WO2005067405A2

WO2005067405A2 - Method for screening responsiveness to drugs effective in treatment or prevention of vitiligo and method for prognosis of vitiligo

Info

Publication number: WO2005067405A2
Application number: PCT/KR2005/000186
Authority: WO
Inventors: Ai Young Lee; Nan Hyung Kim
Original assignee: School Foundation Eulji
Priority date: 2004-01-20
Filing date: 2005-01-20
Publication date: 2005-07-28
Also published as: KR100672853B1; KR20050076690A; WO2005067405A3

Abstract

The present invention relates to methods and kits for screening responsiveness to drugs effective in treatment or prevention of vitiligo, methods and kits for screening active ingredients effective in treatment or prevention of vitiligo, and methods and kits for prognosis of vitiligo using eukaryotic translation initiation factor 4A1 (eIF4A1) gene, ribosomal protein L13 (L13) gene and mediator of RNA polymerase to transcription (MRT) gene.

Description

METHOD FOR SCREENING RESPONSIVENESS TO DRUGS EFFECTIVE IN TREATMENT OR PREVENTION OF VITILIGO AND METHOD FOR PROGNOSIS OF VITILIGO

FIELD OF THE INVENTION The present invention relates to methods and kits for screening responsiveness to drugs effective in treatment or prevention of vitiligo, methods and kits for screening active ingredients effective in treatment or prevention of vitiligo, and methods and kits for prognosis of vitiligo.

BACKGROUD OF THE INVENTION Retinoids have an effect on cell differentiation and proliferation. Retinoids are metabolic or synthetic derivatives of vitamin A, and vitamin A activity is fulfilled by three major compounds, retinol, retinyl aldehyde and retinoic acid (RA) . Combined treatment with topical corticosteroids and topical all- trans-retinoic acid (tretinoin; ATRA) to prevent corticosteroid- induced skin atrophy (1-3) has shown 'more rapid repigmentation in certain patients suffering from a generalized type of vitiligo. It is unclear how ATRA produced a better outcome in certain patients and why the effects of RA were inconsistent. The effects of RA on keratinocytes and fibroblasts have been studied, whereas few and conflicting data are available on the effects of RA on normal human melanocytes (4) . The role of RA in regulating constitutive melanogenesis of normal human melanocytes is inconsistent: RA had no effects on melanogenesis (5,6), inhibited melanogenesis (7,8), or increased pigmentation (9). Although the results showing increased pigmentation were obtained in studies with the micropig or mouse, similar results were confirmed in human volunteers (9) . The variability of the response to RA treatment, inherent in each donor, may require a large sampling from cultures of different sources to define the action of RA on melanocytes (7) . The reported effects of RA on melanocyte numbers are also contradictory. RA inhibited the proliferation of normal human melanocytes grown in serum-free culture medium (5) , whereas it increased the numbers of both constitutive and ultraviolet B-activated mouse melanocytes (9) . The effects of RA on melanogenesis may either be direct (5,7) or through cell-to-cell interaction between melanocytes, keratinocytes and fibroblasts (6,8) . The effects of RA on keratinocytes are relatively consistent. ATRA and/or 9-cis RA caused proliferation in normal human keratinocytes cultured in a submerged culture system (10) . Although the exact molecular mechanisms by which retinoids regulate keratinocyte proliferation are not well understood, heparin-binding EGF-like growth factor (HB-EGF) , a member of the EGF family of growth factors, has been shown to mediate, at least in part, the RA-induced keratinocyte proliferation and epidermal hyperplasia (11) .

DETAILED DESCRIPTION OF THIS INVENTION The present inventors have made intensive study to search biological ingredients which can be used as a marker for responsiveness to vitiligo drugs. As a result, the inventors have found that 3 types of genes are differentially expressed depending on the level of responsiveness. Accordingly, it is an object of this invention to provide a method for screening responsiveness to drugs effective in treatment or prevention of vitiligo. It is another object of this invention to provide a method for screening responsiveness to all-trans-retinoic acid (ATRA) used for treatment of vitiligo. It is st±ll another object of this invention to provide a kit for screening responsiveness to drugs effective in treatment or prevention of vitiligo. It is another object of this invention to provide a biological marker for responsiveness to drugs effective in treatment or prevention of vitiligo. It is another object of this invention to provide a method for screening active ingredients effective in treatment or prevention of vitiligo. It is another object of this invention to provide a kit for screening active ingredients effective in treatment or prevention of vitiligo. It is another object of this invention to provide a method for prognosis of vitiligo. It is another object of this invention to provide a method for prognosis of vitiligo. It is another object of this invention to provide a kit for prognosis of vitiligo. Other objects .and advantages of the present invention will become apparent from examples to follow, appended claims and drawings .

