WO2016022387A2 - A mouse model for multiple sclerosis - Google Patents

Abstract

Disclosed are methods to induce multiple sclerosis-like lesions in the CNS of an animal by intracerebral administration of acellular CSF from a subject with MS into the CNS of the animal. The methods produce animals with demyelinated CNS lesions suitable for study of MS. The disclosed methods can be used to determine the efficacy of treatment for MS. Further disclosed are animals produced by these methods.

Description

A MOUSE MODEL FOR MULTIPLE SCLEROSIS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 62/034,507, filed August 7, 2014, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

[0002] Multiple sclerosis (MS) is an autoimmune disease of unknown origin, characterized by demyelination and axonal loss throughout the central nervous system (CNS) (Compston, et al., Lancet 372: 1502-1517 (2008)). MS often starts out as a clinically isolated syndrome followed by a series of alternating periods of remission and exacerbation, referred to as relapsing-remitting MS (RRMS) (Hafler, Journal of clinical investigation 113:788-794 (2004); Sadiq, Merritt's Textbook of Neurology 11th edition 134:941-962 (2005)). While patients typically return to near normal neurologic function at the end of each episode, over time, failure of the CNS to remyelinate MS lesions (Franklin, Nat Rev Neurosci 3:705-714 (2002)) and regenerate axons (Trapp, et al., N Engl J Med 338:278-285 (1998); Kornek, et al., Am J Pathol 157:267-276 (2000)) can lead to secondary progressive MS (SPMS), an irreversible progression of clinical disability (Ferguson, et al., Brain 120(Pt 3):393-399 (1997); Lovas, et al., Brain 123(Pt 2):308-317 (2000)). In addition, 10 -15 of MS patients will have primary progressive MS (PPMS), a clinical progression from onset without remissions (Miller, et al., Lancet Neurol 6:903-912 (2007)).

[0003] At present the therapeutic options for patients with MS are limited, and no

regenerative treatment exists for such patients. This dismal outlook for patients with PMS is largely due to the fact that there are no good experimental models of investigation for this condition. The commonly used animal model of MS, experimental allergic

encephalomyelitis (EAE), which is pathologically characterized by intense inflammation, is more representative of inflammatory demyelination than the degenerative phase of MS (Gold, et al. Brain: a journal of neurology 129: 1953-1971 (2006)). Moreover, tissue specimens from patients with MS are not generally available, and post-mortem pathology poorly represents the dynamic biological events related to ongoing disease pathogenesis.

Cerebrospinal fluid (CS, which surrounds the brain and spinal cord, is considered the most accurate indicator of CNS biology for MS. This is because any molecules secreted in the CNS from resident or infiltrating cells drain into the CSF, making it a useful tool to monitor disease activity (Awad, et al., J Neuroimmunol 219: 1-7 (2010); Harris, et al., Mult Scler. (2013); Stangel, et al., Nature reviews Neurology 9:267-276 (2013)).

[0004] Attempts to transfer the pathophysiology of MS into animals by the use of patient CSF (cells and/or supernatant) have failed to produce an accepted model. Saeki, et al., PNAS 89:6157-6161 (1992), utilized a single injection of RRMS-CSF cells, collected during disease exacerbation, into the cisterna magna of immunocompromised (SCID) mice, which was reported to transfer MS-like symptoms including necrotic, demyelinated CNS lesions and inflammation, paralysis, and ataxia, into mice. However, attempts to reproduce the results of Saeki et al. by other groups failed (Hao, et al., Neurology 44: 163-165 (1994); Jones, et al., / Neuroimmunol 56: 119-126 (1995)), and the lesions and inflammation in the Saeki et al. studies were postulated to be the result of graft- versus-host response to the transferred cells (Jones, et al., J Neuroimmunol 56: 119-126 (1995)). These originators of these studies abandoned this yet-to-be validated mouse model in favor of continued research in the established EAE model. In a recent paper published on this model (Fujimura et al., Acta Neuropathol. 93:567-578 (1997)), Fujimura et al. noted that cell free CSF of MS individuals did not induce CNS lesions.

[0005] In the last two decades, the introduction of disease-modifying agents has significantly changed the outcome of RRMS patients (Hauser, et al. Ann Neurol (2013)). By contrast, understanding and treating PMS, which is characterized by the steady accumulation of irreversible disability, is still inadequate (Hawker, et al., Ann Neurol 66:460-471 (2009)). This is primarily due to the fact that there are no good clinical or experimental paradigms to predict if and when a transition from RR to SPMS will occur or if a patient will develop PPMS after an initial clinically isolated syndrome (Rovaris, et al., Lancet Neurol 5:343-354 (2006)). Indeed, even serial conventional MRI brain scans which are widely accepted as surrogate markers of disease activity for RRMS are not sensitive enough to monitor the progressive phase of MS (Barkhof, et al., Nature Reviews Neurology 5:256-266 (2009)), and more specific in-vivo markers of neurodegeneration are needed for a reliable assessment of putative new treatment options. Therefore, the immunological and pathological mechanisms involved in PMS are poorly understood. [0006] An animal model to further explore the development of MS, mechanisms of disease progression, and treatment efficacy in patients, is urgently needed.

BRIEF SUMMARY OF THE DISCLOSURE

[0007] Disclosed herein are methods of inducing demyelinating lesions in the central nervous system (CNS) of an animal, such as a mouse. The methods involve administering acellular cerebrospinal fluid (aCSF) from a subject with multiple sclerosis (MS) to an animal by at least one intracerebral injection, such as two to fourteen intracerebral injections. aCSF can be administered, for example, by twice weekly injections. Further provided herein are non- human animals produced by the disclosed methods.

[0008] In the disclosed methods, the subject can have progressive MS (PMS), particularly primary progressive MS (PPMS) or secondary progressive MS (SPMS). Lesions induced by the disclosed methods are identified by one or more of: demyelinated axons; myelin debris; ionized calcium-binding adapter molecule 1- positive (Ibal+) cells; glial fibrillary acidic protein- positive (GFAP+) cells; CD3+ cells; CD138+ cells, CD19+ cells; axonal

degeneration at the lesion periphery; or increased expression of amyloid precursor protein (APP). Lesions can be present in the brain, alone or accompanied by at least one of meningeal inflammation and subpial demyelination in the spinal cord of said animal.

[0009] The disclosed methods include the steps of obtaining a CSF sample from the subject and removing the cells from the CSF sample to obtain an acellular CSF sample, prior to administering aCSF to an animal. In some embodiments, the subject has not been treated for MS. In other embodiments, the subject has been treated for MS. Where the subject has not been treated for MS, the severity of demyelinating lesions induced by administration of aCSF from the subject is increased relative to the severity of demyelinating lesions induced by administration of aCSF from an MS patient who has been treated for MS.

[0010] In some embodiments, the CNS of the lesion-induced animal has increased expression of one or more markers selected from CD3, CD4, CD28, CD11C, granulocyte-macrophage colony-stimulating factor 2 (CSF2), IL17, IL4, CXC-motif chemokine 13 (CXCL13), transforming growth factor beta-1 (TGFP-l); and interferon-gamma (IFNy), relative to control levels. In other embodiments, the spinal cord of a lesion-induced animal has increased expression of at least one of CD3, CD4, or CXCL13, relative to control levels. Control levels can represent the level of expression of one or more of the disclosed markers in an animal that has not received administration of aCSF from a subject with MS.

[0011] Also disclosed herein are methods to determine the effectiveness of a treatment for multiple sclerosis (MS). The methods include (a) administering acellular cerebrospinal fluid (aCSF) from a subject with MS, before the subject is treated for MS, into a first animal set with at least one first animal; (b) administering aCSF from the same subject, after the subject has been treated for MS, into a second animal set with at least one second animal; and (c) determining the efficacy of treatment by comparing one or more CNS characteristics of the first and second animal sets. The one or more CNS characteristics are selected from: the presence or severity of CNS lesions; the presence of inflammatory cells and/or chemokines in the CNS; and expression of pro-inflammatory and/or anti-inflammatory markers in the CNS, according to parameters defined further hereinbelow. CNS lesions are identified by one or more of: demyelinated axons; myelin debris; ionized calcium-binding adapter molecule 1- positive (Ibal+) cells; glial fibrillary acidic protein- positive (GFAP+) cells; CD3+ cells; CD138+ cells, CD19+ cells; axonal degeneration at the lesion periphery; or increased expression of amyloid precursor protein (APP). In these methods, aCSF can be administered to each animal set by two to fourteen intracerebral injections per animal.

[0012] According to these methods of determining treatment effectiveness, reduced presence or severity of CNS lesions in the second animal set, relative to the presence or severity of CNS lesions in the first animal set, indicates that the treatment was effective. In further embodiments, the treatment is determined to be effective if the second animal set has increased expression of CD 17, and/or decreased expression of one or more of CD3, CD4, CD28, CD l lC, granulocyte-macrophage colony- stimulating factor 2 (CSF2), IL4, CXC- motif chemokine 13 (CXCL13), or interferon- gamma (IFNy), relative to expression of these markers in the first animal set.

[0013] Further disclosed herein are methods to diagnose progressive multiple sclerosis (PMS) in a subject. The methods include administering acellular cerebrospinal fluid (aCSF) from a subject to a test animal by at least one intracerebral injection of the aCSF, wherein the presence of at least one lesion in the CNS of the test animal subsequent to the administration indicates PMS in said subject. [0014] Additionally disclosed herein are methods to monitor the progression of MS in a subject, by administering aCSF from a subject with MS, the aCSF being obtained from the subject at different times, into a test animal by at least one intracerebral injection, where increased severity of lesions induced from aCSF obtained from the subject at a later time point, relative to the severity of lesions induced from aCSF obtained from the subject at an earlier time point, indicate that the subject's MS is worsening. It is believed that the methods disclosed herein can lead to improved treatments for MS, to improve the quality of life for subjects with MS.

