Patent Application
20050175998
Pharmaceutical Compositions Comprising A Substance Capable of Activating FcRγ, Agents for Stimulating Myelinogenesis, Agents for Stimulating the Differentiation of Oligodendroglial Precursor Cells, Agents for Activating Fyn Tyrosine Kinase, Agents for Stimulating the Expression of Myelin Basic Protein, A Method of Detecting Oligodendroglias or Oligodendroglial Precursor Cells Capable of Myelinogenesis, A Method of Examining FcRγ Expression in Animal Brain Tissues or Cells, and Reagent Kits
The present invention relates to pharmaceutical compositions comprising a ligand for the γ chain of Fc receptors (FcRγ); agents for stimulating myelinogenesis; agents for stimulating the differentiation of oligodendroglial precursor cells; agents for activating Fyn tyrosine kinase; agents for stimulating the expression of myelin basic protein; a method of detecting myelinogenetic oligodendroglias or precursor cells thereof; a method of examining FcRγ expression in animal brain tissues or cells; and reagent kits.
Myelin, a multi-layered membranous sheath enwrapping individual axons, is required for both the fast conduction of nerve impulses and for axonal function and integrity (1,2). Myelin is synthesized by oligodendroglia in the central nervous system (CNS), a process occurring shortly after the birth. Defects with myelin cause severe diseases in human; multiple sclerosis (MS) is one of such diseases, characterized by severe loss of myelin in the CNS. Although the disease was first described more than a century ago, both the etiology and cure reman largely unknown and uninvestigated (3). Only recently, a population of oligodendroglial precursor cells was first observed in MS lesions (4). Unraveling mechanisms that enable those endogenous precursor cells to be differentiated and/or strengthened so as to regenerate the lost myelin would prove to be an efficient therapeutic target. Therefore, comprehensive studies for elucidating the mechanism of myelinogenesis are of great importance in the treatment and cure of demyelinating diseases.
Myelin is composed of a limited number of myelin proteins. Myelin basic protein (MBP) comprises 30-40% of all myelin proteins within the CNS. MBP is indispensable in myelinogenesis, playing a crucial role in the compaction of the myelin sheath (5). MBP also regulates the expression of other myelin proteins such as myelin-associated glycoprotein (MAG), indicating that MBP is extremely important for myelinogenesis (6). The spontaneous Shiverer mouse, which has a natural knock-out of the MBP gene (7), exhibits severe hypomyelination of CNS axons, leading to premature death within 3 months. Specific isoforms of MBP containing exon 2 of the MBP gene play a regulatory role in myelinogenesis (8), suggesting that the preliminary events of myelinogenesis require MBP.
In the process of myelinogenesis, Fyn tyrosine kinase (Fyn) functions as an essential signaling molecule within oligodendroglia (9), stimulating the expression of MBP (10). Mice deficient in Fyn suffer severe decreases in MBP production, resulting in severe dysmyelination (9-11). Fyn, a non-receptor type tyrosine kinase, requires coupling to an adapter molecule in order to receive extracellular signals. Although myelin-associated glycoprotein (MAG) has been proposed as a candidate molecule responsible for the initial triggering of Fyn signals in myelination (9-13), MAG-deficient mice demonstrated only subtle myelin abnormalities (14,15). No significant differences in MBP expression were observed in MAG-deficient mice (14-16), casting doubt on the validity of MAG as the upstream signaling molecule which links extracellular signals to Fyn. Several researchers postulated that compensatory molecules may function in MAG-deficient mice; such molecules, however, have yet to be identified (17,18).
It was suggested that MAG is not the trigger of myelinogenesis but that hitherto unknown molecules are responsible for triggering (6). In addition, in vitro studies have revealed that the activation of Fyn during the morphological differentiation of oligodendroglia occurs prior to the first expression of MAG (19). It is MBP that regulates the expression of MAG in vivo (6).
It is an object of the present invention to identify the trigger that functionally couples to Fyn to cause the initial expression of MBP, thereby obtaining therapeutic strategies for diseases that result from defects with myelinogenesis. This molecule, i.e. the true trigger of myelinogenesis, must be expressed prior to the expression of MAG (i.e. early in the second week after birth; 6,20). Elucidation of this signal cascade will allow close understanding of the mechanism underlying myelinogenesis.
The present inventors have demonstrated that the γ subunit of the immunoglobulin Fc receptor (FcRγ), an essential signaling molecule in the immune system (reviewed in 21), is involved in myelinogenesis. FcRγ, which couples to Fyn (21), governs the initial expression of MBP in oligodendroglia. The present inventors propose that this FcRγ-Fyn-MBP cascade is essential in myelinogenesis. The present inventors detected Fc receptors, specific for immunoglobulin G, in oligodendroglia, suggesting that antibody-Fc receptor interactions may regulate myelinogenesis. CD45, an Fyn regulating molecule (22), co-expresses with FcRγ in oligodendroglia during myelinogenesis. The involvement of this signaling molecule in the process of myelinogenesis is supported by the discovery of a mutation in CD45 within some populations of patients with MS (23). The findings of the present inventors introduce a connection between the brain and the immune system, clarifying the mechanism of myelinogenesis. Elucidation of this correlation has provided future therapeutic strategies for demyelinating diseases/dysmyelinating diseases including MS. The present invention has been achieved based on these findings.
The present invention can be summarized as follows.
The term “activating FcRγ” used herein means triggering or enhancing an FcRγ-dependent intracellular cascade. As a specific example of the FcRγ-dependent intracellular cascade, FcRγ-Fyn-MBP cascade may be given. It has been demonstrated by the present inventors that in this FcRγ-Fyn-MBP cascade, FcRγ couples to Fyn, regulating the initial expression of MBP in oligodendroglia.
It is possible to activate FcRγ by, for example, cross-linking FcRγ with an antibody. Alternatively, it is possible to activate FcRγ by cross-linking type I Fc receptor γ (hereinafter referred to as “FcγRI”) or type III Fc receptor γ (hereinafter referred to as “FcγRIII”) (Fc receptors for IgG) to which FcRγ is coupled to, with an antibody to FcγRI or FcγRIII. It was also been reported that FcRγ is activated when FcεRI is cross-linked with an antibody to FcεRI (reviewed in H. Turner & J. P. Kinet, Nature 402, B24 (1999); see also the references cited in this review). In the cross-linking of FcεRI, it has been reported that calcium signals by IP3R as well as ERK and JNK are regulated through complicated coupling of Lyn, Syk, PLCγ, etc. (reviewed in H. Turner & J. P. Kinet, Nature 402, B24 (1999); see also the references cited in this review; for FcγRs, see J. V. Ravetch & S. Bolland, Ann. Rev. Immunol. 19:275 (2001) and the references cited therein). Whether it is downstream of FcεRI or FcγRs, the cascade is considered to be entirely FcRγ-dependent. Nevertheless, FcRγ seems to have cascades in a cell-specific manner.