In one aspect of this invention, there is provided a method for screening responsiveness of a vitiligo patient to drugs effective in treatment or prevention of vitiligo, which comprises the steps off : (a) isolating total RNAs from epidermis obtained from the vit±ligo patient treated with the drugs,- (b) synthesizing cDNA with said isolated total RNAs; (c) conducting PCR with at least one type of primer set selected from the group consisting of (i) primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) primer set hybridizing with ribosomal protein L13 (L13) gene and (iii) primer set hybridizing with mediator of RNA polymerase to transcription (MRT) gene, and said cDNA as a template; (d) Obtaining electrophoresis bands of said PCR product by electrophoresis; and (e) deciding that said patient has responsiveness on said drug, provided that the electrophoresis band pattern is substantially identical to that of positive control prepared from normal epidermis. The present inventors found that eIF4Al, L13 and MRT are closely correlated with body responsiveness to drugs effective in treatment and prevention of vitiligo. Among the proteins, the biological functions of eIF4Al are well studied. However, the functions of L13 and MRT are still unknown. 13 is considered to play a role in the elongation stage of mRNA translation as a 60S ribosomal subunit protein (12) . eIF4A is one of the translation factors involved in binding mRNA to the 43S preinitiation complex (13,14). mRNA translation represents an important control point in gene expression. Binding to the cap structure at the extreme 5 end of all cytoplasmic eukaryotic mRNAs may constitute a key regulatory step in mRNA translation. eIF4A is one of the proteins associated with the cap (13) as a subunit of the heterotrimeric translation initiation factor, eIF4F (15-17) . eIF4A, which possesses ATP-dependent RNA helicase activity, also unwinds the secondary structure of mRNAs to facilitate translation (18,19) . eIF4A is more abundant than the other cap-binding proteins and can exist both as part of the eIF4F complex and as free eIF4A. Unlike the other components of the eIF4F complex, eIF4A activity is not regulated by phosphorylation in mammalian cells (20) , but is controlled primarily by modulating mRNA levels (21,22) . eIF4A is encoded by two genes, eIF4Al and eIF4A2. eIF4Al mRNA is synthesized and translated most efficiently in growing cells, whereas eIF4A2 mRNA synthesis and translation is associated preferentially with the growth-arrested state (23) . eIF4Al, L13 and MRT performing these biological functions are closely correlated with body responsiveness to drugs effective in treatment and prevention of vitiligo. The present invention is applicable to vitiligo. The responsiveness to drugs used for treatment or prevention of skin diseases which requires the administration of corticosteroids is investigated in the present invention. Preferably, drugs are selected from the group consisting of all- trans-retinoic acid (ATRA) , vitamin C, trichloroacetic acid, and calcipotriol . Most preferably, the drug is ATRA. In the present method, the step for isolating total RNAs can be carried out in accordance with the conventional procedures for the isolation of total RNAs known to one of skill in the art (Sambrook, J. , et al . , Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) ; Tesniere, C, et al . , Plant Mol . Biol . Rep. , 9:242(1991); Ausubel, F.M. et al . , Current Protocols in Molecular Biology, John illey & Sons (1987); and Chomczynski, P. et al . , Anal. Biochem. , 167:156(1987)). For example, total RNAs are isolated conveniently from cells using Trizol. The second step of the present invention is the step to synthesize cDNA with total RNAs isolated. Since total RNAs of the present invention are isolated from human epidermis, mRNAs have poly-A tails in their end position. This sequence feature enables us to synthesize cDNA with oligo dT primers and reverse trancriptase conveniently (PNAS USA, 85:8998(1988); Libert F, et al . , Science, 244:569(1989); and Sambrook, J. et al . , Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001) ) . Primer set used in PCR step of the present invention is primer set hybridizing with eIF4Al gene, primer set hybridizing with L13 gene, primer set hybridizing with MRT gene or combination thereof. Most preferably, primes set used is primer set hybridizing with eIF4Al gene which has closest correlation with responsiveness. The term "primer" as used herein refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, Such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template . A primer sequence need not reflect the exact sequence of , the template but must be sufficiently complementary to hybridize with a template. Therefore, primer set in the present invention need not to have perfectly complement sequence of such a template as eIF4Al gene, L13 gene or MRT gene, but must be sufficiently complementary to hybridize with these templates. For example, primer set hybridizing with eIF4Al gene is primer set hybridizing with Sequence list no.l, primer set hybridizing with L13 gene is primer set hybridizing with Sequence list no.2 and primer set hybridizing with MRT gene is primer set hybridizing with Sequence list no. 3. The term "primer set" as used herein refers to the primer pairs consisting of forward primer and reverse primer. one skilled in the art may design these primers easily considering template sequence, and For example, may use such a software program for the primer design as PRIMER 3 program. In PCR, hybridizations (annealing) are usually performed under stringent conditions that allow for specific binding between an oligonucleotide and a target nucleic acid. A stringent condition is any suitable buffer concentration and temperature that allow specific hybridization of the oligonucleotide to a complementary nucleic acid (M. Kanehisa, Nucleic Acids Res., 12:203 (1984)). Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5^°C lower than the thermal melting point (T_ra) for the specific sequence In the present invention, PCR can be conducted in accordance with the conventional procedures for PCR. US 4,683,195, US4, 683,202 and US 4,800,159 disclose the detailed procedures for PCR. For example, PCR may be conducted in the suitable buffer comprising Tag polymerase, dNTPs, cDNA template, primer set and MgC12 under programmed temperatures. The resultants produced by PCR amplification are separated by electrophoresis. The intensity of occurred band is evaluated by naked eyes or quantified by a densitometer. If the electrophoresis band pattern is substantially identical to that of a positive control prepared from normal epidermis, it is concluded that the patient has responsiveness on said drug. The positive control is prepared from epidermis isolated from humans who do not suffer from skin diseases. Prepared positive control is processed according to the steps (a) -(d), thereby, the electrophoresis band pattern of it is obtained. The term "electrophoresis band pattern is substantially identical" means that it is concluded that the intensity of the bands prepared from sample is substantially identical to that of the bands prepared normal epidermis when evaluated with naked-eyes, or that the quantified intensity of the bands prepared from sample is substantially identical to that of the bands prepared normal epidermis when quantified with a densitometer. In the quantification with a densitometer, it is preferable to compare the relative ratio of to an internal standard. Internal standards include GAPDH (glyceraldehyder 3- phosphate dehydrogenase) . In another aspect of this invention, there is provided a method for screening responsiveness of a vitiligo patient to all- trans-retinoic acid (ATRA) used for treatment of vitiligo, which comprises the steps of: (a) isolating total RNAs from epidermis obtained from the vitiligo patient treated with ATRA; (b) synthesizing cDNA with said isolated total RNAs; (c) conducting PCR with primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene and said cDNA as a template; (d) Obtaining electrophoresis bands of said PCR product by electrophoresis; and (e) deciding that said patient has responsiveness on said drug, provided that the electrophoresis band pattern is substantially identical to that of positive control prepared from normal epidermis.