[0015] Further disclosed herein are methods to test candidate agents for treatment of MS. The methods include administration of aCSF from a subject with MS to a test animal by at least one intracerebral injection, and administration of the candidate agent to the same animal, where the candidate agent can be administered before, concurrent with, and/or subsequent to administration of aCSF from the subject. The candidate agent is identified as a potential treatment for MS if administration of the candidate agent leads to reduced induction, formation, or severity of CNS lesions in the animal, or if the agent leads to improved healing of induced lesions.

[0016] Further disclosed herein are methods for utilizing the lesion-induced animal to evaluate the effect of MS CSF on microglial activation. Such methods include extracting microglia (brain macrophages) from the brain of an lesion-induced animal, and determining the level of microglia. In some embodiments, microglia are identified based on CD1 lb⁺ and CD45^hl. In certain embodiments, the extent of increase in the level of microglia in an lesion- induced animal as compared to a control is correlated with the disease status, progression or treatment response of the MS patient from whom aCSF sample has been obtained to induce lesions in the animal.

BRIEF DESCRIPTION OF THE FIGURES

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] Figs 1A-1C. Inflammatory demyelinated lesion in the corpus callosum of a mouse injected for 4 weeks with MS-CSF. (A) Luxol fast blue staining of a brain section of a mouse injected with MS-CSF. Black arrow indicates demyelinated lesion. Inset, section showing site of catheter insertion with absence of overt inflammation. (B) Tiles scan reconstruction of immunostained brain lesion. Green, MBP immunostaining. Red, Ibal⁺ macrophages. White, BrdU⁺ proliferative cells. Blue, DAPI. Inset, higher magnification lesioned area in B showing Ibal⁺ macrophage-engulfed myelin debris. (C) Regression analysis between patient EDSS score (x-axis) and percentage of lesioned animals (y-axis). R = correlation coefficient.

[0019] Figs 2A-2E. Axonal loss, astrogliosis, and immune cell infiltration in MS-CSF induced brain lesion. (A) Axonal loss, (B) astrogliosis, and (C) T cells inside the lesion, visualized by immunostaining with neurofilament (red/yellow in A), GFAP (red in B), and CD3 (red/pink in C), respectively. Green = MBP immunostaining. Blue = DAPI. (D)

CD138⁺ plasma cells (black arrows) and (E) CD19⁺ B cells (black arrows) in the lesioned area (solid white lines).

[0020] Figs 3A-3E. Spinal cord inflammation, demyelinating lesions, and locomotor evaluation after 4 weeks of CSF injections. (A-C) Meningeal inflammation visualized by immunostaining for Ibal (red) and BrdU (white) spanning the (A) cervical, (B) thoracic, and (C) lumbar segments of the spinal cord. Arrows indicate foci of subpial demyelination. (D) Immunostained spinal cord lesion. Green, MBP immunostaining. Red, Ibal⁺ macrophages. White, BrdU⁺ proliferative cells. Blue, DAPI. (E) Locomotor performance comparison between mice treated with CTRL-CSF, PPMS-CSF, or SPMS CSF.

[0021] Figs 4A-4E. Inflammatory demyelinated lesion in the corpus callosum of a mouse injected for 2 weeks with MS-CSF. (A) Tiles scan reconstruction of immunostained brain section showing lesion localization. (B) Lesion. Green, MBP immunostaining. Red, Ibal⁺ macrophages. White, BrdU⁺ proliferative cells. Blue, DAPI. (C) Degenerative axons within the lesion visualized by immunostaining for APP (red). (D) CD138⁺ plasma cells (black arrows) and (E) CD19⁺ B cells (black arrows) on the periphery of the lesioned area (solid white lines).

[0022] Figs 5A-5D. RNA level comparisons after 4 weeks of CSF injections. (A) Heat map representation of gene expression in brains and spinal cords of animals treated with PPMS-CSF, SPMS-CSF, or CTRL-CSF. Values are the average of 3 technical replicates normalized to saline (calibrator) and represented on a logarithmic scale. (B-D) Gene expression comparison in spinal cord of CSF-injected animals for (B) CD3, (C) CD4, and (D) CXCL13. Significant P values obtained by one-way ANOVA are reported for each gene [post-hoc analysis: Tukey HSD Test, CD3, CD4, CXCL13: P<.005 for PPMS vs. SPMS or CTRL].

[0023] Figs 6A-6C. Effect of CSF injections on Thl-Thl7 immune responses in the mouse CNS. (A) Protein level comparisons in mouse CSF. Black horizontal bars represent controls value (set as calibrator). Error bars represent SEM. **p = 0.008, one-way ANOVA [post-hoc analysis: Tukey HSD Test, IL6, IL17: P<.001 for: PPMS or SPMS vs. control; IL10, IL12p40: P < .005 for: PPMS or SPMS vs. control]. BDL= below detection limit. N = number of biological replicates examined. SPMS = secondary progressive MS; PPMS = primary progressive MS. (B) T cell subtype comparisons between CSF injected animals with (++) or without (--) in vitro stimulus. (C) Thl7/Thl cell ratio comparisons between in vitro stimulated and unstimulated samples. Error bars represent SEM. *p=0.03, two-way ANOVA [post-hoc analysis: Bonferroni Test (Thl7— : P < .001 for: PP vs. saline; P < .005 for PP vs. SP and CTRL. Thl7++: P < .001 for: PP vs. CTRL) and saline]. N = number of mice examined. SP = secondary progressive; PP = primary progressive; CTRL = control.

[0024] Figs 7A-7B. Protein level comparisons of pro and anti-inflammatory cytokines present in the injected human CSF cohort. (A) Comparisons between CSF samples derived from untreated patients. Error bars represent SEM. Significant P values obtained by one-way ANOVA are reported for each cytokine [post-hoc analysis: Tukey HSD Test, IL17: P<.005 for PPMS, SPMS, or RRMS vs. CTRL; IL6: P<.005 for PPMS vs. CTRL or RRMS; IL8 and ΜΙΡ-1β: P<.005 for PPMS vs. CTRL]. The number of biological replicates examined was: 6 for PPMS and RRMS; 8 for SPMS and CTRL. (B) Comparisons between CSF samples derived from progressive MS patients before and after treatment. Significant P values obtained by student t-test are reported for each cytokine. The number of biological replicates examined was 4 (2 PPMS and 2 SPMS combined post-assay). PPMS = primary progressive MS; SPMS = secondary progressive MS; CTRL = control.

[0025] Figures 8A-8C. Change in the levels of CD1 lb+/CD45 ^hi cells in mouse brain. (A) Average change in the levels of CD1 lb+/CD45 ^hl cells in the brains of mice injected with aCSF from untreated patients with progressive MS ("PROG UNTREATED"), treated patients with progressive MS ("PROG UNTREATED"), or healthy individuals ("CTRL"), or artificial CSF ("ACSF"). (B) Change in the levels of CD1 lb+/CD45 ^hi cells resulting from injections of aCSF from each individual patient with primary progressive MS, before and after treatment. (C) Change in the levels of CD1 lb+/CD45 ^hl cells resulting from injections of aCSF from each individual patient with secondary progressive MS, before and after treatment.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0026] Multiple sclerosis (MS) is an inflammatory disease in which the myelin sheathes surrounding neuronal projections in the CNS (central nervous system) are damaged. Within the CNS, the immune system attacks myelin (the fatty substance that surrounds and insulates axons), as well as the axons themselves. The damaged myelin forms lesions which result in scar tissue (sclerosis) formation, which distorts or interrupts neuronal signaling, leading to a variety of neurological symptoms.

[0027] The term "lesion" as used herein refers to a pathological discontinuity of tissue. The terms "CNS lesion", "demyelinating lesion" or "demyelinated lesion" are used

interchangeably herein to refer to an area of the brain or spinal cord that manifests with abnormal tissue, particularly an area that manifests with demyelinated axons. These lesions can be discrete and defined, such as an area characterized by any one or more of

demyelinated axons, increased presence of immune cells, axonal loss, and/or increased amounts of amyloid precursor protein, within the lesion or at the lesion border. Immune cells can be any one or more of macrophages, microglia, B-cells, T-cells, or other cells associated with an inflammatory response. However, the lesions can also be diffuse and poorly defined, for example, showing both myelinated and demyelinated axons, and/ or immune cells within and adjacent to a lesion-like area.

[0028] Disclosed herein are methods of inducing demyelinating lesions in the CNS of an animal, comprising administering acellular cerebrospinal fluid (aCSF) from a subject with MS to said animal by at least one intracerebral injection. This disclosure establishes for the first time that acellular CSF derived from untreated Progressive MS (PMS) patients can cause typical MS-like pathological lesions in the mouse CNS.

[0029] The inventors have discovered that cell-free spinal fluid derived from patients with MS, injected into the brain of a candidate animal, results in the development of discrete lesions of inflammatory demyelination that closely resemble CNS lesions seen with MS. Time course analysis has revealed that demyelination precedes axonal loss and is associated with macrophage proliferation and T-cell infiltration, followed by complete axonal loss, astrogliosis and B-cell/plasma cell infiltration. Lesions do not occur with inflammatory or non-inflammatory control-CSF or saline injections.