The term “immunoglobulin for intravenous injection” used herein means an immunoglobulin extracted from a pool of many and unspecified immuoglobulins. Such immunoglobulin is polyclonal, and its major component is IgG. Such immunoglobulin is used as medical IVIg.
The term “ligand for the γ chain of Fc receptors” (hereinafter referred to as the “ligand for FcRγ”) means a substance which specifically binds to the γ chain of Fc receptors (hereinafter referred to as “FcRγ”).
The term “stimulating the differentiation of myelin basic protein” used herein refers to inducing any of the morphological/chemical changes observed during the period up to the maturation of oligodendroglial precursor cells into oligodendroglia. Specific examples of such changes include the appearance of processes from oligodendroglial precursor cells, or increase in the expression levels of proteins such as MBP that are indispensable for myelinogenesis.
The term “activating Fyn tyrosine kinase” used herein refers to producing one or both of the following effects: increasing the enzyme activity of Fyn tyrosine kinase (qualitative increase) or increasing the expression level of Fyn itself (mRNA or protein) (quantitative increase). It is speculated that the qualitative increase requires CD45.
The term “stimulating the expression of myelin basic protein” used herein refers to causing or increasing the expression of myelin basic protein; the expression of myelin basic protein may be either at the RNA level or at the protein level.
As myelin basic protein (hereinafter, referred to as “MBP”), isoforms of 14.0, 17.0, 18.5 and 21.5 kD are known. These isoforms are generated by selective use of the seven exons on the MBP gene. Isoforms whose expression is promoted by a ligand for FcRγ are mainly those isoforms which comprise exon 2 (i.e. isoforms of 17.0 and 21.5 kD).
The term “being coupled to FcRγ” used herein refers to co-operating with FcRγ functionally or physically. For example, FcγRI contains FcRγ as a constituent molecule (physical co-operation), and the FcγRI signaling cascade is FcRγ-dependent (functional co-operation).
The term “stimulating myelinogenesis” used herein means: replacing or repairing removed myelin sheaths in demyelinating diseases; completing or repairing by replacement those myelin sheaths which were formed incompletely in dysmyelinating diseases; and stimulating foreign oligodendroglias or oligodendroglial precursor cells introduced into a patient's body so that they actually form myelin sheaths in the body to thereby enhance their forming ability in transplantation/regeneration medicine.
The term “demyelinating diseases” used herein refers to those diseases wherein myelin sheaths once formed are denatured or removed for some reason; the term “dysmyelinating diseases” used herein refers to those diseases wherein myelinogenesis itself is impaired for some reason; the term “transplantation/regeneration medicine” used herein refers to medicine wherein an oligodenroglia prepared by some method is transplanted into patients to thereby replace and repair impaired myelin sheaths or allow formation of myelin sheaths in the patients in order that low or no myelinogenesis is artificially compensated.
Examples of “demyelinating diseases” include, but are not limited to, multiple sclerosis, optical neuromyelitis, and acute diffuse encephalomyelitis. Examples of “dysmyelinating diseases” include, but are not limited to, Krabbe's disease, metachromatic leukodystrophy, and adrenoleukodystrophy. Other diseases where dysmyelination is caused by metabolic disorders are often classified into metabolic diseases in spite of their being dysmyelinating diseases. However, all diseases where myelinogenesis is impaired can be defined as dysmyelinating diseases.
The term “myelinoclasis” used herein refers to physical disintegration for some reason of myelin sheaths, once formed or being formed, and the resultant deprivation of their function. For example, in encephalomyelitis and the like, immune cells generally destroy myelin sheaths in an inflammatory manner.
In the method of the invention for detecting myelinogenetic oligodendroglias or precursor cells thereof, the expression of FcRγ in oligodendroglias or precursor cells thereof is used as an indicator. The expression of FcRγ may be either at the RNA level or at the protein level.
The term “marker” used herein means a substance which may serve as an indicator when the identity of a specific tissue or cell is examined (e.g. FcRγ) or a substance which is useful in searching for the indicator (e.g. anti-FcRγ antibody).
The term “immunohistochemical or immunocytochemical analysis” used herein refers to a means of detecting a specific antigen present in a tissue or cells by an antigen-antibody reaction. For example, a specific antigen present in a tissue or cells may be visualized with a labeled antibody and then observed under a microscope (direct method). Alternatively, a method may be employed in which a primary antibody specific to the antigen and a labeled secondary antibody specific to the primary antibody are used (indirect method). It is also possible to detect the fluorescence of a labeled secondary antibody by flow cytometry (FACS).
The term “gene amplification method” used herein means a method of anplifying DNA in chain reactions using DNA polymerase.
The term “Western blotting” used herein means a method in which a protein of interest separated by electrophoresis is transferred onto a nitrocellulose membrane, PVDF membrane or the like and then detected with an antibody.
In one embodiment of the present invention, a pharmaceutical composition comprising as an active ingredient a substance capable of activating FcRγ may be used to prevent and/or treat at least one disease selected from the group consisting of demyelinating diseases, dysmyelinating diseases and myelinoclasis. In the future, a therapeutic treatment in which neural stem cells, ES cells or subject-derived oligodendroglias are transplanted and regenerated will be practiced. (Experiments to transplant Schwann cells, which form myelin in the peripheral nerve system, into the central verve system have already begun on human subjects in Yale University.) When such a treatment has become possible, the technique of the present invention will be applicable as a booster for myelin formation. In myelinoclasis occurring secondarily (e.g. destruction of nerve axons precedes destruction of myelin sheaths), if an attempt to artificially repair the destructed nerve axons has become possible, oligodendroglial transplantation and myelin regeneration will similarly become necessary. The technique of the present invention will also be applicable to such a case.
In another embodiment of the present invention, it is possible to use FcRγ as a marker for oligodendroglias or precursor cells thereof. FcRγ-positive oligodentrolias may be regarded as cells having the potential for myelinogenesis.
