HK1047549B - Il6ril6 chimera for the treatment of neurodegenerative diseases - Google Patents

Description

IL6RIL6 chimeras for the treatment of neurodegenerative diseases

Technical Field

The present invention relates generally to the field of neurological diseases and disorders, and in particular to neurodegenerative diseases, neuroprotection, neural myelination, and the production of myelinating cells. More specifically, the invention provides IL6RIL6 chimeras for use in the manufacture of a medicament for treating neurological diseases or disorders, particularly for protecting nerves and treating demyelinating diseases, and for enhancing nerve regeneration.

Technical Field

The formation of myelin sheaths is the fundamental process by which the Central Nervous System (CNS) and the Peripheral Nervous System (PNS) form and function. The myelin sheath surrounding axons is necessary for the normal conduction of electrical impulses along nerves. Myelin loss is seen in a number of diseases, among which Multiple Sclerosis (MS) affecting the CNS, acute infectious polyneuritis (Guilain-Barre syndrome) CIDP, etc. (see Abramsky and Ovadia 1997; Trojaborg, 1998; Hartung et al 1998). Although the etiology is various, such as infectious disease origin, or autoimmune attack, demyelinating diseases result in loss of nerve function, possibly leading to paralysis and death. Although current therapeutic drugs reduce inflammatory injury in MS and delay disease progression, there is still a need to develop therapies that result in remyelination and recovery of neurological function (Abramsky and Ovadia 1997, Pohlan et al 1998).

Myelin is synthesized by specific glial cells: oligodendrocytes in the CNS and myelinating schwann cells in the PNS. These two types of cells may be referred to as myelinating cells at their fully differentiated stage. Myelin is a structure composed of lipid membranes containing different proteins. Myelin Basic Protein (MBP) is the major component of CNS and PNS myelin protein (30%). During the final undifferentiated phase of oligodendrocytes and myelinating schwann cells, the expression of the MBP gene and other genes encoding various myelin proteins (e.g., PO, PMP-22, MAG for PNS, PLP, MOG for CNS) becomes active. These cells originate in the embryonic neural crest (Fraser 1991), from which they migrate and undergo a differentiation process with a number of steps. The development of Schwann Cells (SC) appears to involve three major steps: 1) generating precursor cells (pSC) from the transitional cells; 2) proliferated and transformed into an embryonic sc (esc) expressing the S100 protein; 3) the post-natal part of the eSC population eventually differentiates into myeloid SCs, expressing MBP and other myelin proteins (Kioussi and Gruss 1996). Cells that migrate out of the neural crest are not only pSC's, but also sensory and sympathetic neurons, smooth muscle cells and cells that reach the skin and hair follicles, and pigment-producing melanocytes. The fate of neural crest cells is influenced by various induction factors: differentiation into glial, neuronal and muscle cells is promoted by the action of neurotonins such as Glial Growth Factor (GGF), BMP2/4 and TGF-. beta.s, respectively (Anderson 1997). Growth factors, such as bFGF or PDGF or SDF, promote melanocyte differentiation (Stocker et al 1991; Anderson 1997).

Schwann and oligodendrocyte progenitor cells eventually differentiate into active myeloblasts. Myelination itself appears to be dependent on signals generated by the interaction between nerve axons and glial cells (Lemke and Chao1998, Trapp et al 1988). When axonal Schwann cell contact is interrupted, as after nerve injury, the cells reverse to a non-myelinating state and myelin protein gene expression is lost (Jessen and Mirsky 1991). In order to stimulate myelination or remyelination following a neurological disease or trauma, it may be of paramount importance to identify factors that induce myelination.

Acute injury (including trauma, hypoxia and ischemia) induced damage to the CNS can affect neurons and white matter. Although the greatest attention has been given to the processes leading to neuronal death, there is increasing evidence that injury to oligodendrocytes (forming myelinated axons) is also a particular component of CNS injury. Early 3 hours after ischemia in the rat brain showed that oligodendrocytes played a pathological role, suggesting that these cells were even more sensitive to the effects of excitotoxins than neuronal cells (Pantoni et al 1996). One possible candidate factor for mediating cell death is that many acute CNS injuries are accompanied by a significant increase in glutamate concentration (Lipton et al 1994). Indeed, it was found that even oligodendrocytes in addition to neurons express functional glutamate receptors of the AMPA/kainic acid subtype. However, oligodendrocytes exhibit high fragility (vulnerability to injury) to the applied glutamate.

Neurotonins acting on embryonic precursor Schwann cells, such as GGF, are also survival, growth and maturation factors of oligodendrocytes and Schwann cells in the damaged nerve after birth. GGF is one of the mitotic factors provided by axonal contact (Topliko et al 1996). Recombinant hGGF was administered for a prolonged period of time in a mouse model of multiple sclerosis (Cannella et al 1998) or in crush-injured peripheral nerves (Chen et al 1998)₂Can promote myelin production. Another cytokine induced by Schwann cell axonal contact is the ciliary neurotrophic factor CNTF (Lee et al 1995). CNTF and Leukemia Inhibitory Factor (LIF) have been shown to promote the survival of oligodendrocytes from ocular nerves cultured in vitro with bFGF or PDGF and to increase the amount of MBP expressing oligodendrocytes in these cultures (Mayer et al 1994). However, when CNTF and LIF are added to precursor glial cells, they appear to promote differentiation of astrocytes and induce expression of the GFAP marker in astrocytes, which is mainly a survival effect on oligodendrocytes with little effect on the level of MBP gene expression (Kahn and DeVallis 1994, Bonni et al 1997). However, CNTF in combination with brain-derived neurotrophic factor BNDF promotes recovery of injured peripheral sciatic nerves (Ho et al 1998).

CNTF and LIF are cytokines that act through a common receptor system that includes the LIF receptor (LIFR) and the gp130 chain, which is part of the interleukin-6 (IL-6) receptor complex (Ip et al 1992), and thus CNTF and LIF are part of the cytokine IL-6 family. For CNTF and LIF, signal transduction is turned on by LIFR dimerizing with gp130, while IL-6 signals through the two gp130 chains forming dimers (Murakami et al 1993). For binding gp130, IL-6 binds to the IL-6 receptor chain (present on certain cells and is a gp80 transmembrane proteinWhite matter, the soluble form of which, when supplied extracellularly, may also act as an IL-6 agonist) forms a complex (Taga et al 1989, Novick et al 1992). Encoding soluble IL-6 receptor (sIL-6R)_cThe entire coding region of the DNA was fused to IL-6, producing recombinant IL6RIL6 chimera in CHO cells (Chebath et al 1997, WO 99/02552). This IL6RIL6 chimera enhances the bioactivity of the IL-6 type, which binds with much higher efficiency to the gp130 chain compared to mixtures of IL-6 and sIL-6R (Kollet et al 1999).