MS in humans and CNS lesions

[0030] MS can be categorized as progressive MS (PMS, in which symptoms and disease course steadily worsen over time with little or no remission) or relapsing-remitting MS (RRMS). RRMS is characterized by defined attacks of worsening neurologic function, also called relapses, flare-ups or exacerbations, followed by partial or complete recovery periods (remissions), during which symptoms improve partially or completely and there is no apparent progression of disease. The majority of people with MS are initially diagnosed with RRMS. Progressive MS (PMS) encompasses secondary progressive MS (SPMS) and primary progressive MS (PPMS). Most people who are initially diagnosed with RRMS will eventually transition to SPMS, where the disease progresses more steadily (although not necessarily more quickly), with or without relapses. In contrast to SPMS, primary progressive MS (PPMS) is characterized by steadily worsening neurologic function from the first appearance of symptoms. Although the rate of progression may vary over time with occasional plateaus and temporary, minor improvements, there are no distinct relapses or remissions. About 10 percent of people with MS are diagnosed with PPMS.

[0031] MS is associated with the development of discrete lesions in the CNS characterized by demyelination of neurons and infiltration of leucocytes. Ferguson et al., Brain 120:393- 399 (1997), categorizes human MS lesions as acute, active chronic, or chronic. Acute MS lesions are typified by areas of local demyelination, with CD3⁺ T-cells and CD68⁺ macrophages present throughout the lesion. In some cases inclusions containing myelin debris are seen in the interior of macrophages. Acute lesions are further characterized by positive staining for amyloid precursor protein (APP), a marker for axonal damage, throughout the lesion. Active chronic lesions are characterized by T-cells, macrophages, and APP staining present more around the border of the lesion than within. Chronic lesions are characterized by few or no T-cells or macrophages in the lesion, no or low APP staining within or around the lesion, and reduced axon density. In addition, MS and MS lesions are characterized by the presence of B cells, including CD 19+ B cells and CD 138+ plasma cells (Gilden et al., Mult. Scler. 2: 179-183 (1996); Ritchie et al., J. Immunol. 173:649-656 (2004)); and astrogliosis (activation of astrocytes; Gudi et al., Front. Cell. Neurosci. 8(73): 1-24 (2014)). [0032] Neurological symptoms resulting from these CNS lesions include numbness or weakness in one or more limbs, partial or complete loss of central vision, usually in one eye, often with pain during eye movement (optic neuritis), double vision or blurring of vision, tingling or pain in parts of the body, electric- shock sensations that occur with certain head movements, tremors, walking (gait), balance, or coordination problems, slurred speech, fatigue; dizziness, bladder or bowel dysfunction, cognitive dysfunction, emotional changes, sexual dysfunction, depression, spasticity, speech or swallowing problems, hearing loss, seizures, respiration or breathing problems and itching.

Lesions induced by the disclosed methods

[0033] The lesions seen in animals induced by the methods disclosed herein show strong parallels with human MS, including demyelinated foci with immune cells such as CD3+ T cells, CD19⁺ B cells, CD138⁺ plasma cells, activated macrophages/microglia, axonal loss, astrogliosis, and APP⁺ neural projections. The induced lesions also show time course- dependent characteristics similar to acute, chronic active, and chronic lesions in human MS pathology.

[0034] An advantage of the present methods over known methods of inducing MS-like lesions in model animals is that the claimed methods can induce CNS lesions without artificial antigen sensitization. This is in contrast with other models, such as the experimental autoimmune encephalomyelitis (EAE), that are commonly used as a model for MS pathology. EAE is induced by immunization with CNS tissue or myelin peptides, such as myelin basic protein or proteolipid protein, in adjuvant. Sensitization to myelin antigens in EAE typically occurs through the use of adjuvant, which contains immunoirritants such as highly

immunogenic bacterial components. Thus, in EAE, the inducing antigens are known, whereas in MS, there is no unique identified antigen (Constantinescu et al., Br. J. Pharmacol. 164: 1079-1106 (2011)). In the methods disclosed herein, MS lesions are induced by repeated administration of only cell-free CSF fluid, without co-administration of adjuvant. Thus, the model disclosed herein can provide insight into the pathology of MS that avoids the potential errors that can result from using an adjuvant-induced immune response to study a neurodegenerative condition that has no identified antigen-based stimulus. Obtaining acellular CSF

[0035] In the disclosed methods, acellular CSF (aCSF) from a subject is serially administered into the brain of a non-human animal. The repeated administration of aCSF leads to the development of discrete lesions, the development of which can be studied and tested.

[0036] As a first step in the process, a sample of cerebrospinal fluid (CSF) is collected from a subject.

[0037] A subject is defined as a person diagnosed with MS, or suspected of having MS, or suspected of having a neurological disorder. The subject can be a person exhibiting one or more symptoms associated with MS. In one embodiment, the subject is a person with active disease as defined by the presence of at least one of the following criteria in the 6 months preceding CSF sample collection: (1) one or more relapses documented by a neurologist's examination; (2) a change in 0.5 points or greater in the EDSS score; and (3) change in MRI, such as a change in the number or size of lesions or the presence of gadolinium-enhancing lesions. In some embodiments, the subject has not been treated for MS; in other

embodiments, the subject has been treated for MS.

[0038] CSF can be collected by standard methods, such as by lumbar puncture or by access port aspiration of implanted pumps. CSF samples are preferably processed immediately and kept on ice. To remove cells and obtain an acellular CSF (aCSF) sample, the sample can be centrifuged to pellet the cells, or filtered to remove cells and cell debris. For example, samples can be centrifuged at between 150-300xg for 10-20 minutes to separate cells from fluid. Following centrifugation, cell-free supernatant can be decanted, pipetted, or otherwise removed from the centrifuge tube containing the cell pellet. For filtration methods, the filtered solution is kept while the cells are discarded. Aliquots of CSF can be stored at -60 to -100°C, preferably -80°C, until use.

[0039] After removing cells to obtain an aCSF sample, the aCSF is administered to an animal for testing.

Administration of aCSF to a non-human animal

[0040] The animal models of the present invention encompass any non-human vertebrates that are amenable to procedures yielding a neuronal demyelination condition in the animal's nervous systems including the central and peripheral nervous system. Examples of model organisms include but are not limited to mammals, primates, and rodents. Non-limiting examples of the preferred models are rats, mice, guinea pigs, cats, dogs, rabbits, pigs, chimpanzees, and monkeys. The test animals can be wild type or transgenic. In a specific embodiment, the animal is a mouse.

[0041] Animals are subject to intracerebral administration of aCSF. Administration can be by repeat intracranial injection, by injection through an implanted cannula, or by other means that put the aCSF sample into direct contact with the animal CNS. In one embodiment, a cannula is implanted within the network of ventricles in the brain that extend to form the central canal of the spinal cord, preferably within the third ventricle. aCSF is administered to the animal 1, 2, 3, or 4 times per week, for 1, 2, 3, 4, 5, 6, 7, or 8 weeks. At least two administrations of aCSF are performed, preferably 2, 4, 6, 8, 9, 10, 12, 14, or 15

administrations. The amount of aCSF administered depends on the animal used. For example, administration can be between 0.01-50 ml/kg body weight, preferably 0.1-5.0 ml/kg body weight, even more preferably 0.2-2.0 ml/kg body weight. In embodiments, one or more, two or more, three or more, four or more, five or more, or six or more animals are administered portions of the same sample of aCSF from a subject, and the resulting CNS characteristics of each animal can be combined and/or averaged to provide a result for that subject.

[0042] Administration of aCSF from a subject into an animal induces the formation of discrete, demyelinated, MS-like lesions in the CNS of an animal. The presence of lesions indicates the presence of MS, particularly PMS, in an untreated subject. In a subject treated for MS, the absence of lesions, or reduction in number and/or severity of lesions induced by aCSF of a subject, relative to the number and/or severity of lesions induced by aCSF from the same subject prior to treatment, indicates that the treatment is effective.

Characteristics of induced lesions

[0043] Animals administered aCSF from a subject with MS can present with a range of CNS characteristics relative to control animals, including, but not limited to: presence of lesions in the brain; presence of lesions in the spinal cord; meningeal inflammation; subpial demyelination; increased severity of CNS lesions; increased presence of inflammatory cells or chemokines in the CNS; increased levels of microglia (e.g., cells characterized by CDl lb⁺CD45^hl); decreased expression of transforming growth factor beta- 1 (TGFP-l) in the CNS; increased expression of pro-inflammatory markers in the CNS, including increased expression of one or more, two or more, three or more, four or more, or five or more markers selected from CD3, CD4, CD28, CD11C, granulocyte-macrophage colony- stimulating factor 2 (GM-CSF2), IL-17, IL-4, IL-6, IL-8, CXC-motif chemokine 13 (CXCL13), MCP-1/ CCL2 (monocyte chemotactic protein- 1/ chemokine c-c motif ligand 2), ΜΙΡ-1β/ CCL4

(macrophage inflammatory protein- 1β/ chemokine c-c motif ligand 2), and interferon-gamma (IFNy). In some embodiments, the spinal cord of a lesion-induced animal has increased expression of at least one of CD3, CD4, or CXCL13, relative to control levels.

[0044] Lesions are identified by the presence of one or more, two or more, three or more, or four or more of: demyelinated axons; myelin debris; ionized calcium-binding adapter molecule 1- positive (Ibal+) cells; glial fibrillary acidic protein- positive (GFAP+) cells; CD3+ cells; CD 138+ cells, CD 19+ cells; axonal degeneration at the lesion periphery; and increased expression of amyloid precursor protein (APP); relative to control levels.

