, preferably one or more segments of a nerve, most preferably a peripheral nerve such as the sciatic nerve, in any medium that is suitable for culturing mononuclear phagocytes. After removal of the tissue or cells, mononuclear phagocytes are cultured in the stimulatory conditioned medium in order to enhance their capacity to promote axonal regeneration.
• the stimulatory conditioned medium can be stored and later used as desired for stimulating mononuclear phagocytes.
• Such stimulatory conditioned medium can be provided in the form of a commercial kit.
• the stimulatory conditioned medium is preserved during storage, for instance by refrigeration, whether as a liquid or as frozen medium.
• the stimulatory conditioned medium is lyophilized.
• the mononuclear phagocytes are exposed to a tyrosine kinase inhibitor, such as tyrphostine, before, during, or after stimulation, so as to reduce or eliminate undesired mononuclear phagocyte activities, such as secretion of TNF- ⁇ .
• the mononuclear phagocytes can be preserved, e . g. by cryopreservation, either before or after culture.
• Cryopreservation agents which can be used include but are not limited to dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183: 1394-1395; Ashwood-Smith, 1961, Nature 190: 1204-1205) .
• DMSO dimethyl sulfoxide
• glycerol polyvinylpyrrolidone 5 (Rinfret, I960, Ann. N.Y. Acad. Sci.
• the cooling procedure can be carried out by use of,
• a programmable freezing device or a methanol bath procedure.
• Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling.
• Programmable controlled-rate freezers such as
• samples can be cryogenically stored in mechanical freezers, such as freezers that maintain a temperature of about -80°C or about -20°C.
• samples can be cryogenically stored in liquid nitrogen (-196 °C) or its vapor.
• Such storage is greatly facilitated by the availability of highly efficient liquid nitrogen refrigerators, which resemble large Thermos containers with an extremely low vacuum and internal super insulation, such that heat leakage and nitrogen losses are kept to an absolute minimum.
• cryopreservation of viable cells or modifications thereof, are available and envisioned for use, e.g., cold metal-mirror techniques. See Livesey and Linner, 1987, Nature 327:255; Linner et al. , 1986, J. Histochem. Cytochem. 34(9) :1123-1135; see also U.S. Patent No. 4,199,022 by Senken et al., U.S. Patent No. 3,753,357 by Schwartz, U.S. Patent No. 4,559,298 by Fahy. Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-41°C) and chilled immediately upon thawing.
• cryoprotective agent if toxic in humans, should be removed prior to therapeutic use of he thawed mononuclear phagocytes.
• One way in which to remove the cryoprotective agent is by dilution to an insignificant concentration.
• the mononuclear phagocytes are suspended in a sterile pharmaceutically acceptable carrier and administered into the CNS of a mammal, including a human subject, at or near a site of injury or disease.
• the pharmaceutically acceptable carrier is PBS, a culture medium, or any pharmaceutically acceptable fluid in which the mononuclear phagocytes are suspended-
• alternative piiarmaceutically acceptable carriers will readily be apparent to those skilled in the art.
• treatment with inononuclaar phagocytes may optionally be combined with local or systemic anti- inflammatory therapy, for instance administration of (a) a steroid such, dexamethasone or methylprednisolone, (b) a non- ster ⁇ idal anti-inflammatory agent (NSAXD) such as aspirin, indomethacin, ibupro en, fenopro e ⁇ , ketoprofen or naproxen, or (c) an anti-inflammatory peptide, such as Thr-Lys-Pro (TKP) .
• NSAXD non- ster ⁇ idal anti-inflammatory agent
• NSAXD non- ster ⁇ idal anti-inflammatory agent
• NSAXD non- ster ⁇ idal anti-inflammatory agent
• TKP Thr-Lys-Pro
• the present invention encoirpasses the optional use of an anti-inflammatory agent at any dose that is effective in the subject t ⁇ be treated.
• Such effective doses are well known to those skilled in the art and include, for example, standard-dose therapy, such as systemic methylprednisolone 100 mg daily for a human adult, and ⁇ iigh-dose therapy, sxich as systemic methylprednisolone looo g daily for a human adult.
• treatment with mononuclear phagocytes may optionally be combined with concurrent administration to the CNS of one or more adjuvant factors.
• adjuvant factors suitable for this purpose include acidic fibroblast growth factor (aFGF) ; transforming growth factor-beta (TGF-/3) ; interleukin 6 (IL-6) ; neurotrophic factors, e.g.
