WO2001064240A2

WO2001064240A2 - Methods for promoting production of myelin by schwann cells

Info

Publication number: WO2001064240A2
Application number: PCT/US2001/006294
Authority: WO
Inventors: Emma E. Moore; Julia E. Novak
Original assignee: Zymogenetics, Inc.
Priority date: 2000-02-29
Filing date: 2001-02-27
Publication date: 2001-09-07
Also published as: EP1259253A2; WO2001064240A3; CA2401716A1; AU2001241816A1; JP2003525251A

Abstract

A method for promoting the expression of myelin or Protein Zero in Schwann cells using Zcyto7 or IL-17. Zcyto7 or IL-17 are further used to promote myelination of the peripheral nervous system. This is particularly useful in treating diseases dymyelinating diseases such as diabetic neuropathy, Guillain-Barre' Syndrome, chronic demyelinating disease, acute demyelinating polyneuropathy and human immunodeficiency viral demyelinating neuropathy or demyelination caused by trauma.

Description

METHODS FOR PROMOTING PRODUCTION OF MYELIN

BY SCHWANN CELLS

BACKGROUND OF THE INVENTION

The peripheral nervous system (PNS) serves as a bridge between the environment and the central nervous system (CNS). The PNS is comprised of primary afferent neurons, which sends information from sensory receptors to the CNS, somatic motor neurons, which transmit electrical stimuli from the CNS to voluntary muscles, and autonomic motor neurons, which transmit electrical stimuli to cardiac muscle, smooth muscle or glands. A neuron generally has a cell body, and an axon, which is a long nerve cell process extending from the cell body that is capable of rapidly conducting nerve impulses over long distances so as to deliver signals to cells. The axons of many vertebrate neurons are insulated by a myelin sheath, which greatly increases the rate at which an axon can conduct an action potential. Schwann cells are responsible for myelinating nerve cells in the peripheral nervous system. The Schwann cells wrap layer upon layer of their own plasma membrane in a tight spiral around the axon thereby insulating the axonal membrane so that almost no current leaks across it. Unmyelinated axons in the PNS are nonetheless embedded in Schwann cells although they are not ensheathed by myelin.

A number of neuropathies of the PNS are associated with demyelination or failure of the Schwann cells to properly ensheath the axons of the PNS. They are diabetic neuropathy, Guillain-Barre' disease (acute demyelinating polyneuropathy), chronic inflammatory demyelinating polyradiculoneuropathy (CIPD), and HTV inflammatory demyelinating disease. Also axon damage due to physical trauma may result in demyelination of the PNS. Thus, there is a need to discover agents that can be used to promote the production of myelin by Schwann cells. DESCRIPTION OF THE INVENTION

The present invention fills this need by providing for a method for promoting production of myelin or P zero protein by Schwann cell comprising bringing a Zcyto7 polypeptide or IL-17 into contact with Schwann cells. Examples of Zcyto7 polypeptides are the polypeptides of SEQ ID NOs: 2, 7, and 9-28.

Preferably, the mammal treated will be a human and the Zcyto7 will be of the human allotypes. Preferably, the Zcyto7 will be administered in an amount of about 0.1 to 100 micrograms (μg) per kilogram of body weight.

The teachings of all of the references cited in the present specification are incorporated in their entirety herein by reference.

Definitions

The term "effective amount" as used herein regarding the effective amount of Zcyto7 administered in accordance with the present invention means an amount of Zcyto7 that causes increased expression of myelin by Schwann cells. The effective amount of Zcyto7 or IL-17 to be administered is from 0.1 μg to lmg of Zcyto7 or IL-17 per kilogram of body weight per day. More preferably, the effective amount is from 1 μg to 500 μg of Zcyto7 or IL-17 per kilogram of body weight. Zcyto7 should be administered daily until the symptoms of neuropathy dissipate.