[0045] Lesions can be characterized by location within the animal CNS and by location within the animal brain; and/or by total volume of lesioned areas. In one embodiment, lesions are present only in the brain. In other embodiments, lesions are present in the brain and spinal cord. The presence of lesions in the brain and spinal cord of an animal indicate that the subject whose aCSF was administered to the animal has PMS. In another embodiment, the presence of one or more lesions in the corpus callosum of the animal brain indicates the subject has MS. In a further embodiment, the presence of lesions in the corpus callosum combined with one or more lesions in at least one of the dorsal fornix, fasciculus retroflexus, stria medullaris, and/or the primary somatosensory cortex of the animal, indicates that the subject has PMS.

[0046] CNS lesions can also be characterized by total volume of lesioned areas in the CNS. For example, the total volume of lesioned tissue in an animal can be measured in terms of cubic millimeters, with greater volumes of lesioned tissue indicating greater lesion severity. Increased total lesion volume can result from an increased number of lesions, and/or from larger lesioned areas, each of which correlates with increased severity of MS in the subject from which aCSF was obtained. aCSF from a patient without MS will produce no lesions in an animal; however, aCSF from a patient with MS will produce lesions in an animal CNS, such as a mouse CNS or brain, with a total volume between 1 x 10 -^"3 mm 3 to 15 x 10 -^"3 mm 3 , preferably 2 x 10 -^"3 mm 3 to 12 x 10 -^"3 mm 3. Where more than one animal is administered aCSF from a single subject, lesion volumes for multiple animals receiving aCSF from a common subject can be averaged. For example, aCSF from a subject with PMS will produce lesions with an average total volume, over multiple animals, between 4 x 10 -^"3 mm 3 to 10 x

10 -^"3 mm 3 , preferably between 5 x 10 -^"3 mm 3 to 9 x 10 -^"3 mm 3.

[0047] Increased "lesion severity" can be characterized by increased lesion volume, and also by increased amount of demyelinated axons, myelin debris, Ibal+ cells, GFAP+ cells; CD3+ cells; CD138+ cells, CD19+ cells; axonal degeneration; and/or amyloid precursor protein (APP), relative to less severe lesions, which are characterized with less demyelination, more axons present, fewer inflammatory cells, and/or less APP. In embodiments, greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of animals injected with aCSF from a subject develop CNS lesions.

[0048] In some embodiments, the severity of demyelinating lesions induced by said administration is increased relative to the severity of demyelinating lesions induced by administration of CSF from a subject who does not have MS, or a subject with MS who has received treatment for MS. In other embodiments, the severity of demyelinating lesions induced by CSF administration is decreased relative to the severity of demyelinating lesions induced by administration of CSF from the same subject prior to treatment of the subject for MS.

[0049] The "control" can be an animal who has not received aCSF from a subject with MS. The control animal can be, for example, a sham-treated animal administered saline or other sterile solution; an animal administered aCSF from a subject without MS; an animal administered aCSF from a subject with a non-MS neurological condition; or an animal receiving no administration at all or administration with artificial CSF. "Control levels" of a marker represents the level of that marker in an animal that has not received administration of aCSF from a subject with MS.

[0050] As used herein, a "non-MS neurological condition" refers to a condition or disease that can have one or more overlapping symptoms with MS. Examples of non-MS

neurological conditions include, but are not limited to, Neuromyelitis optica, Acute

Disseminated Encephalomyelitis, Subacute combined degeneration of the spinal cord, Neurosarcoidosis, HIV- and/or HTLV1 -associated myelopathy, Polyarteritis nodosa, Neurosyphilis, systemic lupus erythematosus, Behcet disease, Sjogren disease, primary lateral sclerosis, Adrenomyeloneuropathy, paraneoplastic and spinocerebellar syndromes, collagen-vascular diseases, stroke, Parkinson's disease, neurodegenerative diseases and age- related neurodegeneration, and neurological conditions resulting from viral infections, Lyme disease, side effects from high exposure to certain toxic materials, severe vitamin B12 deficiency, and autoimmune conditions.

Methods to determine MS or MS subtype in a subject

[0051] The disclosed methods can be used in a diagnosis of MS, particularly PMS, in a subject. Administration of aCSF from a subject with PMS (that is, either PPMS, or SPMS) typically induces the formation of lesions in an animal CNS. The presence of lesions in the CNS of one or more animals following administration of aCSF from a subject indicates that the subject has MS, particularly PMS. Further, the presence of lesions in the spinal cord of at least one test animal indicates a likelihood that the subject has PMS, particularly PPMS. The methods provided herein can be combined with one or more additional diagnostic criteria to establish a diagnosis of MS. Such additional diagnostics include analysis of a patient's medical history, magnetic resonance imaging (MRI), evoked potentials (EP), visual evoked potentials (VEP), oligoclonal band analysis, and blood tests. These diagnostic methods are known in the art.

[0052] The disclosed methods can further be used to distinguish PMS from RRMS in a subject. Specifically, aCSF from a subject with RRMS induces fewer and/or less severe lesions in animals relative to aCSF from a subject with PMS. Thus, the number and/or severity of CNS lesions in one or more animals administered a portion of an aCSF sample from a subject can be used to distinguish SPMS and PPMS (identified by greater lesion severity) from RRMS (identified by reduced lesion severity). This distinction reflects the known clinical and pathological differences between the two forms of the disease. RRMS is characterized by disease exacerbations and remissions and thus in theory, the CSF in a subject with RRMS can include a pool of pro-inflammatory and anti-inflammatory or regulatory healing factors, whereas in progressive disease, the CSF will have greater amounts of pro-inflammatory cells and cytokines, correlated with the relentless worsening of PMS states. Methods to determine treatment efficacy and monitor MS progression

[0053] Further disclosed are methods to determine the effectiveness of a treatment for multiple sclerosis (MS). In these methods, aCSF from a subject with MS, who has not been treated for MS, is administered to a first animal set of at least one first animal, according to the methods disclosed herein. The results of this administration are compared to the results of administering aCSF from the same subject, after the subject has been treated for MS, into a second animal set of at least one second animal. By comparing one or more CNS

characteristics of the first and second animal sets, the effectiveness of the treatment in the subject can be determined. For example, effective treatment is indicated if there is reduced severity of CNS lesions in the second animal set, relative to the severity of CNS lesions in the first animal set. In another example, the treatment is determined to be effective if the second animal set has increased expression of CD 17, and/or decreased expression of one or more of CD3, CD4, CD28, CD11C, granulocyte-macrophage colony-stimulating factor 2 (CSF2), IL4, CXC-motif chemokine 13 (CXCL13), or interferon-gamma (IFNy), relative to expression of these markers in the first animal set.

[0054] Additionally disclosed are methods to monitor the progression of MS in a subject. In these methods, CSF from a subject with MS is repeatedly obtained, such as every two, four, six, or eight months; once a year; once every two, three, or four years; or any combination of these times. The subject can be undergoing treatment for MS, or untreated. Portions of an aCSF sample from the subject at each time point can be administered to at least one animal. By comparing the CNS characteristics of administration of aCSF obtained at a later time point with the CNS characteristics of administration of aCSF from the same subject at an earlier time point, the progression of MS (or ideally, lack of progression) can be monitored in the subject. For example, if increased severity of CNS lesions, increased levels of proinflammatory markers, and/or decreased levels of anti-inflammatory markers are present in the animal administered aCSF at a later time point, relative to an animal administered aCSF at an earlier time point, MS in the subject is indicated as progressing to a worsened state.

Animal models and methods of use

[0055] Animals with CNS lesions induced by the present methods can be further used for testing a candidate agent for treatment of MS. The method includes the steps of: (a) administering a candidate agent to a test animal with CNS lesions generated by the disclosed methods of inducing CNS lesions in the test animal by administration of aCSF from a subject with MS; and (b) determining the effect of the candidate agent on treating the lesion or preventing the formation of lesions. In embodiments, the agent is administered before, concurrent with, and/or after administration of aCSF, to determine if the agent is able to prevent lesion formation, is able to decrease the severity of lesions induced, or is able to treat existing lesions, for example, by improving re-myelination and/or formation of healthy CNS tissue.

[0056] Further disclosed herein are methods for utilizing a lesion-induced animal disclosed herein to evaluate the effect of MS CSF on microglial activation. Such methods include extracting microglia (macrophages in the brain) from the brain of an lesion-induced animal, and determining the level of macrophages. In some embodiments, brain macrophages are identified based on CD1 lb⁺ and CD45^hl. It has been shown herein that aCSF obtainined from MS patients can induce an increased level of microglia to a larger extent - in other words, can induce microglia activation at a higher level - as compared to control (see, e.g., Figure 8A). Further, it has been shown herein that aCSF from patients responsive to a treatment has a reduced ability to induce microglia activation post treatment as compared to pre-treatment (see, e.g., Figures 8B and 8C). In certain embodiments, the extent of increase in the level of microglia in an lesion-induced animal as compared to a control is correlated with the disease status, progression or treatment response of the MS patient from whom aCSF sample has been obtained to induce lesions in the animal.

[0057] Further provided herein are animals with CNS lesions induced by the disclosed methods.

EXAMPLES

METHODS

Patient Selection and CSF Collection

[0058] Cerebrospinal fluid (CSF) was collected with informed consent from 28 patients, 20 with clinically definite MS (as defined by McDonald et al., 2001), and 8 non-MS controls (Table 1). Of the 18 MS patients, 8 were secondary progressive, 6 primary progressive, and 4 relapsing remitting. Eight non-MS control (CTRL) CSF samples were obtained for diagnostic purpose from untreated patients with other neurological diseases, including inflammatory (human T lymphotropic virus type-I associated myelopathy and transverse myelitis) and non-inflammatory diseases (spinal cord injury, spinal stenosis, and stroke). All of the MS patients in this study had active disease (see below for definition), and none of them received any immunomodulatory treatment for at least 6 months prior to CSF collection (untreated samples). A second CSF collection was performed in 4 patients who appeared to have stabilized as assessed by the expanded disability status scale (EDSS) after treatment with intrathecal methotrexate (ITMTX) (3 patients) or natalizumab (1 patient). CSF was obtained with sterile techniques either by standard lumbar puncture or by access port aspiration of implanted pumps. CSF samples were processed immediately and kept on ice. Samples were centrifuged at 200xg for 15 minutes to separate cells from fluid (acellular CSF or aCSF). aCSF was collected with a sterile pipette and divided in 0.1, 0.5, and 1 ml aliquots. All samples were confirmed to be free of red blood cell contamination. Aliquots of CSF were stored at -80°C until use.