• nerve growth factor NGF
• NT-3 neurotrophic factor 3
• NT-4 neurotrophic factor 4
• NT-5 neurotrophic factor 5
• BDNF brain-derived neurotrophic factor
• Each adjuvant factor can be administered at a dose of 6 to 10 ng/kg, either as a single dose or in repeated doses, e.g. at weekly intervals.
• one or more adjuvant factors are administered into the CNS at or near a site of disease or injury that is treated with mononuclear phagocytes, either together with, or shortly before or after administration of the mononuclear phagocytes.
• one or more adjuvant factors are administered regionally, such as by intraventricular administration for treatment of the brain, by intrathecal administration for treatment of the spinal cord, or by intraocular administration for treatment of the retina or optic nerve. Both native and recombinant adjuvant factors can be used.
• the present invention further encompasses combined treatment with (a) mononuclear phagocytes, (b) steroidal or non-steroidal anti-inflammatory therapy, and (c) one or more adjuvant factors.
• the mononuclear phagocytes are administered immediately following CNS injury and are introduced at the site of CNS injury, for example with a glass micropipette.
• the present invention encompasses administration of mononuclear phagocytes at any time (e.g. within a week, a fortnight, a month, 2 months, 3 months or 6 months) after the CNS sustains injury or disease, and encompasses introduction of the mononuclear phagocytes at or near a site of CNS injury or disease by any neurosurgically suitable technique.
• compositions and methods of the present invention are useful for treating any injury or disease of the CNS that results in or is accompanied by axonal damage.
• the injury or disease may be situated in any portion of the CNS, including the brain, spinal cord, or optic nerve.
• One example of such injury or disease is trauma, including coup or countercoup injury, penetrating trauma, and trauma sustained during a neurosurgical operation or other procedure.
• Another example of such injury or disease is stroke, including hemorrhagic stroke and ischemic stroke.
• Yet another example of such injury or disease is optic nerve injury accompanying optic neuropathy or glaucoma. Still further examples of CNS injury or disease will be evident to those skilled in the art from this description and are encompassed by the present invention.
• the optimal dose of mononuclear phagocytes is proportional to the number of nerve fibers affected by CNS injury or disease at the site being treated. In a preferred embodiment, the dose ranges from about 2.5 x 10 3 to about 10 5 mononuclear phagocytes for treating a lesion affecting about
• nerve fibers such as a complete transection of a rat optic nerve, and ranges from about 2.5 x 10 4 to about 10 6 mononuclear phagocytes for treating a lesion affecting about
• the dose ranges from about 10 4 to about 10 5 mononuclear phagocytes for treating a lesion affecting about 10 5 nerve fibers and ranges from about 10 5 to about 10 6 mononuclear phagocytes for treating a lesion affecting about 10 6 nerve fibers.
• the dose of mononuclear phagocytes can be scaled up or down in proportion to the number of nerve fibers affected at the lesion or site of injury being treated.
• the present invention provides an assay for identifying stimulatory tissues and cells and stimulatory biologically active agents.
• Mononuclear phagocytes are cultured together with the tissue or cells to be tested, in medium conditioned by the tissue or cells to be tested, or in medium to which the biologically active agent or agents to be tested have been added at various concentrations. Thereafter, the mononuclear phagocytes are assayed for phagocytic activity, or nitric oxide production. Mononuclear phagocytes with increased phagocytic activity or increased production of nitric oxide have an enhanced capacity to promote axonal regeneration. In a preferred embodiment, both phagocytic activity and nitric oxide production are measured, and mononuclear phagocyte stimulation is detected by observing an increase in either of these activities, more preferably in both of these activities.
• the phagocytic capacity of the mononuclear phagocytes is increased by at least 10 percent, more preferably by at least 25 percent, still more preferably by at least 50 percent.
• the nitric oxide production of the mononuclear phagocytes is increased by at least 50 percent, more preferably by at least 100 percent, and still more preferably by at least 200 percent.
• phagocytic activity is measured by contacting the mononuclear phagocytes with labeled particles and subsequently determining the amount of label associated with the cells.
• particles can be used for this purpose, including without limitation latex or polystyrene beads and naturally occurring cells, such as red blood cells, yeast and bacteria.
• the particles can be opsonized, for instance with immunoglobulin or complement.