In still another aspect of this invention, there is provided a kit for screening responsiveness of a vitiligo patient to drugs effective in treatment or prevention of vitiligo, which comprises at least one type of primer set selected from the group consisting of (i) primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) primer set hybridizing with ribosomal protein L13 (L13) gene, and (iii) primer set hybridizing with mediator of RNA polymerase to transcription (MRT) gene . The kit in the present invention is used to perform the above-mentioned methods of the present invention. Accordingly, the common descriptions between the method of the present invention and the kit of the present invention are omitted in order to avoid undue redundancy leading to the complexity of this specification. The kit of the present invention may further include reagents for the amplification of DNA: for example, buffer, DNA polymerase, DNA polymerase cofactor, and dNTPs . One skilled in the art can easily determine the optimum amount of the reagents . In further another aspect of this invention, there is provided a method for screening responsiveness of a vitiligo patient to all-trans-retinoic acid (ATRA) used for treatment of vitiligo: (a) preparing a cell homogenate containing total proteins from epidermis obtained from the vitiligo patient treated with ATRA; (b) incubating said cell homogenate with anti-eIF4Al antibody; (c) detecting the signal of antigen-antibody immune response between the proteins in the cell homogenate and anti- eIF4Al antibody; and (d) deciding that said patient has responsiveness on said ATRA, provided that the signal in the step

(c) is substantially more intense than that of negative control prepared from epidermis obtained from a vitiligo patient not treated with ATRA. In still further another aspect of this invention, there is provided a kit for screening responsiveness of a vitiligo patient to all-trans-retinoic acid (ATRA) , which comprises anti-eIF4Al antibody. As described above, the method of the present invention may be carried out on the basis of proteins as well as mRNAs. Where performing the method of the present invention on the basis of protein, basically antigen-antibody immune response is involved. In the present invention, the preparation of a cell homogenate containing total proteins from epidermis can be conducted in accordance with the conventional procedures for it . For example, epidermis is immersed in liquid nitrogen and powdered. The resultant powder is dissolved in buffer containing protease to prepare a cell homogenate. The present method can be carried out quantitatively or qualitatively according to conventional immunoassay procedures based on antigen-antibody reaction (e.g., radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot assay, Western blot assay, inhibition or competition assay and sandwich assay) (Enzyme Immunoassay, E . T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Enzyme-linked immunosorbent assay (ELISA) , in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984). For example, according to the radioimmunoassay method, the radioisotope (e.g., P³² and S^3S) labeled anti-eIF4Al antibody may be used for the qualitative or quantitative detection of eIF4Al protein in a sample by the determination of immune complex formed with anti-eIF4Al antibody. In addition, according to the ELISA method, the example of the present method may comprise the steps of: (i) coating wells in a plate with anti-eIF4Al antibodies; (ii) adding a sample to the wells for reaction and washing; (iii) adding anti-eIF4Al antibodies conjugated to enzyme catalyzing colorimetric reaction or fluorescent material to the wells for reaction; and (iv) measuring the binding of the anti-eIF4Al antibody to the eIF4Al in the sample . The enzyme catalyzing colorimetric reactions includes, but not limited to, alkaline phosphatase, J3 -galactosidase, and horseradish peroxidase. Where using alkaline phosphatase, bromochloroindolylphosphate (BCIP) , ni ro blue tetrazolium (NBT) , naphtol-AS-Bl-phosphate and ECF may be used as a substrate; in the case of using horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, luminol, ABTS (2,2-Azine- di [3-ethylbenzthiazoline sulfonate] ) and o-phenylenediamine (OPD) may be used as a substrate. Meanwhile, where performing the method of the present invention according to western blot, the method of the present invention comprises the steps of: (i) denaturating the proteins obtained from epidermis (e.g., denaturation with SDS and 2- mercaptoethanol) ; (ii) performing SDS-PAGE with the denatured proteins; (iii) transferring the proteins on gel to NC membrane; (iv) reacting the proteins on NC membrane with a primary antibody, anti-eIF4Al; (v) reacting the primary antibody with a secondary antibody conjugated to enzyme catalyzing colorimetric reaction; (vi) inducing the colorimetric reaction by adding a substrate for the enzyme conjugated to the secondary antibody; and (vii) measuring the intensity of color developed. According to the present invention, it is concluded that patient has responsiveness on ATRA, provided that the signal in step (c) is substantially more intense than that of negative control prepared from epidermis obtained from a vitiligo patient not treated with ATRA. This signal intensity can be compared qualitatively (with naked eyes) or quantitatively (by quantification with a densitometer or spectrophotometer) . A kit for screening responsiveness of a vitiligo patient to all-trans-retinoic acid (ATRA) may include other materials required for immune analysis as well as anti-eIF4Al antibody. For example, where the kit of the present invention is ELISA kit and HRP is used as an enzyme catalyzing colorimetric reaction, such substrates as chloronaphtol, aminoethylcarbazol, diaminobenzidine, luminol, ABTS, and OPD can be included. In another aspect of the present invention, there is provided that a biological marker for responsiveness of a vitiligo patient to drugs effective in treatment or prevention of vitiligo, which comprises at least one type of biological marker selected from the group consisting of (i) eukaryotic translation initiation factor 4A1 (eIF4Al) or nucleic acids encoding eIF4Al, (ii) ribosomal protein L13 (L13) or nucleic acids encoding L13 , and (iii) mediator of RNA polymerase to transcription (MRT) nucleic acids encoding MRT . The marker of the present invention is. a novel usage of the nucleic acids encoding eIF4Al, L13 and MRT. The common descriptions between the biological marker and the method of the present invention are omitted in order to avoid undue redundancy leading to the complexity of this specification. In the present invention, the term "nucleic acid" is considered to include DNA (gDNA and cDNA) and RNA. Nucleoside, a basic unit constituting nucleic acid, includes the natural nucleosides as well as the nucleosides having modified base moieties and/or modified sugar moieties (Scheit, Nucleotide Analogs, John Wiley, New York, 1980) ; Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990))