The present specification encompasses the contents of the specification and/or drawings of Japanese Patent Application No. 2001-229553 based on which the present patent application claims priority.
a shows images of cultured OPCs from wild-type mouse and CD45-deficient mouse (CD45−/−) without stimulation (control) or with 24-hr stimulation using anti-FcRγ antibody. While the wild-type cells display morphological differentiation upon stimulation, CD45-deficient cells do not. These results show that CD45 is necessary for the differentiation of OPC. Panel b shows the results of Western blotting of the cells shown in Panel a. This time, cells were stimulated not only with FcRγ but also with IgG. When stimulated with IgG and FcRγ, wild-type OPC displayed increased MBP expression, but CD45-deficient mouse did not show such increase. These results show that CD45 is necessary for the differentiation of OPC. Panel c shows detection by Western blotting of MBP in myelin fractions extracted from brains of wild-type mouse, CD45-deficient mouse and Fyn-deficient mouse (Fyn−/−) at P10. As seen from this image, CD45-deficient mouse and Fyn-deficient mouse show reduced MBP, as compared to the wild-type mouse. This supports the results of analysis of the tissues (d-h), and also suggests that both CD45 and Fyn are important for myelinogenesis (MBP expression). Panels d and e show myelin in the stria tun of wild-type mouse and CD45-deficient mouse at day 10 after birth by immunohistochemical staining of MBP. “LV” represents the lateral ventricle. CD45-deficient mouse shows decreased expression of MBP. Panels f, g and h show immunohistological staining of MAG at P10. Since CD45 is speculated to be responsible for the activation of Fyn, comparison is made with Fyn-deficient mouse. The wild-type mouse shows a great number of MAG-positive oligodendroglias in the corpus callosum (CC) and cerebral cortex (CTX), whereas CD45-deficient and Fyn-deficient mice show decrease of MAG-positive cells to similar extents. These results support that CD45 is involved in the activation of Fyn.
Hereinbelow, the present invention will be described in more detail.
The pharmaceutical compositions of the invention comprise, as an active ingredient, a substance capable of activating FcRγ. Specific examples of the substance capable of activating FcRγ include ligands for FcRγ that produce at least one effect selected from the following (A), (B) and (C):
Anti-FcRγ antibodies may be prepared by conventional methods.
For example, in order to prepare polyclonal antibodies, animals are immunized with FcRγ, the immunogen. Then, an anti-FcRγ antibody-containing material is collected from the animal, and the antibody of interest is separated and purified therefrom. Alternatively, a partial peptide of FcRγ may be used. It is also possible to immunize animals with an artificial peptide synthesized based on the nucleotide sequence encoding FcRγ (GenBank M33195 (human FcRγ)). If a short peptide (generally, 6-18 amino acids in length) is used as an immunogen in such a case, the peptide may be conjugated with a carrier protein such as Keyhole limpet haemocyanin. When the FcRγ protein is administered for immunization, usually 50-100 μg is used.
In order to prepare monoclonal antibodies, animals with high antibody titers are selected from the above-described immunized animals, and the spleen or lymph nodes are removed from them 3 to 5 days after the final immunization. Antibody-producing cells contained therein are fused to myeloma cells, followed by selection of hybridomas capable of stable production of high-titer antibodies. The hybridoma is grown in the abdomen of animals. Then, the monoclonal antibody of interest may be purified from the abdominal dropsy or may be purified from the serum or the like of the animal. Alternatively, the hybridoma may be cultured in a medium to allow antibody production, and then the monoclonal antibody may be purified from the culture supernatant or the like. Methods of preparing monoclonal antibodies are described in P. N. Nelson et al., J. Clin. Pathol. : Mol. Pathol. 53, 111-117 (2000), which is cited herein by reference.
Methods of preparing FcRγ, the immunogen, are described in Takai et al. (58) and the references cited therein, which are cited herein by reference. With respect to human FcRγ, it is easy to prepare a synthetic peptide based on the published sequence for human FcRγ (GenBank M33195).
For the immunization of animals (e.g. mammals), the immmogen FcRγ is administered alone or with a diluent, carrier, etc. to a site capable of antibody production. Preferably, the immunogen is administered by subcutaneous injection. At this time, complete or incomplete Freund's adjuvant may also be administered in order to enhance antibody productivity. Usually, the administration of the immuoogen is performed once a week; for monoclonal antibodies, the administration may be performed once or twice a week, and for polyclonal antibodies, the administration may be performed 3 or 4 times a week.
Specific examples of animals to be immunized include, but are not limited to, monkey, rabbit, dog, guinea pig, mouse, rat, sheep, goat and chicken.
Antibodies are recovered from the blood, abdominal dropsy, etc. of the thus immunized animals. The most common method used for measuring antibody titers is ELISA (Enzyme Linked Immunosorbent Assay). For further examination, immunohistochemistry and Western blotting may be used. For details, see Nelson et al., J. Clin. Pathol.: Mol. Pathol. 53, 111-117 (2000).
The separation and purification of antibodies may be performed according to conventional methods for separation/purification of immuoglobulins (e.g. salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, adsorption & desorption with ion exchangers (such as DEAE), ultracentrifugation, gel filtration, specific purification methods in which a specific antibody is recovered with an antibody-binding material or an active adsorbent such as protein-A and then the binding is dissociated to thereby obtain the antibody).
The thus prepared antibody contains IgG as a major component with minor amounts of other immuoglobulins such as IgM, IgA, etc. This antibody binds to FcRγ specifically.
Anti-FcRγ antibody-producing hybridomas may be prepared by immunizing animals (e.g. mouse) in the same manner as described for polyclonal antibody preparation, selecting those individuals exhibiting high antibody titers from the immunized animals, removing the spleen or lymph nodes from them within one week after the final immunization, and then fusing antibody-producing cells contained therein with myeloma cells. Cell fusion procedures may be performed according to known methods, e.g. the method of Koehler and Milstein (Nature, 256, 495, (1975)). Examples of useful fusion promoters include polyethylene glycol (PEG), Sendai virus, etc. Preferably, PEG is used. Examples of myeloma cells useful in the invention include Sp2/0, NS1, NS0, X63Ag8, etc. Preferably, Sp2 is used. A preferable ratio of the number of antibody-producing cells used (spleen cells) to the number of myeloma cells is from about 1:1 to about 1:2. When PEG (preferably, PEG1000 to PEG6000) is added at a concentration of about 50% and the resultant cell mixture is incubated at 37° C. for about 20-30 minutes, an efficient cell fusion can be achieved.
Separation and purification of the anti-FcRγ monoclonal antibody may be performed in the same manner as described for the polyclonal antibody, according to conventional methods for separation and purification of immuoglobulins.
As long as the anti-FcRγ antibody binds to FcRγ specifically and produces a desired effect, it may be an antibody of any class of IgG, IgA or IgM, or it may be their Fab′ or Fab fraction that is left after removal of Fc or Fc region, or a polymer thereof. Alternatively, a chimeric antibody may be used which is obtained by fusing the variable gene moiety of anti-FcRγ antibody to the human immunoglobulin constant gene and expressing the resultant gene as a recombinant.
The anti-FcRγ antibody may be modified with sugar chains which would be disrupted by enzymes present in the brain.
Examples of the substance capable of activating FcRγ other than anti-FcRγ antibody include, but are not limited to, the following.