• the particles can be labeled with any suitable marker, including without limitation a fluorescent marker (such as fluorescein or rhoda ine) , a radioactive marker (such as a radioactive isotope of iodine, carbon or hydrogen) , and an enzyme.
• the assay can be performed with unlabeled particles (e.g. red blood cells or yeast) ; the unlabeled particles are detected by any suitable method, such as microscopically, with or without staining.
• the mononuclear phagocytes are first contacted with fluorescent polystyrene beads; cell- associated fluorescence is subsequently measured by flow cytometry.
• nitric oxide production is measured by the Griess-reagent assay as described in Hibbs et al., 1987, Science 235:473-476 , which is incorporated herein by reference.
• other assays for nitric oxide production may be used, as are known to those of skill in the art. See, e.g., Packer (ed.), 1996, Methods in Enzymology 268 : 58-247 , which is incorporated herein by reference.
• the assay of the present invention also provides a means of determining the period of culture required in order to stimulate the mononuclear phagocytes.
• Mononuclear phagocytes are cultured for various periods with stimulatory tissue or cells, in medium conditioned by stimulatory tissue or cells, or in medium to which at least one stimulatory biologically active agent has been added. Thereafter, the phagocytic activity or nitric oxide production of the mononuclear phagocytes, or both these properties, are measured.
• Peripheral blood was pooled from adult Sprague-Dawley (SPD) rats. Monocytes were isolated by fractionation on a one-step Percoll gradient as previously described. F. Colotta et al., 1984, J. Immunol. 132:936-944. The monocyte- enriched fraction was recovered from the Percoll interface,
• each bag received 10 ml containing 1 x 10 7 cells.
• monocytes from SPD or Wistar rats were used, and were cultured in polypropylene _ 5 tubes or in Teflon bags.
• Non-stimulated monocytes were prepared by culturing isolated monocytes in a Teflon bag or polypropylene tubes, as
• Sciatic nerve-stimulated monocytes were prepared by culturing monocytes in a Teflon bag or polypropylene tubes for 2-24 hours together with at least one segment of a rat sciatic nerve.
• Optic nerve-stimulated monocytes were prepared by culturing
• the monocytes were 95% pure as determined by morphology and by immunocytochemistry with the monoclonal antibody EDI (Serotec, Oxford, England) as described. Hirschberg et al., 1994, J. Neuroimmunol . 50: 9-16
• Skin was also used to stimulate monocytes.
• 10 6 rat monocytes were cultured with a 1 cm x 1 cm square of skin obtained from germ-free rats by punch biopsy.
• rat skin was cultured in protein-free medium to produce skin-conditioned medium containing skin-derived proteins; 10 6 rat monocytes were then cultured with skin-conditioned medium containing 200 ⁇ g of protein.
• the lipophilic neurotracer dye 4- (4- (didecylamino) styryl) -N- methylpyridinium iodide (4Di-10ASP) (Molecular Probes, Eugene, Oregon, USA) was applied to the injured optic nerve, 2 mm distal to the site of injury.
• the retina was removed, prepared as a flattened whole mount in 4% paraformaldehyde solution, and examined by fluorescence microscopy to detect and count the number of labeled retinal ganglion cells (RGCs) in the entire retina. Only axons that had regrown past the site of injury to the site at which dye was applied could take up the dye and transport it retrogradely to the retinal ganglion cells.
• HRP horseradish peroxidase
• Monocytes from SPD or Wistar rats were suspended in DCCM-1 medium (10 6 cells in 1 ml) and were cultured without further additions or with the indicated number of syngeneic rat sciatic or optic nerve segments or with the addition of medium conditioned by syngeneic rat sciatic or optic nerve at the indicated concentrations of total protein. See Section 4 for details. After the indicated time in culture, the nerve segments (if any) were removed, the samples were centrifuged, and the supernatants were collected.
• Rats were subjected to optic nerve transection and treated at the time of injury with control medium or with 2.5 x 10 3 - 1 x 10 5 non-stimulated (NS) monocytes, 2.5 x 10 3 - 1 x
• the number of labeled retinal ganglion cells (RGCs) in rats from each treatment group is shown in Figure 1 as a percentage of RGCs labeled in normal optic nerves. Rats receiving no cells showed almost no labeling of RGCs. Rats receiving NS monocytes showed labeling of modest numbers of RGCs, while treatment with OS monocytes resulted in labeling of greater numbers of RGCs. In rats receiving SS monocytes, the median number of labeled RGCs was over 5-fold higher than in the rats treated with OS monocytes, and was about 15-fold higher than in the rats treated with NS monocytes.