The term "allelic variant" denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

Zcyto7 and a method for making Zcyto7 polypeptides have been disclosed in International Patent Application No. PCT/US98/08212, Publication No. WO 98/49310. Introduction

The present invention is based upon the discovery that Zcyto7 or IL-17 can induce the production of myelin by Schwann cells. The present invention is also based upon the discovery that Zcyto7 can induce the production of protein zero by Schwann Cells. Protein zero is a major structural protein of peripheral myelin, and and is a homophilic immunoglobulin cell adhesion molecule, which mediates adhesion of Schwann cell membranes as they enwrap axons and generate compact myelin, Spiryda L. B„ J. Neurosci, Res. 54: 137-146 (1998).

The axons of many vertebrate neurons are insulated by a myelin sheath, which greatly increases the rate at which an axon can conduct an action potential. Schwann cells, which are supporting or glial cells, form myelin in the peripheral nerves. The Schwann cells wrap layer upon layer of their own plasma membrane in a tight spiral around the axon, thereby insulating the axonal membrane so that almost no current leaks across it. The sheath is interrupted at regularly spaced intervals called the "nodes of Ranvier', where almost all the Na⁺ channels in the axon are concentrated. Because the ensheathed portions of the axon membrane are so well insulated, a depolarization of the membrane at one node almost immediately spreads passively to the next node. Thus, an action potential propagates along a myelinated axon by jumping from node to node, a process called salutatory conduction. This type of conduction has two main advantages: action potentials travel faster, and metabolic energy is conserved because the active excitation is confined to the small regions of axonal plasma membrane at nodes of Ranvier. Conduction in myelinated axons is characterized by a rapid electronic conduction (because of the decreased time constant for conduction) with little decrement (because of the increased length constant) between the nodes of Ranvier. Only at the nodes is the action potential regenerated. A myelination of an axon increases electronic conduction velocity by sevenfold.

Myelinated axons are also more efficient metabolically than nonmyelinated axons. The sodium-potassium pump extrudes the sodium that enters and re-accumulates the potassium that leaves the cell during action potentials. In a myelinated axon, ionic currents are restricted to the small fraction of the membrane surface at the nodes of Ranvier. For this reason fewer Na⁺ and K⁺ ions traverse a unit area of membrane, and less ion pumping is required to maintain Na⁺ and K⁺ gradients. The present invention is a method for inducing the expression of myelin or Protein zero by Schwann cells. Thus, Zcyto.7 can be administered to treat a number of demyelinating PNS neuropathies, or to induce the production of myelin around regenerating peripheral nerve cells that have been injured by trauma.

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS: 1, and 2 represent a single allele of the human Zcyto7. One can clone allelic variants of these sequences by probing cDNA or genomic libraries from different individuals according to standard procedures.

Acute Demyelinating Polyneuropathy

An example of a demyelinating disease of the PNS is acute demyelinating polyneuropathy. This acute inflammatory polyneuropathy, also known as Guillain-Barre' syndrome (GBS), occurs in all parts of the world and in all seasons. It affects children and adults of all ages and both sexes. A mild respiratory or gastrointestinal infection precedes the neuritic symptoms by 1 to 3 weeks in about 60 percent of the patients. Other less common antecedent events include surgical procedures, viral exanthems and other viral illnesses such as cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus (HIV), bacterial infections, e.g., Mycoplasma pneumoniae, Lyme disease and particularly Campylobacter_/e/_.ra, and lymphoma, particularly Hodgkin's disease.

Symptomatology

The major clinical manifestation of GBS is weakness, which evolves, more or less symmetrically, over a period of several days or a week or two. Proximal as well as distal muscles of the limbs are involved, usually the lower extremities before the upper trunk, intercostals, neck, and cranial muscles are affected later. The weakness can progress to total motor paralysis with death from respiratory failure within a few days. More than half of the patients complain of pain and an aching discomfort in the muscles, mainly those of the hips, thighs, and back. Paresthesias (tingling, burning, numbness) are also a frequent and early symptom but tend to be evanescent; occasionally they are absent throughout the illness. The most important laboratory aids are the cerebrospinal fluid (CSF) examination and electrodiagnostic studies. An increase in CSF protein is probably due to widespread inflammatory disease of the nerve roots. In a few patients (10 percent or less), the CSF protein values are normal throughout the illness. Electrodiagnostic studies may be normal early in the illness. Then there occurs a reduction in conduction velocity or conduction block in motor neurons. Prolonged distal latencies and abnormal F responses (with affection of proximal parts of nerves) are other diagnostic findings.