The combination of IL-6 effects on cells of the central and peripheral nervous systems suggests that this cytokine has a protective effect on neuronal cells and is involved in inflammatory neurodegenerative processes (Gadient and Otten 1997, Mendel et al 1998). The activities of CNTF and LIF on glial cells were much higher than the stimulatory effects of IL-6 on astrocyte differentiation and no effect on myelin protein producing cells (Kahn and De Vellis 1994). IL-6 was found to prevent glutamate-induced cell death in hippocampal gyrus (Yamada et al 1994) and striatal (Toulmond et al 1992) neurons. The mechanism by which IL-6 protects nerves against NMDA-induced toxicity, and selective excitation of NMDA subtype glutamate receptors, remains elusive. IL-6 was indeed found to potentiate NMDA-mediated elevation of intracellular calcium. In transgenic mice expressing higher levels of IL-6 and soluble IL-6R (sIL6-R), accelerated nerve regeneration following sublingual nerve injury was observed as indicated by degeneration of the sublingual nerve nuclei in the brain (Hirota et al 1996). In this study, the addition of IL-6 and sIL-6R to Dorsal Root Ganglion (DRG) cell cultures showed increased axonal extension of neurons, but no effect on myelinating cells was reported.

According to the above data, CNTF, LIF or a mixture of IL-6 and sIL-6R have not been shown to induce the eventual differentiation of glial cells into myelinating cells. However, in summary, stimulation of myelinating cell differentiation may be of great benefit to patients suffering from demyelinating or neurodegenerative diseases.

The citation of documents herein is not an admission that such documents, or the materials described, are prior art with respect to the patentability of the claims of this application, and the citation of any document for its content or date is based on the information available to the applicant at the time of filing the patent application and does not constitute an admission as to the correctness of such citation.

Brief description of the invention

It is an object of the present invention to provide a method for the treatment and/or prevention of neurological diseases or disorders. In particular, it is an object of the present invention to provide a method for stimulating or enhancing differentiation of differentiated glial cells or their precursors to form myelinated cells. The invention is based on the use of recombinant IL6RIL6 chimeric proteins which have a significantly higher affinity for gp130 than mixtures of IL-6 and sIL-6R.

It is another object of the invention to provide a method of stimulating or enhancing myelination or regeneration of damaged nerve fibers, such as axonal remyelination induced after an in vivo sciatic nerve axotomy, in addition to myelination of myelinating genes induced in vitro by IL-6 chimeras.

It is another object of the invention to use IL6RIL6 chimeras to increase the number of Schwann cell developments in Dorsal Root Ganglion (DRG) cultures. In addition, it is an object of the invention to use IL6RIL6 to induce these cells to differentiate and wrap around axons and produce myelin basic protein, as shown by schwann cell lines co-cultured with primary DRG.

It is another object of the invention to use IL6RIL6 chimeras to induce transcription of the Myelin Basic Protein (MBP) gene in a transdifferentiated system. In this transdifferentiation system, cells with a melanocyte phenotype were transformed into schwann myelin sheath phenotype cells, as shown by the effect of IL6RIL6 on mouse melanoma.

It is another object of the invention to use IL6RIL6 chimera as a neuroprotective agent to prevent neuronal cell death in the hippocampal gyrus, the region involved in the memory code, which shows early degeneration in Alzheimer's disease and ischemia.

Another object of the invention is to use IL6RIL6 chimera as a protective agent against the neurotoxic processes induced by excitatory amino acids, such as the protective effect provided by IL-6 chimera on glutamate-induced neurotoxicity in primary cultures of neonatal rat cerebellar neurons.

The invention also proposes a molecular mechanism for IL6RIL6 to induce and inhibit specific transcription factors that induce the differentiation of the MBP gene to the myelinating phenotype.

Accordingly, the present invention provides the use of IL6RIL6 chimera for the manufacture of a medicament for the treatment and/or prevention of neurological diseases and disorders. In particular, the invention provides the use of IL6RIL6 chimera for the manufacture of a medicament for the treatment of traumatic neurodegenerative, demyelinating and/or neurodegenerative diseases of the CNS or PNS.

More specifically, the invention provides the use of IL6RIL6 chimeras in the treatment of Multiple Sclerosis (MS), Alzheimer's disease, Parkinson's disease or ALS.

The invention also provides pharmaceutical compositions for the treatment and/or prevention of neurological diseases and disorders comprising IL6RIL6 chimera and optionally one or more pharmaceutically acceptable excipients. In particular the pharmaceutical composition is for the treatment of traumatic neurodegeneration, demyelinating diseases and/or neurodegenerative diseases of the CNS or PNS.

Preferred uses of the pharmaceutical compositions of the invention are in the treatment of MS, alzheimer's disease, parkinson's disease or ALS.

Brief Description of Drawings

FIG. 1 shows the increase of MBP RNA in Schwann cell cultures treated with IL6RIL6 chimera or Macacan (FSK) or untreated (NT).

FIG. 2 shows the measurement of proliferation of undifferentiated oligodendrocytes after 24, 48 and 72 hours of incubation with various amounts of IL6RIL6 chimera.

FIG. 3 shows that IL6RIL6 strongly induced MBP mRNA (A) and reduced Pax-3mRNA (B) throughout the culture time of F10.9 cells, NT being untreated cells.

FIG. 4 shows a comparison of the neuroprotective effects of IL-6 alone and IL-6 chimera on NMDA-mediated neurotoxicity in hippocampal organotypic slices.

FIG. 5 shows the long-lasting neuroprotective effect of IL-6 chimera and the effect of IL-6 alone in hippocampal organotypic slices.

FIG. 6 shows the effect on neuronal survival by NGF withdrawal following 24 h treatment with IL6RIL6 chimera, as determined by the MTT assay.

Detailed Description

The present inventors have found that the addition of recombinant IL6RIL6 protein to cultured dorsal root ganglion cells or melanotic cells stimulates these cells to differentiate into myelinating cells, and that the addition of recombinant IL6RIL6 protein to co-cultured neurons and Schwann cells induces the latter to form a myelin sheath that regularly surrounds axons. The invention thus relates to the use of IL6RIL6 chimeras for the manufacture of a medicament for producing myelinating cells, or for stimulating, enhancing or accelerating the production of myelinating cells.

This IL-6 chimera also induced remyelination of transected fibers following axonotomy in rat sciatic nerves in vivo. Accordingly, the invention also relates to the use of IL6RIL6 chimeras for the manufacture of a medicament for inducing, enhancing or accelerating remyelination following nerve or axonal trauma or injury.

It has also been found that the addition of IL6RIL6 recombinant protein to organotypic cultures induces long-term protection against nerve agents, and therefore, the present invention also relates to the use of IL6RIL6 chimeras for the manufacture of a medicament capable of inducing, enhancing, prolonging or accelerating neuroprotection against nerve agents, and a medicament capable of inhibiting, reducing or slowing nerve cell death (e.g., likely to die as a result of apoptosis).

The IL6RIL6 chimera (also known as IL6RIL6 or IL-6 chimera) is one kind of recombinant glucoprotein obtained through fusing the coding complete sequence of natural soluble IL-6 receptor delta-Val and the whole mature coding sequence of natural IL-6, both humanized. The IL6RIL6 chimeras can be produced in suitable eukaryotic cells, such as yeast cells, insect cells, and the like, preferably in mammalian cells. Most preferably in genetically engineered CHO cells as described in WO 99/02252. It will be appreciated by those skilled in the art that although the protein is preferably of human origin, similar fusion proteins from other sources may be used in the present invention, provided that it retains the above-described biological activity.

