Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1825—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/06—Radiation therapy using light
  • A61N5/0613—Apparatus adapted for a specific treatment
  • A61N5/062—Photodynamic therapy, i.e. excitation of an agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/06—Radiation therapy using light
  • A61N5/067—Radiation therapy using light using laser light

Definitions

• AD Alzheimer's Disease
• Mahnnow et al., 2006 the etiology of the amyloid pathology is poorly understood.
• AD amyloid precursor protein
• presenilins 1 and 2 drive the uncontrolled formation of amyloid-beta
• the brain's pathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular amyloid plaques, the latter being a product of the amyloidogenic processing of APP and the resulting deposition of amyloid-beta in the brain parenchyma (Benilova et al., 2012; Hardy and Selkoe, 2002; Ittner and Gotz, 2011).
• Increasing aggregation of diffusible amyloid-beta peptides from the ISF and the CSF into toxic oligomeric intermediates and their accumulation in the brain parenchyma are believed to be precipitating factors for different neuroinflammatory abnormalities (Guillot-Sestier et al., 2015; Hong et al., 2016; Matarin et al., 2015), such as the formation of neurofibrillary tangles (Ittner and Gotz, 2011) and the pronounced neuronal dysfunction (Palop et al., 2007; Sun et al., 2009; Walsh et al., 2002) in the AD brain.
• MS Multiple Sclerosis
• Organs generally function less effectively with age. For example, skin becomes less elastic, muscle tone is lost, and heart function declines. Aging is a substantial risk factor for numerous neurological diseases, including neurodegenerative diseases and inflammatory neurological diseases.
• Modulating lymphatic vessels in accordance with some embodiments, are used to treat, prevent, or ameliorate symptoms of neurodegenerative diseases such as Alzheimer's disease (AD) and inflammatory neurological diseases such as multiple sclerosis (MS).
• AD Alzheimer's disease
• MS multiple sclerosis
• Some embodiments include a method of increasing flow of fluid in the central nervous system of a subject.
• the method comprises determining (e.g., identifying) the subject to be in need of increased fluid flow in the central nervous system, and administering an effective amount of a composition comprising, consisting essentially of, or consisting of a flow modulator such as a VEGFR3 agonist and/or Fibroblast Growth Factor 2 (FGF2) to a meningeal space of the subject in need, so that the amount of VEGFR3 agonist and/or FGF2 increases the diameter of a meningeal lymphatic vessel of the subject.
• the method increases fluid flow in the central nervous system of the subject.
• determining the subject to be in need of increased fluid flow comprises determining the subject to have a neurodegenerative disease, determining the subject to have a risk factor for the neurodegenerative disease, or both.
• the neurodegenerative disease is selected from the group consisting of one or more of the following: Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
• determining the subject to be in need of increased fluid flow comprises determining the subject to have Alzheimer's disease. In some embodiments, determining the subject to be in need of increased fluid flow comprises determining the subject to have a risk factor for AD selected from the group consisting of at least one of the following: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM), a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, familial AD, or a symptom of dementia.
• apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
• PICALM phosphatidylinositol-binding clathrin assembly protein
• the VEGFR3 agonist and/or FGF2 is administered selectively or otherwise localized to the meningeal space of the subject.
• the VEGFR3 agonist and/or FGF2 is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and/or FGF2, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and/or FGF2, or a combination of any of the listed routes.
• CSF cerebral spinal fluid
• the VEGFR3 agonist is administered. In some embodiments, the VEGFR3 agonist is selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant, or fragment thereof. In some embodiments, the effective amount of VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the risk factor for the neurodegenerative disease. In some embodiments, the effective amount of VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the neurodegenerative disease.
• the diameter of the meningeal lymphatic vessel is increased by at least 5%, 10%, 15%, 20%, 30%, 50% or more (e.g., when post-administration is compared to pre-administration). In some embodiments, the diameter of the meningeal lymphatic vessel is increased in a manner that increases flow of fluid in the CNS (e.g., brain), for example by at least 10%, 20%, 30%, 50%, or more (e.g., when post- administration is compared to pre-administration). In some embodiments, increasing fluid flow in the central nervous system of the subject comprises increasing a rate of perfusion of fluid throughout an area of the subject's brain.
• the central nervous system of the subject comprises amyloid-beta plaques, and wherein increasing the fluid flow reduces the quantity of amyloid-beta plaques. In some embodiments, increasing the flow reduces the quantity of accumulated amyloid-beta plaques by at least 5%. In some embodiments, at least some of the accumulated amyloid-beta plaques are in the meninges of the subject's brain. In some embodiments, increasing the fluid flow in the CNS increases clearance of soluble molecules in the brain of the subject (e.g., by at least 10%, 20%, 30%, 50%, or more).
• administration of the composition comprising the flow modulator in some embodiments increases the fluid flow in the CNS and increases clearance of soluble molecules in the CNS (e.g., brain, CSF) by more than about 10%, 20%, 30%, 50%, or more as compared to pre-administration.
• increasing the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
• CSF cerebral spinal fluid
• ISF interstitial fluid
• Some embodiments include the composition for use in increasing flow of fluid in the central nervous system of the subject.
• Several embodiments include a method of reducing a quantity of accumulated amyloid-beta plaques in a subject having a neurodegenerative disease or a risk factor therefor.
• the method comprises determining the subject to have the neurodegenerative disease or the risk factor, and administering a composition comprising, consisting of, or consisting essentially of a VEGFR3 agonist and/or FGF2 to a meningeal space of the subject, so that fluid flow in the central nervous system of the subject is increased.
• the method can reduce the quantity of accumulated amyloid-beta plaques in the subject.
• at least some of the accumulated amyloid-beta plaques are in the meninges of the subject's brain.
• the quantity of accumulated amyloid-beta plaques is reduced by at least 5%, 10%, 20% or more.
• the increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject's brain.
• administering the composition comprising, consisting of, or consisting essentially of the VEGFR3 agonist and/or FGF2 increases the diameter of a meningeal lymphatic vessel of the subject's brain by at least 5%, 10%, 15%, 20%, 30%, 50% or more, thus increasing fluid flow.
• flow of fluids in the CNS (e.g., brain) of the subject is increased by at least 10%, 20%, 30%, 40%, 50%, or more.
• the subject has the neurodegenerative disease.
• the method further comprises determining the subject to have the neurodegenerative disease.
• the neurodegenerative disease is selected from the group consisting of one or more of the following: Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychaitric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
• the subject has the risk factor for the neurodegenerative disease.
• the method further comprises determining the subject to have the risk factor for the neurodegenerative disease.
• the risk factor comprises a risk factor for Alzheimer's disease selected from the group consisting of one or more of the following: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM) , a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, familial AD, or a symptom of dementia.
• apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
• PICALM phosphatidylinositol-binding clathrin assembly protein
• CR3 complement receptor 1
• Siglee-3 variant in CD
• Some embodiments include a method of increasing clearance of molecules from a central nervous system (CNS) of a subject, comprising administering a composition comprising, consisting of, or consisting essentially of VEGFR3 agonist and/or FGF2 to a meningeal space of the subject, so that fluid flow in the CNS of the subject is increased.
• the method can thus increase clearance of molecules from the CNS of the subject.
• the increased clearance of molecules from the CNS of the subject comprises an increased rate of movement of molecules from the CNS to deep cervical lymph nodes.
• the increased clearance of molecules from the CNS of the subject reduces accumulation of the molecules in the brain.
• amyloid-beta plaques are cleared from the CNS of the subject.
• At least some amyloid-beta plaques are cleared from meningeal portions of the central nervous system of the subject.
• a quantity of accumulated amyloid-beta plaques in the CNS is reduced by at least 5%, 10%, 15%, 20%, or more.
• the increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject's brain.
• cognitive function of the subject for example in short- or long-term memory task, is improved.
• the method further comprises determining the subject to have a neurodegenerative disease, or a risk factor for a neurodegenerative disease.
• the neurodegenerative disease is selected from the group consisting of at least one of the following: Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
• AD Alzheimer's disease
• dementia dementia
• Parkinson's disease dementia
• cerebral edema amyotrophic lateral sclerosis
• PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections
• meningitis meningitis
• hemorrhagic stroke hemorrhagic stroke
• autism spectrum disorder ASD
• brain tumor and epilepsy.
• the method comprises determining the subject to have Alzheimer's disease.
• the method comprises determining the subject to have a risk factor for Alzheimer's disease selected from the group consisting of one or more of the following: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM) , a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, familial AD, or a symptom of dementia.
• the VEGFR3 agonist is administered.
• the VEGFR3 agonist is selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant, or fragment thereof.
• the VEGFR3 agonist and/or FGF2 is administered selectively to the meningeal space of the subject.
• the VEGFR3 agonist and/or FGF2 is administered to the subject by a route selected from the group consisting of one or more of the following: nasal administration, transcranial administration, contact cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and/or FGF2, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and/or FGF2, or a combination of any of the listed routes.
• the effective amount of VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the risk factor for the neurodegenerative disease.
• the effective amount of VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the neurodegenerative disease.
• the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
• CSF cerebral spinal fluid
• ISF interstitial fluid
• Some embodiments include the composition for use in increasing clearance of molecules from a central nervous system (CNS) of the subject.
• Some embodiments include a method of decreasing immune cell migration through a meningeal lymphatic vessel in a subject.
• the method comprises (a) administering a composition comprising, consisting of, or consisting essentially of a VEGFR3 antagonist to a meningeal space of the subject, or (b) ablating a meningeal lymphatic vessel of the subject, or a combination of (a) and (b).
• the method can thus decrease immune cell migration through the meningeal lymphatic vessel in the subject.
• the lymphatic vessels are selectively ablated by ligation, optical activation of visudyne in the lymphatic vessel, or both.
• the VEGFR3 antagonist is administered selectively to a meningeal space of the subject.
• the VEGFR3 antagonist is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 antagonist, or expression in the subject of a nucleic acid encoding the VEGFR3 antagonist, or a combination of any of the listed routes.
• CSF cerebral spinal fluid
• the VEGFR3 antagonist is administered to a subject who does not have a disease characterized by increase angiogenesis, for example cancer or a tumor.
• the VEGFR3 antagonist comprises an antibody specific for VEGFR3 or VEGF-c or VEGF-d.
• the method further comprises determining the subject to have an inflammatory neurological disease or a risk factor therefor.
• the risk factor is selected from the group consisting of at least one of the following: familial multiple sclerosis, infection, advanced age, suspicion that the subject has multiple sclerosis, or at least one symptom of inhibited neuromotor function.
• the inflammatory neurological disease comprises or consists essentially of a demyelinating disease of the central nervous system.
• the inflammatory neurological disease comprises or consists essentially of multiple sclerosis.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of immune cells from the parenchyma of the subject's brain to deep cervical lymph nodes of the subject.
• the movement is decreased by at least 5%, for example at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more (e.g., the amount of cells that migrate pre- and post-administration).
• inflammation in the CNS (e.g., brain) of the subject is decreased.
• the immune cell migration comprises migration of lymphocytes.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises decreasing a density of lymphocytes in the meningeal lymphatic vessel.
• the lymphocytes comprise or consist essentially of T cells.
• the density is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more (e.g., as a comparison of pre- and post- administration).
• inflammation in the CNS (e.g., brain) of the subject is decreased.
• decreasing immune cell migration through the meningeal lymphatic vessels decreases a quantity of activated T cells in the deep cervical lymph nodes that have a migratory phenotype.
• the migratory phenotype comprises a CDl la+ phenotype, a CD49d+ phenotype, or both.
• decreasing immune cell migration through the meningeal lymphatic vessel decreases a quantity of in T cells in the central nervous system that produce inflammatory cytokines.
• the inflammatory cytokines comprise IL-17, IFN-gamma, or both.
• neuromotor function of the subject is improved.
• Some embodiments include the composition for use in decreasing immune cell migration through a meningeal lymphatic vessel in the subject.
• Some embodiments include a method of reducing inflammation in the nervous system of a subject having an inflammatory neurological disease of the central nervous system, or a risk factor therefor.
• the method can comprise (a) administering a composition comprising, consisting of, or consisting essentially of a VEGFR3 antagonist to a meningeal space of the subject; or (b) ablating a meningeal lymphatic vessel of the subject; or a combination of (a) and (b), in which the VEGFR3 antagonist, ablation, or both, decrease immune cell migration through the meningeal lymphatic vessel in the subject.
• the method can thus reduce inflammation in the central nervous system.
• the inflammatory neurological disease comprises or consists essentially of a demyelinating disease of the central nervous system. In some embodiments, the inflammatory neurological disease comprises or consists essentially of multiple sclerosis. In some embodiments, the subject has the inflammatory neurological disease. In some embodiments, the subject has the risk factor for the inflammatory neurological disease. In some embodiments, the method further comprises determining that the subject has the risk factor for the inflammatory neurological disease. In some embodiments, the risk factor is selected from the group consisting of at least one of the following: familial multiple sclerosis, suspicion that the subject has multiple sclerosis, infection, advanced age, or at least one symptom of inhibited neuromotor function.
• the lymphatic vessels are selectively ablated by ligation, optical activation of visudyne in lymphatic vessels, or both.
• the VEGFR3 antagonist is administered selectively to a meningeal space of the subject.
• the VEGFR3 antagonist is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 antagonist, or expression in the subject of a nucleic acid encoding the VEGFR3 antagonist, or a combination of any of the listed routes.