Treatment

Most of the evidence suggests that the clinical manifestations of this disorder are the result of a cell-mediated immunologic reaction directed at peripheral nerves. In general administration of Zcyto7 should begin upon diagnosis of the disease at the dosages listed below. However, treatment with Zcyto7 may be delayed until the underlying inflammatory condition has subsided. Plasma exchange or intravenous administration of immunoglobulin can be employed to alleviate the inflammatory condition. For best results this should be done within two weeks of onset of symptoms. The usual plasma exchange regimen removes 200 to 250 mlJkg in four to six treatments on alternate days. The usual replacement fluid is saline and 5 percent albumin.

Alternatively immunoglobulin can be administered at 0.4g kg/day for 5 consecutive days. Zcyto7 can be administered at the onset of the disease, during treatment to alleviate the inflammatory process or after the treatment. Administration of Zcyto7 should be continued on a regular basis, at least 1-3 times a week until full neurologic recovery by the patient.

In chronic inflammatory demyelinating polyradiculoneuropathy, other immunosuppressants and plasmapheresis can be used to alleviate the inflammatory condition and Zcyto7 can be administered to promote remyelination.

Diabetic Neuropathies

Zcyto7 can also be used to promote myelination expression in Schwann cells to treat diabetic neuropathies. The demyelination that occurs in diabetes mellitus is believed to be caused by ischemia. The patient should be administered Zcyto7 1 to 3 times a week at the dosages listed below. Use of Zcyto7 to Promote Re-myelination of Peripheral Neurons Injured by Trauma

Unlike the central nervous system the neurons of the PNS have the ability to regenerate or repair themselves after injury caused by trauma. After an axon of the PNS is lost through trauma, the proximal stump of the damaged axon develops sprouts. In the PNS these sprouts elongate and grow along the path of the original nerve if this route is available. The Schwann cells in the distal stumps of the nerve not only survive the degeneration of the neuron, but they also proliferate and form rows along the course previously taken by the axons. Growth cones of the sprouting axons find their way along the rows of Schwann cells and may eventually reinnervate the original peripheral target structures. The Schwann cells then remyelinate the axons. To expedite the generation of the myelin sheath around the newly formed axon, Zcyto7 can be administered, preferably every one to three days until it becomes apparent the nerve is regenerated by means of the appropriate neurological test.

Modes of Administration

For pharmaceutical use, the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a Zcyto7 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., (Mack Publishing Co., Easton, PA, 19th ed., 1995). Therapeutic doses will generally be in the range of 0.1 to 100 μg kg of patient weight per day, preferably 0.5-20 μg/kg per day, with the exact dose determined by the clinician according to accepted standards determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. Nucleic Acid-based Therapeutic Treatment

Zcyto7 can be also administered by means of gene therapy. In one embodiment, a gene encoding a Zcyto7 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl) vector [Kaplitt et al, Molec. Cell. Neurosci.2: 320-330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford- Pemcaudet et al., J. Clin. Invest. 90 :626-630 (1992), and a defective adeno-associated virus vector [Samulski et al, J. Virol, 57:3096-3101 (1987); Samulski et al J. Virol, 55:3822-3828 (1989)].

In another embodiment, the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al, Cell, 33:153 (1983); Te in et al, U.S. Patent No. 4,650,764; Temin et al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol. 52:1120 (1988); Temin et al, U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al. and Blood, S2:845 (1993).

Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner et al, Proc. Natl Acad. Sci. USA, 84:7413-7417 (1987); see Mackey et al, Proc. Natl. Acad. Sci. USA, 35:8027-8031 (1988)]. The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

It is possible to remove the cells from the body and introduce the vector as a naked DNA plasmid and then re-implant the transformed cells into the body. Naked DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter [see, e.g., Wu etal, J. Biol Chem., 257:963-967 (1992); Wu et al, J. Biol Chem., 253:14621-14624 (1988)].

Example 1 Cloning of Zcyto7

Zcyto7 was identified from expressed sequence tag (EST) 582069 (SEQ

ID NO: 3) by its homology to Interleukin-17. The EST582069 cDNA clone was obtained from the IMAGE™ consortium Lawrence Livermore National Laboratory through Genome Systems, Inc. The cDNA was supplied as an agar stab containing E. coli transfected with the plasmid having the cDNA of interest and then streaked out on an LB 100 μg/ml a picillin and 100 μg/ml methicillin plate. The cDNA insert in EST582069 was sequenced. The insert was determined to be 717 base pairs long with a 180 amino acid open reading frame and a 22 amino acid signal peptide.

Example 2 Construction of Zcyto7 Expression Vectors

A 473 bp Zcyto7 PCR DNA fragment was generated with 1 μl of a dilution of the EST582069 plasmid prep of Example 2 and 20picomoles (pm) of primer SEQ ID NO: 4 and 20 pm primer SEQ ID NO: 5. The digested reaction mixture was electrophoresed on a 1 % TBE gel; the DNA band was excised with a razor blade and the DNA was extracted from the gel with the Qiaquick« Gel Extraction Kit (Qiagen). The excised DNA was subcloned into plasmid nfpzp9, which had been cut with Bam and Xho. Nfpzp9 is a mammalian cell expression vector comprising an expression cassette containing the mouse metallothionein-1 promoter, a sequence encoding the tissue plasminogen activator (TPA) leader, then multiple restriction sites. These were followed by the human growth hormone terminator, an E. coli origin of replication and a mammalian selectable marker expression unit containing the S V40 promoter, enhancer and origin of replication, a dihydrofolate reductase gene (DHFR) and the SV40 terminator.

Zcyto7 was purified by means of affinity chromatography using anti- Zcyto7 antibodies.

Example 3 Cloning of Murine Zcyto7

Mouse Zcyto7 was identified from an expressed sequence tag (EST)

660242 (SEQ ID NO: 8) by its homology to human Zcyto7. EST660242 cDNA clone was obtained from the IMAGE consortium Lawrence Livermore National Laboratory through Genome Systems, Inc. The cDNA was supplied as an agar stab containing E. coli transfected with the plasmid having the cDNA of interest and then streaked out on an LB 100 μg/ml ampicillin, 25 μg/ml methicillin plate. The cDNA insert in EST660242 was sequenced. The insert was determined to be 785 base pairs with an open reading frame of 180 amino acids and a putative 20 amino acid signal peptide. The sequences are defined by SEQ ID NO: 7 and SEQ ID NO: 6.

Example 4

Induction of Myelin Expression by Zcyto7 OBJECT

The object of the present experiment was to determine if Zcyto7 could induce the expression of myelin by Schwann cells.

TEST SYSTEM

Primary Schwann cell cultures and dorsal root ganglia (DRG)-Schwann cell co-cultures were established. Rat primary Schwann cells were isolated as described by Einheber et al, J. Cell Biol. 123:1223-1236 (1994). Schwann cells were grown in 100 mm poly-L-lysine coated plates in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), pituitary extract (lmg/ml) and 10 μM forskolin; up to 80-90% confluency.

Dissociated neuronal cultures from normal rats were established as described by Kleitman et al, Tissue culture methods for the study of myelination, in Culturing Nerve Cells, Banker and Goslin, Eds, pp. 338-378(MIT Press, Cambridge, MA, 1992). Briefly, DRG were dissected out from the rats, treated with 0.25% trypsin, mechanically dissociated, washed, and resuspended in LI 5 medium with 10% fetal calf serum. The neurons were plated onto 12-mm glass coverslips coated with ammoniated rat tail collagen (Biomedical Technologies, Inc.) for microscopic evaluation. Starting one day following the dissection, cultures were treated over a 2-week period with three cycles of 5-fluorodeoxyuridine and uridine (both at 10 μM) in standard medium to assure a pure neuronal population. Standard medium consisted of minimum essential medium (MEM) supplemented with 10% FBS, 2mM glutamine, 0.4% glucose, and 50ng/ml β- nerve growth factor (β-NGB).