In still another aspect of the present invention, there is provide a method for screening active ingredients effective in treatment or prevention of vitiligo, which comprises the steps of: (a) treating candidate drugs or biological molecules to epidermis separated from a vitiligo patient; (b) evaluating expression level of at least one gene selected from eukaryotic translation initiation factor 4A1 (eIF4Al) gene , (ii) ribosomal protein L13 (L13) gene and (iii) mediator of RNA polymerase to transcription (MRT) gene in said treated epidermis; and (c) deciding that said candidate drugs or biological molecules are active ingredients for treatment or prevention of vitiligo, provided that said expression level evaluated is substantially identical to that of positive control prepared from normal epidermis . The present invention relates to the biological marker of the present invention, i.e., a novel usage of the nucleic acids encoding eIF4Al, L13 and MRT. Since eIF4Al, L13 and MRT show distinctive expression levels depending on certain drug candidates, they can be used for screening active ingredients effective in treatment or prevention of vitiligo. The common descriptions between the screening method of the present invention and the responsiveness screening method of the present invention as described above are omitted in order to avoid undue redundancy leading to the complexity of this specification. The evaluation of expression level of eIF4Al, L13 and MRT may be carried out on the basis of nucleotide and protein. Where nucleotide is targeted, the analysis of the expression level may be performed by above-mentioned RT-PCR. Where protein is targeted, the antibodies against eIF4Al, L13 and MRT are used. These antibodies (polyclonal and monoclonal antibody) is easily prepared following the procedures known to the art to which the present invention pertains (Harlow, E. and Lane, D., Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1988; Zola, H., Monoclonal Antibodies : A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991) . Preferably, the antibody used in the present invention is monoclonal antibody. The analysis of the expression level, i.e., the analysis of the protein level in cells, can be performed quantitatively or qualitatively according to conventional immunoassay procedures {Enzyme Immunoassay, E. T. Maggio, ed. , CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Enzyme-linked immunosorbent assay (ELISA) , in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed. , Humana Press, NJ, 1984). For example, according to the radioimmunoassay method, the radioisotope (e.g., P³² and S³⁵) labeled antibody of the present invention may be used for the qualitative or quantitative detection of eIF4Al, L13 or MRT protein in a sample by the measurement of immune complex formed with the antigen i.e. eIF4Al, L13 or MRT protein in the sample. In addition, according to the ELISA method, the example of the present method may comprise the steps of: (i) coating wells in a plate with antibodies capable of binding to eIF4Al, L13 or MRT protein; (ii) adding a sample to the wells for reaction and washing; (iii) adding antibodies of the present invention to the wells for reaction; (iv) adding secondary antibodies conjugated to enzyme catalyzing colorimetric reaction or fluorescent material to the wells for reaction; (v) measuring the binding of the secondary antibodies. The enzyme catalyzing colorimetric reactions includes, but not limited to, alkaline phosphatase, β -galactosidase, and horseradish peroxidase. Where using alkaline phosphatase, bromochloroindolylphosphate (BCIP) , nitro blue tetrazolium (NBT) , naphtol-AS-Bl-phosphate and ECF may be used as a substrate; in the case of using horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, luminol, ABTS (2 , 2-Azine-di [3-ethylbenzthiazoline sulfonate] ) and o-phenylenediamine (OPD) may be used as a substrate. Finally, it is concluded that said candidate drugs or biological molecules are active ingredients for the treatment or prevention of vitiligo, provided that the expression level analyzed is substantially identical to that of positive control prepared from normal epidermis .

In still further another aspect of the present invention, there is provided a kit for screening active ingredients effective in treatment or prevention of vitiligo, which comprises at least one primer set selected from the group consisting of (i) primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) primer set hybridizing with ribosomal protein L13 (L13) gene and (iii) primer set hybridizing with mediator of RNA polymerase to transcription (MRT) gene. In another aspect of the present invention, there is provided a kit for screening active ingredients effective in treatment or prevention of vitiligo, which comprises at least one antibody selected from the group consisting of (i) antibody capable of binding to eukaryotic translation initiation factor 4A1 (eIF4Al) , (ii) antibody capable of binding to ribosomal protein L13 (L13), and (iii) antibody capable of binding to mediator of RNA polymerase to transcription (MRT) . The kit in the present invention is used to perform the above-mentioned screening methods. Accordingly, the common descriptions are omitted in order to avoid undue redundancy leading to the complexity of this specification.

In still another aspect of the present invention, there is provided a method for prognosis of vitiligo, which comprises the steps of: (a) isolating total RNAs from normal epidermis of a vitiligo patient or vitiligo epidermis of said vitiligo patient ,- (b) synthesizing cDNA with said isolated. total RNAs; (c) conducting PCR with primer set hybridizing with eIF4Al gene and said cDNA as a template,- (d) Obtaining electrophoresis bands of said PCR product by electrophoresis; and (e) deciding (i) that the possibility of vitiligo development in said normal epidermis is high, provided that the intensity of the PCR bands from said normal epidermis of said vitiligo patient is more reduced than that of PCR bands from positive control obtained from normal epidermis or (ii) that vitiligo has been improved, provided that the intensity of PCR bands from said vitiligo epidermis of said vitiligo patient is more increased than that of the previously prepared PCR bands . In further another aspect of the present invention, there is provided a method for prognosis of vitiligo, which comprises the steps of: (a) preparing a cell homogenate from normal epidermis of a vitiligo patient or vitiligo epidermis of said vitiligo patient ,- (b) incubating said cell homogenate with anti-eIF4Al antibody; (c) detecting the signal of antigen-antibody immune response between the proteins in the cell homogenate and anti-eIF4Al antibody; and (d) deciding (i) that the possibility of vitiligo development in said normal epidermis is high, provided that the intensity of the signal from said normal epidermis of said vitiligo patient is more reduced than that of the signal from positive control obtained from normal epidermis or (ii) that vitiligo has been improved, provided that the intensity of the signal from said vitiligo epidermis of said vitiligo patient is more increased than that of the previously detected signal. In another aspect of the present invention, there is provided a kit for prognosis of vitiligo, which comprises primer set hybridizing with eIF4Al gene. In another aspect of the present invention, there is provided a kit for prognosis of vitiligo, which comprises an anti-eIF4Al antibody. The term "prognosis" means an estimate of the future of vitiligo . According to the prognosis method of the present invention performed on the basis of RNA, the PCR band finally obtained is analyzed, and then, it is concluded that (i) the possibility of vitiligo development in said normal epidermis is high, provided that the intensity of the PCR bands from said normal epidermis of said vitiligo patient is more reduced than that of PCR bands from positive control obtained from normal epidermis. Namely, it is appreciated that vitiligo is likely to be expanded to normal epidermis of the vitiligo patient showing such band intensity. In addition, among the final PCR bands, it is concluded that (ii) vitiligo has been improved, provided that the intensity of PCR bands from said vitiligo epidermis of said vitiligo patient is more increased than that of the PCR bands previously prepared through the same procedures prior to the preparation of the PCR band, or that vitiligo has become worse, provided that the intensity is more reduced. According to the prognosis method of the present invention performed on the basis of protein, among the signal of antigen- antibody immune response obtained finally, it is concluded that (i) the possibility of vitiligo development in said normal epidermis is high, provided that the intensity of the signal from said normal epidermis of said vitiligo patient is more reduced than that of PCR bands from positive control obtained fr-om normal epidermis . In addition, it is concluded that (ii) vitiligo has been improved, provided that the intensity of the signal from said vitiligo epidermis of said vitiligo patient is more increased than that of the signal previously detected through the same procedures prior to the detection of the signal, or that vitiligo bias become worse, provided that the intensity is more reduced. According to the method of the present invention, it is possible to estimate the future of vitiligo relatively accurately.