• FIG. 3 Representative fluorescence micrographs of labeled RGCs in retinas after treatment with SS monocytes or control medium are shown in Figure 3.
• the absence of labeled RGCs following treatment with control medium indicates that transection was complete and that the labeled RGCs represent regenerating axons that traversed the site of transection and not merely fibers that escaped the experimental injury.
• the photomicrographs in Figure 4 further verify that regrowth has occurred.
• nerves treated with control medium (E) no labeled fibers could be seen distal to the site of HRP application.
• nerves treated with SS monocytes (A-D) labeled fibers were seen emerging from the proximal part of the nerve, crossing the site of transection (ST) and extending distally. Structures resembling growth cones (gc) were observed at the tips of these labeled fibers.
• Both rat and mouse sciatic nerve stimulated the capacity of monocytes to promote axonal regeneration.
• Rat monocytes were suspended at 2.5 x 10 5 cells in 1 ml
• DCCM-1 medium and were cultured for 2-24 hours without further additions (CONTROL) , with 1 segment of rat sciatic nerve (1SS) , with 2 segments of rat sciatic nerve (2SS) , or with 4 segments of rat sciatic nerve (4SS) .
• the phagocytic activity of the 2SS and 4SS preparations after 2 hours in culture is shown in Figure 7 relative to the phagocytic activity of CONTROL monocytes. After culture for 2 hours with two segments of sciatic nerve, the monocytes showed increased phagocytic activity; after culture for 2 hours with four segments of sciatic nerve, the monocytes showed a greater increase in phagocytic activity.
• the phagocytic activity of the 1SS and 4SS preparations after 24 hours in culture is shown in Figure 8 relative to the phagocytic activity of CONTROL monocytes.
• the monocytes After culture for 24 hours with one segment of sciatic nerve, the monocytes showed increased phagocytic activity; after culture for 24 hours with four segments of sciatic nerve, the increase in phagocytic activity was even greater.
• the 4SS preparation showed a greater increase in phagocytic activity after 24 hours than after 2 hours.
• Rat monocytes were suspended at 2.5 x 10 5 cells in 1 ml DCCM-1 medium and were cultured for 2-24 hours without further additions (CONTROL) or with 4 segments of rat optic nerve (40S) .
• the phagocytic activity of the 40S preparations after 2 hours in culture is shown in Figure 9 relative to the phagocytic activity of CONTROL monocytes. After culture for 2 hours with four segments of optic nerve, the monocytes showed a decrease in phagocytic activity.
• the phagocytic activity of the 40S preparations after 24 hours in culture is shown in Figure 10 relative to the phagocytic activity of CONTROL monocytes. After culture for 24 hours with four segments of optic nerve, the monocytes showed a decrease in phagocytic activity similar to that seen after 2 hours.
• Optic nerve conditioned medium was prepared by culturing 10 segments of rat optic nerve for 2 hours in 1 ml DCCM-1 medium. While fresh DCCM-1 medium is protein-free, the optic nerve conditioned medium contained protein. Rat monocytes were suspended at 2.5 x 10 5 cells in 1 ml DCCM-1 medium and were cultured for 24 hours with 1-6 segments of rat sciatic nerve without further additions (0) or with optic nerve conditioned medium at a total protein concentration of 10 ⁇ g/ml (10), 1 ⁇ g/ml (1) or 0.1 ⁇ g/ml (0.1).
• Figure 11 presents the phagocytic activity of monocytes cultured with sciatic nerve in the presence of optic nerve conditioned medium relative to the phagocytic activity of monocytes cultured with sciatic nerve in the absence of optic nerve conditioned medium.
• Addition of optic nerve conditioned medium attenuated the enhancement in phagocytic activity caused by culture with sciatic nerve. This attenuation was most marked in the preparation that received 0.1 ⁇ g/ml optic nerve conditioned medium.
• Similar results (not shown) were obtained when optic nerve segments were cultured in DCCM-1 medium for 8 hours and the resulting supernatants were dialyzed overnight at 4°C against PBS and subsequently stored at -20°C or -70°C.