• the VEGFR3 antagonist comprises or consists essentially of an antibody specific for VEGFR3 or VEGF-c or VEGF-d.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of immune cells from the parenchyma of the subject to deep cervical lymph nodes of the subject. In some embodiments, decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of lymphocytes from cerebral spinal fluid in the subject to deep cervical lymph nodes of the subject. In some embodiments, decreasing immune cell migration through the meningeal lymphatic vessel comprises decreasing a density of the immune cells in the meningeal lymphatic vessel. In some embodiments, the density is decreased by at least 10% (e.g., when comparing pre- and post- administration). In some embodiments, the immune cells comprise lymphocytes. In some embodiments, the lymphocytes comprise or consist essentially of T cells.
• reducing inflammation in the central nervous system comprises decreasing a quantity of activated T cells in the deep cervical lymph nodes that have a migratory phenotype.
• the migratory phenotype comprises a CDl la+ phenotype, a CD49d+ phenotype, or both.
• decreasing immune cell migration through the meningeal lymphatic vessels decreases a quantity of in T cells in the central nervous system that produce inflammatory cytokines.
• the inflammatory cytokines comprise IL-17, IFN-gamma, or both.
• the method further comprises ameliorating a neuromotor symptom in the subject.
• neuromotor function of the subject is improved.
• FIGs. 1A-M are a series of microscope images and graphs showing albuminal distribution of meningeal T cells and identification of Lyve- 1 expressing vessels adjacent to the dural sinuses.
• FIG. 1A is a schematic representation of the whole-mount dissection of the dura mater. SSS, superior sagittal sinus; TS, transverse sinus.
• FIG. IB is a representative images of CD3e labelling in whole-mount meninges (scale bar, 2,000 ⁇ ). Insets, higher magnification of the boxes highlighted in b (scale bar, 90 ⁇ (top inset) or 150 ⁇ (bottom inset)).
• DAPI 4',6-diamidino-2-phenylindole.
• FIG. 1A is a schematic representation of the whole-mount dissection of the dura mater. SSS, superior sagittal sinus; TS, transverse sinus.
• FIG. IB is a representative images of CD3e labelling in whole-mount meninges (scale bar,
• FIG. 1C is a schematic representation of a coronal section of whole-mount meninges.
• FIG. ID is a representative image of a coronal section of whole-mount meninges (scale bar, 200 ⁇ ).
• FIG. IE is a representative images of CD3e and CD31 immunolabelling in a coronal section of whole- mount meninges. Scale bar, 100 ⁇ . Inset, higher magnification of the box highlighted in the left panel (scale bar, 30 ⁇ , inset, 4 ⁇ ) shows CD31 labeling in the lumen and CD3e labeling outside the lumen.
• FIG. ID is a representative image of a coronal section of whole-mount meninges (scale bar, 200 ⁇ ).
• FIG. IE is a representative images of CD3e and CD31 immunolabelling in a coronal section of whole- mount meninges. Scale bar, 100 ⁇ . Inset, higher magnification of the box highlighted in the left panel (scale bar, 30 ⁇ , inset,
• FIG. 1G is a series of panels that show CD3e and MHCII express.
• the left panel shows representative images of CD3e and MHCII-expressing cells around the superior sagittal sinus (meningeal cartoons here and elsewhere depict the location of the presented images; scale bar, 50 ⁇ ).
• the middle panel shows higher magnification of the box highlighted on the left (scale bar, 10 ⁇ ).
• FIG. 1H is a representative image of CD31 and CD3e labelling around the superior sagittal signal (scale bar, 30 ⁇ ). Arrowheads indicate CD3e labeleing.
• FIG. 1J is a representative image of Lyve - 1 labelling on whole-mount meninges (scale bar, 1,000 ⁇ ).
• FIG. 1L is a representative image of CD31 and Lyve-1 labelling of a coronal section of the superior sagittal sinus (scale bar, 70 ⁇ ). CD31 and Lyve-1 labeling are each observed to the left of the dashed line, but generally do not overlap, and the sinusal lumen is shown with an arrowhead.
• FIG. 1M is an image and a corresponding inset showing higher magnification of a Lyve-1 positive vessel presenting a conduit-like structure (scale bar, 50 ⁇ ). Inset, xl.7 magnification of the Lyve-1 + vessel presented in the panel to the left; arrowhead points to the lumen of the vessel.
• FIGs. 2A-H are a series of microscope images and graphs showing Molecular and structural characterization of meningeal lymphatic vessels.
• FIG. 2A is a series of representative images of Proxl expression in the nuclei of Lyve-1 + vessels in the dural sinuses of Proxl tdT mice (scale bars, 10 ⁇ )( ⁇ order of left-to-right, the four panels show Lyve-1, Proxl -tdTOMATO, DAPI, and overlay).
• FIG. 2B is a series of representative images of podoplanin and Lyve-1 labelling on dural sinuses (scale bar, 40 ⁇ ) (in order of left-to-right, the three panels show Lyve-1, Podoplanin, and overlay).
• FIG. 2A is a series of representative images of Proxl expression in the nuclei of Lyve-1 + vessels in the dural sinuses of Proxl tdT mice (scale bars, 10 ⁇ )( ⁇ order of left-to-right, the four panels show Lyve
• FIG. 2C is a series of representative images of VEGFR3 and Lyve- 1 staining on dural sinuses (scale bar, 20 ⁇ )( ⁇ order of left-to-right, the three panels show VEGFR3, Lyve-1 , and overlay).
• FIGs. 2D and 2E Adult mice were injected i.c.v. (cisterna magna) with 4 ⁇ g of rhVEGF-c (Cysl56Ser) or with PBS. Meninges were harvested 7 and 14 days after the injection.
• FIG. 2D is a set of representative images of Lyve-1 and Proxl labelling of meninges at day 7 after injection (scale bars, 30 ⁇ ) (the panel on the left shows injection with PBS, and the panel on the right shows injection with rhVEGF-c).
• FIG. 2F and 2G are a series of representative images of smooth muscle cells (alpha-smooth muscle actin, oc-SMA) and Lyve- 1 labelling on dural sinuses (scale bars, 50 ⁇ (g) or 20 ⁇ )).
• the top panel shows Lyve-1 and the bottom panel shows oc-SMA.
• FIG. 2H is a representative low power micrograph (transmission electron microscopy) of a meningeal lymphatic vessel (scale bar, 5 ⁇ ). Inset, higher magnification of the box highlighted in FIG. 2H. Yellow arrowheads 1 show basement membrane; red arrowheads 2 show anchoring filaments (collagen fibres); and green arrowheads 3 show cellular junction. [0015] FIGs.
• FIGs. 3A-J are a series of microscope images and graphs showing functional characterization of meningeal lymphatic vessels. Representative z-stacks of the superior sagittal sinus of adult mice injected intravenously (i.v.) with fluorescein and intracerebroventricularly (i.c.v.) with QDot655 (n-3 mice).
• FIGs. 3A and 3B are low- magnification images showing fluorescein labelling in a meningeal blood vessel and in the superior sagittal sinus (scale bars, 20 ⁇ in FIGs. 3A and 3B). In contrast, QDot655 labelling (arrowheads) is prominent in the perisinusal vessel.
• FIG. 3C and 3D are coronal section of the z-sack presented in FIGs. 3A and 3B (scale bars, 20 ⁇ in FIGs. 3C and 3D).
• CSF cerebrospinal fluid.
• the arrowhead in FIG. 3E shows a CSF-filled vessel.
• FIG. 3E is a set of panels of a representative z-stack of cerebrospinal fluid-filled vessel from a mouse injected i.c.v. with both QDot655 and Alexa488-conjugated anti-Ly
• 3F is a set of panels showing image of immunolabelling for CD3e and MHCII along with Lyve-1 in the meninges.
• the top panel is a representative image of immunolabelling for CD3e and MHCII along with Lyve-1 in the meninges (scale bar, 15 ⁇ ).
• the bottom panel is a representative image of 3D reconstruction of the meningeal lymphatic vessels showing the luminal localization of the CD3e and MHCII-expressing cells (scale bar, 20 ⁇ ).
• FIGs. 3G and 3H adult mice were injected i.c.v. with 5 ⁇ of 10% Evans blue.
• Superficial cervical lymph nodes FIG. 3G, arrowheads
• deep cervical lymph nodes FIG.
• FIGs. 4A-G show meningeal immunity and lymphatic vessels in the dural sinuses.
• FIG. 4A is a representative image of CD31 staining in whole-mount meninges (scale bar, 2,000 ⁇ ).
• FIG. 4B is a representative images of T cells (CD3e, arrowheads) in the dura-arachnoid, pia, dural sinuses, and choroid plexus (scale bars, 70 ⁇ ).
• FIG. 3 J mean ⁇ s.e.m.
• FIG. 4E adult mice injected i.v. with 100 ⁇ of DyLight 488 lectin 5 min before euthanasia to enable labelling of the cardiovascular system.
• FIG. 4F adult mice were injected i.v. with 10 ⁇ g of FITC-conjugated anti-CD45 antibody or FITC-conjugated isotype antibody. Meninges were harvested one hour after the injection and labelled with anti-CD3e.
• FIG. 4F is a series of representative images of CD3e immunolabelling around dural sinuses are shown.
• FIG. 4G shows a representative 3D reconstruction of the lymphatic vessels localization around the superior sagittal sinus.
• FIG. 5 is a series of microscope images and graphs showing identification, characterization and validation of the expression of classical lymphatic endothelial cell markers by the meningeal lymphatic vessels.
• FIG. 5 A is a representative image of Proxl labelling on meningeal Lyve-1 expressing vessels (n-3 mice; scale bars, 10 ⁇ ).
• FIG. 5B is a schematic representation of the whole-mount dissection of the diaphragm.
• FIG. 5C shows characterization of the specificity of the podoplanin antibody.
• FIG. 5D shows characterization of the specificity of the VEGFR3 antibody.
• FIG. 5E shows quantification of the number of Proxl+ nuclei per mm2 of lymphatic vessel (mean ⁇ s.e.m.; n - 4 animals each group).
• FIGs. 6A-B is a series of graphs showing identification of meningeal lymphatic endothelial cell population by flow cytometry. FACS analysis of the lymphatic endothelial cells in diaphragm, skin (ear), and dural sinuses.
• FIG. 6A shows gating strategy employed to identify lymphatic endothelial cells (CD31+ podoplanin+). Lymphatic endothelial cells are identified as singlet, live cells, CD45- and CD31+ podoplanin+.
• FIG. 6B depicts representative dot plots for lymphatic endothelial cells (CD31+ podoplanin+) in the diaphragm, skin, and dura mater of adult mice.
• FIGs. 7A-E is a series of microscope images showing pilot identification of lymphatic vessels in human dura.
• FIG. 7A is a representative image of a formalin-fixed coronal section of human superior sagittal sinus.
• FIGs. 7B and 7C are each representative images of Lyve-1 staining on coronal section of human superior sagittal sinus (scale bar, 100 ⁇ ). The box in c highlights the presence of Lyve-1 -expressing macrophages in human meninges, as seen in mice.
• FIG. 7D is a set of representative images of Lyve-1 (left panel) and CD68 (right panel) staining of coronal sections of human superior sagittal sinus. Note the absence of CD68 positivity on Lyve-1 positive structures (scale bars, 50 ⁇ ).
• FIG. 7E is a representative images of podoplanin (right panel) and Lyve- 1 (left panel) staining of coronal sections of human superior sagittal sinus (scale bars, 50 ⁇ ).
• FIGs. 8A-J are a series of microscope images and diagrams showing initial lymphatic features of meningeal lymphatic vessels.
• FIG. 8A is a representative images of CCL21 (middle panel) and Lyve-1 (left panel) labelling of the meningeal lymphatic vessels (scale bars, 10 ⁇ ). Overlay is shown in the right panel
• FIGs. 8B and 8C are each representative images of VE-Cadherin and Lyve- 1 staining on meningeal blood vessels (FIG.
• FIGs. 8D-F are each representative images of Claudin-5 and Lyve-1 staining on meningeal blood (FIG. 8D) and lymphatic (FIG. 8E, for which left panel shows Claudin-5 and right panel shows Lyve-1) vessels, and diaphragm lymphatic vessels (FIG. 8F, for which left panel shows Claudin-5 and right panel shows Lyve-1); arrowheads point to Claudin-5 aggregates (scale bars, 10 ⁇ ).
• FIGs. 8D-F are each representative images of Claudin-5 and Lyve-1 staining on meningeal blood (FIG. 8D) and lymphatic (FIG. 8E, for which left panel shows Claudin-5 and right panel shows Lyve-1) vessels, and diaphragm lymphatic vessels (FIG. 8F, for which left panel shows Claudin-5 and right panel shows Lyve-1); arrowheads point to Claudin-5 aggregates (scale bars, 10 ⁇ ).
• FIG. 8G and 8H are each representative images of integrin-oc9 and Lyve-1 labelling on skin (FIG. 8G; ear) and meninges whole mount (FIG. 8H).
• FIG. 8G lower right panel shows Lyve-1
• the upper right panel shows integrin-oc9
• the left panel shows overlay.
• Scale bars 40 ⁇ .
• No integrin-oc9 expressing valves were detected in the meningeal lymphatic vessels.