BRIF DESCRIPTION OF THE DRAWINGS Figure 1 is a photograph showing differential display of ATRA-treated and vehicle-treated vitiligo epidermides using kits incorporating an annealing control primer (ACP) . Different levels of certain mRNA expression between normal (N) and vehicle-treated epidermis (V) were partially or completely restored after treatment with ATRA (A) . Although the bands (arrow) and their expression levels were variable in different patients (as in clone 3) , 10 bands from six patients showed the restoration of expression levels up to normal levels with ATRA. Figure 2 is DNA sequence and primers of eIF4Al. One of the mRNA bands that showed restored expression level with ATRA application was cloned. The sequence between 841 and 1261

(underline) has more than 99% homology with eukaryotic translation initiation factor 4A1 (eIF4Al) , whose size is approximately 1.4 kb . A set of primers was designed for RT-PCR; one of them is shown in capital bold characters, and the other shown in bold character. Figure 3a is a photograph showing semi-quantitative RT-PCR analysis and relation to clinical responsiveness. RT-PCR was performed in five patients with primers specific for the four known genes, i.e. eukaryotic translation initiation factor 4A1 (eIF4Al) , ribosomal protein L13 (L13) , mediator of RNA polymerase to transcription (MRT) , and ribosomal phosphoprotein PO (PO) with GAPDH as an internal standard. The levels of eIF4Al mRNA expression in vehicle-treated depigmented epidermis (V) were lower than those in normally pigmented epidermis (N) . With ATRA treatment (A) , the levels were elevated to near normal levels in three patients (patients #1, #2, #3) who showed a favorable clinical response to ATRA, whereas no change was observed in the other two patients (patients #4, #5) who showed no difference between the clinical response from ATRA and those from vehicle treatment. L13, MRT, and RO mRNAs showed similar correlation in certain patients . Figure 3b is a graph showing semi-quantitative RT-PCR analysis and relation to clinical responsiveness. The relative amount of mRNA was accomplished by calculating the ratio of the amount of each mRNA relative to the amount of GAPDH. The ratios of normalized densitometric values between the depigmented and normally pigmented epidermis were analyzed in vehicle-treated and ATR-treated specimens. These ratios examined the relation to clinical response, and correlated with eIF4Al mRNA in all five patients. Fig 4 is a graph showing the result of Real-time PCR of eIF4Al. Fig 5 is a photograph showing western blot analysis for eIF4Al protein expression. N is vehicle-treated normally pigmented epidermis, V is vehicle-treated depigmented epidermis and A is ATRA-treated depigmented epidermis. Recombinant eIF4Al protein was used as a positive control, β-actin was used for an internal standard. Fig 6 is a photograph showing western blot analysis for the expression pattern of eIF4Al protein in normally pigmented and depigmented epidermis.

EXAMPLES

MATERIALS AND METHODS Patients Six patients (two men and four women) diagnosed with a generalized type of vitiligo were included in this study. Their ages were between 21 and 52 years (mean age, 38 years) . No patients had received treatment in the previous month. A placebo- controlled, paired-comparison, left-right study using ATRA and vehicle was performed for 3-6 months with all patients. After informed written consent had been given, epidermal specimens were obtained from the patients using suction. The normally pigmented epidermis from the same patient was taken as a control. Negative pressure, between 200 mmHg and 250 mmHg, was applied to the normally pigmented epidermis and the depigmented epidermis with or without ATRA treatment to make suction blisters . The roofs of the suction blisters from all patients were used for RNA isolation.

GeneFishing™ reverse transcription-polymera.se chain reactio Total RNAs from the suction-blistered epidermides were isolated by gentle homogenization using Trizol (GibcoBRL, 15596- 026, NY) . Reverse transcription (RT) was conducted using the GeneFishing™ DEG kits (Seegene, DEK 3101 & 3104, South Korea) as follows : 3 μg of the extracted RNAs was put into a tube containing RNase-free water and 10 μM dT-ACPl with a final volume of 9.5 μL using a DNA Thermal Cycler 9600 (Perkin Elmer) . Equal amounts of RNA were compared to identify differentially expressed bands between the samples. The mixture was incubated at 80 °C for 3 min and spun briefly after chilling on ice. Twenty microliters of reaction solution, consisting of 5x RT buffer, 2 mM dNTP, RNase inhibitor, and M-MLV reverse transcriptase (Promega, M170B, WI) , was added into the mixture. The tube was incubated at 42 °C for 90 min, heated at 94 °C for 2 min, chilled on ice and spun briefly. The synthesized first-strand cDNA was diluted by adding 80 μL of RNase-free water. The cDNA samples were stored at -20 °C until use. PCR amplification was conducted using the same GeneFishing™ DEG kits (Seegene, DEK 3101 & 3104, South Korea) in 50 μL of reacting solution, consisting of lOx buffer without MgCl₂, 25 mM MgCl₂, 5 μM arbitrary ACPs, 10 μM dT-ACP2, 2 mM dNTP, 2.5 U Taq DNA polymerase (Genecraft, GC-002-250, Fance) , and 50 ng of first-strand cDNA, using a DNA Thermal Cycler 9600 (Perkin Elmer) . Each kit comprises 20 different arbitrary annealing control primers. The thermal cycler was preheated to 94 °C before the tubes were placed in it. The program of PCR amplification was as follows; 1 cycle at 94 °C for 5 min, 50 °C for 3 min, and 72 °C for 1 min, 40 cycles at 94 °C for 40 s, 65 °C for 40 s, and 72 °C for 40 s, and 72 °C for 5 min. The DNA fragments produced by PCR were separated by electrophoresis on a 2% agarose gel. The bands were photographed using Polaroid film under ultraviolet light after ethidium bromide staining and analyzed by a densitometry (Pharmacia) .