More particularly, the invention relates to the use of IL6RIL6 chimeras to stimulate the differentiation of differentiated glial cells or their precursors into myelinating cells. As shown herein, IL6RIL6 induces differentiation processes in myelinating cells, including activation of genes required for the formation of myelination around nerve axons, and inhibition of both genes required for the maintenance of the non-myelinating phenotype.

The invention discovers that the addition of IL6RIL6 chimera to cultured embryonic dorsal root ganglion (eDRG) cells (isolated from 14-15 day gestational mouse embryos) has a significant effect on the development of precursor Schwann cells in the presence of DRG: after 2-5 days of culture, the number of embryonic Schwann cells increased significantly, their phenotype changed significantly, their membranes began to wrap around DRG axons, and MBP induced. The present invention also found that IL-6 chimeras induced a significant increase in Schwann cell binding along unmyelinated axons within 5 hours in Schwann cell and neuron cocultures. Schwann cells labeled with fluorescein gold elongate for several days, and their fluorescent cytoplasm is seen to form a regular sheath around the axon. Without the chimera or with NGF, Schwann cells bound much less and did not form a sheath.

Accordingly, the invention also relates to the use of IL6RIL6 chimera for the manufacture of a medicament for inducing Schwann cell proliferation or/and differentiation and myelination of Schwann cells in the peripheral nervous system.

The invention also shows that IL-6 chimeras can induce myelination in peripheral nerves in vivo. Following axotomy of the rat sciatic nerve and apposition of the proximal and distal stumps, the IL-6 chimeras induced peripheral nerve regeneration and remyelination of transected fibers in vivo. It was found that remyelination of distal end fibers is increased 4-fold with an increase in the number and thickness of myelinated fibers formed in the presence of an IL-6 chimera, and thus the present invention also relates to the use of IL6RIL6 chimera for the manufacture of a medicament capable of inducing remyelination of the peripheral nervous system.

In addition, the present invention has discovered that IL-6 chimeras are capable of inducing expression of genes encoding myelin protein components, such as MBP, PLP and PO groups, in myelinating cells of the peripheral nervous system (e.g., Schwann cells) and in cells of the central nervous system (e.g., oligodendrocytes).

Accordingly, the invention also relates to the use of IL6RIL6 chimera for the manufacture of a medicament for inducing myelination and/or remyelination of oligodendrocytes in the central nervous system.

The invention also discovers that the expression of MBP gene is induced within 6-12 hours by adding the IL6RIL6 chimeric substance into the cultured B16/F10.9 mouse melanoma cell line. Other genes encoding myelin proteins, such as the cnpase gene, were also induced, but gene expression involved in melanogenesis (melanin pigment formation), such as tyrosinase, was strongly inhibited, and F10.9 cells treated with IL6RIL6 also underwent significant morphological changes, resulting in a schwann-like phenotype. Phenotypic changes and induction of specific myelin genes support the following hypotheses: that is, IL6RIL6 caused these cells to transdifferentiate from the melanoma cell state to the myelinating state. Because in the embryo, cells that migrate out of the neural crest can give rise to melanoma cells or myelinating schwann cells and oligodendrocytes. This suggests that IL6IL6 may affect the homing of these cells and promote myelinating cell formation. Accordingly, the invention also relates to the use of IL6RIL6 chimeras for the manufacture of a medicament for inducing, promoting, enhancing or accelerating the formation of myelinating cells in the central and peripheral nervous system and/or for inducing the transdifferentiation of melanoma cells into myelinating cells.

Furthermore, the present invention shows that IL6RIL6 acts to down-regulate the homeobox gene Pax-3, a gene expressed in embryonic neural crest cells before they differentiate into myelinating Schwann cells (Kioussi and Grass 1996). It is known that Pax-3 inhibits the MBP gene. Thus, it appears that Pax-3 inhibition is critical in the final maturation of myelinating cells, and therefore IL6RIL6 acts on a critical differentiation switch (i.e., Pax-3 inhibition).

Pax-3 is a transactivator of the micropalmia-associated transcription factor MITF, which in turn induces and maintains the expression of tyrosinase and other genes responsible for the melanocyte phenotype. The finding that IL6RIL6 rapidly inhibits Pax-3 may explain the molecular activities that promote myelinating activity of neural crest derived cells. After nerve injury, myelinated axons undergo demyelination as part of the Wallerian degeneration. During this period schwann cells reduced the expression of the MBP gene and other related myelin protein genes. With the upregulation of Pax-3 and GFAP, this meant a reversal from myelinating SCs to non-myelinating and proliferating SCs (Kioussi and Gruss 1996). It appears that IL6RIL6 chimera is a potent cytokine that inhibits Pax-3 and induces SCs to restore their myelinating activity, thereby reversing Wallerian neurodegeneration, according to the present invention. The same considerations apply to brain demyelinating diseases, since, as in trauma, the demyelination process triggered by macrophages and other inflammatory cells exacerbates neurodegeneration in these diseases. Accordingly, the invention also relates to the use of IL6RIL6 chimeras for the manufacture of a medicament for the treatment of nerve injury and/or traumatic neurodegeneration and/or axonal injury.

Mice immunized with MBP to induce autoimmune demyelination (a model system for chronic relapsing multiple sclerosis) were injected with IL6RIL6(Cannella et al 1998). Using this pharmaceutical paradigm, the ability of IL6RIL6 to induce myelin protein gene expression and myelinating cell differentiation in vivo was observed. The invention therefore also relates to the use of IL6RIL6 chimera for the production of a medicament for the treatment and/or prophylaxis of autoimmune demyelinating diseases, in particular for the treatment and/or prophylaxis of multiple sclerosis.

IL-6 chimeras have also been shown to have neuroprotective effects, to prevent loss of neuronal viability and inhibit early degeneration in memory coding regions caused by toxic substances in Alzheimer's disease and ischemia. IL-6 chimeras have been found to protect nerves from the neurotoxic effects of glutamate, and the present invention is therefore concerned with the manufacture of a medicament for protecting nerve cells from death (particularly due to neurotoxic substances) and for the treatment and/or prevention of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease or ALS.

Other neurological disorders that may be treated with IL6RIL6 chimeras are stroke, movement disorders, epilepsy, pain, and the like.

"pharmaceutically acceptable" is intended to include any carrier that does not interfere with the biologically active effects of the active ingredient and is not toxic to the host. For example, for parenteral administration, IL6RIL6 chimera can be formulated in injectable unit dosage forms in excipients such as saline, dextrose solution, serum albumin, and Ringer's solution.

IL6RIL6 chimeras can be administered to a patient in need of treatment in a variety of ways including intradermal, transdermal (in sustained release formulations), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical and intranasal routes. Other therapeutically effective routes of administration may also be employed, for example, by absorption through epithelial or endothelial tissue, or gene therapy in which a DNA molecule encoding a chimeric IL6RIL6 is administered to a patient (via a vector) resulting in the expression and secretion of the chimeric IL6RIL6 in vivo. Furthermore, IL6RIL6 chimeras can be administered with other biologically active ingredients, such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles.