Table 1.

CSF =cerebro spinal fluid. EDSS = expanded disability status scale. PP = primary

progressive. SP = secondary progressive. RR = relapsing, remitting. CTRL = controls.

Both mice were injected with CSF obtained from a natalizumab treated patient. Clinical Assessment of MS Patients

[0059] All patients in the study had a complete neurological examination at the onset of the study. In addition, routine brain MRI scans were performed on all study subjects 1-2 weeks before CSF collection. Active disease was defined by the presence of any one of the following criteria in the 6 months preceding CSF sample collection: (1) one or more relapses documented by a neurologist's examination; (2) change in 0.5 point or greater in the EDSS score; and (3) change in MRI, specifically a change in the number or size of lesions or the presence of gadolinium-enhancing lesions.

Cannula Implantation

[0060] Mice (C57/BL6, males) were purchased from The Jackson Laboratories (Bar Harbor, ME). At 8 weeks of age, animals were anesthetized with a solution of ketamine (lOOmg/kg) and xylazine (lOmg/kg). They were positioned in a Knopf Small Animal Stereotactic device such that their heads were stable and immobile. The skull was exposed and a small hole was drilled at the following stereotactic coordinates in reference to the bregma: -1 mm

(anteroposterior axis), 0 mm (medial-lateral axis). A custom made cannula (26 gauge, 5mm pedestal, cut 3.5 mm below the pedestal; PlasticsOne, Roanoke, VA) was inserted for its entire length at this position to reach the dorsal third ventricle. The cannula was glued in place using Loctite454 instant adhesive and then with dental cement. Two to three sutures were used to hold the skin around the incision together. A dummy (PlasticsOne), made to fit the length and gauge of the cannula, was screwed into the guide to keep the passage unobstructed. All animal experiments were approved by IACUC committee of the St Luke's Roosevelt Hospital Center of New York and conformed to NIH guidelines.

CSF Injections

[0061] Mice were allowed to recover for one week before injections to ensure proper healing of the wound and stability of the cannula. Prior to the injection, the dummy was unscrewed from the guide. An injection needle (PlasticsOne) was attached to polyethylene tubing (Becton Dickinson, Sparks, MD) and rinsed first with 100% ethanol and then with sterile saline. 22 μΐ of sterile CSF was loaded into the tubing using a Hamilton Syringe. CSF or saline was injected over the course of 10 minutes. The needle was kept in place for an additional 5 minutes to avoid liquid backflow. Injections were given twice a week, for 1, 2, or 4 weeks. Mice were injected daily with BrdU (lOmg/mL) 5ul/g of body weight until sacrificed.

Table 2. CSF injections

Mouse Sample Collection

[0062] CSF collection: Anesthetized mice were positioned in a Knopf Small Animal Stereotactic device so that their heads were stable. The skin and muscle covering the cisterna magna were cut and pulled to the side to reveal the membrane of the cisterna magna. A fire- pulled borosilicate glass capillary tube (Sutter Instrument, O.D. 1.5mm, 0.75mm) was inserted approximately 1mm into the membrane and CSF was collected by capillary suction (5-8 μΐ per sample). Samples were transferred to an Eppendorf tube, flash frozen in liquid nitrogen, and stored at -80 °C until use.

[0063] Protein and RNA extractions: Brain and spinal cord were removed and cut in half longitudinally. One half was placed in RIPA buffer (Cell Signaling, Danvers, MA) with HALT Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific,

Rockford, IL) and then sonicated for protein extraction. The other half was processed for RNA extraction using QIAZOL and RNEASY Lipid Tissue Midi Kit (Qiagen).

[0064] Animal Perfusion: Animals were intracardially perfused first with saline and then with 4% paraformaldehyde (PFA) in 0.1M PBS, pH 7.4. Brains and spinal cords were post- fixed in formaldehyde for at least 24 hours, then processed and embedded in a paraffin cassette. Tissues were sliced at 5μιη with a microtome and mounted onto UNIFROST PLUS Microscope Slides (VWR, Chester, PA). Slides were dried overnight before staining.

Tissue Staining

[0065] Immunofluorescence: Microscope slides were deparaffinized with a xylene-alcohol scale and antigen retrieval was performed using antigen unmasking solution from Vector Laboratories (Burlingame, CA). Slides were washed and blocked in 10% normal goat serum (NGS) with 0.2% Triton X-100 for 1 hour. Following blocking, tissue was incubated with primary antibodies or the appropriate control (negative control, isotype control, antibody preabsorbed with the relative immunogenic peptide) for 2 hours at room temperature.

Antibodies used included mouse anti-myelin basic protein (MBP, 1: 1000), mouse anti- neurofilament SMI312 (1 : 1000), mouse anti- tubulin-β-ΙΙΙ (all from Covance, Denver, PA), rabbit anti-glial fibrillary acidic protein (GFAP, 1:500) (Dakocytomation, Glostrup,

Denmark), rabbit anti-Ibal (1:200) (Wako Chemicals, Richmond, VA), rabbit anti-CD3 (1: 1000) (Abeam, Cambridge, MA), rabbit anti-amyloid precursor protein (APP, 1: 100) (Life Technologies, Carlsbad, CA), mouse anti BrdU (1 :400) (Developmental Studies Hybridoma Bank, Iowa City, IA). After PBS washes, slides were incubated for 2 hours at RT in the dark with the appropriate ALEXAFLUOR conjugated secondary antibodies (Life Technologies) diluted in blocking solution. Slides were washed for 5 minutes in 4',6-diamidino-2- phenylindole (DAPI) diluted in PBS (1:5000), followed by two PBS washes. Tissue was sealed under a cover slip using FLUOROMOUNT (Sigma-Aldrich, St. Louis, MO). Samples were analyzed using a Zeiss LSM 510 Meta Confocal microscope running ZEN imaging software at lOx, 20x, 40x, and 63x magnification.

[0066] Immunohistochemistry: After incubation with primary antibody (rabbit anti CD 138 at 1:400, and rabbit anti CD19 at 1:400 dilution, Abbiotec, San Diego, CA), slides were incubated with horseradish peroxidase-conjugated secondary antibodies (1 hr at RT). After developing with DAB (Vector Laboratories, Burlingame, CA) and counterstaining with Hematoxylin (Sigma- Aldrich, St, Louis, MO), slides were sealed with CYTOSEAL mounting media (Thermo Scientific, Portsmouth, NH).

[0067] Luxol Fast Blue staining: Microscope slides were baked for 30 minutes at 37°C to dry, and deparaffinized with a xylene-alcohol scale. Slides were immersed in 2 changes of 95% ethanol for 1 minute each, and then placed in Luxol Fast Blue solution (0.1% Solvent Blue 38 (Sigma) in 95% Ethanol, + 5ml 10% glacial acetic acid, filtered) at 58°C for at least 3 hours. Following staining, slides were immersed in 95% alcohol and then distilled water to remove excess stain. Differentiation was accomplished by submerging the slides in 0.05% lithium carbonate for 10-20 seconds, followed by two changes (1 minute each) in 70% ethanol. These steps were repeated if further differentiation was required. Slides were washed in distilled water and then stained for 5 minutes in Cresyl Violet Solution (0.25% Cresyl violet acetate (Sigma) + 5 drop 10% glacial acetic acid per 300 ml solution, filtered). Slides were then washed twice with 95% ethanol (1 minute each) and 100% ethanol (2 minutes each), cleared in 3 changes of xylene, dried, and then mounted using CYTOSEAL (Thermo Scientific, Portsmouth, NH).

Quantitative real time PCR (q-RT-PCR)

[0068] q-RT-PCR was performed on the Applied Biosystems 7900 HT Fast Real-Time PCR System using individual TAQMAN probes or customized TAQMAN low density array and TAQMAN Gene Expression MASTER MIX (Applied Biosystems, Foster City, CA). For array PCR, samples were loaded and centrifuged twice at room temperature for 1 minute at 1200rpm. Fold change in RNA levels was calculated using the ΔΔ Ct method (relative quantitation), with the saline group used as calibrator, and 18S rRNA expression as internal control. Data were represented on a heat map generated with R (The R Project for Statistical Computing). Luminex assay

[0069] For mouse CSF analysis, samples were pooled together from mice injected with the same patient CSF and mixed with BIO-PLEX PRO™ Mouse Cytokine Thl7 Panel A 8-Plex (Bio-Rad Laboratories, Hercules, CA). For human CSF analysis, individual samples were mixed with BIO-PLEX PRO™ Human Cytokine 17-Plex Assay (Bio-Rad). CSF samples that were found to be blood-contaminated were excluded from the analysis. Both types of CSF samples were run on a BIOPLEX 200 system (Bio-Rad) according to the manufacturer's protocol. Briefly, 50 μΐ of each CSF supernatant and various concentrations of each cytokine standard (Bio-Rad) were added to 50 μΐ of antibody-conjugated beads (Bio-Rad) in a 96-well filter plate (Millipore, Billerica, MA). After a 30 min incubation, the plate was washed and 25 μΐ of a biotinylated antibody solution (Bio-Rad) was added to each well, followed by another 30 min incubation. The plate was then washed and 50 μΐ of streptavidin-conjugated phycoerythrin (PE; Bio-Rad) was added to each well and incubated for 10 min. Following a final wash, the contents of each well were resuspended in 125 μΐ of assay buffer (Bio-Rad) and analyzed using a BIO-PLEX Array Reader (Bio-Rad). The cytokine concentrations were calculated by reference to a standard curve for each cytokine derived using various concentrations of the cytokine standards (0.2, 0.78, 3.13, 12.5, 50, 200, 800 and 3200 pg ml) assayed in the same manner as the CSF samples. The lower detection limit for each cytokine was: 0.19 pg/ml for ILip, 0.06 pg/ml for IL2, 0.02 pg/ml for IL-4, 0.22 pg/ml for IL-5, 0.17 pg/ml for IL-6, IL-7, and TNF-a, 0.13 pg/ml for IL-8, IL-17, and ΜΙΡ-Ιβ, 0.23 pg/ml for IL- 10, 0.27 pg/ml for IL-12(p70), 0.09 pg/ml for IL-13, 0.06 pg/ml for G-CSF, 0.31 pg/ml for GM-CSF, 0.24 pg/ml for IFN-y, and 0.12 pg/ml for MCP-1. All samples were analyzed undiluted in duplicate.