Myelinating co-cultures were established by seeding the purified neurons with rat primary Schwann cells as described Einheber, et al, id.

TEST ARTICLE AND FORMULATION METHODS

Four solutions of Zcyto7, A258F, A258G, A275F and A31 IF, were analyzed all of which had been expressed in Baby Hamster Kidney (BHK) cells. Solution A258F contained human Zcyto7 at a concentration of 0.603 mg/ml of phosphate buffered saline (PBS) at pH 6.0. Solution A258G contained human Zcyto7 at a concentration of 0.089 mg/ml of PBS + 0.1 % BS A at a pH of 6.0. Solution A311 contained human Zcyto7 that had been dimerized by fusing an Immunoglobulin (Ig) Fc portion to the carboxy terminus of the polypeptide; the concentration of the Zcyto7 fusion protein was at 0.45 mg/ml of PBS at pH 7.0. Solution A275F contained murine Zcyto7 at a concentration of lmg/ml of PBS at pH 6.0. A control of PBS was also tested. Also tested was a solution of IL-17 at a concentration of 50 μg/ml of PBS, pH 7.0.

A solution of progesterone (Sigma) at a concentration of 1 mg/ml of ethanol was also prepared.

METHODS EXPERIMENT 1

EFFECT OF ZCYTO7 IN PURE SCWANN CELLS

The effect of Zcyto7 was tested in pure Schwann cell culture and in a co- culture of DRG and Schwann cells. Primary Schwann cells were grown to 80-90% confluency in 100mm plates as described above. 12 h before treatment media was changed to 10 ml of N2 (defined medium) with or without 1 μM progesterone. The Zcyto7 solutions were added as 1.1 ml of 1 OX solution in N2 media, and cells were incubated for 48 hours (h). Cells were washed once with Hanks balanced salt solution (HBSS). The cell suspension was frozen on dry ice, thawed, pelleted and resuspended in 100 μl of lysis buffer, 125 mM tris (hydroxymethyl) aminomethane HC1 (Tris HC1) pH7.0, 15% sucrose, 4% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA). Lysates were boiled for 5 minutes (min) on a water bath and cooled on ice. The protein concentration was determined using 4 μl in a BioRad BCA assay. Each sample was then supplemented with dithiothreitol (DTT) to a final concentration of 10 mM phenylmethylsulfonyl fluoride (PMSF) - to 2 mM, pepstatin and leupeptin - to 10 μg/ml.

60 μg of protein from each sample were separated on 4-12% Tris-Glycin NOVEX gels at 125 V constant for 1.5 h. Each gel contained vehicle control, vehicle supplemented with progesterone control, and randomly chosen samples from treatment groups. Transfer to poly (vinylidine difluoride) (PVFD) membrane was carried out according to the NOVEX electrotransfer protocol, at 25 V, for 40 min. Membranes were rinsed with tris buffered saline (TBS) for 3 min, and blocked with TBS/0.1% TWEEN- 20/2% BSA for 1 h. Membranes were then incubated with 1:1000 dilution of anti-MBP mouse monoclonal IgG (Boehringer, Mannheim) in TBS/0.1% TWEEN-20, 0.5% BSA overnight, at 4°C. After washing 4 times for 30 min with TBS/0.1% TWEEN-20, membranes were incubated with anti-mouse horseradish peroxidase conjugate (1 :20,000 dilution in TBS/0.1% TWEEN-20, incubated with SUPERSIGNAL chemiluminescent HRP substrate (Pierce Chemical Co., Rockford, ILL) and exposed to Kodak X-Ray film for 15-20 min.