• FIG. 81 is a representative low power micrographs (transmission electron microscopy) of the meningeal lymphatic vessels (scale bar, 2 ⁇ ); I, lumen; SC, supporting cell; BEC, lymphatic endothelial cell; BEC, sinusal endothelial cell.
• FIGs. 9A-C are a series of microscope images showing drainage of cerebrospinal fluid into the meningeal lymphatic vessels.
• FIG> 9A is a representative z-stack of QDot655 filled cerebrospinal fluid drainage both in the blood vasculature (sinus) and in the meningeal lymphatic vessels after i.c.v. injection (scale bar, 20 ⁇ ).
• FIG. 9B is a representative image of CD31 and Lyve-1 immunostaining on whole-mount meninges.
• Adult mice were injected i.c.v. with 2.5 ⁇ g of Alexa 488 conjugated anti-Lyve-1 antibody. Thirty minutes after the injection, the meninges were harvested and stained with anti-CD31.
• FIG. 9C is a representative z-stack of the superior sagittal sinus of adult mice injected i.v. with QDot655 and i.c.v. with Alexa488 conjugated anti-Lyve-1 antibody.
• panel "i" is a coronal section of the z-stack presented in panel c. The signal from the remaining skull and/or collagen-rich structure above the meninges was recorded (blue).
• FIG. 9C, panel ii is a reconstruction of the z-stack presented in panel c showing the localization of the meningeal lymphatic vessels under the skull (scale bars, 50 ⁇ ).
• FIGs. 10A-F are a series of microscope images and graphs showing meningeal lymphatic vessels carrying immune cells.
• FIG. 10A is a representative images of T cells (CD3e) and lymphatic endothelial cells (Lyve-1) on dural sinuses (scale bar, 20 ⁇ ).
• Panels ii and iii of FIG. 10A are, Orthogonal sections representing T-cell localization around Panel ii and within Panel iii the Lyve-1 structures (scale bars, 5 ⁇ ).
• FIG. 10C-D show representative images of Lyve-1 staining on dural meninges from CDl lcYFP mice (scale bars, 20 ⁇ ).
• CD 11c positive cells most probably dendritic cells
• FIG. 10E is a representative image of B220+ cells and lymphatic endothelial cells (Lyve-1) immunolabelling in the meninges (arrowheads indicate B220+CD11C- cells; scale bar 20 ⁇ ).
• FIG. 10F is a representative dotplot of B220+ cells (gated on singles, live, CD45+) within the dural sinuse expressing CD19; -40% of the B220+ cells express CD 19.
• FIGs. 11A-E are a series of microscope images and graphs showing draining of Evans blue from the meningeal lymphatic vessels but not the nasal mucosa into the deep cervical lymph nodes.
• FIGs. 11A-C Adult mice were injected i.c.v. with 5 ⁇ of 10% Evans blue. The meninges were harvested 30 min after injection and Evans blue localization was assessed by confocal microscopy.
• FIG. 1 1A is a representative image of Evans blue localization in both
• FIG. 1 1B is a representative profile of Evans blue (31) and Lyve-1 (32) relative fluorescence intensity on a cross-section of the image presented in FIG. 1 1A
• FIGs. 1 1D-E adult mice were injected intranasally with 5 ⁇ of 10% Evans blue.
• the successful targeting of the nasal mucosa (FIG. 11D) and the lack of accumulation of Evans blue in the deep cervical lymph nodes (FIG. 1 IE) 30 min after the injection are demonstrated.
• FIGs. 12A-H are a series of microscope images and graphs showing effects of deep cervical lymph node resection and of the lymphatic vessels ligation on the meningeal immune compartment.
• FIGs. 12A-E show the deep cervical lymph nodes were resected (xDCLN) or sham-operated. Three weeks after resection, the meninges were harvested, single cells isolated, and analysed for T-cell content by flow cytometry.
• FIG. 12A shows gating strategy to analyse meningeal T cells. Meningeal T cells are selected for singlets, CD45+, live cells and TCR +.
• FIG. 12B is a representative dot plot for CD8+ and CD4+ cells in meninges of sham and xDCLN mice.
• FIG. 12F shows representative images of the ligation surgery. To highlight the lymph vessels, Evans blue was injected i.c.v. before the surgery. Black arrowhead points to the node, yellow arrowhead points to the ligated Evans blue-filled vessels.
• FIG. 12G shows sham-operated or ligated animals were injected i.c.v. with 5 ⁇ of 10% Evans blue. The deep cervical lymph nodes were harvested 30 min after the injection and analysed for Evans blue content. Representative images of the Evans blue accumulation in the deep cervical lymph nodes of the sham-operated and ligated animals are presented.
• FIG. 12F shows representative images of the ligation surgery.
• FIGs. 13 A and 13B are schematic diagrams showing connection between the glymphatic system and the meningeal lymphatic system.
• FIGs. 14A-C are a series of microscope images and graphs showing photoablation of meningeal lymphatics.
• FIG. 14A shows schematic of the experiment.
• FIG. 14B shows Adult mice injected i.c.v. (cisterna magna) with 5 or 2( ⁇ g of Visudyne (or PBS as control), which was activated using 690nm laser (FIG. 14A). Meninges were stained for Lyve- 1 , Prox 1 and CD31 24 hrs post-ablation. Disruption of lymphatics in superior sagittal (FIG. 14B) and transverse sinuses (cFIG. 14C). In FIG.
• the top panel refers to a control
• the middle mane refers to 20,000 ng of Visudyne
• the bottom panel refers to 5,000 ng of Visudyne.
• FIGs. 15A-E are a series of diagrams, microscope images, and graphs showing modulation of the meningeal lymphatic affects drainage into the cervical lymph nodes.
• FIG. 15 A shows a scheme of the measurement of lymphatic drainage. Fluorescent microbeads (0.5 ⁇ in diameter - 5 ⁇ 1) were injected in the lateral ventricle of mice at a rate of 0.5 ⁇ 1/ ⁇ . 30min later, the deep cervical lymph nodes were harvested, sliced and immunostained for the presence of fluorescent beads.
• FIG. 15B is a pair of representative sections of deep cervical lymph nodes from sham operated (left) and ligated (right) mice immunostained for lymphatic vasculature (Lyve-1), fluorescent microbeads and Lyve-1.
• FIG. 15C shows quantification of the coverage of dCLN section by fluorescent beads in the sham and ligated mice.
• N 3-4 mice per group, Student-t- test.
• d Quantification of the coverage of dCLN section by fluorescent beads in PBS and Visudyne injected mice (24h after ablation).
• N 3-5 mice per group, *p ⁇ 0,05, Student-t-test.
• FIG.s 16A-B are a series of graphs showing impairment of lymphatic drainage in aged (24 months) and in J20 mice.
• FIGs. 17A-D are a series of graphs and microscope images showing meningeal immunity and meningeal lymphatic vessels during EAE.
• FIG. 17A C57B16/J mice were immunized with CFA/Mog35-55 and their meninges were dissected and analyzed at different time points after immunization. While on days 3 and 5, no change in diameter of meningeal lymphatic vessels was observed, on day 7 a significant increase was evident.
• FIG. 17B meninges excised from CFA/Mog35-55 immunized mice were also labeled for T cell (CD3) contents and cell numbers were enumerated. Significant decrease in cell counts was observed on day 7 with dramatic increase on day 13, at the onset of the disease.
• CD3 T cell
• FIG. 17C mice underwent survival surgery for deep cervical lymph nodes removal. Sham-operated animals and naive mice were used as controls. Three weeks after excision, animals were immunized with CFA/Mog35-55 and EAE was followed. Excision of deep cervical lymph nodes ameliorated EAE development and its severity, in line with previously published works39.
• FIG. 17D dult mice were subjected to deep cervical lymph node afferent lymphatic ligation or sham operated. On the same day, EAE was induced by subcutaneous injection of 200 ⁇ g emulsified MOG35-55 peptide in complete Freund adjuvant. Mice were scored daily to assess disease progression. Repeated measure 2-way ANOVA was used for statistical analysis. Panel ii of FIG. 17D shows an image of deep cervical lymph nodes from the indicated surgical procedure.
• FIGs. 18A-D are a series of microscope images and graphs showing photoablation of meningeal lymphatic vessels.
• FIGs. 19A-C are a series of images showing in vivo photoconversion of meningeal T cells expressing KikGR.
• FIG. 19 A SCID mice were reconstituted with CD4+ T cells expressing the fluorophore KikGR. Two weeks later the skull above the sagittal sinus was thinned and meningeal T cells were imaged by two-photon imaging in the anesthetized animal. After taking the pre-conversion image, the thinned mouse skull was exposed to an ultraviolet light source for 2-3 minutes before reimaging the same region post-conversion. The laser was tuned to lOOOnm (KikGR-GFP) or 1075nm (KikGR-RFP) for imaging.
• FIG. 19 A SCID mice were reconstituted with CD4+ T cells expressing the fluorophore KikGR.
• FIG. 19B-C show photoconversion via unthinned skull.
• FIG. 19B shows CD4+ KikGR reconstituted SCID mouse was exposed to focused UV light for 5 minutes with some areas protected from light by aluminum foil. The black box roughly denotes the imaging area.
• FIG. 19C shows the dura mater was isolated and immediately imaged by confocal microscopy. Photoconversion was observed in regions that received UV light (above the dotted line was shielded, below the dotted line was unshielded). Scale bar represents 50 ⁇ .
• FIGs. 20A-C are a series of images showing meningeal T cell depletion.
• Adult mice were transcranially injected with 15 ⁇ g of anti-mCD3e f(ab')2 or control f(ab')2 every other day for 6 days. Meninges were harvested 4 days after the last injection.
• FIG. 20A is a representative dot-plot of the meningeal CD4 T cells.
• FIG. 20B shows meningeal CD4 T cell quantification. As shown in FIG.
• OTI-GFP mice were transcranially injected with 15 ⁇ g of anti-mCD3e f(ab')2 of control f(ab')2 once, and the meninges were harvested 24h after the injection. Representative dot plot of the GFP+ populations in the meninges of control or injected mice are shown.
• FIGs. 21A-B are a series of images and a graph showing assessment of lymphatic drainage efficiency.
• Adult C57B16 mice were ligated or sham operated. Fifteen hours after the ligation, 5 ⁇ 1 of 0.5 ⁇ diameter fluorescent beads were injected into the right lateral ventricle at a rate of 0.5 ⁇ 1/ ⁇ . 30 min after the injection, the deep cervical lymph nodes were harvested, sliced (30 ⁇ thick) and strained for DAPI and lymphatic vasculature. The whole lymph nodes were imaged and the coverage of bead was measured.
• FIG. 21 A is a set of representative consecutive slices of deep cervical lymph node in sham (left series) and ligated (right series) animals. Notably, beads are not observed in the deep cervical lymph nodes of the ligated series.
• FIG. 21B shows quantification of the beads coverage in the sham and ligated animals. Each color represents one animal, each dot being one lymph node.
• FIG. 22 is a graph showing ablation of the meningeal lymphatic decreases EAE score.
• ICV cisterna magna
• Visudyne that was then photoconverted
• MOG 35-55 emulsion subcutaneously.
• mice were scored prior to sacrifice (brain, meninges, and spinal cord were harvested for IHC to measure the amount of demyelination and the infiltrate). This experiment also demonstrates that the ablation of the meningeal lymphatic system decreases disease severity.
• FIGs. 23A-H are a series of microscope images and graphs showing that that impairing meningeal lymphatic drainage in adult mice in accordance with some embodiments affects brain fluid homeostasis.
• FIGs. 24A-E are a series of microscope images and graphs showing that impairing meningeal vessels significantly decreases drainage into deep cervical lymph nodes.
• FIGs. 25A-B are a series of graphs showing that ablation of meningeal lymphatic vessels in old mice does not further aggravate influx of a CDF tracer in the brain.
• FIG. 26 is a graph showing that transcranial treatment with gel+VEGF- C156S had a significant effect on meningeal lymphatic vessel diameter.
• FIGs. 27A-D are a series of microscope images and graphs showing that transcranial application of VEGF-C in accordance with some embodiments leads to improved CSF influx into brain and memory in aged subjects.
• FIGs. 28A-C are a series of graphs showing that expression of an exogenous VEGF-C transgene by cells in the CNS increases flow.
• FIGs. 29A-F are a series of graphs showing that expression of an exogenous VEGF-C transgene by cells in the CNS improves cognitive performance as tested in the Morris water maze test.
• FIGs. 30A-B are a series of microscope images showing meningeal amyloid-beta deposition in models of Alzheimer's disease.
• FIGs. 31A-B are a series of graphs showing quantification of the total area of LYVE-1+ lymphatic vessels (FIG. 31A) and of the area occupied by ⁇ aggregates (FIG. 3 IB) in the meningeal whole-mounts of adult C57BL/6 mice.
• FIGs. 32A-C are a series of graphs showing meningeal lymphatic ablation increases amyloid-beta ( ⁇ ) aggregates in 5xFAD mice.
• FIGs. 33A-C are a series of graphs showing that meningeal lymphatic ablation exacerbates dementia symptoms in an AD model.
• FIGs. 34A-C are a series of graphs showing that expression of VEGF-C in the CNS ameliorates dementia symptoms in an AD model.
• FIG. 35 is a graph showing the quantification of lymphatic vessels immunostained by i.c.m. injected antibody and total lymphatic area (inset) at different time point post injection.
• FIGs. 36A-B are a series of graphs showing characteristics of meningeal lymphatic vessel structures.