CSF stimulation of cultured T cells and FACS analysis

[0070] Cervical, axillary, and inguinal lymph nodes were harvested from CSF injected animals after 8 injections. Samples from the same animal were pooled together and single cell suspensions were prepared using a 70 μΜ cell strainer. CD4+ cells were isolated using DYNABEADS FLOWCOMP™ Mouse CD4 kit (Life Technologies), plated at 4 x 105 cells/well in 96-well culture plates and cultured in RPMI containing 10% FBS, 1% minimum essential medium, 0.1% beta-mercaptoethanol with (stimulated) or without (unstimulated) 10% CSF derived from the same sample that was injected into the animal. CSF was replenished every other day. After 2 weeks cells were treated with brefeldin A (GolgiPlug; BD Bioscience, San Jose, California), harvested, pre-incubated for 15 minutes with staining buffer containing BD Fc Block (^g/10⁶ cells in 100 μΐ), and surface-stained with anti-CD4- V450 (BD Bioscience). After being fixed and permeabilized with BD

CYTOFIX/CYTOPERM™ Fixation/ Permeabilization Solution Kit (BD Bioscience), cells were stained with IFN-y-APC and anti-IL17A-PE (both from BD Bioscience). Isotype- matched IgG were used as a negative control. The stained cells were analyzed using a FACS ARIA from BD Bioscience.

Statistical analysis

[0071] SPSS was used for statistical analysis. Data were presented as mean + standard error of the mean (SEM) and one or two-way ANOVA with post-hoc analysis (Tukey HSD or Bonjerroni Test) or student t-test were used to assess the significance of the data. P-values < 0.05 were considered statistically significant.

RESULTS

MS-CSF induces inflammatory, demyelinating lesions in the mouse brain.

[0072] CSF samples were collected and their cell count, total protein content, and albumin levels were determined. No significant differences were found among groups (Table 1). The inventors injected CSF into the third ventricles of mice (Table 2). After four weeks of twice weekly injections, 16 out of 24 animals injected with PPMS-CSF and 16 out of 25 injected with SPMS-CSF developed discrete demyelinating lesions at distinct locations in the brain, such as the corpus callosum (Fig. 1A), the cingulum, the dorsal fornix, the fasciculus retroflexus, the lateral ventricles, the primary somatosensory cortex, and the stria medullaris (Table 3). The total volume of lesioned tissue per animal was greater for PPMS- CSF (6.87 +/- 2.1 x 10^"3 mm³) and SPMS- CSF (7.38 +/- 2.5 x 10^"3 mm³) than for RRMS-CSF (3.51 +/- 1.22 x 10^"3 mm³) (Table 3). Table 3. Brain lesion distribution among lesioned animals

r- e roilexas; LV = lateral veatneles; SM = stria, mesfeillaris

[0073] Serial section analysis revealed that lesions in the corpus callosum were the most extensive, spanning 200 to 350 μιη on the medial-lateral axis and 100 to 150 μιη on the anteroposterior axis, localized 600 to 1000 μιη (on the anterior-posterior axis) away from the injection site. Due to their size and frequency the inventors chose to further characterize specifically these lesions. Immunostaining revealed the presence of BrdU⁺ proliferative cells and Ibal⁺ (ionized calcium-binding adapter molecule 1) macrophages (Fig. IB) with engulfed myelin debris (Fig. IB, inset) inside the lesions. Only 2 mice out of 15 injected with RRMS- CSF developed similar lesions (Table 3). No lesions were observed when mice were injected with either control-CSF or saline. Regression analysis (Fig. 1C) showed a correlation between the percentage of lesioned animals and patient EDSS score (R² = 0.3445). No correlation was found between percentage of lesioned animals and patient age (R² = 0.0013).

PMS- CSF induces axonal loss and astrogliosis in the mouse brain

[0074] In PMS, demyelinated CNS lesions are often characterized by the loss of axons and the presence of a glial scar. To determine if these key pathological features were also present in the mouse CSF-induced lesions, the inventors stained consecutive brain sections containing a demyelinated area with markers for axonal integrity (Fig. 2A) and astrogliosis (Fig. 2B). Neurofilament staining revealed a complete loss of axonal projections within the demyelinated area (Fig. 2A). In addition, degenerating axons were present around the lesion edges but not inside, confirming the complete axonal loss at the lesion's core. Furthermore, an elaborate net of GFAP⁺ (glial fibrillary acidic protein) astrocytes resembling a glial scar filled completely the demyelinated area (Fig. 2B), a pre-requisite for sclerotic scar development in MS brain pathology (Smith, et al., Prog Brain Res 94:411-422 (1992);

Williams, et al., Glia 55: 1300-1312 (2007)).

T and B cells are involved in CSF-induced lesion pathology

[0075] To investigate whether infiltration of peripheral immune mediators was involved in the lesion pathology, the inventors looked for the presence of B and T lymphocytes in lesioned brains. Numerous CD3⁺ T-cells were found throughout the lesion area (Fig. 2C) but were virtually absent in the normal appearing white matter. Similar data were obtained when looking for CD138⁺ antibody-producing plasma B-cells (Fig. 2D), and to a lesser extent, for CD19⁺ immature B-cells (Fig. 2E). MS-CSF rarely induces demyelinating lesions in mouse spinal cord despite extensive inflammation

[0076] The vast majority of animals that developed brain lesions also presented with extensive meningeal inflammation (BrdU⁺/Ibal⁺ cells) and foci of subpial demyelination spanning every segment of the spinal cord (Figs. 3A-3C). However, only a small percentage of those mice developed discrete demyelinated, inflammatory lesions in their spinal cord (Fig. 3D and Table 4). These lesions were much smaller than the ones found in the brain but exhibited similar characteristics such as demyelination, BrdU⁺ cells, macrophages, axonal loss, astrogliosis, and CD3⁺ T-cell infiltration. However, neither CD138⁺ plasma cells nor CD19⁺ B-cells were found in this type of lesion. Interestingly, discrete spinal cord lesions were observed only in mice injected with PPMS-CSF and no spinal cord inflammation was detected in animals injected with RRMS-CSF (Table 4). Locomotor performance, evaluated by Rotarod testing, did not vary among treated groups (Fig. 3E). These data are consistent with the fact that, in the vast majority of treated animals, the spinal cord was spared from severe injury.

Demyelination precedes axonal loss and glial scar formation

[0077] To further characterize the lesion genesis, the inventors performed a time course experiment, in which animals were injected biweekly with PMS-CSF and sacrificed 3 days after the second or fourth injection (Table 2, "Time Course"). For this experiment, the inventors selected patient CSF based on the percentage (80% or higher) of mice that had previously developed lesions at 4 weeks post treatment. After 1 week of treatment (2 injections), no lesions were detected in either the brain or spinal cord of treated animals. After 2 weeks (4 injections), lesions were seen with demyelination, proliferative

macrophages, and T-cells (Fig. 4B). However, axonal loss within the lesion was incomplete, as documented by the presence of numerous APP⁺ degenerating axons (Fig. 4C), and GFAP⁺ astrocytes were found surrounding the lesion but not inside, suggesting that glial scar formation had not been initiated. CD138⁺ plasma cells (Fig. 4D) and CD19⁺ B-cells (Fig. 4E) were also found mainly outside the area of demyelination. Table 4. Lesioned animals among surviving mice- four week CSF injections

Patient CSF code Diagnosis lesioned SC lesioned animals/total animals/total surviving surviving animals animals

CSF injections over 4 weeks (twice a week)

1PP Primary 0/4 0/4

progressive MS

2PP Primary progressive MS 4/5 1/5

3PP Primary progressive MS 2/3 0/3

4PP Primary progressive MS 3/4 1/4

5PP Primary progressive MS 3/4 0/4

6PP Primary progressive MS 4/5 1/5

Total for primary progressive MS 16/25 3/25

ISP Secondary progressive MS 2/2 0/2

2SP Secondary progressive MS 1/2 0/2

3SP Secondary progressive MS 3/4 0/4

4SP Secondary progressive MS 0/4 0/3

5SP Secondary progressive MS 4/4 0/5

6SP Secondary progressive MS 2/3 0/3

7SP Secondary progressive MS 1/2 0/2

8SP Secondary progressive MS 3/3 0/3

Total for secondary progressive MS 16/24 0/24

1RR Relapsing remitting MS 0/3 0/3

2RR Relapsing remitting MS 1/2 0/2

3RR Relapsing remitting MS 0/2 0/2

4RR Relapsing remitting MS 0/3 0/3

5RR Relapsing remitting MS 1/3 0/3

6RR Relapsing remitting MS 0/2 0/2

Total for relapsing remitting MS 2/15 0/11 Table 5. Lesioned animals among surviving mice- Time course/ Treated patients