The anti-FcγRI antibody in 1) and the anti-FcγRIII antibody in 2) may be prepared based on the above-described preparation method for anti-FcRγ antibody, using FcγRI and FcγRIII, respectively, as an immunogen.
The antagonist to FcγRII in 3) may be any substance which has an effect of inhibiting a function that acts on FcRγ inhibitorily (i.e. negatively).
Since FcγRII (type II Fc receptor) acts inhibitorily upon the activity of FcRγ, it is believed that stimulation of FcRγ would produce more effect if FcγRII is blocked with an antagonist in advance.
The artificial compound that is presumed to have the ability to bind to FcRγ in view of the structure of FcRγ mentioned in 4) may be any compound as long as it is capable of activating FcRγ. The ability to bind to FcRγ may be estimated by IP-Western blotting or the like. For example, the compound may be reacted with oligodenroglia, followed by immunoprecipitation (IP) with anti-FcRγ antibody. If the recovered substance is the artificial compound, the compound is estimated to have the ability to bind to FcRγ. The artificial compound thus estimated to have the binding ability may be a commercial product or may be synthesized chemically.
As subclasses of IgG having a high binding property to FcγRI among IgG, there may be enumerated IgG3, IgG1, IgG4, IgG2, etc. for human and IgG2a, IgG1, IgG2b, IgG3, etc. for mouse. FcγRI is a high-affinity IgG receptor, and FcγRIII a low-affinity IgG receptor. For the activation of FcγRI, an IgG subclass with a high binding ability to FcγRI (e.g. IgG3 for human) may be administered selectively. For a relevant review, see J. E. Gessner et al., Ann. Hematol. 76:231-248 (1988). These IgG subclasses may be prepared by mixing the IgGs obtained from subclass-specified hybridomas to make them polyclonal, or by obtaining an antibody to a non-reactive hybridoma.
The anti-FcεRI antibody of 6) above may be prepared based on the above-described preparation method for anti-FcRγ antibody, using FcεRI as an immunogen.
The above-described substances capable of activating FcRγ may be used independently or in combination.
The substance capable of activating FcRγ may be administered either alone or as a pharmaceutical composition comprising pharmaceutically acceptable carriers, diluents or excipients, to mammals (e.g. human, rabbit, dog, cat, rat, mouse) orally or parenterally. Dose levels may vary depending upon the subject to be treated, target disease, symptom, administration route, and so on. When used for preventing or treating adult patients with demyelinating diseases (e.g. multiple sclerosis), a substance capable of activating FcRγ (e.g. anti-FcRγ antibody) is usually administered at about 150-500 mg/kg body weight, preferably about 400-500 mg/kg body weight per dose, about once or twice a month. Preferably, the above-mentioned dose is administered by intravenous injection continuously for 2-3 days at the beginning of the treatment. For other parenteral administration routes and oral administration route, similar doses may be administered. When the condition of the patient is particularly severe, the dose may be increased.
As compositions for oral administration, solid or liquid preparations may be used. More specifically, tablets (including sugar-coated, tablets and film-coated tablets), pills, granules, powder, capsules (including soft capsules), syrups, emulsions, suspensions, or the like may be enumerated. These preparations may be formulated by conventional methods and may contain carriers, diluents or excipients conventionally used in the field of pharmaceutical manufacturing. For example, carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate, etc.
As compositions for parenteral administration, injections and suppositories may be mentioned, for example. Injections may be in such forms as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Such injections may be formulated by conventional methods. Specifically, they are prepared by dissolving, suspending or emulsifying a substance capable of activating FcRγ in an aseptic aqueous or oily liquid. Examples of aqueous liquids for preparing injections include physiological saline and isotonic solutions containing glucose and other auxiliary agents. These aqueous liquids may be used in combination with appropriate auxiliary solubilizers such as alcohol (e.g. ethanol), polyalcohol (e.g. propylene glycol, polyethylene glycol), or nonionic surfactant (e.g. Polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Examples of oily liquids for injection include sesame oil, soybean oil, etc. They may be used in combination with an auxiliary solubilizer such as benzyl benzoate, benzyl alcohol, etc. Usually, the prepared injections are filled in appropriate ampoules. Suppositories for rectal administration may be prepared by mixing a substance capable of activating FcRγ with a conventional suppository base.
The above-described pharmaceutical compositions for oral or parenteral administration may be prepared into unit dosage forms so that appropriate amounts of active ingredients are administered. Such unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc.
The above-described pharmaceutical composition may contain other active ingredient unless undesirable interaction will emerge from formulation with the substance capable of activating FcRγ. For example, an antagonist to FcγRII may be administered in order to enhance the effect of the composition.
The substance capable of activating FcRγ is not only useful in such pharmaceutical compositions, but also useful in other applications. For example, the substance may be used as a reagent for experiments in order to stimulate myelinogenesis, stimulate the differentiation of oligodendroglial precursor cells, activate Fyn tyrosine kinase, or stimulate the expression of myelin basic protein; or the substance may be used as an additive for use in an artificial culture system for oligodendroglial precursor cells.
In the method of the invention of detecting myelinogenetic oligodendroglias or precursor cells thereof, FcRγ expression in oligodendroglias or precursor cells thereof is used as an indicator. The expression of FcRγ in oligodendroglias or precursor cells thereof may be examined by the methods to be described later in Examples (e.g. immunohistochemical or immunocytochemical analysis, gene amplification methods, Western blotting, etc.). Further, the expression of at least one marker selected from the group consisting of PDGFαR, MAG, A2B5, 04, 01, MBP, PLP, DM-20, CNPase, MOG, NG2 and AN2 in oligodendroglias or precursor cells thereof may be used as another indicator. With the use of the expression of these markers as indicators, more accurate detection becomes possible. Among the above markers, 04, PDGFαR, NG2 and DE20 (which are markers for immature oligodendroglia) are preferably selected.
Hereinbelow, one example of procedures for examining FcRγ expression in animal brain tissues or cells by immunohistochemical or immunocytochemical analysis is described below.
When immunohistochemical analysis is used, brain tissues removed from animals are first fixed, and then paraffin sections are prepared. After deparaffination, a primary antibody is added to the section dropwise and reacted at an appropriate temperature for an appropriate period. After the primary reaction, the section is washed and then reacted with a secondary antibody at an appropriate temperature for an appropriate period. When a biotin-labeled antibody is used as the secondary antibody, the section is further reacted with HRP-labeled streptavidin at an appropriate temperature for an appropriate period. After washing, the section is stained with a solution containing DAB. It is preferable to perform counter staining with methyl green or the like.
When immunocytochemical analysis is used, fixed tissues can be reacted with a primary antibody without preparation of sections. Generally, when cells are to be examined, a secondary antibody is labeled with a fluorescent dye (such as FTTC) and the fluorescence is observed with a fluorescent microscope.