For parenteral administration (intravenous, subcutaneous, intramuscular), IL6RIL6 can be formulated as a solution, suspension, emulsion, or lyophilized powder, along with pharmaceutically acceptable gastrointestinal excipients (e.g., water, saline, dextrose solution) and additives that maintain osmolality (e.g., mannitol) or chemical stability (e.g., preservatives and buffers). The formulations are sterilized by conventional techniques.

An "effective amount" refers to an amount of the active ingredient sufficient to affect the course and severity of the above-mentioned disease, resulting in a reduction or alleviation of the disease. The effective amount depends on the route of administration and the condition of the patient.

The dosage to be administered to a patient in a single dose or multiple doses will vary depending on various factors, including the pharmacokinetic properties of the IL6RIL-6 chimera, the route of administration, the condition and characteristics of the patient (sex, age, body weight, health and size of the head), the extent of the disease, concurrent therapy, frequency of treatment and the desired effect. It is well within the ability of the skilled artisan to determine the adjustment and control of dosage ranges, and in vitro and in vivo methods of measuring remyelination of nerves.

While the invention has been described in conjunction with specific embodiments, it will be understood that further modifications may be made. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. The principles of the invention and without departing from the disclosure herein may be practiced using art-recognized or customary methods as set forth in the appended claims.

All references cited herein, including journal articles or abstracts, published or unpublished patent applications, publications, or foreign patents or other references, are incorporated by reference herein, including all data sheets, figures, and articles, and the entire contents of the documents cited in these references are also incorporated by reference herein.

The invention will be described in more detail by means of the following non-limiting examples and the accompanying drawings.

Examples

Example 1 Effect of IL6RIL6 on myelination and remyelination in vitro

The dorsal roots in the spinal cord contain primarily sensory neurons that synapse in the Dorsal Root Ganglia (DRGs). DRG at mouse embryogenesis (e 14-e 15 days) is a convenient source of neurons and embryonic Schwann cells that have not differentiated into myelinating SCs. Li (1998) describes a method for obtaining DRG explants cultured in vitro, which were plated on coverslips in Costar plate wells and incubated with F12/DMEM medium (Glbco) with substantially similar effect of coating the coverslips with collagen or with poly-D-lysine. The culture was carried out in a medium without addition of growth factors or cytokine supplements, in a medium supplemented with nerve growth factor (NGF 40. mu.g/ml) or with IL6RIL6 chimeric recombinant protein (3. mu.g/ml). Cultures were examined daily with an Olympus inverted microscope attached to a photographic imaging system (Lecia LIDA system). The sections of the coverslips were fixed with paraformaldehyde and the MBP protein was labeled with a monoclonal primary antibody against myelin basic protein and a secondary antibody conjugated to fluorescein, the neuronal cell bodies and axons were stained with an antibody against neurofilament protein, and some of the coverslips were examined by scanning Electron Microscopy (EM).

After 2-5 days, DRG exosomes cultured with the additive showed that the cells from which the explants grew appeared polygonal or ovoid. However, upon addition of NGF, oval cells outgrow long axonal processes, forming a fine network that can be stained by anti-neurofilament antibodies. Some axons were long-bifurcated but no schwann cells were seen along the axon. In contrast, cultures with IL6RIL6 showed not only neuronal cells with neurite-staining axons, but also flattened schwann cells with bipolar long stretches of terminal branches that did not stain as neuropilins. Scanning electron microscopy clearly observed that these schwann cells were wrapped around the axon starting with a membrane with a wavy edge along the axon process.

anti-MBP staining showed positive staining of schwann cells in IL6RIL 6-treated cultures, particularly in cell arrays arranged one after the other, while little MBP-specific fluorescence was seen in NGF-treated cultures without IL6RIL 6.

Similar results were observed in DRG cultures of rat embryos at day 15 of gestation.

Schwann cells from the mouse sciatic nerve were cultured in vitro with 1.4g/ml IL6RIL-6 and MBP RNA transcript levels were determined. In contrast, treatment of the same culture with mao monkey 20M is a chemical that artificially increases cellular AMP levels and induces cMBP (Lemke and Chao 1998). The results showed that IL6RIL6 induced MBP gene expression 3 days after culture as efficiently as mao monkey hormone, and was more effective in maintaining MBP RNA levels (FIG. 1).

In a study of 18 day embryonic Dorsal Root Ganglion (DRG) cells, IL-6 chimera was found to induce MBP and POmRNA expression within three days, while Pax-3mRNA was strongly inhibited in the same cell system. The dose-dependent profile of the effect of IL-6 chimera on PO and MBP gene expression shows that IL-6 chimera exhibits the greatest effect at 500ng/ml and thus IL-6 chimera appears to be an important inducer of normal glial gene expression.

Schwann cell lines from rat sciatic nerve or rat dorsal root ganglion (ORG) cells were prepared for further study of the effects of IL-6 chimeras.

As determined by RT-PCR and Northern blot analysis, the Schwann cell line from rat sciatic nerve was found to induce 2-3 fold of PO gene (expression) in response to IL-6 chimera, and the protein product formed about 50% of the myelin sheath of peripheral nerve.

To investigate the transcriptional activation of myelin gene components by the IL-6 chimera, the MBP promoter was introduced into an expression vector upstream of the luciferase reporter gene and detected in rat sciatic nerve Schwann cells. Up to 7-fold induction of MBP promoter transcriptional activity was observed in these cells in response to IL-6 chimera in the absence of Macacan. In other reporter assays, IL-6 chimera was found to induce 2.5-fold transcriptional activity of the PO gene promoter in these cells.

The above results suggest that IL-6 chimeras have a direct effect on myelin gene transcription in Schwann cells of interest.

Another Schwann cell line, designated CH cell line, was isolated from rat Dorsal Root Ganglion (DRG) and was found to grow at a slow rate, depending on the IL-6 chimera (14 ng/ml). Interestingly, CH cells are astrocytes, similar to oligodendrocytes. This cell shows the function of myelin production induced by the IL-6 chimera. The cells express PO and MBP and die when cultured in serum-containing media lacking the chimera.

Example 2: inhibition of oligodendrocyte proliferation in vitro

The effect of IL-6 chimeras on central nervous system cells was investigated. It has been established that differentiation of oligodendrocytes is accompanied by inhibition of their proliferation, which promotes the formation of sheaths and myelin sheaths by the oligodendrocytes.

Thus, the ability of IL6RIL6 chimeras to inhibit oligodendrocyte proliferation in vitro was determined. This experiment used an immortalized mouse primary oligodendrocyte ("oligo-neu" cell) line with a t-neu oncogene. Jung et al (1993) describe the establishment, properties and culture conditions of this cell line.

1. After 2 and 3 days, proliferation responses of undifferentiated oligo-neu cells were measured for different doses of IL6R/IL6 chimera (1g/ml, 500ng/ml, 250ng/ml, 125ng/ml and 0ng/ml (control)), and growth rates were quantified by measuring cellular metabolic activity with a spectrofluorimetric/colorimetric growth indicator, Alamar blue. The reagent contains a redox indicator and can show double changes of fluorescence and color in the reaction of chemical reduction of the growth medium caused by cell growth. The reagents and assays are described by Ahmed et al (1994) and U.S. Pat. No. 5,501,959.