• FIGs. 37A-F are a series of microscope images showing that show that T cells accumulate in meningeal lymphatics.
• FIG. 38 is a microscope image showing that exogenously injected T cells (CFSE) located with the meningeal lymphatics (Lyve-1) of the transverse sinus (CD31).
• CFSE exogenously injected T cells
• FIG. 39 is a microscope image showing that the exogenously injected DC (TAMRA - red) located within the meningeal lymphatics.
• FIGs. 40A-B are a series of graphs showing quantification of the percentage of KiKR CD4 T cells in the meninges, blood and nasal mucosa (FIG. 40A) and dCLN, sCLN and ILN (FIG. 40B).
• FIGs. 41A-B are a series of graphs showing activation and migration of T cells into the deep cervical lymph nodes.
• FIG. 42 is a graph showing density of T cells per mm of dCLN.
• FIGs. 43A-D are a series of graphs showing that meningeal T cells circulate into the cervical lymph nodes in a CCR7-CCL21 dependent manner.
• FIGs. 44A-E are a series of graphs showing that meningeal T cells circulate into the cervical lymph nodes in a CCR7-CCL21 dependent manner.
• FIGs. 45A-C are a series of graphs showing that meningeal dendritic cells circulate into the cervical lymph nodes.
• FIGs. 46A-G are a series of graphs showing that meningeal lymphatics is the main route for immune cells and macromolecules circulation into the cervical lymph nodes.
• FIG. 47 is a graph showing that exogenously-labeled T cells cycle in meningeal lymphatics.
• FIGs. 48A-D are a series of graphs showing that meningeal vasculature ablation in accordance with some embodiments herein affects immune cell size and coverage in the CNS.
• FIGs. 49A-D are a series of graphs showing that T cell migration is inhibited by the ablation of meningeal lymphatic vessels.
• FIGs. 50A-H are a series of graphs showing a lack of inflammation- induced lymphangiogenesis of the meningeal lymphatic endothelial cells.
• FIGs. 51A-D are a series of graphs showing that ablation of lymphatic drainage ameliorate MOG-specific T cells activation in the deep cervical lymph nodes resulting in ameliorated disease development.
• the central nervous system was viewed as being immune privileged, and was believed to lack a classical lymphatic drainage system.
• a lymphatic system is present in meningeal spaces, and functions in draining macromolecules, immune cells, and debris from the central nervous system (CNS).
• CNS central nervous system
• modulating drainage by the meningeal lymphatic drainage can affect certain diseases of the brain and central nervous system, but the effect of a given modulation is dependent on the particular disease (e.g., experiments described herein show that reducing meningeal lymphatic drainage can ameliorate some neurological diseases, while exacerbating others).
• reducing drainage by meningeal lymphatic vessels can reduce the flow in fluids of the CNS such as, cerebral spinal fluid (CSF) and interstitial fluid (ISF), and can exacerbate symptoms of neurodegenerative diseases characterized by increases in concentration and/or accumulations of molecules in the central nervous system, for example, Alzheimer's disease (AD).
• CSF cerebral spinal fluid
• ISF interstitial fluid
• Modulating lymphatic vessels to increase flow in accordance with some embodiments herein can alleviate symptoms of AD, including cognitive symptoms, and accumulation of amyloid-beta plaques.
• inhibiting immune cell migration through meningeal lymphatic vessels can ameliorate physiological and motor symptoms of inflammatory neurological diseases such as multiple sclerosis (MS).
• MS multiple sclerosis
• methods, compositions, and uses for treating, preventing, inhibiting, or ameliorating symptoms of neurodegenerative diseases associated with increased concentration and/or the accumulation of macromolecules, cells, and debris in the CNS for example, AD, which is associated with the accumulation of amyloid-beta plaques
• the methods, compositions, and uses can increase drainage by lymphatic vessel, and thus increase flow in CSF and ISF.
• methods, compositions, and uses for treating, preventing, inhibiting, or ameliorating symptoms of inflammatory neurological diseases such as MS are described.
• the methods, compositions, and uses can reduce and/or inhibit immune cell migration through lymphatic vessels.
• Several embodiments herein are particularly advantageous because they include one, several or all of the following benefits: (i) increased flow in the CNS; (ii) decreased accumulation of macromolecules, cells, or debris in the CNS (for example, decreased accumulation of amyloid-beta); (iii) maintenance of or improvement in cognitive function (for example memory function) in a subject suffering from, suspected of having, and/or at risk for dementia (such as in a neurodegenerative disease such as AD); (iv) decreased migration of activated immune cells (for example T cells) in the CNS; (v) decreased inflammation in the CNS; (iv) decreased immune-modulated destruction of myelinating cells such as oligodendrocytes; and/or (vii) maintenance of or improvement in motorneuron function in a subject suffering from, suspected of having, and/or at risk for an inflammatory neurological disease such as MS.
• flow shall be given its ordinary meaning and shall also refer to a rate of perfusion through an area of the central nervous system of a subject. Flow in some embodiments, can be measured as a rate at which a label or tracer in CSF perfuses through a particular area of the central nervous system (see, e.g., Example 1). As such, flow can be compared between two subjects or two sets of conditions by ascertaining how quickly an injected label or tracer perfuses throughout a particular area or volume of the brain and/or other portion of the CNS.
• flow modulators shall be given its ordinary meaning and shall also broadly refer to classes of compositions that can increase or decrease the passage of substances into and out of meningeal lymphatic vessels, and thus can modulate flow in CSF and ISF, and/or, can modulate immune cell migration within, into, and out of the meningeal lymphatic vessels.
• flow modulators increase the diameter of meningeal lymphatic vessels, which increases drainage, resulting in increased flow in the CSF and ISF. In some embodiments, flow modulators increase the number of meningeal lymphatic vessels, thus increasing net drainage, resulting in increased flow in the CSF and ISF.
• suitable flow modulators for increasing flow include, but are not limited to, VEGFR3 agonists, for example VEGF-c and VEGF-d, and Fibroblast Growth Factor 2 (FGF2), and functional fragments, variants, analogs, and mimetics of these molecules.
• VEGFR3 agonists for example VEGF-c and VEGF-d
• FGF2 Fibroblast Growth Factor 2
• reducing the size, diameter, accessibility, or quantity of meningeal lymphatic vessels can reduce migration of immune cells through the meningeal lymphatic vessels (see Example 2 and FIG. 24).
• limiting access to meningeal lymphatic vessels by immune cells limits migration of immune cells into and out of the meningeal lymphatic vessels, and thus limits their migration from one area to another. For example, migration of immune cells from the brain to or from the deep cervical lymph nodes via the meningeal lymphatic vessels can be restricted.
• flow modulators that decrease the diameter, size, quantity or function of meningeal lymphatic vessels, or by surgical procedures that minimize, limit access to, or ablate meningeal lymphatic vessels.
• suitable flow modulators for limiting access, size (e.g. decreasing diameter), quantity, function, or diameter of meningeal lymphatic vessels (and thus decreasing flow and drainage) in accordance with various embodiments herein include, but are not limited to, VEGFR3 antagonists, as well as compositions for ablating and inhibiting meningeal lymphatic vessels, for example visudyne.
• mechanically ablating or neutralizing meningeal lymphatic vessels can reduce flow and/or migration by immune cells into and/or out of the meningeal lymphatic vessels.
• a flow modulator e.g., VEGFR3 agonists, VEGFR3 antagonists, or FGF
• a flow modulator comprises or consists essentially of a polypeptide or protein that comprises a modification, for example a glycosylation, PEGylation, or the like.
• a composition or composition for use in accordance with methods and uses described herein comprises or consists essentially of one or more flow modulators (e.g., VEGFR3 agonists, VEGFR3 antagonists, FGF, or visudyne), and a pharmaceutically acceptable diluent or carrier.
• flow modulators e.g., VEGFR3 agonists, VEGFR3 antagonists, FGF, or visudyne
• suitable pharmaceutically acceptable carriers and formulations are described in "Remington: The Science and Practice of Pharmacy” 22nd Revised Edition, Pharmaceutical Press, Philadelphia, 2012, which is hereby incorporated by reference in its entirety.
• the composition comprises or consists essentially of a unit dose of a flow modulator effective for increasing flow of CNS fluids, increasing clearance of molecules in the CNS, reducing a quantity of accumulated amyloid-beta plaques, reducing immune cell migration, or reducing inflammation in accordance with methods or uses as described herein.
• the composition comprises, or consists essentially of a single unit dose of flow modulator effective for increasing flow, increasing clearance reducing accumulate amyloid- beta plaques, reducing immune cell migration, or reducing inflammation.
• the effective amount of flow modulator is about 0.00015 mg/kg to about 1.5 mg kg (including any other amount or range contemplated as a therapeutically effective amount of a compound as disclosed herein), is less than about 1.5 mg/kg (including any other range contemplated as a therapeutically effective amount of a compound as disclosed herein), or is greater than 0.00015 mg/kg (including any other range contemplated as a therapeutically effective amount of a compound as disclosed herein).
• VEGFR3 also known as FLT4, is a receptor tyrosine kinase, and its signaling pathway has been implicated in embryonic vascular development, and adult lymphangiogenesis. Upon binding of ligand, VEGFR3 dimerizes, and is activated through autophosphorylation. It is shown herein that VEGFR3 agonists are a class of flow modulators that increase the diameter of meningeal lymphatic vessels, and which increase drainage and the flow of CSF and ISF in accordance with some embodiments herein (see Examples 4-6, FIGs. 26, 27A-D, 28A, 28C).
• VEGFR3 agonists are suitable for methods, compositions, and uses for treating, ameliorating, reducing the symptoms of, or preventing neurodegenerative diseases associated with accumulation of molecules in the brain, for example AD, in accordance with some embodiments herein.
• a flow modulator comprises, consists of, or consists essentially of a VEGFR3 agonist.
• VEGFR3 agonist in accordance with methods, compositions, and uses herein can be understood in terms of its ability to increase meningeal vessel diameter, by its ability to increase flow of CSF or ISF, or by its ability to treat, ameliorate, or prevent, by its ability to increase clearance of substances from the CNS, symptoms of a neurodegenerative disease such as AD, for example quantities of beta-amyloid plaques or measurements of cognitive function.
• an effective amount of VEGFR3 agonist increases meningeal vessel diameter by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, including ranges between any two of the listed values.
• an effective amount of VEGFR3 agonist increases flow of the CSF or ISF by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, including ranges between any two of the listed values.
• Example VEGFR3 agonists suitable for methods, uses, and compositions in accordance with some embodiments herein include the polypeptides VEGF-c and VEGF-d, the amino acid sequences of which are shown in Table 1, below, as well as variants and analogs of VEGF-c and/or VEGF-d.
• VEGF-c in accordance with some embodiments herein has been demonstrated to increase the diameters of meningeal lymphatic vessels, and to increase drainage, CSF and ISF flow, and clearance in the CNS. See Example 4.
• a VEGFR3 agonist comprises, consists of, or consists essentially of VEGF-c.
• a VEGFR3 agonist comprises, consists of, or consists essentially of VEGF-d.
• VEGF-c and VEGF-d together agonize VEGFR3, and can be provided in a single composition, or in separate compositions.
• a VEGFR3 agonist comprises, consists or, or consists essentially of an analog, variant, or functional fragment, such as a mutant, ortholog, fragment, or truncation of VEGF- c or VEGF-d, for example a polypeptide comprising, or consisting essentially of an amino acid sequence having at least about 80% identity to SEQ ID NO: 1 or 2, for example at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity, including ranges between any two of the listed values.
• exogenous nucleotides encoding a VEGFR3 agonist can also be suitable for methods, uses, and compositions in accordance with some embodiments herein. Accordingly, in some embodiments, a nucleotide encoding VEGF-c or VEGF-d as describe herein is expressed in a subject in order to administer the VEGFR3 agonist to a subject.
• an exogenous vector such as a retroviral, lentiviral, adenoviral, or adeno-associated viral vector comprising or consisting essentially of a nucleic acid encoding a VEGFR agonist as described here can be inserted into a host nucleic acid of the subject (for example in the genome of a somatic cell of the subject).
• the vector further comprises transcriptional machinery to facilitate the transcription of the nucleic acid encoding the VEGFR agonist, for example, a core promoter, transcriptional enhancer elements, insulator elements (to insulate from repressive chromatin environments), and the like.
• the VEGFR3 agonist comprises a modification, for example a glycosylation, PEGylation, or the like.
• a composition for use in accordance with the methods described herein comprises the VEGFR3 agonist (e.g. VEGF-c and/or VEGF-d), and a pharmaceutically acceptable diluent or carrier.
• a flow modulator comprises, consists of, or consists essentially of a VEGFR3 antagonist.
• the flow modulator can comprise or consist of, or consist essentially of a VEGFR antagonist.
• a VEGFR3 antagonist includes an antibody specific for VEGFR3 or VEGF-c or VEGF-d.
• the antibody can comprise or consist essentially of a monoclonal antibody that binds specifically to VEGFR3 or VEGF-c or VEGF-d.
• antibodies can be generated against VEGFR3 or VEGF-c or VEGF-d in a host organism, such as a rodent, clones can be produced using hybridoma technology, and screens can be performed to identify hybridomas that produce monoclonal antibodies with suitable binding to VEGFR3 or VEGF-c or VEGF-d.