For details of procedures for the method of examining FcRγ expression in animal brain tissues or cells by immunohistochemical or immunocytochemical analysis, see Examples described later.
Hereinbelow, one example of procedures for examining FcRγ expression in animal brain tissues or cells by a gene amplification method is described.
Total RNA is extracted from animal brain tissues or cells. The RNA is subjected to RT-PCR using an appropriate pair of primers to thereby detect mRNA of FcRγ. The appropriate pair of primers is so designed that a specific region of the nucleotide sequence of human FcRγ-mRNA (GenBank M33195), for example, can be amplified specifically. The specific region to be amplified is determined considering the following points:
In one Example described later, a pair of primers (SEQ ID NOS: 1 and 2) is designed by making predictions based on the mouse FcRγ-mRNA sequence of GenBank NM010185 (SEQ ID NO: 17). The primer represented by SEQ ID NO: 1 is so designed that a sequence from positions 178 to 195 in the FcRγ-mRNA sequence shown as SEQ ID NO: 17 is recognized. The primer represented by SEQ ID NO: 2 is so designed that a sequence from positions 284 to 303 in the same FcRγ-mRNA sequence is recognized.
For details of procedures for the method of examining FcRγ expression in animal brain tissues or cells by a gene amplification method, see Examples described later.
Hereinbelow, one example of procedures for examining FcRγ expression in animal brain tissues or cells by Western blotting is described.
Protein sample solutions are prepared from animal brain tissues or cells and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The resultant SDS-PAGE gel is transferred onto a membrane and blocked. After washing, the membrane is reacted with a primary antibody and an enzyme-labeled secondary antibody in this order. After appropriate washing, a substrate for the enzyme is added for color formation.
For details of procedures for the method of examining FcRγ expression in animal brain tissues or cells by Western blotting, see Examples described later and C. Seiwa, I. Sugiyama, T. Yagi, T. Iguchi, H. Asou, Neurosci. Res. 37, 21 (2000).
As the primary and the secondary antibody useful in the above-described immunohistochemical or immunocytochemical analysis and Western blotting, those listed in item 35) of the Notes provided later may be given.
The oligodendroglias or precursor cells thereof where FcRγ expression has been confirmed by the above-described method are judged to be capable of myelinogenesis. This judgment is applicable to the diagnosis of the degree of progress or the prognosis of treatment of diseases such as demyelinating diseases, dysmyelinating diseases and myelinoclasis. Briefly, if FcRγ-positive cells (oligodendroglias or precursor cells thereof) remain, the fact suggests that there is a possibility of myelin regeneration. Therefore, it is believed that stimulation treatment using a substance capable of activating FcRγ (e.g. anti-FcRγ antibody) would be effective. Or, when demyelination is so severe that repeated regeneration is hardly effective against continuous demyelination, it is considered that oligodendroglias or precursor cells thereof might possibly die out. Under such conditions, it is expected that FcRγ-positive cells have disappeared and that a stimulation treatment would not produce much effect. The more the FcRγ-positive cells are, the better the prognosis will be. However, those cases where mutations or abnormalities are found in the FcRγ-mediated signaling pathway are excluded as exceptions. In such cases, it is reasonable to consider that prognosis is bad because the pathway is not expected to function even if FcRγ-positive cells are present.
The present invention provides a method of examining FcRγ expression in animal brain tissues or cells by any of the following methods: immunohistochemical or immunocytochemical analysis, a gene amplification method, or Western blotting. These methods of the present invention are useful in detecting myelinogenetic oligodendroglias or precursor cells thereof. The oligodendroglias or precursor cells thereof where FcRγ expression has been confirmed are judged to be capable of myelinogenesis. This judgment is applicable to the diagnosis of the degree of progress or the prognosis of treatment of diseases such as demyelinating diseases, dysmyelinating diseases and myelinoclasis.
When FcRγ expression in animal brain tissues or cells is examined by immunohistochemical or immunocytochemical analysis or Western blotting, it is preferable to further examine the expression of at least one marker selected from the group consisting of PDGFαR, MAG, A2B5, 04, 01, MBP, PLP, DM-20, CNPase, MOG, NG2 and AN2 in oligodendroglias or precursor cells thereof.
Further, the present invention provides immunohistological or cell-staining reagent kits and Western blotting reagent kits, both types of kits comprising an anti-FcRγ antibody. These reagent kits may further comprise at least one antibody selected from the group consisting of anti-PDGFαR antibody, anti-MAG antibody, A2B5 antibody, 04 antibody, 01 antibody, anti-MBP antibody, anti-PLP antibody, anti-DM-20 antibody, anti-CNPase antibody, anti-MOG antibody, NG2 antibody and AN2 antibody. These antibodies may be prepared by the methods described in the review article of N. Baumann & D. Pham-Dinh, Physiol. Rev., 81:871-927 (2001).
Anti-FcRγ antibody, anti-PDGFαR antibody, anti-MAG antibody, A2B5 antibody, 04 antibody, 01 antibody, anti-MBP antibody, anti-PLP antibody, anti-DM-20 antibody, anti-CNPase antibody, anti-MOG antibody, NG2 antibody and AN2 antibody may be labeled with a fluorescent dye, enzyme, heavy metal or the like (direct method). Alternatively, instead of labeling these antibodies (primary antibodies), secondary antibodies specific thereto may be labeled with a fluorescent dye, enzyme, heavy metal or the like (indirect method).
The immunohistological or cell-staining reagent kits of the invention may further comprise color formers (e.g. DAB), aqueous hydrogen peroxide (to be used for eliminating endogenous peroxidase and activating DAB), buffers (e.g. PBS (phosphate buffered saline), counter-staining dyes (e.g. methyl green), cover slips (plastic covers to be put on sections for reducing antibody consumption) and so on.
The Western blotting reagent kits of the invention may further comprise transferring buffers, blocking reagents, washing solutions, and so on.
The present invention also provides gene amplification reagent kits comprising at least a pair of primers capable of specifically amplifying the mRNA of FcRγ. The primers may be so designed that a specific region of the nucleotide sequence of FcRγ-mRNA of the relevant animal species (GenBank M33195 for human (SEQ ID NO: 15) and GenBank NM 010185 for mouse (SEQ ID NO: 17)) can be amplified specifically.
The primers are 15-30 nucleotides, preferably 20-25 nucleotides, in length. Specific examples of primers include a primer having the nucleotide sequence as shown in SEQ ID NO: 1 and a primer having the nucleotide sequence as shown in SEQ ID NO: 2.
The gene amplification reagent kits of the invention may further comprise reverse transcriptases, DNA polymerases, RNase-free water, buffers, control mRNAs, control primer sets, dNTP mix, and so on.
By using the reagent kit of the invention, it is possible to examine FcRγ expression in animal brain tissues or cells.