Density of myelin basic protein (MBP) bands was measured on LYNX

Densitometer (Applied Imaging) with ILLUMA System and expressed as percent progesterone-induced control MBP.

EXPERIMENT 2 PRODUCTION OF MYELIN IN DRG-SCHWANN CELL (DRG-SC) CO-CULTURES

After establishing the myelinating co-cultures, Schwann cells were allowed to proliferate for one week attached to the neurons. The medium was then changed to 15% and 50 μg/ml ascorbate was added to promote basal lamina formation and to initiate myelination. The effect of the Zcyto7 was evaluated for 3.5 weeks in the presence of ascorbate, with the medium changed every two days. Cultures were then fixed in 3.7% formaldehyde for 10 minutes and made permeable in 100% methanol for 15 minutes at -20°C. Following with PBS having 3% BSA, the samples were incubated with mouse anti-MBP antibody (Boehringer-Mannheim) overnight, washed and incubated with Cy3-labeled anti-mouse IgG. Samples were washed, mounted in VECTASHIELD and examined by epifluorescence with a Zeiss AXIOSKOP microscope.

Myelin production was quantitatively and qualitatively evaluated, and myelin density was determined for each of the five replicates per condition. Six representative regions in each sample were captured using a low-level light COHUE digital camera, and the density of fluorescent signal determined using the National Institutes of Health (NDH) Scion Image. The mean and standard deviation of myelin density was calculated for each group and converted to a percentage by dividing by the density seen in the appropriate control culture. Morphometric measurements were carried out using double blind analysis.

EXPERIMENTAL DESIGN

In experiment 1, the effect of the 4 different solutions of Zcyto7 on MBP accumulation was evaluated in pure Schwann cell cultures, with and without progesterone pretreatment. Solution A275F was evaluated in experiment 1 at 1 ng/ml, 10 ng/ml, 100 ng/ml and 1 μg/ml.

Four controls were included in each experiment: test vehicle alone, vehicle supplemented with progesterone, vehicle supplemented with IL-17, and vehicle supplemented with progesterone and IL-17 [See J. Immunol, 250:5445-5456 (1993)]. Three replicates were set up per each condition.

Solution A258F was chosen on the basis of the results of experiment 1 was evaluated in experiment 2 for myelin formation in DRG-Schwann co-cultures.

Concentrations of A258F of 1 ng/ml, 10 ng/ml, and 100 ng/ml were tested without the addition of progesterone, and a sample having a concentration of 100 ng/ml with the addition of progesterone was tested. Five controls were included: test vehicle alone, vehicle supplemented with ascorbate, vehicle supplemented with ascorbate plus progesterone and vehicle supplemented with ascorbate plus IL17, at 10 ng/ml and 100 ng/ml. RESULTS

Table 1 shows the mean induction + standard deviation (SD) for each solution of Zcyto7. Treatment of pure Schwann cell cultures with Zcyto7 no matter from which solution resulted in various degrees of MBP induction. The best results were obtained with solution A258F. It induced MBP accumulation in dose-dependent manner, with maximal effect at 100 ng/ml. hi contrast to the other compounds, culture-to-culture variability was minimal.

Pretreatment of the Schwann cell cultures with progesterone appears to mediate the induction of MBP in all cases. However, even in the absence of progesterone pretreatment, solutions A258F, A258G and A275F significantly induced MBP accumulation at 100 ng/ml.

Table 1. Mean MBP induction after treatment with Zcyto7 (as percent of progesterone induction)

Table 2. MBP signal density in various DRG-Schwann cell co-culture treatment groups

Table 3 Mean MBP induction after treatment with IL-17 (as percent of progesterone induction)

Example 5 Induction of the Expression of Protein Zero by Zcyto7

The present example shows that Zcyto7 induces Schwann cells to produce Protein Zero. .