• a particular monoclonal antibody against VEGFR3 or VEGF-c or VEGF-d be further screened for variants which desired properties, for example higher affinity to VEGFR3 or VEGF-c or VEGF-d.
• a screen can be performed using techniques known to the skilled artisan, for example randomly mutating nucleic acid sequences encoding hypervariable regions of the antibody, and using phage display technology to screen for high affinity variants.
• the VEGFR3 or VEGF-c or VEGF-d antibody comprises or consists essentially of a chimeric, humanized, or fully human antibody.
• the VEGFR3 or VEGF-c or VEGF-d antibody binds specifically to an extracellular domain of VEGFR3 or VEGF-c or VEGF-d.
• An example polypeptide sequence of human VEGFR3 is available as Uniprot Accession No. P35916, and is provided herein as SEQ ID NO: 3.
• An example polypeptide sequence of human VEGF-c is available as Uniprot Accession No. P49769, and is provided herein as SEQ ID NO: 1.
• An example polypeptide sequence of human VEGF-d is available as Uniprot Accession No. 043915, and is provided herein as SEQ ID NO: 2.
• molecules that functionally have the same or similar effects as a VEGFR3 antagonist can be used instead of a VEGFR3 antagonist, even if these molecules do not directly interact with VEGFR3.
• molecules that neutralize VEGFR ligands such as VEGF-c and/or VEGF-d can reduce VEGFR3 signaling.
• an antibody specific for VEGF-c or VEGF-d can be used in the place of a VEGFR3 antagonist.
• a decoy molecule functions to inhibit VEGFR3 signaling, and can be a VEGFR3 antagonist in accordance with methods, compositions, and uses herein.
• an inactive VEFGR3 fragment or mutant can be used to reduce or inhibit VEGFR3 signaling.
• a shorter secreted isoform of VEGFR3, "isoform 3" (SEQ ID NO: 4) has been shown to inhibit VEGFR3 signaling by binding to VEGFR3 agonists like VEGF-c and VEGF-d, thus reducing the amount of ligand available to activate functional VEGFR3.
• the flow modulator comprises or consists essentially of Fibroblast Growth Factor 2 (FGF2).
• FGF2 Fibroblast Growth Factor 2
• FGF2 can increase drainage (and flow) of CSF or ISF in meningeal lymphatic vessel, for example by increasing the diameter of meningeal lymphatic vessel.
• An example of a suitable FGF2 amino acid sequence in accordance with some embodiments is provided as Unitprot Accession No. P09038 (human FGF2) (SEQ ID NO: 5).
• Visudyne is a substance which can accumulate in meningeal lymphatic vessels, and, upon activation with 689nm non-thermal red light, can release oxygen species, ablating or destroying meningeal lymphatic vessels. Accordingly, Visudyne can be suitable as a flow modulator in accordance with some embodiments herein as an inhibitor of meningeal lymphatic vessels, which in turn reduces or inhibits passage of substances such as immune cells through meningeal lymphatic vessels, and/or flow. In some embodiments, the flow modulator comprises or consists essentially of visudyne.
• Flow modulators in accordance with methods, compositions for use, or uses of embodiments herein can be administered to a subject using any of a number of suitable routes of administration, provided that the route of administration administers the flow modulator to the meningeal space of a subject. It is noted that many compounds do not readily cross the blood-brain barrier, and as such, some routes of administration such as intravenous will not necessarily deliver the flow modulator to the meningeal space (unless the flow modulator can readily cross the blood-brain barrier).
• administering to the meningeal space of a subject it is not necessarily required that a flow modulator be administered directly to the meningeal space, but rather, this term encompasses administering a flow modulator directly and/or indirectly to the meningeal space. It is contemplated that administering the flow modulator so that it is in fluid communication with the meningeal space of the subject in accordance with some embodiments herein (typically by administering the flow modulator on the "brain" side of the blood-brain barrier), the flow modulator will be administered to the meningeal space. Accordingly, in some embodiments, the flow modulator is not administered systemically.
• the flow modulator is not administered systemically, but rather is administered to a fluid, tissue, or organ in fluid communication with the meningeal space, and on the brain side of the blood-brain barrier. In some embodiments, the flow modulator is not administered systemically, but rather is administered to the CNS. In some embodiments, the flow modulator is administered to the CNS, but is not administered to any organ or tissue outside of the CNS. In some embodiments, the flow modulator is not administered to the blood. In some embodiments, the flow modulator is not administered to a tumor, or to the vasculature of a tumor.
• the flow modulator is administered nasally.
• the flow modulator can be provided in a nasal spray, or can be contacted directly with a nasal mucous membrane.
• the flow modulator is administered through contacting with CSF of the subject.
• the flow modulator can be directly injected into CSF of a patient (for example into a ventricle of the brain).
• Suitable apparatuses for injection can include a syringe, or a pump that is inserted or implanted in the subject and in fluid communication with CSF.
• a composition comprising or consisting essentially of the flow modulator, for example a slow-release gel, is implanted in a subject so that it is in fluid communication with CSF of the subject, and thus contacts the CSF.
• the flow modulator is administered transcranially.
• a composition comprising or consisting essentially of the flow modulator such as a gel can be placed on an outer portion of the subject's skull, and can pass through the subject's skull.
• the flow modulator is contacted with a thinned portion of the subject's skull to facilitate transcranial delivery.
• the flow modulator is administered by expressing a nucleic acid encoding the flow modulator in the subject.
• a vector comprising or consisting essentially of the nucleic acid for example a viral vector such as a retroviral vector, lentiviral vector, or adenoviral vector, or adeno-associated viral vector (AAV) can be administered to a subject as described herein, for example via injection or inhalation.
• expression of the nucleic acid is induced in the subject, for example via a drug or optical regulator of transcription.
• the flow modulator e.g. the VEGFR3 agonist, FGF2, or VEGFR3 antagonist
• the flow modulator is administered selectively to the meningeal space of the subject, or is for use in administration selectively to the meningeal space of the subject.
• administered "selectively" and variations of the root term indicate that the flow modulator is administered preferentially to the indicated target (e.g. meningeal space) compared to other tissues or organs on the same side of the blood brain barrier.
• direct injection to meningeal spaces of the brain would represent "selective" administration, whereas administration to CSF in general via a spinal injection would not.
• the flow modulator is administered selectively to the meningeal space, and not to portions of the CNS outside of the meningeal space, nor to any tissues or organs outside of the CNS. In some embodiments, the flow modulator is administered selectively to the CNS, and not to tissue or organs outside of the CNS such as the peripheral nervous system, muscles, the gastrointestinal system, musculature, or vasculature.
• a flow modulator can be administered in a single administration, or in two or more administrations, which can be separated by a period of time.
• the flow modulator as described herein can be administered via a route of administration as described herein hourly, daily, every other day, every three days, every four days, every five days, every six days, weekly, biweekly, monthly, bimonthly, and the like.
• the flow administration is administered in a single administration, but not in any additional administrations.
• Some embodiments include methods of making a composition or medicament comprising or consisting essentially of a flow modulator as described herein suitable for administration according to a route of administration as described herein.
• a composition comprising or consisting essentially of a VEGFR3 agonist is prepared for nasal administration, administration to the CSF, or transcranial administration.
• a composition comprising or consisting essentially of a VEGFR3 antagonist is prepared for nasal administration, administration by contacting with CSF, or transcranial administration.
• Methods, uses, and compositions in accordance with some embodiments herein can be useful for treating, preventing, inhibiting, ameliorating, or reducing the symptoms of one or more neurodegenerative diseases, or compositions for use in these methods.
• These diseases can occur in subjects, for example humans, as well as non-human animals, such as non-human mammals, and non-human primates in particular.
• neurodegenerative, neurodevelopmental, neuroinflammatory, or neuropsychiatric diseases associated with accumulation of macromolecules, cells, and debris in the CNS are treated, prevented, inhibited, or reduced by methods, uses, or compositions that increase flow, drainage, and/or clearance in meningeal lymphatic vessels.
• neurodegenerative diseases associated with accumulation of macromolecules, cells, and debris in the CNS are treated, prevented, inhibited, or reduced.
• neurodgenerative diseases include Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
• AD Alzheimer's disease
• Parkinson's disease dementia
• cerebral edema amyotrophic lateral sclerosis
• PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections
• meningitis meningitis
• hemorrhagic stroke hemorrhagic stroke
• autism spectrum disorder ASD
• brain tumor and epilepsy.
• the neurodegenerative disease can be prevented, treated, or ameliorated prophylactically.
• a subject having one or more risk factors for the neurodegenerative disease can be determined to be in need of receiving a method, use, or composition described herein.
• a subject may have accumulated amyloid-beta plaques in their CNS, and may benefit from increased flow, increased drainage, increased clearance and/or reduction of amyloid-beta plaques, even if they do not yet have an AD diagnosis based on cognitive symptoms.
• a number of risk factors for AD are suitable as risk factors in accordance with methods, compositions, and uses of some embodiments herein, for example familial AD, a genetic marker for AD, or a symptom of AD such as early dementia.
• the foremost risk factor for sporadic AD is age.
• increased risk of this form of AD has also been attributed to diverse genetic abnormalities.
• diploidy for apolipoprotein-E84 ( ⁇ - ⁇ 4) widely viewed as a major genetic risk factor promoting both early onset of amyloid-beta aggregation and defective amyloid-beta clearance from the brain (Deane et al., 2008; Zlokovic, 2013).
• the risk factor for AD is selected from the group consisting of at least one of the following: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM), a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, or a symptom of dementia.
• apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
• PICALM phosphatidylinositol-binding clathrin assembly protein
• CR3 complement receptor 1
• Siglee-3 a variant in CD33
• TREM2 myeloid cells 2
• Methods, uses, and compositions in accordance with some embodiments herein can be useful for treating, preventing, inhibiting, ameliorating, or reducing the symptoms of one or more inflammatory neurological diseases including but not limited to, demyehnating diseases of the central nervous system and multiple sclerosis (MS). These diseases can occur in subjects, for example humans, as well as non-human animals, such as non-human mammals, and non-human primates in particular. Without being limited by theory, it is contemplated, according to several embodiments herein, that meningeal lymphatic vessels function in regulation of tissue immune surveillance in addition to removing macromolecules, and debris. As shown herein, immune cells are found in, and pass through the meningeal lymphatic vessels. Examples 14-22.
• methods, uses, or compositions are for treating a subject suffering from, suspected of having, or at risk for an inflammatory neurological disease.
• methods, uses, or compositions are for treating a subject suffering from, suspected of having, or at risk for an inflammatory neurological disease who does not have cancer.
• methods, uses, or compositions are for treating a subject suffering from, suspected of having, or at risk for an inflammatory neurological disease who does not have a tumor.
• methods, uses, or compositions are for treating a subject suffering from, suspected of having, or at risk for an inflammatory neurological disease who does not have a disease characterized by increased angiogenesis such as, for example, a cancer or tumor.
• inflammatory neurological diseases are treated, prevented, inhibited, or reduced by methods, uses, or compositions that reduce, inhibit, or prevent migration of immune cells through meningeal lymphatic vessels.
• diseases include inflammatory diseases, in which the activation and proliferation of immune cells such as T cells into to the CNS is facilitated by migration of these cells meningeal lymphatic vessels.
• diseases include inflammatory diseases in the central nervous system, for example demyelinating diseases such as MS.
• the inflammatory disease (such as MS) can be prevented, treated, or ameliorated prophylactically, and as such, a subject having risk factors for MS can be determined to be in need of receiving a method, use, or composition described herein.
• a subject may have T cell infiltration in their CNS, and may benefit from decreasing the migration of immune cells through meningeal lymphatic vessels, for example by decreasing access to, diameter of, and/or quantity of meningeal lymphatic vessels, even if they do not yet have any large-scale demyelination, substantial motor impairment symptoms, or a classical MS diagnosis.
• a number of risk factors for MS are suitable as risk factors in accordance with some embodiments herein, for example familial MS, a genetic marker for MS, demyelination, a reduction in oligodendrocytes, infection, advanced age, or a symptom of MS such as loss of motor neuron function.
• Some aspects include methods of, compositions for use, or uses for increasing flow in fluid in the central nervous system of a subject, or compositions for use in these methods.
• the methods or uses can include determining whether the subject is in need of increased fluid flow in the central nervous system. If the subject is in need of increased fluid flow, the method or use can include administering an effective amount of VEGFR3 agonist to a meningeal space of the subject.
• the amount of VEGFR3 agonist can increase flow for example, by increasing the diameter of a meningeal lymphatic vessel of the subject, by increasing the quantity of meningeal lymphatic vessels of the subject, and/or by increasing drainage through meningeal lymphatic vessels of the subject.
• the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
• the VEGFR3 agonist comprises VEGF-c or VEGF-d or an analog, variant, or fragment thereof. It is also contemplated that for methods and uses in some embodiments herein, FGF2 can be substituted for the indicated VEGFR3 agonist in order to increase flow, or can be used in addition to a VEGFR3 agonist in order to increase flow.
• compositions for, or use for increasing fluid flow in the CNS can be useful for treating, preventing, or ameliorating the symptoms of neurodegenerative diseases associated with the increased concentration and/or accumulation of molecules or cells or debris in the CNS.
• a subject can be determined to be in need of increased fluid flow by determining whether the subject has a neurodegenerative disease, or is at risk of developing a neurodegenerative disease.