Cloning and sequencing Differentially expressed bands were extracted from the agarose gel using the GENECLΞAN II kit, a GLASSMILK gel extraction kit (Q-BIOgene, #1001-400, UK) . Each DNA fragment was cloned using the TOPO TA Cloning kit (Invitrogen, K4500-01, CA) as follows: the PCR product with a single 3 adenine overhang was inserted into a pCR^*2.1-TOPθ^" vector and the recombinant vector transformed into competent E. coli . The amplified DNA was extracted using an AccuPrep Plasmid Extraction kit (Bioneer, #K-3030-l, South Korea) . Sequencing of the DNA was performed by Macrogen Company (South Korea) .

Primer sequence The DNA sequence of each gene was compared to and confirmed by comparison with sequences in GenBank (NIH, MD) . The primer sequence of each gene was designed using the PRIMER 3 program (MIT, MA) . The synthesis of primers was performed by Bioneer Company (South Korea) . Seni-guantitative RT-PCR analysis cDNA was synthesized using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Boehringer Mannheim, 1483 188, Germany) with the RNAs that had been extracted from five of the six vitiligo patients. Sense and antisense primers were designed based on subcloned cDNA fragment sequences (Figure 1) . Primers for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as an internal standard. The GAPDH sense primer was 5 - ACCACAGTCCATGCCATCAC-3 and the anti-sense primer was 5 - TCCACCACCCTGTTGCTGTA-3 ,- these generated a 452-bp product. PCR amplification was performed in a 40 μL reaction, consisting of lOx reaction buffer, 2.5 mM MgCl₂, 250 μM dNTPs, 100 ng PCR primers, 0.5 U Taq DNA polymerase, and 20-50 ng of DNA, using a DNA Thermal Cycler 9600 (Perkin Elmer) . The PCR amplification for the three primer sets (3-1, 64-1 and 77-4) consisted of 25 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The program for 14-1 was 30 cycles at 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1.5 min. A program of 25 cycles at 94 °C for 1 min, 59 °C for 1 min, and 72 °C for 1 min was used for GAPDH. The DNA fragments produced by PCR were separated by electrophoresis on 2% agarose gels. The bands were photographed using Polaroid film under ultraviolet light after ethidium bromide staining. The optical density of each band was analyzed by densitometry (Pharmacia) . The relative amount of mRNA was accomplished by calculating the ratio of the amount of each mRNA relative to the amount of GAPDH.

Real - time PCR Total RNAs isolated from seven vitiligo patients were used. cDNA was synthesized from RNAs using the First Strand cDNA

Synthesis Kit (Boehringer Mannheim, Germany) . The TaqMan^® universal PCR master mix kit (PE Applied Biosystems) was used in the amplification reaction of the TaqMan^® PCR. Oligonucleotide PCR primers and TaqMan^® probes, which contained FAM as the 5 reporter and TAMRA as the 3 quencher, were designed using the Primer- Express program (Perkin Elmer) and synthesized commercially by MuenBio Inc. (Baltimore, MD, USA) . The sequences were as follows: eIF41 sense primer 5 - CTGGCCAGAGGCATTGATGT-3 and eIF41 antisense primer 5 - CCACACCTTTACGGCCAAAC-3 , with eIF41 TaqMan^® probe 5 - TTTCTTTAGTCATCAACTATGACCTTCCCACCAA-3 . By using an endogenous control as an active reference, quantitation of an mRNA target can be normalized for differences in the amount of total RNAs added to each reaction. β-Actin mRNA was assessed using the Human hu β-actin (20X) kit (PE Applied Biosystems) . Primers and TaqMan^® fluorogenic probes were added to a final concentration of 0.9 μM and 0.25 μM, respectively. Total PCR volume was 20 μL, including 1 μL of the cDNA, which is equal to 50 ng total RNAs. Triplicate reaction tubes were set up for each sample and transcript under investigation. The tubes were placed in an ABI Prism 7900HT System programmed for 40 sequential cycles, each comprising 1 cycle of 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were analyzed using the SDS 2.1 program (PE Applied Biosystems) . The comparative Ct method uses arithmetic formulas to achieve the same result for relative quantitation. The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by 2^"ΔΔcτ. The results from triplicate reaction were analyzed using Wilcoxon' s Signed Ranks Test to examine the differences between the ATRA-treated and vehicle-treated samples from each patient. A p value of less than 0.05 was considered statistically significant. Synthesis of anti -rheIF4Al antibody For cloning of recombinant human eIF4Al (rheIF4Al) , human keratinocyte cells were isolated as previously described (Boyce ST.,et al., J^". Invest Der atol 81 : 33s-40s (1986) ) . Total RNAs were extracted from primary cultured human keratinocyte cells by the TRIZOL (Invitrogen) method. cDNA was generated by reverse transcription using oligo-dT primer and MMLV RT-PCR kit (Promega) . eIF4Al cDNA was amplified by PCR using 5"-ATGTCTGCGAGCCAGGATTCC- 3" as the forward primer and 5"-TCAGATGAGGTCAGCAACATTG-3 " as the reverse primer, using the following cycling parameters: 95 °C for 10 min, and 35 cycles of 30 s at 95 °C, 30 s at 58 °C, and 1.5 min at 72 °C, followed by an incubation at 72 °C for 10 min. The PCR product was subsequently cloned into PCR2.1-TOPO vector (Invitrogen, Frederick, MD) . The cloned gene was sequenced and confirmed to correspond to the published sequence for human eIF4Al (gene bank accession number BC009585) . A polyclonal anti- eIF4Al antibody was synthesized using the cloned amplified rheIF4Al gene (Labfrontier Co., Korea) .