The results are shown in FIG. 2. The addition of the IL6R/IL6 chimera to the oligodendrocyte medium resulted in a significant decrease in growth after 48 hours, with growth decreasing to 40-50% after 72 hours compared to the control. Interestingly, the highest amount (1g) of IL6R/IL6 chimeric was less potent than the lower amount (500-125 ng/ml).

This experiment shows that the IL6R/IL6 chimera strongly inhibits the in vitro proliferation of oligodendrocytes, indicating that it is an enhancer of the differentiation of the cells. Since oligodendrocytes are myelinating glial cells in the CNS, the IL6R/IL6 chimera may be a substance that actively promotes myelination in vivo.

Example 3: effect of IL6R/IL6 chimera on oligodendrocyte morphology

To investigate the effect of IL-6 chimera on oligodendrocyte morphology, Oli-neu cells were cultured with 1g/ml IL6R/IL6 chimera (see example 2) for 3 days.

Dibutyl cAMP (dbcAMP) is a substance known to induce differentiation of oligodendrocytes (see, e.g., Jung et al 1995) to investigate how the addition of IL6R/IL6 chimera affects the morphological effects of dbcAMP, oligo-neu cells were pretreated with dbcAMP for 3 days, followed by addition of IL6R/IL6 chimera with dbcAMP. Parallel experiments with IL6R/IL-6 chimeric (not pretreatment) treatment of cells.

Morphological changes were assessed either with phase contrast microscopy or by immunohistochemical staining of the oligodendrocyte markers cnpase (2, 3-cyclized nucleoside 3' -phosphodiesterase) and GalC (sialoganglioside and sulfatide) with the guide antibody A2B5 to the myelin lipid galactocerebroside.

The addition of IL6R/IL6 chimera to Oli-neu cells induced significant morphological changes. The cell bodies assemble into rows and become longer, and the cells themselves elongate and appear to fuse together, indicating entry into the late stage of differentiation.

Immunohistochemistry showed expression of the oligodendrocyte marker, positive for the oligodendrocyte specific marker A2B5 antigen and cnpase, but negative for the astrocyte marker GFAP, indicating that the oligodendrocyte phenotype was indeed maintained.

Pretreatment with DbcAMP and IL6R/IL6 chimera treated with dbcAMP for 3 days increased the number of oligodendrocytes produced by the sheath. Except for elongated cells seen with IL6R/IL6 chimera alone, these cells appeared morphologically more like differentiated cells. Lamellar cells are visible, indicating late stage differentiation.

The conclusion is that: in vitro treatment with the IL6R/IL6 chimeric resulted in morphological changes in oligodendrocytes, which further supported their role as inducers of oligodendrocyte differentiation and myelination.

Example 4: effect of IL-6 chimeras on neuronal Schwann cell interactions

By culturing mouse DRG in the presence of the DNA synthesis inhibitors arabinose c (arac) and fluorodeoxyuridine (FudR), it was seen that neuronal cells produced axons and glial cell proliferation was inhibited. These DRG cultures can be used as a source of unmyelinated axonal neuronal cells. Schwann cells were exogenously added to these cultures to study the interaction between neurons and schwann cells.

Rat sciatic nerve Schwann cell line cells were labeled with gold fluoride, a marker that penetrates the membrane lipid bilayer and remains for long periods of time without affecting cell function. Co-culturing neuronal cells and these Schwann cells in the presence of IL-6 chimera resulted in a significant increase of Schwann cells bound along the axon within 5 hours. These cells elongate and after a few days it can be seen that their fluorescent cytoplasm forms a regular sheath around the axon. Without this chimera or with NGF, Schwann cell binding was greatly reduced and no sheath was formed. These results show that IL-6 chimeras have a very rapid effect on glial cell-neuron interaction, suggesting that adhesion molecules are induced or activated. This system allows the investigation of the various steps in myelination that are affected by IL-6 chimeras.

Example 5: effect of IL6R/IL6 chimera on myelin formation in Mixed cultures of neurons and oligodendrocytes

To further investigate the myelination-inducing activity of the IL6R/IL6 chimera, an anatomical culture of the cerebral hemisphere of mouse E15 (15 days embryo) was prepared. Preparation and culture conditions were as described by Lubetzki et al (1993). The primary cells were maintained in culture for 8 days, followed by addition of IL6R/IL6(1.5g/ml) for 11 days.

MBP (myelin basic protein) and the oligodendrocyte-specific protein PLP (proteolipid protein) are markers of mature and/or myelinating oligodendrocytes and are a major component of CNS myelin. They can therefore be used as markers for assessing active production of myelin in cell culture.

No myelination occurred at day 4 under this culture condition, so day 4 mRNA was used as a standard control in this experiment. Myelination was observed in cell culture at day 19 under this condition.

On days 4 and 19, mRNA was extracted from treated and untreated wells for quantitation of MBP and PLPmRNA.

mRNAs for MBP, PLP and GAPDH were measured by real-time quantitative RT-PCR (Medhurst et al 2000) as internal control RNAs using a PE Applied Biosystems Prism model 7700 sequencer.

The results are summarized in table 1 below. Two independent experiments were performed. Indicating that mRNA expression was several-fold higher than control RNA expression at day 4. The IL6R/IL6 chimera significantly increased expression of MBP and PLP 2-fold in both experiments, again supporting the effect of the IL6R/IL6 chimera as an inducer or enhancer of myelination.

Table 1:expression of MBP and PLP in Primary cortical cultures

Day 4 controls Day 19 controls Day 19

IL6R/IL6 chimeras

Experiment I

MBP 1 72 181

PLP 1 1265 2157

Experiment II

MBP 1 187 2157

PLP 1 1884 4198

And (4) conclusion: examples 1-5 show that IL6R/IL6 chimeras have antiproliferative, inducible, and differentiative myelinating activity. This activity of the IL6R/IL6 chimera strongly suggests a beneficial role for the IL6R/IL6 chimera in both peripheral and central nervous system myelination. This effect is useful in diseases such as myelination deficits, demyelination and insufficient myelination of CNS and PNS neurons. IL6R/IL6 chimeras may be useful agents for the treatment of demyelinating diseases and/or remyelination, such as multiple sclerosis.

Example 6: effect of chimeras on peripheral nerve regeneration in vivo

In vivo experiments, rat sciatic nerve axonotomy was docked at the proximal and distal stumps to induce peripheral nerve regeneration and transverse fiber remyelination (Sahenk et al, 1994). The effect of IL-6 chimera on peripheral nerve remyelination after axotomy was examined. Rats (7) were given 100mcg/kg intraperitoneal doses of sIL-6/IL-6 chimera every 2 days for 12 days, starting on the day of surgery. Control animals (9) were injected with PBS. Sections of the regenerated nerve 2.5 and 5mm below the axonotomy were analyzed after 12 days by transmission electron microscopy and the number of restored fibers was evaluated.