• the disease can be associated with the increased concentrations and/or accumulation of molecules or cells or debris in the CNS, for example Alzheimer's Disease (AD).
• AD Alzheimer's Disease
• the subject can be determined to be at risk for the disease, for example through having familial occurrence of the disease, by having one or more genetic markers associated with the disease, through advanced age, or by exhibiting symptoms of the disease, for example early dementia in the case of AD.
• advanced age refers to an age characterized by a decrease in memory function, decrease in CSF production, substantial increases in neuronal senescence, and in the context of some embodiments, can include at least 65 years of age in a human, for example, at least 60, 65, 70, 75, 80, or 85, including ranges between any of these values.
• determining whether the subject is in need of increased fluid flow comprises determining the subject to have a neurodegenerative disease such as AD. In some embodiments, determining whether the subject is in need of increased fluid flow comprises determining the subject to have a risk factor for the neurodegenerative disease associated with the increased concentration and/or accumulation of molecules or macromolecules or cells or debris in the CNS as described herein. In some embodiments, determining whether the subject is in need of increased fluid flow comprises determining the subject to have a risk factor, and also determining the subject to have the disease itself.
• the neurodegenerative disease is selected from the group consisting of at least one of the following: Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
• AD Alzheimer's disease
• dementia dementia
• Parkinson's disease cerebral edema
• ALS amyotrophic lateral sclerosis
• PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections
• ASD autism spectrum disorder
• brain tumor and epilepsy.
• the neurodegenerative disease is Alzheimer's disease.
• the risk factor is a risk factor for Alzheimer's disease as described herein.
• the VEGFR3 agonist and/or FGF2 is administered to the subject after determining that the subject has a risk factor for the neurodegenerative disease (even if the subject does not necessarily have the disease itself), for example for prophylactic treatment or prevention. In some embodiments, the VEGFR3 agonist and/or FGF2 is administered to the subject after determining that the subject has the neurodegenerative disease.
• VEGFR3 agonist and/or FGF2 are administered selectively to the meningeal space of the subject.
• the VEGFR3 agonist and/or FGF2 is administered to the meningeal space, but is not administered outside the CNS.
• the VEGFR3 agonist and/or FGF2 is administered to the meningeal space, but is not administered to the blood.
• the VEGFR3 agonist and/or FGF2 is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and/or FGF2, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and/or FGF2, or a combination of any of the listed routes.
• it is the VEGFR3 agonist that is administered.
• the VEGFR3 agonist is selected from the group consisting of at least one of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
• the administration of the VEGFR3 agonist results in an increase in CNS fluid flow, meningeal lymphatic vessel diameter, meningeal lymphatic vessel number, meningeal lymphatic vessel drainage, or amelioration of symptoms of a neurodegenerative disease.
• the administration of the VEGFR3 agonist increases diameter of the meningeal lymphatic vessel is increased by at least about 5%, for example at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including ranges between any two of the listed values.
• an average diameter of a population of meningeal lymphatic vessels of the subject is increased by a value noted herein.
• the administration of the VEGFR3 agonist increases fluid flow in the central nervous system of the subject, comprising increasing a rate of perfusion of fluid throughout an area of the subject's brain.
• the administration of the VEGFR3 agonist increased the ISF flow, which in turn reduces the quantity of amyloid-beta plaques in the subject's CNS.
• the quantity of accumulated amyloid-beta plaques can be reduced by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, including ranges between any two of the listed values. It is shown herein that some brains of humans with AD have structures resembling amyloid-beta plaques in the meninges (see FIG. 30B). Accordingly, in some embodiments, at least some of the accumulated amyloid-beta plaques are in the meninges of the subject's brain.
• increasing the fluid flow increases clearance of soluble molecules in the brain of the subject. Clearance of soluble molecules can be ascertained, for example, by monitoring the retention of a particular compound, molecule, or label over an area of the brain over a particular period of time. In some embodiments, increasing the fluid flow increases clearance of soluble molecules in the brain of the subject by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, including ranges between any two of the listed values.
• Some aspects include methods, compositions for use, and uses for reducing a quantity of accumulated amyloid-beta plaques, or decreasing the rate of accumulation of amyloid-beta plaques, in a subject having a neurodegenerative disease or a risk factor for such a disease, or compositions for use in such methods.
• the methods or uses can include determining the subject to have the neurodegenerative disease or the risk factor.
• the methods or uses can include administering a VEGFR3 agonist and/or FGF2 to a meningeal space of the subject, so that fluid flow (e.g., flow of ISF, CSF, or both) in the central nervous system of the subject is increased.
• the quantity of accumulated amyloid-beta plaques in the subject can be reduced, or the rate of accumulation can be reduced.
• at least some of the accumulated amyloid-beta plaques are in the meninges of the subject's brain.
• the quantity of accumulated amyloid-beta plaques, or the rate of accumulation is reduced by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% including ranges between any two of the listed values.
• the VEGR3 agonist and/or FGF2 is administered selectively to the meningeal space. In some embodiments, the VEGR3 agonist and/or FGF2 is administered to the CNS, but not outside the CNS. In some embodiments, the VEGR3 agonist and/or FGF2 is administered to the CNS, but not blood. In some embodiments, the VEGFR3 agonist is selected from the group consisting of at least one of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
• administering the VEGFR3 agonist and/or FGF2 increases the diameter of a meningeal lymphatic vessel of the subject's brain by at least 2%, for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including ranges between any two of the listed values, thus increasing flow in ISF.
• increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject's brain.
• the subject is known to have the neurodegenerative disease, for example AD, dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, or epilepsy.
• the method further includes determining that the subject has the neurodegenerative disease. In some embodiments, for example if the method or use is prophylactic, the method included determining whether the subject has the risk factor for the neurodegenerative disease, even if the subject does not necessarily have a diagnosis for the disease itself.
• risk factors for AD that are useful in accordance with methods, compositions, and uses of some embodiments herein include diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM), a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), familial AD, advanced age, or a symptom of dementia.
• apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
• PICALM phosphatidylinositol-binding clathrin assembly protein
• CR3 complement receptor 1
• Siglee-3 a variant in CD33
• TREM2 myeloid cells 2
• Some aspects include a method, use, or composition for use in increasing clearance of molecules from the central nervous system of a subject.
• the method or use can comprise administering a composition comprising, consisting of, or consisting essentially of a flow modulator (e.g., VEGFR3 agonist and/or FGF2) to a meningeal space of the subject, in which fluid flow in the central nervous system of the subject is increased.
• a flow modulator e.g., VEGFR3 agonist and/or FGF2
• Increased clearance of molecules from the CNS of the subject can comprise an increased rate of movement of molecules from the CSF to deep cervical lymph nodes, and thus can be ascertained by monitoring the rate of movement of molecules and/or labels in the CNS to deep cervical lymph nodes.
• the VEGR3 agonist and/or FGF2 is administered selectively to the meningeal space.
• the compostion comprising, consisting of, or consisting essentially of the flow modulator (e.g., VEGR3 agonist and/or FGF2) is administered to the CNS, but not outside the CNS.
• the VEGR3 agonist is administered to the CNS, but not blood.
• the VEGFR3 agonist is selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
• increasing flow by increasing the diameter of, increasing drainage by, and/or increasing the quantity of meningeal lymphatic vessels as described herein can increase clearance of molecules from the CNS of the subject, and thus reduces the concentration and/or accumulation of the molecules in the CNS and brain in accordance with some embodiments herein. Accordingly, in some embodiments, increasing clearance of molecules in the CNS reduces concentration and/or accumulation of the molecules in the CNS and brain. For example, if amyloid-beta plaques are present in the CNS of the subject, increasing clearance can reduce amyloid beta plaques, or decrease the rate of their accumulation.
• amyloid beta plaques can diminish, or the rate of increase can be reduced.
• decreases of amyloid-beta plaques can represent a decrease in an etiology of a disease caused by amyloid-beta plaques, and, more generally can indicate an increase in fluid flow in the CNS, for example via drainage by meningeal lymphatic vessels.
• a quantity of accumulated amyloid-beta plaques in the central nervous system, or the rate of accumulation thereof, is reduced by at least 2%, for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% including ranges between any two of the listed values
• amyloid-beta plaques are cleared from meningeal portions of the central nervous system of the subject.
• increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject's brain.
• methods, uses, and compositions for increasing clearance of molecules from the CNS can be useful in treating, preventing, or ameliorating symptoms of neurodegenerative diseases, for example diseases associated with accumulation of macromolecules, cells, or debris in the CNS. Accordingly, in some embodiments, the method or use further includes determining the subject to have such a neurodegenerative disease, or a risk factor for such a neurodegenerative disease.
• Example neurodegenerative diseases include Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
• AD Alzheimer's disease
• Parkinson's disease dementia
• cerebral edema amyotrophic lateral sclerosis
• PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections
• meningitis meningitis
• hemorrhagic stroke hemorrhagic stroke
• autism spectrum disorder ASD
• brain tumor and epilepsy.
• the subject is determined to have a risk factor for the neurodegenerative disease, indicating that the subject is in need of, and/or may benefit from increased clearance of molecules from the CNS.
• the subject can have a risk factor for AD as noted herein.
• a VEGFR3 agonist as described herein can be administered.
• the VEGFR3 agonist is selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant or functional fragment of either of these.
• the VEGFR3 agonist and/or FGF2 is administered selectively to the meningeal space of the subject.
• the VEGFR3 agonist and/or FGF2 is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and/or FGF2, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and/or FGF2, or a combination of any of the listed routes.
• the VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the risk factor for the neurodegenerative disease.
• the VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the neurodegenerative disease.
• the VEGFR3 agonist and/or FGF2 can be administered in an effective amount.
• Some aspects include methods, uses, or compositions for use in decreasing immune cell migration through meningeal lymphatic vessels in a subject, or compositions for use in such methods.
• some inflammatory neurological diseases such as MS
• some aspects include a method or use of decreasing immune cell migration through meningeal lymphatic vessels in a subject (e.g., to or from the brain or deep cervical lymph nodes).
• the method or use can include administering a VEGFR3 antagonist to a meningeal space of the subject or ablating a meningeal lymphatic vessel of the subject, or a combination of these.
• the method or use can thus decrease immune cell migration through meningeal lymphatic vessels in the subject.
• the VEGR3 antagonist is administered selectively to the meningeal space.
• the VEGR3 antagonist is administered to the CNS, but not outside the CNS.
• the VEGR3 antagonist is administered to the CNS, but not blood.
• the VEGFR3 antagonist comprises or consists essentially of an antibody specific for VEGFR3 or VEGF-c or VEGF-d.
• the VEGFR3 antagonist is administered to a subject who does not have a disease characterized by increased angiogenesis, for example a cancer or tumor.
• the VEGFR3 antagonist is administered to the meningeal space of the subject. In some embodiments, the VEGFR3 antagonist is administered selectively to a meningeal space of the subject. In some embodiments, the VEGFR3 agonist is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 antagonist, or expression in the subject of a nucleic acid encoding the VEGFR3 antagonist, or a combination of any of the listed routes.
• CSF cerebral spinal fluid
• the meningeal lymphatic vessels are selectively ablated by ligation, optical activation of visudyne in the lymphatic vessel, or both.
• the ligation can be performed surgically.
• visudyne is used to selectively ablate meningeal vessels.
• the visudyne can administered to the subject (via a route of administration noted for flow modulators herein), and the administered visudyne can then be optically activated to selectively ablate meningeal lymphatic vessels.
• the VEGFR3 antagonist comprises or consists essentially of an antibody specific for VEGFR3 or VEGF-c or VEGF-d.
• the method further includes determining the subject to have an inflammatory neurological disease or a risk factor for such a disease.
• Example diseases can include demyelinating diseases of the central nervous system, for example MS.
• the method is performed prophylactically, the method is performed on a subject who has a risk factor for MS, but does not necessarily have a diagnosis for MS.
• the risk factor can include familial multiple sclerosis, suspicion that the subject has multiple sclerosis, infection, advanced age, or at least one symptom of inhibited neuromotor function.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of from the parenchyma to deep cervical lymph nodes of the subject.
• the cells include lymphocytes, for example T cells.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of lymphocytes from cerebral spinal fluid to deep cervical lymph nodes of the subject.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises decreasing a density of immune cells (e.g., lymphocytes) in the meningeal lymphatic vessel.
• the density can be decreased by at least 5%, for example at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including ranges between any of the listed values.
• the lymphocytes comprise or consist essentially of T cells.
• decreasing migration of immune cells through the meningeal lymphatic vessels decreases a quantity of activated T cells in the deep cervical lymph nodes that have a migratory phenotype.
• Activated T cells can be identified, for example, by a CD62L- CD44+ phenotype.
• the migratory phenotype can be identified as CDl la+, a CD49d+, or both.
• decreasing migration of immune cells through the meningeal lymphatic vessel decreases a quantity of in T cells in the central nervous system that produce inflammatory cytokines.
• Example inflammatory cytokines that can be reduced in accordance with some embodiments include IL-17, IFN-gamma, or both.
• Some aspects include methods, uses, and compositions for use in reducing inflammation in the central nervous system, for example in inflammatory neurological diseases, or compositions for use in such methods.
• the method or use can reduce inflammation in the nervous system of a subject having an inflammatory disease of the central nervous system, or a risk factor for the inflammatory disease of the central nervous system.