The reagent kit of the invention may contain written instructions and may be wrapped. The instructions may teach how to use the kit and how to analyze results.
Hereinbelow, the present invention will be described specifically with reference to the following Examples. These Examples are provided for the purpose of illustration, and should not be construed as limiting the scope of the invention.
In the immune system, the activation of Src family tyrosine kinase signaling involves the interaction between molecules with cytoplasmic domains containing immunoreceptor tyrosine-based activation motifs (ITAM) (24; reviewed in 25). Src family tyrosine kinase phosphorylates the tyrosine residues within the ITAM, triggering intracellular signaling (26). Fyn, a member of the Src family, is critical in the initial events of myelination (9). This evidence suggests that an ITAM-bearing molecule, expressed in oligodendroglia during the initial events of myelination, may be involved in myelinogenesis. FcRγ, containing a cytoplasmic ITAM shown to be phosphorylated by Fyn (27), was examined as a candidate but this immunological molecule has not been previously shown to occur within oligodendroglial cells.
The following is the first report detailing the existence of FcRγ within the oligodenoroglial lineage. Total RNA was extracted from cultured oligodendroglial precursor cells (OPC) (28,29) isolated from the mouse CNS but MAG had not yet been expressed at that stage (30). Significant expression of FcRγ mRNA, however, was detected by RT-PCR (31) at levels comparable to mast cells (
The inventors have also detected the expression of Fcγ in A2B5-positive oligodendroglial precursor cells (33;
The expression of FcRγ within OPC and immature oligodendroglia, considering that this molecule couples to Fyn via ITAM (27), suggests that Fcγ may be involved in myelinogenesis in oligodendroglia.
In order to examine Fcγ expression within the CNS in vivo, the inventors performed immunohistochemistry on the brains of neonatal mice at variable ages (36,38,39). FcRγ-positive cells were detectable at birth (P0) within the subventricular zone (germinal matrix areas adjacent to the cerebral ventricles; SVZ) (
In order to confirm that those FcRγ-expressing cells are derived from the oligodendroglial lineage in vivo, the inventors labeled FcRγ-expressing cells with several antibodies and determined their identity (42). At P0, the FcRγ-expressing cells were distinct from both GFAP-positive cells (cells of the astroglial lineage) and Ox-42-positive cells (43; cells of the microglial lineage) (
In the adult CNS, FcRγ-positive cells were observed within a restricted area of the white matter (
FcRγ is expressed both in vitro and in vivo prior to MAG expression in the oligodendroglial lineage. However, under microscopic examination, the expression of MAG and FcRγ did not co-localize completely within oligodendroglia at the pre-myelinating stage in vivo. MAG stained the edges of cellular processes strongly; FcRγ stained the cell membrane of the cellular body (
Based on the assumption that FcRγ initially triggers myelinogenesis (i.e. triggers the Fyn-MBP cascade; 6), the inventors expected the expression of FcRγ to be similar to that of both Fyn and MBP. By examining serial sections, the inventors observed similar staining patterns for FcRγ, Fyn, and MBP within enlarging myelin sheaths at P7 (
In order to examine the effect of FcRγ stimulation on the expression of MBP, the inventors artificially generated FcRγ signaling in OPC (28,29) by administration of an anti-FcRγ rabbit polyclonal antibody (47). Following 24 hours of the stimulation, cultured OPC with few processes demonstrated remarkable morphological changes to acquire well-developed processes (
The stimulation of MBP expression predominantly up-regulated the exon 2-containing isoforms (
The inventors observed similar up-regulation of exon 2-containing MBP isoforms and dramatic morphological differentiations following administration of the 2b isoform of mouse immunoglobulin G (IgG2b) in place of the anti-FcRγ antibody (
The inventors have further stimulated OPC derived from FcRγ-deficient mice (58). Similarly administered IgG2b, however, failed to differentiate those OPC in contrast to the changes observed in wild type OPC (
FcRγ and Fyn may be considered to co-operate in oligodendroglia to stimulate MBP expression. Phosphorylation of the FcRγ ITAM requires interactions with Src family tyrosine kinases (26). Among the Src family members, only five are expressed within the CNS (51). Of those five members, only Src, Fyn, and Lyn are expressed within oligodendroglia (52). Fyn, however, is the only Src family member essential for myelinogenesis (52). Fyn has been shown to be responsible for the ITAM phosphorylation of FcRγ (27). Therefore, the activation of FcRγ within oligodendroglia, which resulted in the up-regulation of MBP expression (
In order to further examine the FcRγ-Fyn-MBP cascade in vivo, the inventors analyzed mutant mice deficient in either FcRγ, Fyn, or MBP (39). The most intense period of oligodendroglial proliferation occurs during the first 9 days after birth (53); oligodenroglias predominantly express exon 2-containing MBP at this age (8). The in vitro stimulation results of the inventors suggest that analysis of the myelination program at this age may reveal defects in myelinogenesis. The inventors therefore analyzed the brains of mice 10 days after birth and examined the initial stages of myelination. Immunohistochemical analysis of the white matter of FcRγ-, Fyn-, and MBP-deficient mice (36,38,39) revealed a similar delay in myelinogenesis in the three mutants, as compared with age-matched wild type mice. The restricted expression of MBP and/or MAG observed in all mutants (
Quantitation of the Western blotting results (54) revealed that the total amount of MBP in the adult CNS had decreased to 40.3% and 60.8% of the wild-type level in Fyn-deficient and FcRγ-deficient animals, respectively. The expression of exon 2-containing MBP (the sum for 21.5 kD and 17.0 kD) was found to be more severely affected than the expression of the two other isoforms; levels of these isoforms had decreased to 31.9% and 40.0% of the wild-type level in Fyn-deficient and FcRγ-deficient mice, respectively. The similar patterns observed in FcRγ- and Fyn-deficient mice support the present inventors' theory that these molecules are involved in the signaling cascade that regulates myelinogenesis. The predominant decrease in exon 2-containing MBP levels in both mutants supports the in vitro results of the inventors, also suggesting these isoforms in this process (
Histological comparison of MAG expression at P10 in the mutants revealed that the distribution of MAG-positive immature oligodendroglia (6) is more reduced in MBP-deficient mice than in FcRγ-deficient mice (FIG. 5A-e, f, g) Fyn-deficient mice displayed an intermediate phenotype. In the three mutants, the number of MAG-positive immature oligodendroglias that had migrated into the cerebral cortex from the corpus callosum by P10 (55) was approximately 44%, 25%, and 12% of the wild type level for the FcRγ-, Fyn-, and MBP-deficient mice, respectively (
In order to examine myelination in the absence of FcRγ, the inventors analyzed FcRγ-deficient mice at several ages. The disturbance in myelination observed at P10 (
In order to determine whether the defect observed at P10 (
These observations show that FcRγ is essential in myelinogenesis, not in gliogenesis and support the inventors' theory that FcRγ triggers myelinogenesis both in vitro and in vivo.