Western blot analysis for eIF4Al The epidermal specimens were frozen by dipping them into liquid nitrogen. The frozen tissues were ground to make a powder and homogenized in ice-cold homogenization buffer containing 50 mM Tris-base (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20, and protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 5 μg/ml aprotinin, and 5 μg/ml leupeptin) . The homogenates were centrifuged at 12,000 X g for 30 min at 4°C, and the supernatants collected. The protein concentration in the supernatant was determined using a DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) . Equal amounts of proteins (20 μg) were resolved using 7% SDS-PAGE and transferred to nitrocellulose membranes . The membranes were incubated in a blocking solution of 5% non-fat dry milk in Tris-buffered saline (TTBS) containing 10 mM Tris (pH 7.6), 150 mM NaCl, and 0.1% Tween-20 for 1 h at room temperature . The membranes were incubated with the rabbit polyclonal anti-eIF4Al antibody diluted 1:4,000 in the blocking solution, overnight at 4 °C. Following incubation, the membranes were washed in TTBS and further incubated with an anti-rabbit horseradish peroxidase-conjugated antibody (Pharmingen, San Diego, CA) at a 1:2,000 dilution in the blocking solution for 1 h at room temperature. After the membranes were washed in TTBS for 30 min, they were treated with an enhanced chemiluminescence solution (ECL kit; Amersham Life Science, Buckinghamshire, England) for 1 min and exposed to an X-ray film (Hyperfilm, Amersham life science, Buckinghamshire, England) . To monitor the amount of protein loaded into each lane, the membranes were reprobed with an antibody against β-actin. After stripping the membranes with a stripping buffer (0.1 M glycine, pH 2.5), they were incubated with a 1:5,000 dilution of mouse monoclonal anti-actin antibody (Sigma, USA) in the blocking solution at room temperature for 1 h. Subsequently, the membranes were incubated with a secondary antibody and processed as described above . The films were developed immediately and the protein bands were analyzed densitometrically .

Comparision of eIF4Al protein expression in normally pigmented and depigmented epidermis The difference of eIF4Al protein expression levels in between in normal and depigmented epidermis was analyzed without concerning the response related to ATRA treatment. Normally pigmented and depigmented epidermis of 22 patients were used as specimens, and analysis method was almost similar to western blot analysis described above. β-actin was used for an internal standard.

RESULTS

Differentially expressed amplified cDNA products The expression levels of certain mRNAs were compared among normally pigmented epidermis, depigmented epidermis treated with vehicle, and depigmented epidermis treated with ATRA from the same patients . The levels of approximately 10 mRNAs expressed were different between the normally pigmented and depigmented epidermis. The levels were usually lower in the depigmented epidermis than in the normally pigmented epidermis, although the degree of difference was varied between patients (Figure 1) . The mRNA expression levels, which were decreased in the vehicle-treated epidermis compared with the normally pigmented epidermis, were partially or completely restored after treatment with ATRA (arrow) , whereas those of others were decreased (arrow head) (Figure 1) .

Detection of gene from cloning and sequencing The mRNAs of the bands showing distinct results were amplified using the TOPO TA Cloning kit (Invitrogen, K4500-01, CA) . The sequences from the four genes corresponded to parts of known genes. They were matched with eukaryotic translation initiation factor 4A1 (eIF4Al) (Figure 2) , ribosomal protein L13 (L13) , mediator of RNA polymerase to transcription (MRT) , and ribosomal phosphoprotein PO (PO) gene sequences (Table 1) .

Table 1. Characteristics of genes with elevated expression levels following ATRA treatment in vitiligo.

Clone PCR primer (5' -3' ) Fragment mRNA size Homology size (bp) (kb)

Results from RT-PCR and Correlation with clinical responses The levels of eIF4Al mRNA expression in depigmented epidermis were lower than those in normally pigmented epidermis. The results were similar with two sets of primers (Figure 2) , one of which amplified the full sequence (approximately 1.3 kb) . The levels were partially restored with ATRA treatment in three (patients #1, #2, #3) of five patients who showed favorable clinical responses to ATRA. The levels of eIF4Al mRNA expression, on the other hand, were not changed with ATRA in the two patients (patients #4, #5) who showed no difference between the clinical responses from ATRA and those from the vehicle treatment (Figure 3a and 3b). L13, MRT, and PO mRNAs showed a similar correlation in one, and two patients respectively (Figure 3a and 3b) .

Real - time PCR results and western blot analysis of eIF4Al Real-time PCR was performed with specimens obtained from seven vitiligo patients. #1, #2, #4 and #5 patients showed favorable clinical responses to ATRA, and #6, #7 and #8 patients showed no clinical responses to ATRA in figure 4. The results from real-time PCR of eIF4Al between the normally pigmented epidermis and the vehicle-treated depigmented epidermis did not always agree with those from semi-quantitative RT-PCR. The results of difference between ATRA-treated and vehicle-treated epidermis were more remarkable than those from semi-quantitative RT-PCR. The amount of eIF4Al mRNA in ATRA-treated epidermis was higher than that in vehicle-treated epidermis of the four (patients #1, #2, #4, #5) patients with favorable outcome to ATRA, whereas it was lower or similar in the other three patients (patients #6, #7, #8) . Western blot analysis was done in two patients (patients #9, #10) . In both patients, the vehicle-treated normally pigmented epidermis expressed a higher amount of eIF4Al protein compared to the vehicle-treated depigmented epidermis (Figure 5) . One of them (patient #9) , with a favorable response to ATRA, showed a higher amount of 47 kDa eIF4Al protein expression in the ATRA-treated epidermis, whereas the other patient with no response to ATRA (patient #10) , had similar amounts of eIF4Al protein expression.

Comparision of eIF4Al protein expression in normally pigmented and depigmented epidermis As shown in Figure 6, eIF4Al protein expression level in normal pigmented epidermis was higher than that in normal pigmented epidermis. Consequently, it showed that the determination of eIF4Al protein expression level enable could be applied for prognosis of vitilgo.

REFERENCES

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Claims

What is claimed is: 1. A method for screening responsiveness of a vitiligo patient to drugs effective in treatment or prevention of vitiligo, which comprises the steps of: (a) isolating total RNAs from an epidermis obtained from the vitiligo patient treated with the drugs,- (b) synthesizing cDNA with said isolated total RNAs; (c) conducting PCR with at least one type of primer set selected from the group consisting of (i) primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) primer set hybridizing with ribosomal protein L13 (L13) gene and (iii) primer set hybridizing with mediator of RNA polymerase to transcription (MRT) gene, and said cDNA as a template; (d) obtaining electrophoresis bands of said PCR product by electrophoresis; and (e) deciding that said patient has responsiveness on said drug, provided that said electrophoresis band pattern is substantially identical to that of positive control prepared from normal epidermis.

2. The method according to claim 1, wherein the drug is selected from the group consisting of all-trans-retinoic acid (ATRA), vitamin C, trichloroacetic acid and calcipotriol .