In the presence of IL-6 chimera, a 2.5-fold increase in the number of myelinated fibers 2.5mm below the axonal incision was found compared to PBS-treated controls. The chimera was also found to increase remyelination of more distant fibers, increasing the number of myelinated fibers 5.2-fold at 5mm from the incision. This is important because remyelination decreases with increasing distance from the incision. The effect of CNTF was observed at 0.5mm from the incision, but the effect of the IL-6 chimera appeared to be more extended, with greater effect at 5mm distance than at 2.5 mm. The thickness of the myelinated fibers also increased by more than 2-fold after treatment with the chimeras. In summary, IL-6 chimeras appear to induce remyelination of 10% of the fibers compared to intact, non-axotomized rats.

Example 7: IL6RIL6 induces the MPB gene in melanoma B16-F10.9 cell cultures

The embryonic source of skin melanocytes and schwann cells, which produce black pigment, is a common precursor cell that migrates out of the embryonic neural crest of 8-day mice. Melanoma is a malignant tumor in the skin that arises from melanocytes and is therefore also derived from neural crest progenitor cells. The B16 cell line is derived from Balb/c mice spontaneous melanoma. The F10.9 clone isolated from B16 has a highly malignant metastatic phenotype. Like the other B16 cells, the F10.9 cells also produced black pigment and were enriched for tyrosinase: the 1 st enzyme of the melanin production pathway (Bertolotto, 1996).

P10.9 cells were seeded at 30,000 cells/well in 96-well microtiter plates and cultured in DMEM supplemented with 10% FCS for 3 days, with or without IL6RIL6 at a concentration of 0.3-1 g/ml. Total cellular RNA was extracted and analyzed by Northern blot analysis using cDNA probes for MBP. At 48 hours IL6RIL6 induced MBP mRNA very strongly in F10.9 cells (fig. 3A). Time course studies showed that the increase in MBP RNA began 12 hours after the addition of IL6RIL6 chimera to the cell culture.

The IL6RIL6 chimera induces not only MBP gene expression but also another component of myelin and a marker for differentiated schwann cells: expression of 2 '3' AMP phosphodiesterase or CNPase. These cells also produce extensions at the two poles of the cell body, which are arranged in long arrays typical of Schwann cells in culture.

Surprisingly, IL6R/IL6 was able to switch the phenotype of F10.9 cells from melanin-producing cells to myelin-producing Schwann cells. Tyrosinase activity and melanin production were completely lost 48 hours after the addition of IL6RIL6 chimera to the cells.

MITF is a transcription factor that activates the tyrosinase gene (Bertolotto et al, 1996). Treatment of F10.9 cells with IL6RIL6 strongly inhibited the expression of the MITF gene, which itself is activated by the homologous transcription factor Pax-3 (Watanabe et al, 1998). Pax-3mRNA was measured in IL6RIL6 treated F10.9 cells, showing a decrease in Pax-3 expression starting from 6 to 48 hours (FIG. 3). Pax-3 is known to inhibit the MBP gene (Kioussi and Gruss, 1996). Thus, the effect of IL6RIL6 chimera may be attributed to gene regulation of the Pax-3 homeobox gene (which is expressed prior to myelination during Schwann cell embryonic development and must be suppressed during myelination). However, in degenerated demyelinated nerves, when Schwann cells stop producing MBP, Pax-3 is re-expressed in this cell. Therefore, most importantly, IL6RIL6 both inhibited Pax-3 and caused schwann cells to differentiate into myelinating cells by inducing myelin protein genes.

Example 8: injection of IL6RIL6 in a mouse model of chronic relapsing multiple sclerosis

SJL/J mice were immunized with 0.4mg of bovine MBP plus Freund's incomplete adjuvant (containing 60 micrograms M.tuberculosis H37Ra, Difco) and developed Experimental Autoimmune Encephalomyelitis (EAE). This disease was passively transferred to syngeneic recipient mice by 30,000,000 lymphocytes obtained 12 days after immunization by intravenous injection. Clinical paralysis appeared after one week to 10 days. After the acute phase, there is remission and relapse. These mice (weighing approximately 25 grams) were each injected intraperitoneally or subcutaneously with 1, 3 and 5 microgram doses of IL6RIL 6.4 injections were administered weekly for at least 3 weeks, starting 3 or 7 days after passive transfer. Animals were clinically scored and graded as follows: 1) the tail strength disappears; 2) the hind limbs are weak; 3) paralysis of one limb; 4) paralysis of both limbs; 5) and death. The effect of IL6RIL6 on lowering clinical grade and reducing white matter demyelination was confirmed by optical microscopy of animals' brains and spinal cords after staining myelin with Luxol fast blue.

Example 9: protective Effect of IL6RIL6 chimera on neuronal and oligodendroglial cytotoxicity

Tissue type cultures provide a unique approach to examine many aspects of brain physiology and pathology. Long-term cultures of hippocampal gyrus (a region involved in memory coding and which shows early degeneration in alzheimer's disease and ischemia) slices are particularly valuable in this regard because of their expression of synaptic plasticity mechanisms (such as long-term potency) and response to pathological assaults (such as excitotoxicity).

Cultures were prepared as described by Bahr et al (Bahr 1995) with minor modifications. Hippocampus gyrus of 8-day-old Wistar rats were dissected and 40-micron-thick sections were placed on a porous transparent Millicell-CM membrane (Millipore) and cultured in 6-well multi-well plates. 4 sections were placed on each membrane and the medium was incubated with 50% minimal essential medium (+25mM HEPES, +4mM NaHCO)₃+ NaOH pH7.2), 25% horse serum and 25% Hank's solution, glucose (6.4g/L), penicillin/streptomycin (10ml/L), L-glutamine (2 mM). Cultured in 5% CO before use₂Incubators were changed weekly at 37 ℃ for at least 3 weeks.

Hippocampus slices were exposed to IL6 or IL6 chimera at concentrations ranging from 10pg/ml to 10. mu.g/ml for 15 minutes, followed by NMDA addition at 50. mu.M for an additional 30 minutes. Cultures were placed in fresh medium with or without IL6 or IL6 chimera. At least 4 sections were used per experimental point. After experimental injury of this culture in fresh medium containing propidium iodide (PI 5. mu.1/ml) for 1 hour, NMDA-induced cell death was evaluated for 1 or 3-7 days. Due to the hydrophilic nature of PI, it enters the disrupted cell and attaches to chromatin. The emitted red fluorescence (marker of cell death) was examined with an inverted fluorescence microscope attached to an intensified coupled charge camera device. Total fluorescence was quantified using an imaging analysis system (Image Pro Plus).

Both IL6 and IL6 chimeras were found to protect against NMDA-induced hippocampal cell death. After 1 day of incubation with IL6 at concentrations of 0.1-10ng/ml, they were found to protect 50-30% of the neuronal cells, respectively, whereas IL6 chimera at concentrations of 0.05-1pg/ml protected 40-75% of the neuronal cells. The maximum protection of IL6 chimera was observed at a concentration of 0.5pg/ml (75% cells protected) (FIG. 3).