FcRγ is common to several immunoreceptor (21). Elucidation of the receptors involved in FcRγ activation in oligodendroglia is essential to understanding the molecular mechanism of myelinogenesis. FcRγ-deficient mice have been shown to be unable to express the alpha chain (i.e. ligand binding subunits) of both the IgE Fc receptor (FcεRI) and the IgG Fc receptors (FcγRI/III) on the cell surface (58). The disturbed myelinogenesis in the mutant may have resulted from the lack of both FcRγ and those alpha chains. RT-PCR analysis of immature oligodendroglia in wild type mice (31) could not detect the alpha chain of FcεRI. Alternatively, the alpha chains of FcγRI and FcγRIII were easily detectable (
CD45, a protein tyrosine phosphatase, regulates FcRγ signaling (reviewed in 61). CD45 dephosphorylates the inhibitory site of Src family tyrosine kinases, including Fyn. Without CD45, Fyn remains inactive due to phosphorylation of the inhibitory site (61); signaling via FcRγ would not proceed in the absence of this signaling molecule. CD45, also known as leukocyte common antigen, is expressed specifically within the cells of immune system. The expression of this molecule within oligodenroglia has yet to be reported. The inventors have immunohistochemically double-stained the brains of mice at 7 days after birth (P7), at 10 days (P10) or adult mice (36,38,42) and discovered cells that co-expressed CD45 and MAG (
Purpose) It is known in immunology that FcRγ and Fyn co-operate with each other. If they actually co-operate in myelinogenesis in the brain, FcRγ-Fyn double-deficient animals would exhibit severer symptoms. Based on this prediction, the inventors performed experiments. There are two interpretations for double deficiency. First, contemplate a cascade of A→B→C. If this is truly one cascade, the result (e.g. decrease in C) would be the same whether A or B or both A and B are knocked out. In actual organisms, however, it is considered that a number of cascades are mixed, or if one mechanism is knocked out, it will be compensated by another mechanism. Based on this assumption, deficiency of B would lead to severer symptoms than deficiency of A (since B is closer to C, compensation mechanisms will have less chance to work); or deficiency of both A and B would lead to the severest symptoms.
Methods) FcRγ-deficient mice (39, 58) were mated with Fyn-deficient mice (39, 46) to prepare FcRγ+/−Fyn+/− mice (F1 generation). Mating among the resultant F1 mice should yield FcRγ−/−Fyn−/− double-deficient mice (F2 generation) -at a probability of 1/16. However, the probability was too low to yield the double deficiency. Therefore, FcRγ−/−Fyn+/− and FcRγ+/−Fyn−/− mice were selected from the F2 mice (genotypes were confirmed by extracting DNA from the tail vein and performing PCR), and mated with each other. The resulting F3 generation is to have FcRγ−/−Fyn−/− mice at a probability of 1/4. Since one mouse produces about 7 offspring mice per delivery, it is expected that same members of a litter would be the double deficiency mice. Actually, probably due to serious immunodeficiency (it is expected that FcRγ-Fyn double-knockout would destroy the immune system, causing embryonic or neonatal death) or perhaps due to cerebral malformation, the double-deficient mouse could be obtained at the chance of once in several deliveries, though the probability was considerably low. At present, the inventors are trying to obtain double-deficient F4 generation mice at a probability of 1/1 by mating F3 double-deficient mice among themselves but the efficiency is rather low. F3 mice were used in the experiment. Briefly, the resultant double-deficient mice at P10 were fixed with acid-alcohol, embedded in paraffin and then sliced into sections 10 μm thick. The expressions of MBP and MAG were examined by immunohistochemical techniques. Nissl staining for endoplasmic reticula was also performed to examine the structure of the brain roughly. Similar experiments were also conducted on mice at 1.5 months after birth (young adult).
Results) The results of comparison of wild-type (Wt), Fyn−/−, FcRγ−/− and FcRγ−/−Fyn−/− at P10 showed that the expression levels of both MBP and MAG decreased in the following order: Wt >FcRγ−/− >Fyn−/− >FcRγ−/− Fyn−/− (
Conclusion) It was confirmed that FcRγ and Fyn also co-operate with each other in myelinogenesis in the brain.
Purpose) CD45 is expressed in oligodendroglia, as confirmed in Example 6, and CD45 is known in immunology as an activator of Fyn. Therefore, the inventors speculated that CD45 might play some role in myelinogenesis in oligodendroglia. In addition, since genetic mutations of CD45 were found in multiple sclerosis (MS), the inventors also speculated that abnormalities in oligodendroglia might be in part the etiology of MS. (However, the CD45 abnormalities found in MS so far are not mutations that eliminate CD45 completely but they are mutations that disturb the expression pattern of some isoforms of CD45 or reduce the total amount of expression of CD45.) With respect to the CD45 mutations in MS patients, negative papers have later been submitted, and the issue has not yet been settled. In order to leave no question about this issue, the present inventors attempted to examine whether the myelinogenesis in oligodendroglia would be inhibited in CD45-deficient mice.
Methods) Mice lacking exon 6 of CD45 (thus expressing almost no CD45) were a kind gift from Dr. Kenji Kishihara, Kyushu University. This mouse was originally described in the following article.
Brains from these mice were fixed at P10, sliced and had MBP and MAG stained by immunohistochemical techniques to examine expression levels of myelin. Further, brains were ground and subjected to Western blotting to examine expression levels of MBP. Brains from Fyn-deficient mice (P10) were also fixed and sliced in the same manner, followed by immunohistochemical staining for MAG so as to examine whether they would exhibit reduced myelin expression as in CD45-deficient mice (if CD45 is an activator of Fyn, similar reduction is expected). Further, oligodendroglial precursor cells (OPC) from the CD45-deficient mice were cultured and stimulated with an anti-FcRγ antibody or IgG so as to observe any responses (morphological changes and increase in MBP expression). In addition, in order to demonstrate that CD45 was actually expressed in oligodendroglia, staining of the cultured OPC and histological staining (double-staining with MAG, a marker for oligodenroglia) were performed. In order to confirm that the antibody to CD45 actually reacted with CD45, causing no cross-reaction with something else, CD45 staining was performed on the CD45-deficient mice.
Results) The CD45-deficient mice at P10 showed apparent decrease in myelinogenesis (
Conclusion) Being expressed in OPC and immature oligodendroglia, CD45 was confirmed to be necessary at the stage when OPC is differentiated into myelin-forming oligodendroglia.