• Figure 13 illustrates nitric oxide production of monocytes cultured for 72 hours with medium conditioned by rat sciatic nerve or rat optic nerve.
• Sciatic nerve- conditioned medium produced a statistically significant increase in nitric oxide production, whereas optic nerve- conditioned medium had no statistically significant effect. This result demonstrates that stimulation of mononuclear phagocytes by sciatic nerve is mediated by one or more soluble factors.
• monocytes administered at a site of CNS injury promoted axonal regeneration. All monocytes tested were effective at promoting axonal regeneration. However, monocytes were stimulated (i.e., showed an enhanced capacity to promote axonal regeneration) by culture with a nerve segment, especially with a segment of a peripheral nerve, e.g. sciatic nerve from rat or mouse. This stimulation was evident after all periods of culture tested, i.e. from 2-24 hours. For treating a total transection of a rat optic nerve, which contains about 10 5 nerve fibers, optimal results were obtained by administering about 5 x 10 3 monocytes. However, every dose tested showed a beneficial effect on axonal regeneration.
• Peripheral blood from adult Sprague-Dawley (SPD) rats was drawn into 10 ml syringes coated with heparin (5000 u/ml, Calbiochem, La Jolla, CA) , diluted with an equal volume of PBS, and subjected to fractionation on a one-step gradient of Percoll (1.077 g/ml, Pharmacia, Sweden) by centrifugation at 291 x g for 25 min at 30°C. See Colotta et al., 1984, J. Immunol. 132 : 936-944.
• the monocyte-enriched fraction was recovered from the Percoll interface, washed once with PBS to remove traces of Percoll, and resuspended at 1 x 10 6 cells/ml in DCCM-1 medium (Beit Ha ⁇ emek Ltd., Kibbutz Beit Ha'emek, Israel) .
• the cells were incubated in polypropylene tubes or Teflon bags at 37 °C, 5% C0 2 , with freshly excised segments of rat sciatic nerve (0.5 to 1.0 cm long) for 2 to 24 hours (0.4 - 5.0 x 10° cells/nerve segment).
• T8-T9 vertebrae Male Sprague-Dawley rats (Hebrew University, Jerusalem, Israel) , 200-300 g, were anesthetized with ketamine 40 mg/kg and xyline 100 mg/kg and incised dorsally to expose the T8-T9 vertebrae.
• the muscular insertions on the posterior and transverse vertebral processes were dissected and cut with a monopolar electrocautery device.
• T8 laminectomy was performed with a bone rongeur, without contusive injury to the underlying spinal cord.
• the spinal cord was transected with microscissors, and any remaining fibers were cut with a microknife.
• the underlying vertebral body was exposed through a gap of approximately 3 mm between the cut ends of the spinal cord.
• the exposed surface of the vertebral body and the lateral recesses were checked under high magnification to ensure that no fibers remained uncut. During the procedure, bleeding was controlled with bipolar electrocautery and by application of sterile gelfoam sponge material ( SPONGOSTANTM , Upjohn Co., Kalamazoo, MI). Experimental protocols and procedures were in accordance with NIH guidelines for animal research.
• Syngeneic peripheral blood monocytes were purified by one-step Percoll fractionation and co-cultured with segments of rat sciatic nerve, as described above. Prior to implantation, the sciatic nerve segments were removed and the cells were washed once and resuspended in fresh DCCM-1 medium and their viability determined. In one group of animals, fibrin glue from a commercial kit (Octacol-FI5, OMRIX Biopharmaceuticals SA, Brussels, Belgium) was applied to the gap created between the cut ends of the spinal cord.
• BAC component which contains human fibrinogen 50 mg/ml and other human plasma proteins, as well as tranexa ic acid 92 mg/ml
• thrombin component which contains human thrombin 1000 IU/ml and CaCl 2 40 mM
• monocyte suspension containing the indicated number of cultured monocytes (or control medium) were administered into the spinal cord parenchyma, distal (i.e. caudal) to the site of injury, with a Hamilton syringe.
• fibrin glue was not used, and the monocyte suspension (or control medium) was injected partly into the gap and partly into the distal parenchyma. No significant difference was observed between these fibrin and non-fibrin treatment groups, which were amalgamated for purposes of data analysis.
• aFGF (7.5 ⁇ g/ml, 5 ⁇ l/rat, Calbiochem Megapharm, Cat # 341580) was injected into the distal parenchyma. In all cases, the site of injury was covered with a film of SPONGOSTANTM and the wound was closed in layers.