IL-6 protection was found to diminish over time. After treatment, neuronal cell viability decreased, IL-6 protection was maintained only at 0.1ng/ml after 2 days, and only on 30% of cells at day 5. In contrast, IL-6 chimera was protective for a long period of time at 0.5ng/ml, with approximately 60% cells maintained for 5 days (FIG. 4).

Immunohistochemical staining was performed with Gal-C, a marker specific for oligodendrocytes, to determine the presence of oligodendrocytes in the organotypic slices. After 2-3 weeks, the organotypic slices were exposed to 50 μ M NMDA for 30 minutes in vitro to induce necrotic death in these cultures. In fact, all MBP expression in these cultures, which was different from oligodendrocytes, was abolished after NMDA treatment as determined by RT/PCR.

Activation of glutamate ionization receptors represents an initial manifestation of excitotoxicity processes triggered by excitatory amino acids. Primary cultures of neonatal rat cerebellar neurons (containing > 90% neurons) were used to study the effect of IL-6 chimera on glutamate-induced neurotoxicity and compared to the effects of IL-6.

Primary cultures of cerebellar granulosa cells from 8-day-old Sprague-Dawley pups were prepared as described previously (Pizzi et al, 1993). Cells were seeded on poly-L-lysine coated plates and cultured in Eagle's basal medium containing 10% heat-inactivated fetal bovine serum, glutamine (2mM), gentamicin (50. mu.g/ml) and KCl (25mM) at a density of 2.5X 10⁵Cells/cm². Cytosine arabinoside (10M) was added to the cultures 18 hours after inoculation to prevent proliferation of non-neuronal cells. The experiment was performed 10 days after the neuronal cells were cultured.

Unless otherwise stated, the cultures were contacted to be Mg-free²⁺Locke's solution 50. mu.M glutamic acid for 15 min. IL6 or IL6 chimera was added 5 minutes prior to glutamate treatment, and the plates were re-used conditioned medium at 37 deg.C, 95% air/5% CO₂Culturing, and measuring the cell viability after 18-24 hours.

Cell viability was assessed 18 hours after vital staining with fluorescein diacetate and propidium iodide mixtures as previously described (Pizzi et al, 1993). From micrographs of three representative fields of view per monolayer of neurons, the ratio between fluorescein diacetate (green viable cells) and fluorescein diacetate + propidium iodide was evaluated to calculate the percentage of viable neurons in the monolayer. Values were taken from three sister plates.

Although IL6 at doses of 0.1-10ng/ml failed to induce protection against rat cerebellar granule cells, IL6 chimeras at 0.1pg/ml and 1pg/ml protected 40-20% of neuronal cells from glutamate-induced neurotoxicity, respectively.

Example 10: effect of IL6R/IL6 chimeras on survival of upper cervical ganglion neurons following NGF withdrawal

The neuroprotective effect of IL6R/IL6 chimera in a peripheral neuronal culture system was also investigated.

Neurons from the cervical ganglia (SCG) of P0-P3 rats (postnatal day 0-3) were isolated and stored for 4 days in culture medium containing 5% rat serum and NGF. NGF is essential for the survival of this neuron, while removal of NGF leads to cell death by induction of apoptosis. The previous medium was replaced with NGF-free medium containing anti-growth factor neutralizing antibodies. At this point, 0.1 or 1g/ml IL6R/IL6 chimera (formulated in 1% final concentration of DMSO) was added. The culture was kept at 37 ℃ for 24 hours.

The effect of IL6R/IL6 chimera on NGF deprived neuron survival after 24 hours of treatment was assessed by microscopic examination and MTT assay to determine the mitochondrial activity of the cells. Mosmann (1983) describes this assay in detail. The concentrations used IL6R/IL6 did not show any toxic or morphological effects on SCG neurons treated with NGF.

MTT assay (FIG. 6) results show that the addition of IL6R/IL6 chimera at 100ng/ml and 1g/ml concentrations rescued up to 40% of SCG neurons from death due to NGF shedding. Therefore, IL6R/IL6 has a neuroprotective effect on peripheral neurons, suggesting that it is active in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and ALS (amyotrophic lateral sclerosis).

Having now fully described this invention, it will be apparent to those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without undue experimentation and without departing from the spirit and scope of the invention.

Reference to the literature

Abramsky, o.and Ovadia, H. (1997): new field of multiple sclerosis, clinical research and therapy, Martin Duntiz publishers, London.

Ahmed s.a., Gogal, r.m., jr. A rapid and simple nonradioactive assay for monitoring and determining lymphocyte proliferation³H thymidine incorporation, J.of Immunol. methods 170.211-224

Anderson, D.J (1997): cell and molecular biology of determinants of neural crest cell lines, Trends Genet.13, 276-380.

Bahr, B.A. (1995): long-term survival hippocampal gyrus sections: a model system for studying synaptic mechanisms and pathological processes, J Neurosci Res, 42, 294-.

Bertolotto, c., Bille, k., Ortonne, j.p. and Ballotti, r. (1996): regulation of tyrosinase gene expression by cAMP in B16 melanoma involves two CATGTG motifs around the TATA box, j.cell.biol.134, 747-.

Bonni A, Sun Y, Nadal-Visens M, Bhatt A, Frank DA, Rozovsky I, StahlN, Yancopoulos GD, Greenberg ME (1997): the JAK-STAT signaling pathway regulates glial formation in the central nervous system, Science 278, 477-483

Gannells, b., Hobam, c.j, Gao, y.l.et al (1998): neuregulin, glial growth factor 2, reduces autoimmune demyelination and promotes myelination in a chronic relapsing model of multiple sclerosis, proc. natl. acad. sci. usa, 95, 10100-.

Chebath, j., Fischer, d., Kumar, a., Oh, j.w., Kollet, o., Lapidot, t., Fischer, m., Rose-John, s., Nagler, a., Slavin, s.and revl, M. (1997): the interleukin 6 receptor-interleukin 6 fusion protein has enhanced interleukin 6 type pleiotropic activity. Eur. cytokine Net.8, 359-365.

Chen, l.e., Liu, k.seeber, a.v., katragada, s., Kirk, C and urbaniak, j.r. (1998): recombinant human glial growth factor 2(rhGGF2) enhanced functional recovery of ruptured peripheral nerves (double blind studies). Inte, 33, 341-.

Fraser, S.E.and Bronner-Fraser, M. (1991). Neurocrista cells migrating in avian embryos are pluripotent cells, Development 112, 913-920.

Game.r.a.and Otten, U.H. (1997): interleukin 6(IL-6), a molecule with both beneficial and destructive capabilities. Prog, neurobiol, 52, 379-.

Hartung, H.P., van der Meche, F.G., Pollard, J.D (1998) acute infectious polyneuritis, CIDP and other chronic immune-mediated neurological diseases, Curr.Opin.neuron, 11, 497-Buck 513

Hirota, h., Kiyama, h., Kishimoto, t.and Taga, T. (1996): the expression of interleukin 6 and interleukin 6 receptors is up-regulated after trauma to accelerate the nerve regeneration of mice. Med., 183, 2627-.