• the method or use includes administering a VEGFR3 antagonist to a meningeal space of the subject, ablating a meningeal lymphatic vessel of the subject, or a combination of the two.
• the VEGFR3 antagonist, ablation, or both can decrease migration of immune cells through the meningeal lymphatic vessel in the subject, thus reducing inflammation in the central nervous system.
• the method or use comprises ameliorating a neuromotor symptom in the subject.
• the VEGFR3 antagonist is administered selectively to a meningeal space.
• the VEGFR3 antagonist is administered to the CNS, but not administered outside the CNS.
• the VEGFR3 antagonist is administered to the CNS, but not administered to blood.
• the VEGFR3 antagonist comprises or consists essentially of an antibody specific for VEGFR3 or VEGF-c or VEGF-d, or a VEGFR3 decoy molecule.
• the VEGFR3 antagonist is administered to a subject in need of reduced inflammation in the CNS, but who does not have a disease characterized by increased angiogenesis, such as a tumor or cancer.
• the inflammatory disease comprises or consists essentially of a demyelinating disease of the central nervous system, for example, MS.
• the method is performed on a subject who has the inflammatory disease. Accordingly, in some embodiments, the method includes determining that the subject has the inflammatory disease. In some embodiments, for example if the method is performed prophylactically, the subject can have a risk factor for the inflammatory disease. Accordingly, in some embodiments, the method includes determining that the subject has the risk factor for the inflammatory disease. In some embodiments, the risk factor comprises or consists essentially of familial multiple sclerosis, infection, advanced age, suspicion that the subject has multiple sclerosis, or at least one symptom of inhibited neuromotor function.
• the method includes ablating meningeal lymphatic vessels chemically, surgically, or both. In some embodiments, the method includes selectively ablating meningeal lymphatic vessels by ligation, optical activation of visudyne in lymphatic vessels, or both.
• the method includes administering a VEGFR3 antagonist selectively to a meningeal space of the subject.
• the VEGFR3 antagonist can inhibit migration of immune cells through meningeal lymphatic vessels, for example by decreasing the size and/or quantity of the vessels.
• the VEGFR3 antagonist comprises or consists essentially of an antibody specific for VEGFR3 or VEGF-c or VEGF-d.
• the VEGFR3 antagonist comprises or consists essentially of a VEGFR3 decoy molecule.
• the VEGFR3 antagonist or visudyne is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 antagonist, or a combination of any of the listed routes.
• the VEGFR3 antagonist is administered by expressing a nucleic acid encoding the VEGFR3 antagonist in the subject.
• decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of molecules in cerebral spinal fluid in the subject to deep cervical lymph nodes of the subject. In some embodiments, decreasing immune cell migration through the meningeal lymphatic vessel comprises a decrease in movement of lymphocytes from the parenchyma to deep cervical lymph nodes of the subject. In some embodiments decreasing the immune cell migration decreases a density of lymphocytes in the meningeal lymphatic vessel. For example, the density can be decreased by at least 5%, for example at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including ranges between any of the listed values. In some embodiments, the lymphocytes comprise or consist essentially of T cells.
• reducing T-cell mediated inflammation in the central nervous system in accordance with some embodiments comprises decreasing a quantity of activated T cells in the deep cervical lymph nodes that have a migratory phenotype, as described herein.
• reducing T-cell mediated inflammation in the central nervous system can decrease a quantity of in T cells in the central nervous system that produce inflammatory cytokines.
• Example inflammatory cytokines include IL-17, IFN- gamma, or both.
• lymphatic vessels e.g., meningeal lymphatic vessel(s)
• lymphatic vessels e.g., meningeal lymphatic vessel(s)
• a DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA.
• a DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, or a DNA-targeting RNA; also called “non-coding” RNA or "ncRNA”).
• the term "about” is used herein to provide literal support for the exact number that it precedes, as well as a number differs from the given number by less than 10%. In other embodiments, the term “about” indicates that the number differs from the given number by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
• Some aspects provide methods of treating a condition with a neurological pathology in a subject comprising administering to the subject a therapeutically effective amount of a compound that modulates one or more of a) drainage of the meningeal lymphatic vessel(s); b) diameter of the meningeal lymphatic vessel(s); c) lymphangiogenesis of the meningeal lymphatic vessel(s); d) contractility of the meningeal lymphatic vessel(s); and/or e) permeability of the meningeal lymphatic vessel(s).
• the present disclosure also provides methods of treating AD in a subject by administering to the subject a compound that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s) to increase drainage.
• the present disclosure also provides methods of treating a brain tumor in a subject by administering to the subject a compound that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s) to increase drainage.
• the present disclosure further provides methods of treating MS in a subject by administering to the subject a compound that decreases drainage of the meningeal lymphatic vessel(s), decreases the diameter of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to decrease drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s).
• the present disclosure also provides the identification and description of the meningeal lymphatic vascular system that serves as both tissue clearance and immune-cell trafficking functions of the brain.
• Some aspects include methods of treating a condition with a neurological pathology in a subject by administering to the subject a therapeutically effective amount of a compound that modulates one or more of a) drainage of the meningeal lymphatic vessel(s), b) diameter of the meningeal lymphatic vessel(s), c) lymphangiogenesis of the meningeal lymphatic vessel(s), d) contractility of the meningeal lymphatic vessel(s); and/or e) permeability of the meningeal lymphatic vessel(s).
• the method further comprises identifying a subject in need of said treatment.
• the subject in need of said treatment is susceptible to or suffering from the disorder selected from the group consisting AD, dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), epilepsy, brain tumor, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), MS, and myasthenia gravis.
• a therapeutically effective amount of said compound is administered.
• said compound is a vasodilator.
• said compound is a growth faction.
• said growth factor is selected from the group consisting of VEGF-c, VEGF-d, and FGF2.
• said compound is noradrenaline.
• said compound is a vasoconstrictor.
• said compound is selected from the group consisting of nitric oxide competitor NG-monomethyl L-arginine, cyclo-oxygenase inhibitors, and phosphatidylcholine.
• said therapeutically effective amount of the compound is about 0.00015 mg kg to about 1.5 mg/kg. In further embodiments, said therapeutically effective amount of the compound is about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025
• said therapeutically effective amount of the compound is less than about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025 mg/kg, about 0.03 mg/kg, about 0.035 mg/kg, about 0.040 mg/
• said therapeutically effective amount of the compound is more than about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025 mg/kg, about 0.03 mg/kg, about 0.035 mg/kg, about 0.040 mg/
• the compound is provided in soluble form. In some embodiments, the compound is provided absorbed in nanogels for slow and constant release. In certain embodiments, the compounds are provided on viral vectors which encode for the reagent that is a RNA or polypeptide.
• the compound is administered into the cerebrospinal fluid (CSF) of the subject.
• CSF cerebrospinal fluid
• an ointment comprises said compound and the ointment is administered via application of the ointment to the head of the subject.
• AD Alzheimer's disease
• lymphangiogenic growth factors such as, VEGFC, VEGFD, FGF2
• VEGFC vascular endothelial growth factor
• VEGFD vascular endothelial growth factor
• FGF2 recombinant protein
• the provided data indicates that single injection of recombinant VEGF-c into the CSF is sufficient to increase diameter of meningeal lymphatic vessels and increase drainage efficacy; (FIG. 2e). Lymphatic drainage (using multiphoton microscopy), disease pathology (quantification of ⁇ deposition in the meninges and the brain), and behavior (open field and Morris Water Maze) are assessed during and after treatment with the lymphangiogenic factor/s.
• Some embodiments include methods of treating AD in a subject by administering to the subject a compound that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s).
• the present disclosure further provides methods of treating a brain tumor in a subject by administering to the subject a compound that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s).
• Some aspects include methods for reducing the number and/or volume of existing amyloid plaques or other misfolded proteins comprising administering to a subject a therapeutic effective amount of a compound that increases drainage of and/or increases the diameter of the meningeal lymphatic vessels.
• the subject is selected from the group consisting of subjects identified as being susceptible to Alzheimer's disease and subjects suffering from Alzheimer's disease.
• the method further comprises identifying a subject in need of said treatment.
• the subject in need of said treatment is susceptible to or suffering from the disorder selected from the group consisting of AD and brain tumors.
• Identification of such subjects may be made using techniques known to a person of ordinary skill in the art.
• a therapeutically effective amount of said compound is administered.
• said compound is a vasodilator.
• said compound is a growth faction.
• said growth factor is selected from the group consisting of VEGF-c, VEGF-d, and FGF2.
• said compound is noradrenaline.
• said therapeutically effective amount of the compound is about 0.00015 mg/kg to about 1.5 mg/kg. In further embodiments, said therapeutically effective amount of the compound is about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.0
• said therapeutically effective amount of the compound is less than about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025 mg/kg, about 0.03 mg/kg, about 0.035 mg/kg, about 0.040 mg/
• said therapeutically effective amount of the compound is more than about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025 mg/kg, about 0.03 mg/kg, about 0.035 mg/kg, about 0.040 mg/
• the compound is provided in soluble form. In some embodiments, the compound is provided absorbed in nanogels for slow and constant release. In certain embodiments, the compounds are provided on viral vectors which encode for the reagent that is a RNA or polypeptide.
• the compound is administered into the cerebrospinal fluid (CSF) of the subject.
• an ointment comprises said compound and the ointment is administered via application of the ointment to the head of the subject.
• Proxcre-ERT2 mice were made available.
• Prox-lcre-ERT2::DTASTOP-lox mice will be treated i.c.v. with tamoxifen (TAM) to induce expression of intracellular diphtheria toxin (DTA) in Prox-1 expressing lymphatic endothelial cells that will kill the cells.
• TAM tamoxifen
• DTA diphtheria toxin
• wild type mice will be injected i.c.v.
• FIG. 8 An alternative to ablation approach, is a ligation of lymphatic vessels. As demonstrated in FIG. 7d, this method is feasible and has an effect on EAE. A more efficient method for ligation/ablation will likely yield more robust effect on EAE.
• T cells from mice will be used, ubiquitously expressing a photoconvertible fluorescent protein, and transfer them into T cell deficient hosts to study the kinetics of T cell migration into the meninges (Nowotschin, S. & Hadjantonakis, A. K. Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos. BMC developmental biology 9, 49, doi: 10.1186/1471-213X-9-49 (2009)). T cells will be photo-labeled (green-to-red fluorescence; FIG. 9) in the sinusal area through the skull, or in the deep cervical lymph nodes, using survival surgery procedure.
• T cells After labeling, dynamics of T cell migration into the meninges will be studied during the resting state, and after EAE induction using two-photon microscopy, histological examination and flow cytometry.
• EAE induction using two-photon microscopy, histological examination and flow cytometry.
• T cells We expect T cells to recirculate between meninges and the deep cervical lymph nodes at a certain rate in healthy mice. This rate is expected to change upon EAE induction, when T cells are supposedly leaving meningeal spaces for massive proliferation in the deep cervical lymph nodes and then return and attack the brain.
• PD-L1 is highly expressed on brain lymphatic endothelial cells (data not shown) and we suggest it mediates tolerance to brain antigens. Mice will be injected with anti-PD-Ll neutralizing antibodies i.c.v. with EAE induction.
• TAM injection will be performed immediately prior to CFA/Mog immunization, 7 days after immunization (the time point when T cells are seen leaving the meningeal spaces), and day 10 post immunization (T cell numbers in the meningeal spaces were exploded when assessed at day 13 post immunization) to ensure prolonged ablation.
• Immune response at the spinal cord and cerebellum, and immune response in the meninges and the deep cervical lymph nodes will be assessed, including Teff activation and proliferation, Treg expansion, and intracellular cytokine expression (Thl and Thl7 profile of T cells) in all groups will be assessed at day 15 post immunization (early into clinical signs). Another group of mice will be kept for 3 weeks for behavioral evaluation and then sacrificed for a histological examination of the CNS.
• Photoablation will be performed immediately prior to CFA/Mog immunization, 7 days after immunization (the time point when T cells are seen leaving the meningeal spaces), and day 10 post immunization (T cell numbers in the meningeal spaces were exploded when assessed at day 13 post immunization).
• Immune response at the spinal cord and cerebellum, and immune response in the meninges and the deep cervical lymph nodes will be assessed, including Teff activation and proliferation, Treg expansion, and intracellular cytokine expression (Thl and Thl 7 profile of T cells) in all groups will be assessed at day 15 post immunization (early into clinical signs).
• mice Another group of mice will be kept for 3 weeks for behavioral evaluation and then sacrificed for a histological examination of the CNS. We expect the mice with photoablated lymphatics to exhibit reduced T cell activation, decreased number of Thl/Thl7 cells, and ameliorated disease progression. [0157] A specific depletion of meningeal T cells will be performed by transcranial application of a depleting anti-CD3e antibodies, an efficient procedure (FIG. 11).
• Some aspects include methods of treating MS in a subject by administering to the subject a compound that decreases drainage of the meningeal lymphatic vessel(s), decreases the diameter of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to decrease drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s).
• the method further comprises identifying a subject in need of said treatment.
• the subject in need of said treatment is susceptible to or suffering from MS. Identification of such subjects may be made using techniques known to a person of ordinary skill in the art.
• a therapeutically effective amount of said compound is administered.
• said compound is a vasoconstrictor.
• said compound is selected from the group consisting of nitric oxide competitor NG-monomethyl L-arginine, cyclo-oxygenase inhibitors, and phosphatidylcholine.