Purpose) In Example 3, it was shown that FcRγ is the trigger of myelinogenesis but in the actual adult body, FcRγ is one of the signaling molecules of immunoglobulin Fc receptors such as FcγRI or FcγRIII. Therapeutically, FcRγ may be stimulated directly, but under physiological environments, the intended mechanism is expected to work as follows: IgG binds to FcγRI or FcγRIII, thereby stimulating FcRγ. IgG is supplied from the mother's body via the placenta, or contained in mother's milk. Since the blood-brain barrier (BBB) between the brain and vessels is immature at fetal to infantile stages, IgG is capable of entering the brain. Conversely, an adult has BBB and IgG does not enter the brain, probably preventing excessive myelination. As a matter of fact, the BBB is destroyed in MS patients, probably reflecting demyelination. The destruction may have occurred in order to introduce IgG into the brain and regenerate myelin. In order to say such stories are valid, it is necessary to prove that IgG actually has such a function. Certainly, it has been confirmed in immunology that FcRγ co-operates with FcγRI/RIII and that both FcγRI and FcγRIII are detected in oligodendroglia. Thus, coupling to IgG is expected in all likelihood. However, there is a possibility that some other molecule couples to FcγRI/RIII in the brain. In order to prove the necessity of IgG, the inventors analyzed mice that had been genetically engineered to lose the IgG producing ability. Fyn and FcRγ of the mice are normal in function and expression.
Methods) IgG-deficient μMT mice were purchased from Jackson Laboratory (it appears that the mice are also IgM-deficient; this, however, would not effect the experiment since IgM does not pass through the placenta nor is it little contained in mother's milk.) The original mouse is described in the following reference.
These mice were immunohistochemically stained for MAG at P7, P10 and P50, and compared with wild-type (Wt) mice.
Results) As expected, myelin decreased coed to Wt at any of P7, P10 and P50 (
Conclusion) The importance of FcRγ for in vivo myelinogenesis in oligodendroglia is as described above. The experiment revealed that IgG is a physiological trigger of the myelinogenesis.
A model summarizing the mechanism of myelinogenesis proposed by the present inventors is shown in
IgG2b promotes the expression of MBP and morphological differentiations of OPC in vitro (
Type IIB FcγR (FcγRIIB) is a unique inhibitory Fc receptor that interacts with IgG (reviewed in 67). This receptor may negatively regulate the FcRγ-Fyn-MBP cascade, resulting in diminished ability of polyclonal IVIg to promote myelinogenesis. The inventors confirmed the expression of FcγRIIB in cultured oligodenroglia by both RT-PCR and flow cytometry (data not shown). On the other hand, the inventors cannot exclude the possibility that the putative ligand for FcγRs is not an immunoglobulin. However, as demonstrated by the administration of the antibody to FcRγ (
The expression of exon 2-contaning MBP was detected within MS lesions in which a certain degree of remyelination had proceeded (68). The activation of FcRγ resulted in an up-regulation of exon 2-containing MBP levels, and dramatic morphological differentiation (
In conclusion, the observations of the inventors show that the CNS myelinogenesis is triggered by FcRγ and this uncovers a new connection between the brain and the immune system.
31) Total RNA was extracted from cultured OPC with Trizol reagent (GIBCO BRL). RNA was resuspended in 20 μl TE. For each sample, 1 μl of RNA was reverse-transcribed, using a first strand cDNA synthesis kit (Boehringer Mannheim) to achieve a final volume of 20 μl. For standardization, mouse β-actin was amplified in parallel. An annealing temperature of 52 degrees was used for the amplification of mouse FcRγ, CD3ζ, FcεRIα, FcγRIα, and β-actin. An annealing temperature of 53 degrees was used for the amplification of FcγRIIIα and FcγRIIb. cDNA samples were amplified for 35 cycles (30 sec at 94 degrees, 30 sec for annealing, and 30 sec at 72 degrees). The following primers were used:
All publications, patents and patent applications cited herein are incorporated herein by reference in their entity.
The pharmaceutical compositions and agents of the present invention are capable of stimulating the differentiation of oligodendroglial precursor cells. The pharmaceutical compositions and agents of the present invention are also capable of increasing the expression of Fyn tyrosine kinase. Further, the pharmaceutical compositions and agents of the present invention are capable of stimulating the expression of myelin basic protein.
The pharmaceutical compositions and agents of the present invention are useful in stimulating myelinogenesis.
The pharmaceutical compositions and agents of the present invention are useful in preventing and/or treating at least one disease selected from the group consisting of demyelinating diseases, dysmyelinating diseases and myelinoclasis.
In the future, therapeutic treatments will be practiced in which neural stem cells, ES cells or subject-derived oligodendroglia is transplanted and regenerated. When such treatments have become possible, the technique of the present invention will be applicable as a booster for myelinogenesis.
According to the present invention, a method of using FcRγ as a marker for oligodendroglias or precursor cells thereof is provided. FcRγ-positive oligodendroglias can be regarded as cells having the potential of myelinogenesis.
SEQ ID NO: 1 shows the nucleotide sequence of FcRγ sense primer.
SEQ ID NO: 2 shows the nucleotide sequence of FcRγ antisense primer.
SEQ ID NO: 3 shows the nucleotide sequence of CD3 ζsense primer.
SEQ ID NO: 4 shows, the nucleotide sequence of CD3ζ(antisense primer.
SEQ ID NO: 5 shows the nucleotide sequence of FcεRIα sense primer.
SEQ ID NO: 6 shows the nucleotide sequence of FcεRIα antisense primer.
SEQ ID NO: 7 shows the nucleotide sequence of FcγRIα sense primer.
SEQ ID NO: 8 shows the nucleotide sequence of FcγRIα antisense primer.
SEQ ID NO: 9 shows the nucleotide sequence of FcγRIIIα sense primer.
SEQ ID NO: 10 shows the nucleotide sequence of FcγRIIIα antisense primer.
SEQ ID NO: 11 shows the nucleotide sequence of FcγRIIB sense primer.
SEQ ID NO: 12 shows the nucleotide sequence of FcγRIIB antisense primer.
SEQ ID NO: 13 shows the nucleotide sequence of β-actin sense primer.
SEQ ID NO: 14 shows the nucleotide sequence of β-actin antisense primer.
SEQ ID NO: 15 shows the nucleotide sequence of human FcRγ-mRNA.
SEQ ID NO: 16 shows the amino acid sequence encoded by the nucleotide sequence of human FcRγ-mRNA as shown in SEQ ID NO: 15.
SEQ ID NO: 17 shows the nucleotide sequence of mouse FcRγ-mRNA.
SEQ ID NO: 18 shows the amino acid sequence encoded by the nucleotide sequence of mouse FcRγ-mRNA as shown in SEQ ID NO: 17.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001-229553 | Jul 2001 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP02/07378 | 7/22/2002 | WO |