• each rat underwent implantation of screw electrodes extradurally over the sensorimotor cortex of each cerebral hemisphere.
• rats were maintained under anesthesia (loading dose of ketamine 40 mg/kg and xylazine 10 mg/kg, administered ip and supplemented with one-third of the loading dose every 30 min) .
• a ground needle electrode was inserted transdermally near the dorsum of the neck.
• Contralateral muscle motor evoked potentials were elicited by stimulating the corresponding sensorimotor cortex with twin pulses of anodal stimulation from a Grass SD9 stimulator, applying 10 mA of constant current for 0.1 msec (with the cathodic electrode in the hard palate) .
• At least two motor-evoked potential traces averaging 50 sweeps were recorded from each muscle.
• a longitudinal skin incision was made along the anterior surface of each hindlimb.
• the aponeurotic layers were dissected to expose the gastrocnemius, tibialis anterior, quadriceps, adductors and biceps femoris.
• Monopolar needle electrodes were inserted in the exposed muscles to capture the evoked EMG signals, which were amplified and filtered (Microelectrode AC Amplifier, model 1800, A-M Systems, Everett, WA; 100 Hz to 5 kHz bandpass) ; then digitized (LABVIEW® for Macintosh, National Instruments, Austin, TX) ; and then stored. After the procedure, the skin was sutured and prophylactic antibiotics were administered for the next few days.
• GFAP glial fibrillary acidic protein
• GAP-43 growth-associated protein
• Rats were perfused transcardially with saline and 4% paraformaldehyde/PBS solutions. The spinal cords were removed, postfixed in paraformaldehyde, and immersed overnight in 20% sucrose in PBS. Cryosections (20 ⁇ m) of macrophage-treated and control spinal cords were prepared, placed on gelatin-coated slides, and dried at room temperature.
• Sections were fixed in absolute ethanol for 5 min at room temperature, washed several times in double- distilled water, and incubated for 5 min with 0.5% Tween-20 (Sigma, Israel) in PBS to enhance the permeability of the tissue. Sections were incubated at 37 °C with 5% bovine serum albumin in PBS for 30 min, then for 1-2 hours at room temperature with anti-GFAP antibody (Sigma, Israel; 1:100 dilution) , anti-GAP-43 antibody (Boerhinger-Mannheim, Germany; 1:100 dilution), or antibody raised against a mixture of 68 kDa and 200 kDA neurofilament proteins
• Rats were subjected to complete spinal cord transection and treated at the time of injury with (1) stimulated monocytes and aFGF; (2) stimulated monocytes alone; (3) (4) control medium and aFGF; and (4) control medium alone.
• the number of animals in each treatment group and the results obtained are summarized in Table 1.
• mice in the control groups showed either no movement of their hindlimbs or slight movement of one or two joints; the BBB locomotor score was mostly 2 or less, occasionally 4, and never exceeded 5.
• significant recovery was seen in 12 of the 22 animals in the monocyte-treated groups (groups 1 and 2) (ANOVA, p ⁇ 0.001). Recovery was manifested by extensive movement of all the joints of the hindlimbs, by plantar placement of the paws, and by weightbearing.
• BBB locomotor score 8 (i.e., sweeping movements with no weightbearing or plantar placement of the paw without weightbearing) and two treated animals attained a BBB locomotor score of 9 (i.e., plantar placement of the paw with weightbearing dorsal stepping and no plantar stepping, and a low-threshold contact placing response, which is considered to be a cortically-integrated reflex) .
• BBB locomotor score 9 (i.e., plantar placement of the paw with weightbearing dorsal stepping and no plantar stepping, and a low-threshold contact placing response, which is considered to be a cortically-integrated reflex) .
• group 1 showed recovery of motor activity
• FIG. 1 A and B illustrates progressive recovery of motor function after spinal cord transection in rats treated with monocytes. Additional experiments (data not shown) suggest that the added benefit of aFGF is less marked when animals are treated with greater numbers of monocytes.
• fibrin glue was placed in the gap between the cut ends of spinal cord; monocytes and aFGF were then administered into the fibrin (and not into the distal parenchyma) . No recovery was seen in this treatment group, suggesting that recovery depends on an adequate number of monocytes being accessible to the transected nerve at the site of the lesion and distal to the site of injury.

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