3. The method according to claim 1, wherein the primer set hybridizes with eIF4Al gene.

4. The method according to claim 1, wherein the step (e) is performed by comparing the intensity of electrophoresis-bands with naked eyes or with a densitometer.

5. A method for screening responsiveness of a vitiligo patient to all-trans-retinoic acid (ATRA) used for treatment of vitiligo, which comprises the steps of: (a) isolating total RNAs from epidermis obtained from the vitiligo patient treated with ATRA; (b) synthesizing cDNA with said isolated total RNAs; (c) conducting PCR with primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene and said cDNA as a template; (d) obtaining electrophoresis bands of said PCR product by electrophoresis; and (e) deciding that said patient has responsiveness on said drug, provided that the electrophoresis band pattern is substantially identical to that of positive control prepared from normal epidermis.

6. A kit for screening responsiveness of a vitiligo patient to drugs effective in treatment or prevention of vitiligo, which comprises at least one type of primer set selected from the group consisting of (i) primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) primer set hybridizing with ribosomal protein L13 (L13) gene, and (iii) primer set hybridizing with mediator of RNA polymerase to transcription (MRT) gene.

7. The kit according to claim 6, wherein the drug is selected from the group consisting of all-trans-retinoic acid (ATRA) , vitamin C, trichloroacetic acid and calcipotriol .

The kit according to claim 6, wherein the primer set hybridizes with eIF4Al gene.

9. A biological marker for responsiveness of a vitiligo patient to drugs effective in treatment or prevention of vitiligo requiring administration of corticosteroid, which comprises at least one type of biological marker selected from the group consisting of (i) eukaryotic translation initiation factor 4A1 (eIF4Al) or nucleic acids encoding eIF4Al, (ii) ribosomal protein L13 (L13) or nucleic acids encoding L13, and (iii) mediator of RNA polymerase to transcription (MRT) nucleic acids encoding MRT.

10. The biological marker according to claim 9, wherein the drug is selected from the group consisting of all-trans-retinoic acid (ATRA), vitamin C, trichloroacetic acid, and calcipotriol .

11. The biological marker according to claim 9, wherein the biological marker is eIF4Al or nucleic acids encoding eIF4Al.

12. A method for screening responsiveness of a vitiligo patient to all-trans-retinoic acid (ATRA) used for treatment of vitiligo : (a) preparing a cell homogenate containing total proteins from epidermis obtained from the vitiligo patient treated with ATRA; (b) incubating said cell homogenate with anti-eIF4Al an ibody; (c) detecting the signal of antigen-antibody immune response between the proteins in the cell homogenate and anti-eIF4Al antibody; (d) deciding that said patient has responsiveness on said ATRA, provided that the signal in step (c) is substantially more intense than that of negative control prepared from epidermis obtained from a vitiligo patient not treated with ATRA.

13. A kit for screening responsiveness of a vitiligo patient to all-trans-retinoic acid (ATRA) , which comprises anti-eIF4Al antibody.

14. A method for screening active ingredients effective in treatment or prevention of vitiligo, which comprises the steps of: (a) treating candidate drugs or biological molecules to epidermis separated from a vitiligo patient; (b) evaluating the expression level of at least one gene selected from eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) ribosomal protein L13 (L13) gene and (iii) mediator of RNA polymerase to transcription (MRT) gene; and (c) deciding that said candidate drugs or biological molecules are active ingredients for treatment or prevention of vitiligo, provided that said expression level evaluated is substantially identical to that of positive control prepared from normal epidermis .

15. The method according to claim 14, wherein the drug is selected from the group consisting of all-trans-retinoic acid

(ATRA), vitamin C, trichloroacetic acid, and calcipotriol .

16. The method according to claim 1, wherein the step (c) is performed by comparing the intensity of electrophoresis bands with naked eyes or with a densitometer.

17. A kit for screening active ingredients effective in treatment or prevention of vitiligo, which comprises at least one primer set selected from the group consisting of (i) primer set hybridizing with eukaryotic translation initiation factor 4A1 (eIF4Al) gene, (ii) primer set hybridizing with ribosomal protein L13 (L13) gene and (iii) primer set hybridizing with mediator of RNA polymerase to transcription (MRT) gene.

18. A kit for screening active ingredients effective in treatment or prevention of vitiligo, which comprises at least one antibody selected from the group consisting of (i) antibody capable of binding to eukaryotic translation initiation factor 4A1 (eIF4Al) , (ii) antibody capable of binding to ribosomal protein L13 (L13) , and (iii) antibody capable of binding to mediator of RNA polymerase to transcription (MRT) .

19. A method for prognosis of vitiligo, which comprises the steps of : (a) isolating total RNAs from normal epidermis of a vitiligo patient or vitiligo epidermis of said vitiligo patient; (b) synthesizing cDNA with said isolated total RNAs; (c) conducting PCR with primer set hybridizing with eIF4Al gene and said cDNA as a template; (d) obtaining electrophoresis bands of said PCR product by electrophoresis; and (e) deciding (i) that the possibility of vitiligo development in said normal epidermis is high, provided that the intensity of the PCR bands from said normal epidermis of said vitiligo patient is more reduced than that of PCR bands from positive control obtained from normal epidermis or (ii) that vitiligo has been improved, provided that the intensity of PCR bands from said vitiligo epidermis of said vitiligo patient is more increased than that of the previously prepared PCR bands .

20. A method for prognosis of vitiligo, which comprises the steps of : (a) preparing a cell homogenate from normal epidermis of a vitiligo patient or vitiligo epidermis of said vitiligo patient; (b) incubating said cell homogenate with anti-eIF4Al antibody; (c) detecting the signal of antigen-antibody immune response between the proteins in the cell homogenate and anti-eIF4Al antibody; and (d) deciding (i) that the possibility of vitiligo development in said normal epidermis is high, provided that the intensity of the signal from said normal epidermis of said vitiligo patient is more reduced than that of the signal from positive control obtained from normal epidermis or (ii) that vitiligo has been improved, provided that the intensity of the signal from said vitiligo epidermis of said vitiligo patient is more increased than that of the previously detected signal.

21. A kit for prognosis of vitiligo, which comprises primer set hybridizing with eIF4Al gene.

22. A kit for prognosis of vitiligo, which comprises anti eIF4Al antibody.