Material and Methods

Primary Schwann cells were prepared as described above using 1-day old CD rat neonates. Schwann cells were grown in DMEM, 10% fetal bovine serum (FBS), plus bovine pituitary extract (PEX) at 20 μg/ml and Forskolin at 2μM. The cells were weaned down to a low serum medium ("Li" medium with 1% FBS added to it). Li medium was comprised of DMEM/F12, 10 μg/ml tranferrin, 5 μg/ml insulin, 2 nM progesterone, 2 μM Forskolin, 20 μg/ml PEX, and 20 ng/ml Heregulin-beta-1 (R & D Systems).

The Schwann cells were grown to confluency in the low serum growth medium on poly-L-lysine-coated 10 cm tissue culture dishes. The cell medium was changed to N2 medium + 1 μM progesterone with either Forskolin (Calbiochem) (10 μM and 25 μM) or 100 ng/ml of human Zcyto7. Control medium was Schwann cells in N2 + 1 μM progesterone for the whole time. Cells were harvested and placed in lysis buffer (125 mM Tris HC1, pH 7.0, 15% sucrose, 4% SDS, 10 mM EDTA, plus added Roche Complete-EDTA-free, protease inhibitor cocktail).

Western Blot Analysis:

Protein concentrations of cell lysates were determined by Pierce BCA protein assay (Pierce Chemical Co., Rockford, Illinois). Forty micrograms of each cell lysate sample was run on a 4-12% Tris-Glycine gel (Novex) at 125 V for 1 hour 30 minutes, and transferred onto a PVDF membrane in a Hoeffer unit a 550 mAmps for 1 hour. Mouse monoclonal antibody to protein zero was used at 1:1000 dilution (1 μg/ml final concentration) to probe the blot. A Vectastain biotin/streptavidin-HRP 2^nd antibody system was used for amplication. The blot was developed with Pierce SuperSignal West Pico chemiluminescent substrate and scanned on a Boehringer- Mannheim Lumi-Imager. Results:

The results indicate that 100 ng/ml of human Zcyto7 induces the expression of protein zero by primary rat Schwann cells. Values calculated by the LumiAnalyst program showed induction of protein zero by Zcyto7 to be increased over the control cell culture medium. Designating the amount of P zero protein in the band for the media control as 100%, the relative percentage for human Zcyto7-treated Schwann cells was 173%. The positive control of Forskolin also showed an induction of P zero protein having a relative percentage of P zero protein of 199% at 10 μM, and 30% at 25 μM relative to the cell culture control medium.

Example 6 Hybridization Studies

The expression of myelin basic protein and protein zero was also determined by measuring the mRNA levels that encode myelin basic protein and protein zero. The results showed that the mRNA that encodes for myelin basic protein and the mRNA that encodes for protein zero were upregulated by Zcyto7.

Claims

CLAIMSWe claim:

1. A method for promoting the expression of myelin comprising bringing a Zcyto7 polypeptide into contact with Schwann cells.

2. The method of claim 1 wherein the Zcyto7 polypeptide is selected from the group consisting of SEQ ID NOs: 2, 7, and 9-28.

3. A method for treating demyelinating diseases of the peripheral nervous system (PNS) comprising administering to an individual having a demyelinating disease of the PNS a therapeutically useful amount of Zcyto7.

4. The method of claim 3 wherein the demyelinating disease is selected from the group consisting of diabetic neuropathy, Guillain - Barre' Syndrome, chronic demyelinating disease, acute demyelinating polyneuropathy and human immunodeficiency viral demyelinating neuropathy.

5. A method for promoting the expression of myelin comprising bringing an interleukin-17 (IL-17) polypeptide into contact with Schwann cells.

6. A method for treating demyelinating diseases of the peripheral nervous system (PNS) comprising administering to an individual having a demyelinating disease of the PNS a therapeutically useful amount of IL-17.

7. A method for promoting the expression of P zero protein by Schwann cells comprising bringing a Zcyto7 polypeptide or IL-17 into contact with Schwann cells.

8. A method for promoting the expression of myelin basic protein by Schwann cells comprising bringing a Zcyto7 polypeptide or IL--17 into contact with Schwann cells.