Patient CSF code Diagnosis lesioned SC lesioned animals/total animals/total surviving surviving animals animals

Time course 2 injections (1 week)

2PP Primary progressive MS 0/5 0/5

6PP Primary progressive MS 0/5 0/5

Total for primary progressive MS 0/10 0/10

5SP Secondary progressive MS 0/5 0/5

8SP Secondary progressive MS 0/5 0/5

Total for secondary progressive MS 0/10 0/10

Time course 4 injections (2 weeks)

2PP Primary progressive MS 3/4 0/5

6PP Primary progressive MS 4/5 0/5

Total for primary progressive MS 7/9 0/9

5SP Secondary progressive MS 4/5 0/5

8SP Secondary progressive MS 4/5 0/5

Total for secondary progressive MS 8/10 0/10

Treated-CSF injections

2PP Primary progressive 0/9 0/9

MS/ITMTX

6PP Primary progressive 2/9 0/9

MS/Natalizumab

5SP Secondary progressive 1/9 0/9

MS/ITMTX

8SP Secondary progressive 0/9 0/9

MS/ITMTX

Total for treated progressive MS 3/36 0/36 PMS-CSF mice have altered CNS expression of immune-system related genes

[0078] To determine whether treatment with CSF, derived from PMS patients or control individuals, affects gene expression in the mouse CNS, RNA was extracted from the brain and spinal cord of a subset of mice that received 8 CSF injections. After collection, individual RNA samples were pooled together for each treated group and analyzed in triplicate (technical replicates) by q-RT-PCR using a custom TAQMAN low density array containing pre-hybridized probes to detect the RNA expression of 47 genes. Compared to saline, CSF groups showed a similar transcriptional profiling in the brain, whereas in the spinal cord, gene expression profiling of mice injected with PPMS-CSF was unique (Fig. 5A). In both brain and spinal cord, genes that presented the largest variation among groups (+ 1 fold compared to saline) were associated with the immune system and included: T-cell (CD3, CD4, CD28), B-cell (CD19), and macrophage (CD 11c, Csf2) receptors; cytokines (IL17, IL4); chemokines (CXCL13); transforming growth factor beta-1 (TGFP-l); and interferon-gamma (IFNy). For most of these genes their expression changes were greater in the PMS-CSF injected groups compared to control.

[0079] To perform statistical analysis for the above selected genes, the inventors ran individual monoplex q-PCR assays for each gene. For this experiment samples derived from mice injected with the same patient CSF were pooled together before the assay such that each sample represented an individual patient for a total of 6 PP, 8 SP, and 8 control samples (biological replicates). For both brains and spinal cords, similar expression trends to the ones reported in Fig. 5A were observed for all the genes examined. However, in brain samples due to high intra-group variations no statistical significance was achieved. Interestingly, in the spinal cords, significant upregulation of T cell specific genes CD3 and CD4, and the B cell chemoattractant CXCL13 was reported for the PPMS group compared to SPMS and CTRL (Figs. 5B-5D).

PMS-CSF mice have increased Thl7 cell activity in the CNS and in its periphery

[0080] Based on immuno-pathology and RNA data suggesting T cell involvement in the above described mouse pathology, the inventors investigated the CNS and peripheral expression of Thl and Thl7 cells, which are believed to be key modulators in MS. In the CNS, the expression of cytokines associated with the Thl/Thl7 pathway was analyzed by Luminex-ELISA in brains, spinal cords, and mouse CSF. As described above for the individual monoplex assay, samples derived from mice injected with the same patient CSF were pooled together before the assay. No significant differences between CSF-injected groups were found in either brains or spinal cords. However, when the mouse CSF was analyzed, a significant increase in the expression of IL6 and IL17, two Thl7 associated proteins, and of IL12p40, an antagonist of Thl activation (Kato et al., 1996; Rothe et al., 1997; Kalinski et al., 2001), was observed in animals injected with CSF derived from patients with PMS compared to controls (Fig. 6A). In addition, although higher expression of IL10, which is secreted by several types of T cells including Thl, was detected in the PMS-CSF treated animals, the expression of Thl associated proteins IL12p70 and interferon- gamma (IFN-y) was found below the assay detection limit. These data suggest that, in this animal model of PMS, Thl 7 rather than Thl cells were active in the mouse CNS.

[0081] To investigate if activation of the Thl7 pathway was also present in the peripheral immune system, the inventors analyzed the ratio between Thl 7 and Thl cells by

immuno staining and FACS analysis of cultured lymph nodes cells derived from CSF injected animals and stimulated in vitro with the same patient CSF injected into the animal. Thl7 cells were significantly more abundant in samples derived from mice injected with PPMS- CSF compared to control CSF or saline with or without in vitro stimulus (Fig. 6B). A similar trend was also reported for the SPMS samples after in vitro CSF- stimulation although these differences were not statistically significant. A post-hoc analysis for each individual patient's CSF revealed that animals that developed CNS lesions had a higher Thl7 cell count, with or without stimulus, compared to those that did not. In addition, the Thl7/Thl ratio was increased in both PMS-CSF treated groups after in vitro stimulation (Fig. 6C) whereas control and saline groups remained unaltered.

CSF-induced brain lesions are drastically reduced by clinical treatment and correlate with patient CSF levels of pro-inflammatory cytokines

[0082] To determine whether long term treatment of MS patients had an effect on the above described lesion pathology, the inventors collected CSF from four treated patients whose untreated CSF induced lesions in the vast majority of injected mice. CSF from these four patients after treatment was used in the time course experiment described above. Three of these patients were treated for a year with ITMTX, while the last one received natalizumab for 18 months prior to CSF collection (Table 5, "Treated-CSF injections"). None of the mice developed spinal cord lesions. Only 1 out of 27 animals injected with ΓΓΜΤΧ treated CSF and 2 out of 9 animals injected with natalizumab treated CSF showed distinct brain lesions.

[0083] To investigate which factor(s) in the untreated patient's CSF might have been involved in the mechanism behind lesion formation, the inventors compared the expression level of 17 human cytokines associated with either pro or anti-inflammatory immune responses in our CSF cohort of untreated and treated patients by Luminex-ELISA. The expression of 10 cytokines (IL-Ιβ, IL-2, IL-4, IL-5, IL-10, IL-12Pp70, G-CSF, GM-CSF, IFN-y, and TNF-a) was found below the assay detection limit in the entire cohort. In the untreated cohort (Fig. 7A), of the 7 detectable cytokines IL-17 was significantly higher in the control group compared to each MS subtype with PPMS being the lowest. The opposite trend was found for IL-6, IL-8, MCP-1/ CCL2 (monocyte chemotactic protein- 1/ chemokine c-c motif ligand 2) and ΜΙΡ-1β/ CCL4 (macrophage inflammatory protein- 1β/ chemokine c-c motif ligand 2) with significant differences found between PPMS vs. CTRL or RRMS for IL- 6 and PPMS vs. CTRL for IL-8 and MIP-Ιβ expression. IL-7 and IL-13 showed higher expression in the SPMS but differences among groups were not significant. In the untreated cohort, data from PPMS and SPMS samples were grouped together before comparisons to increase statistical power and then compared with their matched untreated samples.

Interestingly, the CSF levels of IL-6, IL-8, MCP-1, and MIP-Ιβ were significantly reduced after patient's treatment to those of controls (Fig. 7A), whereas CSF levels for IL-17, IL-7, and IL-13 were unchanged (Fig. 7B).

[0084] Characterization of the cellular basis of this lesion formation showed that the Thl7 but not the Thl pathway was stimulated by CSF treatments in the mouse CNS and lymph nodes. This data is consistent with the emerging literature that shows that pathogenic Thl 7 cells play a major role in MS disease pathogenesis (Matusevicius, et al., Mult Scler 5: 101-104 (1999); Kebir, et al. Nature medicine 13: 1173-1175 (2007); Han, et al., Nature 451: 1076- 1081 (2008); Nylander, et al„ The Journal of clinical investigation 122: 1180-1188 (2012)). The inventors further found that the CSF from MS treatment responders showed markedly reduced formation of lesions, suggesting that the factors involved in lesion formation are favorably altered with treatment.

[0085] Cytokine profiling of the injected treated and untreated CSF revealed that these factors include pro-inflammatory molecules, such as CCL2, CCL4, IL-6, and IL-8, which were found to be higher in both types of PMS-CSF compared to RRMS- and control-CSF, which were found to be reduced to control level after treatment. These findings are in agreement with what has been reported by others. For instance, CSF levels of CCL4 has been found to be higher in PPMS (but not in RRMS) vs. non-inflammatory neurological diseases (Matsushita, et al., PLoS One 8:e61835 (2013)) and strong staining of this cytokine has been shown in macrophages within MS plaques (Simpson, et al., J Neuroimmunol 84:238-249 (1998)). Similar reports have been found for the CSF expression of CCL2, IL-6 and IL-8 (Navikas, et al., J Neuroimmunol 64:63-69 (1996); Stelmasiak, et al., Med Sci Monit 6: 1104-1108 (2000); Ishizu, et al., Brain : a journal of neurology 128:988-1002 (2005); Edwards, et al., PLoS One 8:e81007 (2013)). The fact that the highest levels of these cytokines were found in PPMS-CSF, together with higher gene expression of the proinflammatory chemokine CXCL13 in the PPMS-injected mouse spinal cord, could explain the known propensity for spinal cord involvement in PPMS.