• said therapeutically effective amount of the compound is about 0.00015 mg/kg to about 1.5 mg/kg. In further embodiments, said therapeutically effective amount of the compound is about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.0
• said therapeutically effective amount of the compound is less than about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025 mg/kg, about 0.03 mg/kg, about 0.035 mg/kg, about 0.040 mg/
• said therapeutically effective amount of the compound is more than about 0.00015 mg/kg, about 0.00030 mg/kg, about 0.00045 mg/kg, about 0.00060 mg/kg, about 0.00085 mg/kg, about 0.001 mg/kg, about 0.0015 mg/kg, about 0.002 mg/kg, about 0.0025 mg/kg, about 0.003 mg/kg, about 0.0035 mg/kg, about 0.004 mg/kg, about 0.0045 mg/kg, about 0.0050 mg/kg, about 0.0055 mg/kg, about 0.006 mg/kg, about 0.0065 mg/kg, about 0.007 mg/kg, about 0.0075 mg/kg, about 0.008 mg/kg, about 0.0085 mg/kg, about 0.009 mg/kg, about 0.0095 mg/kg, about 0.01 mg/kg, about 0.015 mg/kg, about 0.02 mg/kg, about 0.025 mg/kg, about 0.03 mg/kg, about 0.035 mg/kg, about 0.040 mg/
• the compound is provided in soluble form. In some embodiments, the compound is provided absorbed in nanogels for slow and constant release. In certain embodiments, the compounds are provided on viral vectors which encode for the reagent that is a RNA or polypeptide.
• the compound is administered into the cerebrospinal fluid (CSF) of the subject.
• CSF cerebrospinal fluid
• an ointment comprises said compound and the ointment is administered via application of the ointment to the head of the subject.
• a method of treating a condition with a neurological pathology in a subject comprising administering to the subject a therapeutically effective amount of a compound that modulates one or more of a) drainage of the meningeal lymphatic vessels; b) diameter of the meningeal lymphatic vessels; c) lymphangiogenesis of the meningeal lymphatic vessels; d) contractility of the meningeal lymphatic vessels; and/or e) permeability of the meningeal lymphatic vessels.
• the administration is into the cerebrospinal fluid (CSF) of said subject.
• an ointment comprises said compound and wherein the administration is via application of the ointment to the head.
• the method further comprises identifying a subject in need of said treatment.
• the subject in need of said treatment is susceptible to or suffering from a disorder selected from the group consisting of Alzheimer's disease (AD), dementia, Parkinson's disease, cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy, or a combination of any of the listed disorders.
• the administration increases drainage and/or increases diameter of the meningeal lymphatic vessels.
• the administration increases drainage and/or increased diameter of the meningeal lymphatic vessels; and wherein the subject in need of said treatment is susceptible to or suffering from a disorder selected from the group consisting of Alzheimer's disease (AD) and brain tumor, or a combination of the two.
• the compound is a vasodilator.
• the administration decreases drainage and/or decreases diameter of the meningeal lymphatic vessels.
• the administration decreases drainage and/or decreases diameter of the meningeal lymphatic vessels; and wherein the subject in need of said treatment is susceptible to or suffering from multiple sclerosis (MS).
• MS multiple sclerosis
• the compound is a vasoconstrictor.
• a method of treating Alzheimer's disease in a subject comprising administering to the subject a therapeutically effective amount of a growth factor into the cerebrospinal fluid (CSF) of the subject, wherein the growth factor is selected from the group consisting of VEGF-c, VEGF-d, and FGF2.
• the method further comprises identifying a subject in need of said treatment.
• a viral vector is administered into the CSF and said viral vector encodes the growth factor.
• the viral vector is soluble.
• the viral vector is absorbed in a nanogel prior to administration.
• a method of treating MS in a subject can comprise ligating one or more meningeal lymphatic vessel in said subject. In some embodiments, the method further comprises identifying a subject in need of said treatment.
• Visudyne (verteporfin) or vehicle (as control) were injected into the cisterna magna (intra-cisterna magna, ICM) of anesthetized adult C57BL/6 mice (3 months of age), followed by a photoconversion step that was achieved by shining a non-thermal red light (689 nm) in 5 points above the skull.
• mice were transcardially perfused with saline and meninges, deep cervical lymph nodes (dcLNs) and brain were collected for analysis. Meningeal whole-mounts (scale bar, 1 mm) from vehicle (FIG. 23A) or visudyne (FIG. 23B) injected groups were stained for lymphatic vessel, endothelial hyaluronan receptor 1 (LYVE- 1 , green) and the blood vascular endothelial cell marker CD31 (red). Visudye alone is shown in the inset in the upper right corner of FIGs. 23A and 23B. A significant decrease in the area of LYVE-1 + vessels was observed in the visudyne group FIG.
• FIG. 23C Staining for 4',6-diamidino-2-phenylindole (DAPI) and LYVE-1 in dcLNs showed significantly less drainage of ⁇ 42 (red) in the visudyne group (FIG. 23E for the vehicle group, FIG. 23F for the visudyne group).
• DAPI 4',6-diamidino-2-phenylindole
• LYVE-1 alone is shown in the inset in the lower right corner of each of FIGs. 23E and 23F.
• GFAP glial fibrillary acidic protein
• Example 2 Impairing meningeal vessels significantly decreases drainage into deep cervical lymph nodes.
• Example 3 Ablation of meningeal lymphatic vessels in old mice does not further aggravate influx of a CDF tracer in the brain.
• Example 4 Transcranial application of VEGF-C in old mice leads to improved CSF influx into the brain [0175] 1 mL of a gel matrix alone (vehicle) or containing 200 ng of recombinant human VEGF-C156S was applied every two weeks on top of a thinned skull of old C57BL/6 mice. One month after the initial treatment, OVA-A647 was injected ICM and mice were transcardially perfused with saline 2 h post injection. Staining for LYVE-1 in meningeal lymphatic vessels from old mice revealed that treatment with gel+VEGF-C156S had a significant effect on vessel diameter (FIG. 26).
• FIGs. 27A-C Representative images of dcLNs depicting OVA-A647 (red), and stained with DAPI (blue) and against LYVE-1 (green), show that decreased drainage of OVA-A647 into the dcLNs of old mice was increased by delivery of VEGF-C156S (FIGs. 27A-C).
• OVA-A647 alone is shown in the inset in the upper right corner of each of FIGs. 27A and 27B
• LYVE-1 alone is shown in the inset in the lower right corner of each of FIGs. 27A and 27B.
• Example 5 Expression of an exogenous VEGF-C transgene by cells in the CNS increases flow
• Example 6 Expression of an exogenous VEGF-C transgene by cells in the CNS increases flow and improves cognitive performance
• mice Young adult (2 months of age) and middle-aged (12-14 months) C57BL/6 mice were injected with 10 12 genome copies (GC)/mL (ICM) of either AAV1-CMV-EGFP (or EGFP) or AAVl-CMV-mVEGF-C (or m VEGF-C). 1 month after, mice learning and memory capabilities were assessed in the Morris water maze (MWM) test (FIGs. 29A-C). No differences between the two groups of young adult mice were observed in the acquisition, probe trial and reversal tasks of the MWM (FIGs. 29D-F).
• MWM Morris water maze
• Example 7 Meningeal amyloid-beta deposits in AD patients.
• Non-AD cortical and AD cortical brain sections containing the respective meningeal layers attached, were stained with DAPI (cell nuclei), for the astrocyte marker GFAP and with an antibody recognizing human N-terminal amyloid beta ( ⁇ ) 3 _ 42 residues (clone D54D2).
• Amyloid deposition was observed in the AD (FIG. 30B), but not in the non-AD (FIG. 30A), brain parenchyma, as well as in the meningeal vasculature of the cortex (scale bars, 500 ⁇ ; inset scale bars, 200 ⁇ ).
• Example 8 Meningeal lymphatic (dvs)function modulates amyloid pathology in models of Alzheimer's disease
• mice C57BL/6 adult mice were anesthetized and injected ICM with visudyne to induce meningeal lymphatic vessel ablation, or vehicle as a control. After the photoconversion step, mice were allowed to recover for 72 h. Then, catheters were implanted in the cisterna magna of all mice and 2.5 ⁇ g of ⁇ 42 were injected every 24 h into the CSF for a total of 5 days. Staining with LYVE-1, ⁇ , and the macrophage marker IBA1 in meningeal whole-mounts showed macrophage activation in response to formation of ⁇ 42 aggregates. Quantification was performed of the total area of LYVE-1 + lymphatic vessels (FIG.
• Example 10 Meningeal lymphatic ablation exacerbates dementia symptoms in an AD model
• Example 11 Expression of VEGF-C in the CNS ameliorates dementia symptoms in an AD model
• Example 12 In vivo flow from the cisterna magna to meningeal lymphatic vessels
• Example 13 Characteristics of meningeal lymphatic vessel structures
• Example 15 Accumulation of endogenous T cells in meningeal lymphatics
• Example 16 Density of exogenous T cells in meningeal lymphatics
• Example 18 Quantification of the percentage of KiKR CD4 T cells are shown in the dCLN, sCLN and ILN
• Example 19 Activation and migration of T cells into the deep cervical lymph nodes
• Isolated CD4 T cells were incubated for 2h with Xng of pertussis toxin prior to i.c.m. injection in C57B16 mice.
• Images of control and PTX-treated T cells in the dCLN of WT mice 12h were taken after i.c.m. injection.
• Density of T cells per mm2 of dCLN was quantified (expressed as a percentage of the control condition)(FIG. 42).
• Example 20 Meningeal T cells circulate into the cervical lymph nodes in a CCR7-CCL21 dependent manner
• FIG. 43B A representative dot plot of GFP expression by CD4 T cells in the meninges of C57B16 mice and CCR7 GFP mice is shown in FIG. 43C (representative of 3 independent mice).
• FIG. 43D A representative contour plot of phenotype CCR7 + and CCR7 " CD4 T cells in the meninges of CCR7 GFP mice is shown in FIG. 43D.
• CCR7GF CCR7 expression
• Example 21 Meningeal dendritic cells circulate into the cervical lymph nodes
• KiKGR mice meninges were converted for 2min with violet light every 12h for 2 days. After 2 days, mice were injected i.c.m. with Poly(LC) with peptide. Tissue were harvested 24h after Poly(LC) injection. Samples were gated to identify dendritic cells. Representative dot plots are shown for B6 controls, which KiKG+ and KiKR+ dendritic cells (FIG. 45A) and for KiKGR control mice (FIG. 45B). Representative dot plots of KiKR+ dendritic cells in the dCLN, sCLN and ILN 24h after Poly(LC) injection in converted mice. Representative of 4 mice.
• Example 22 Meningeal lymphatics is the main route for immune cells and macromolecules circulation into the cervical lymph nodes
• mice were injected i.c.m. with 1 million of exogenously labeled T cells, Meninges and nasal cavity were harvested and analyzed at the indicated time points. Representative images of the cribriform plate region after 2 and 12h post i.c.m. injection of CFSE-labeled T cells (green). I.c.m. injected T cells (Deep Red Cell Tracker - red) were detected in the lymphatic of the cribriform plate, but also in and around the lymphatic at the base of the nose. Intralymphatic T cells and perilymphatic T cells were observed.
• Example 24 Effects of meningeal vasculature ablation of immune cell size and coverage
• FIG. 48C Images were obtained of exogenously injected T cells (4 days after ablation) (CFSE) in the dCLN of laser, Visudyne (i.c.m.) and Visudyne (i.c.m.) + laser treated mice 12h after i.c.m. injection.
• Example 26 Lack of inflammation-induced lvmphangiogenesis of the meningeal lymphatic endothelial cells.
• FIG. 50C transverse sinuses
• FIG. 50D transverse sinuses
• Images of T cells (CD3e) in and around the meningeal lymphatics (Lyvel) of the superior sagittal sinus of CFA and MOG immunized mice were obtained at D13 after immunization.
• the density of T cells on the superior sagittal and transverse sinuses of naive, CFA and MOG immunized mice was quantified at different time after immunization (FIG. 50E).
• Example 27 Ablation of lymphatic drainage ameliorate MOG-specific T cells activation in the deep cervical lymph nodes resulting in ameliorated disease development.
• Incidence of EAE development day mice reach a score of 1 or above) in laser, Visudyne (i.n.) + laser and Visudyne (i.c.m.) + laser treated mice.
• the method, use, or composition comprises various steps or features that are present as single steps or features (as opposed to multiple steps or features).
• the method includes a single administration of a flow modulator, or the composition comprises or consists essentially of a flow modulator for single use.
• the flow modulator may be present in a single dosage unit effective for increasing flow (or decreasing immune cell migration).
• a composition or use may comprise a single dosage unit of a flow modulator effective for increasing flow (or inhibiting migration of immune cells) as described herein. Multiple features or components are provided in alternate embodiments.
• the method, composition, or use comprises one or more means for flow modulation.
• the means comprises a flow modulator.
• compositions for use in the method are expressly contemplated, uses of compositions in the method, and, as applicable, methods of making a medicament for use in the method are also expressly contemplated.
• flow modulators for use in the corresponding method are also contemplated, as are uses of a flow modulator in increasing flow according to the method, as are methods of making a medicament comprising the flow modulator for use in increasing flow.
• a range includes each individual member.
• a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
• a group having 1-5 cells refers to groups having 1 , 2, 3, 4, or 5 cells, and so forth.

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