Ho, p.r., coa, g.m., Cheng, e.t., Niell, c., tar, d.m., Shou, h., Sierra, d.and Terris, D.J. (1998) repair of collagen tubule connections with brain neurotrophic factors and ciliary neurotrophic factors in rat models of sciatic nerve injury, arch.orlaryngol.headn eck surg., 124, 761-.

Ip NY, Nye SH, Boulton TG, Davis S, Taga T, Li Y, Birren SJ, YasukawaK, Kishimoto T, Anderson DJ, et al (1992): CNTF and LIF act on neuronal cells through a common signaling pathway involving the IL-6 signaling receptor components gp130, Cell 69: 1121-32

Jessen, k.r.and Mirsky, R. (1991): precursor Schwann cells and their development, Glia 4, 185-194.

Jung, m., Kramer, e., Grzenkowsko, m., Blakemore, w., Aguzzi, a., Khazaiu, k., Chlichlia, k., von Blankenfeld, g., Kettenmann, and Trotter, j. (1995): the activated neu tyrosine kinase immortalizes mouse oligodendroglial precursor cell lines, showing a different degree of interaction with axons in vitro and in vivo, Eur.J. of Neuroscience 7, 1245-1265

Kahn, m.a. and De Vellis, j. (1994): the interleukin 6 cytokine family regulates the oligodendrocyte progenitor cell line, glia.12, 87-98.

Kollet, o., Aviram, r., Chebath, j., ben-Hur, h., Nagler, a., Shultz, l., travel, m.and Lanidor, t. (1999): soluble IL-6 receptor/IL-6 fusion protein enhances human CD34⁺CD38^-the/low/SCID cells (SRC) were maintained and propagated in vitro. Blood, published.

Kioussi, c.and groups, P. (1996): schwann cells, Trends Genet, 12, 84-86 were produced.

Lee, d.a., zuraawel, r.h.and Windebank, A.J (1995): axonal contact induces the expression of ciliary neurotrophic factor in schwann cells, j. neuroehom, 65, 564-568.

Lemke, g.and Chao, m. (1988): axons regulate the expression of schwann cell major myelin and NGF receptor genes. Development 102, 499.

Li, r. (1998): culture methods for selective growth of normal rat and human Schwann cells, Meth, cell. biol., 57, 167-.

Linton, s.a., and rosenberg.p.a. (1994): excitatory amino acids, the ultimate common pathway of neurological disease, N Engl J Med, 330, 613-22.

Lubetzki, c., Demerens, c., Anglade, p., villarroya.h., Frankfurther, a., lee, v.m.y., And ang Zalc, b. (1993): even in culture, oligodendrocytes produce only myelin sheaths of axons, PNAS 90, 6820-.

Mayer, m., Bhakoo, k.and Noble, m. (1994) P: ciliary neurotrophic factor and leukemia inhibitory factor promote the production, maturation and survival of oligodendrocytes in vitro, Development, 120, 143-153.

McDonald, j.w., althromsons, s.p., Hyrc, k.l., Choi, d.w., and goldberg, M.P (1998): oligodendrocytes in the forebrain are highly susceptible to AMPA/kainic acid receptor-mediated excitotoxicity, Nat Med, 4, 291-7.

Medhurst, a.d., Harrison, dbcAMP, Read, s.j., Campbell, c.a., Robbins, m.j.and Pangalos, M.N. (2000): genes expression in CNS tissues and disease models were semi-quantitatively analyzed using a TagMan RT-PCR assay, j.

(1998): function of interleukin 6 in autoimmune encephalomyelitis: studies in gene-targeted mice. Eur.J.Immunol.28, 1727-1737.

Mogmann, T. (1983): rapid colorimetric assay for cell growth and survival: applied to proliferation and cytotoxicity assays, J.Immunol.methods 65, 55-63

Murakami, m., ibi, m., Nakagawa, n., Nagakawa, t., Yasukawa, k., Yamanishi, k., Taga, t.and Kishimoto, t. (1993): IL-6 induces the activation of gp130 homo-dimeric tyrosine kinase, Science 260, 1808-1810.

Novick, d., Shulman, l.m., Chen, l.and revl, m. (1992): the mouse soluble IL-6 receptor enhances the cytostatic effects of IL-6 on human breast cancer and is reversed by monoclonal antibodies, Cytokine, 4, 6-11.

Pantoni, i., Garcia, j.h., and Gutierrez, J.A (1996): white matter of the brain is highly vulnerable to ischemia, Stroke, 27, 1641-6.

Pizzi, m., Fallacara, c., arighi, v., memo.m., and spandex, P.F, (1993): metabolic glutamate receptor activators attenuate excitatory amino acid toxicity and establishment of primary cultures of cerebellar granule cells, J Neurochem, 61, 683-9.

Pohlau, d., Aktas, o., Epplen, c.hartung, h.p., hoffmann.v.andprzuntek, H. (1998): promotion of remyelination is the future principle of treatment of multiple sclerosis, Nervenarzt, 69, 841-850.

Sahenk, z., Seharaseyon, j., and Mendell, J.R. (1994): CNTF enhances the regeneration of peripheral nerves, Brain Res, 655, 246-50.

Stocker, k.m., Sherman, l., Ree, s.and moment, g. (1991): basic FGF and TGF-influence the commitment of melanogenesis in avian embryonic neural crest cells, Development, 111, 635-.

Taga, t., Hibin m., Hirata, y., Yamasaki, k., Yasukawa, k., Matsuda, t., Hirano, t.and Kishimoto, t. (1989): interleukin 6 triggers the binding of its receptor to the possible signal transduction protein gp130, Cell, 58, 573-581.

Topliko, P., Murphy, P.and Charnay, P. (1996): embryonic development of schwann cells: many of the actions of neurotonins along pathways, mol.

Toulmond, s., mage, x., fare, d., and Benavides, J. (1992): local infusion of interleukin-6 attenuated the neurotoxic effects of NMDA on cholinergic neurons in the striatum of rats, Neurosci Lett, 144, 49-52.

Trapp, b.d., Hauer, p.and Lemake, g. (1988): axonal modulation of myelin protein mRNA levels in actively myelinating schwann cells, j. neurosci.8, 3515.

Trojaborg W (1998): acute and chronic neurological disorders: new aspects of acute infectious polyneuritis and chronic inflammatory demyelinating polyneuropathy, reviewed in Electroencephalogr ClinNeuropysiol, 107, 303-.

Watanabe, a., Takeda, k., Plopi s, b.and Tachibana, M. (1998): the superior relationship between Watenberg syndrome genes MITF and Pax 3, Nature Genet, 18, 283-.

Yamada.m., and Hatanaka, h. (1994): IL-6 protects cultured rat hippocampal gyral neurons against glutamate-induced cell death Brain Res, 643, 173-80.

Claims (3)

1. Use of IL6RIL6 chimera for the preparation of a medicament for the treatment and/or prevention of traumatic neurodegeneration, demyelinating and/or neurodegenerative diseases of the central or peripheral nervous system.
2. 2. The use of claim 1, wherein the demyelinating disease is multiple sclerosis.
3. 3. The use of claim 2, wherein the neurodegenerative disease is selected from alzheimer's disease, parkinson's disease and ALS.