[0086] Without being limited, it is believed that the synergistic effects of these human cytokines induce inflammatory pathways such as Thl7, ultimately causing demyelinating pathology in mice. Based on this hypothesis, the higher level of human IL-17 found in control CSF, which never caused lesions, can be explained by the inability of this human cytokine to cross react with the mouse or the lack of other synergistic factors compared to PMS-CSF necessary to induce lesion pathology.

In this disease model no discernible locomotor or other impediments were observed in mice. However, the data shows the development of definite pathological lesions seen in the white matter of their brains at locations distinct from the injection sites of CSF. These findings are novel and provide a biological model that will help to better understand the mechanisms underlying lesion formation and the pathophysiology associated with MS disease

progression. Furthermore, this model has application to evaluate treatment outcomes in mice and guide patients' therapeutic regimens, ultimately leading to the development of specific therapies targeting PPMS and SPMS.

CSF from progressive-MS patients stimulates microglial activation pathways

[0087] Microglia are resident immune cells in the CNS that act as macrophages when activated by injury or disease. This activation is a characteristic of inflammatory diseases like multiple sclerosis (MS). [0088] In this study, the effect of MS cerebrospinal fluid (CSF) on microglial activation was investigated using the mouse model disclosed herein. Mice develop inflammatory demyelinating brain lesions after serial intracerebroventricular injections of acellular CSF obtained from untreated progressive MS patients. In the experiments for this example, mice were injected with aCSF from 5 primary progressive (PP), 7 secondary progressive (SP), MS patients, as well as from 4 non-MS inflammatory controls (IC) and 2 healthy individuals (HC). See Table 6. Microglia/macrophages were extracted from mice brains (n=3 per patient) with a Percoll gradient for FACS analysis. Compared to naive mice or mice injected with artificial CSF, animals in all other groups displayed an upregulation of

CD1 lb⁺/CD45^hlgh macrophages (Figure 8A). In addition, the number of these cells was significantly higher in the PP and SP groups than controls. Remarkably, CSF derived from progressive MS patients who were clinically stable following therapy had greatly diminished capacity to activate microglia/macrophages compared to the paired untreated samples (Figures 8B-8C).

Table 6

PP SP CTRL ACSF

A: Untreated Patient Demographics

# of patients (CSF) 5 7 7 NA

Age at sample collection, mean (SD), 51.4 (10.7) 56.1 (10.2) 56.4 (16.5) NA years

EDSS at sample collection, mean (SD) 6.7 (0.81) 5.8 (1.8) NA NA

Disease duration at sample collection, 11.9 (6.87) 22.6 (6.37) NA NA mean (SD), years

CSF total protein, mean (SD), μg/ml NA

CSF total albumin, mean (SD), μg/ml NA

CSF cell count, mean (SD), /ml 3000 (665.7) 1744 (1495) 864.1 (723.8) NA

B: Treated Patient Demographics

(From Untreated Cohort Above)

# of patients (CSF) 5 7

# patients treated with ITMTX 5 4

# patients treated with natalizumab 0 1 Age at sample collection, mean (SD), 54.1 (10.1) 52.4 (7.1)

years

EDSS at sample collection, mean (SD) 6.9 (0.85) 5.9 (1.2)

Disease duration at sample collection, 14.6 (7.8) 21.4 (4.5)

mean (SD), years

CSF total protein, mean (SD), μg/ml

CSF total albumin, mean (SD), μg/ml

CSF cell count, mean (SD), /ml 4126 (2538) 675.9 (426.5)

C: CSF injections over 4 weeks (twice a

week)

# of mice injected 36 36 24 9

# of mice dead due to treatment 0 0 0 0

# of mice sacrificed/dead early due to 3 0 0 0 surgical complication

# of mice surviving 8 injections 33 36 24 9

# of surviving mice sacrificed for RNA 28 30 18 9 FACS extraction

Claims

What is claimed is:

1. A method of inducing demyelinating lesions in the central nervous system (CNS) of an animal, comprising administering acellular cerebrospinal fluid (aCSF) from a subject with multiple sclerosis (MS) to said animal by at least one intracerebral injection.

2. The method of claim 1, wherein said aCSF is administered by two to fourteen intracerebral injections.

3. The method of claim 2, wherein said aCSF is administered by twice weekly injections.

4. The method of claim 1, wherein said subject has progressive MS (PMS), primary progressive MS (PPMS), or secondary progressive MS (SPMS).

5. The method of claim 1, wherein said animal is a mouse.

6. The method of claim 1, wherein said lesions are identified by one or more of: demyelinated axons; myelin debris; ionized calcium-binding adapter molecule 1- positive (Ibal+) cells; glial fibrillary acidic protein- positive (GFAP+) cells; CD3+ cells; CD138+ cells, CD 19+ cells; axonal degeneration at the lesion periphery; or increased expression of amyloid precursor protein (APP).

7. The method of claim 1, wherein said lesions are present in the brain.

8. The method of claim 7, wherein at least one of meningeal inflammation and subpial demyelination are further present in the spinal cord of said animal.

9. The method of claim 1, further comprising the steps of obtaining a CSF sample from said subject and removing the cells from the CSF sample to obtain an acellular CSF sample, prior to administering aCSF to said animal.

10. The method of claim 1, wherein said subject has not been treated for MS.

11. The method of claim 10, wherein the severity of demyelinating lesions induced by said administration is increased relative to the severity of demyelinating lesions induced by administration of aCSF from an MS patient who has been treated for MS.

12. The method of claim 1, wherein the CNS of the lesion-induced animal has increased expression of one or more markers selected from CD3, CD4, CD28, CD11C, granulocyte-macrophage colony-stimulating factor 2 (CSF2), IL17, IL4, CXC-motif chemokine 13 (CXCL13), transforming growth factor beta-1 (TGFP-l); and interferon- gamma (IFNy), relative to control levels.

13. The method of claim 12, wherein the spinal cord of said lesion-induced animal has increased expression of at least one of CD3, CD4, or CXCL13, relative to control levels.

14. The method of claim 12, wherein said control levels represent the level of expression of said one or more markers in an animal that has not received administration of aCSF from a subject with MS.

15. A non-human animal produced by the method of claim 1.

16. A method for determining the effectiveness of a treatment for multiple sclerosis (MS), comprising administering acellular cerebrospinal fluid (aCSF) from a subject with MS before said subject is treated for MS into a first animal set comprising at least one first animal, administering aCSF from the same subject after said subject has been treated for MS into a second animal set comprising at least one second animal, and determining the efficacy of treatment by comparing one or more CNS characteristics of the first and second animal sets.

17. The method of claim 16, wherein said one or more CNS characteristics are selected from: the presence or severity of CNS lesions; the presence of inflammatory cells and/or chemokines in the CNS; and expression of pro-inflammatory and/or anti-inflammatory markers in the CNS.

18. The method of claim 17, wherein a reduced presence or severity of CNS lesions in the second animal set, relative to the presence or severity of CNS lesions in the first animal set, indicates that said treatment was effective.

19. The method of claim 16, wherein aCSF is administered to each animal set by two to fourteen intracerebral injections per animal.

20. The method of claim 17, wherein said CNS lesions are identified by one or more of: demyelinated axons; myelin debris; ionized calcium-binding adapter molecule 1- positive (Ibal+) cells; glial fibrillary acidic protein- positive (GFAP+) cells; CD3+ cells; CD138+ cells, CD19+ cells; axonal degeneration at the lesion periphery; or increased expression of amyloid precursor protein (APP).

21. The method of claim 17, wherein the treatment is determined to be effective if the second animal set has increased expression of CD 17, and/or decreased expression of one or more of CD3, CD4, CD28, CD11C, granulocyte-macrophage colony- stimulating factor 2 (CSF2), IL4, CXC-motif chemokine 13 (CXCL13), or interferon- gamma (IFNy), relative to expression of these markers in the first animal set.

22. A method for diagnosing progressive multiple sclerosis (PMS) in a subject, comprising administering acellular cerebrospinal fluid (aCSF) from said subject to a test animal by at least one intracerebral injection, wherein the presence of at least one lesion in the CNS of said test animal subsequent to said administration indicates PMS in said subject.

23. A method for diagnosing progressive multiple sclerosis (PMS) in a subject, comprising administering acellular cerebrospinal fluid (aCSF) from said subject to a test animal by at least one intracerebral injection, determining the increase in the level of microglia in the brain of said test animal, wherein a larger increase in the level of microglia as compared to control indicates PMS in said subject.

24. A method for determining the effectiveness of a treatment for multiple sclerosis (MS), comprising administering acellular cerebrospinal fluid (aCSF) from a subject with MS before said subject is treated for MS into a first animal set comprising at least one first animal, administering aCSF from the same subject after said subject has been treated for MS into a second animal set comprising at least one second animal, and determining the efficacy of treatment by comparing one or more CNS characteristics of the first and second animal sets.

25. A method for determining the effectiveness of a treatment for multiple sclerosis (MS), comprising administering acellular cerebrospinal fluid (aCSF) from a subject with MS before said subject is treated for MS into a first animal set comprising at least one first animal, and determining the increase in the level of microglia in the brain of said animal resulting from the administration; administering aCSF from the same subject after said subject has been treated for MS into a second animal set comprising at least one second animal, and determining the increase in the level of microglia resulting from the administration; and determining the effectiveness of the treatment based on comparing the increase in the level of microglia from the second animal set and the increase in the level of microglia from the first animal set, wherein a reduction in the increase from the second animal set indicates that the treatment is effective.